ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
111 Clinical electrophysiology
Sunday, May 04, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 330–351/C0101–C0122
Organizing Section: Visual Neuroscience
Program Number: 330 Poster Board Number: C0101
Presentation Time: 8:30 AM–10:15 AM
Assessment of retinal structural and functional characteristics in
eyes with autoimmune retinopathy
Nithya Rajagopalan1, Kathleen E. Guinn1, Mohammad A. Sadiq1,
Mostafa S. Hanout1, Salman Sarwar1, Jose Maya1, Liz J. Zapata1,
Stuart G. Coupland2, Quan Dong Nguyen1, Yasir Sepah1. 1Ocular
Imaging Research and Reading Center, Stanley M. Truhlsen Eye
Institute, University of Nebraska Medical Center, Omaha, NE;
2
Ophthalmology, Cellular and Molecular University of Ottawa,
Ottawa, ON, Canada.
Purpose: To evaluate the thicknesses of individual retinal layers, and
the correlation between structural changes and functional loss using
spectral domain optical coherence tomography (SD-OCT) scans and
electroretinograms (ERG).
Methods: The Heidelberg Spectralis HRA+OCT device was
utilized to obtain SD-OCT raster scans of 20 eyes from 10 patients
serologically diagnosed with AIR.
The Heidelberg Heyex software (version 5.2) was used to segment
selected retinal layers along a 5 mm horizontal scan passing through
the fovea. Retinal layers analyzed include full retinal thickness
(FRT), retinal pigment epithelium (RPE), photoreceptor layer (PRL),
bipolar cell layer (BPL), combined ganglion cell and inner plexiform
layers (GCL+), nerve fiber layer (NFL), and combined GCL+ and
NFL layers (GCL+/NFL).
Changes in the thicknesses of the layers were assessed in 0.5 mm
increments along the B-scan in the central, nasal, and temporal
regions. These recorded values were compared to corresponding
values of 51 eyes from 51 subjects with no known ocular pathology.
Full-field ERGs were obtained at corresponding visits and were
interpreted by a grader blinded to the diagnoses and OCT findings.
Results: Three patients (6 eyes) were excluded from the analysis
due to the presence of confounding ocular pathologies. For the
remaining 7 patients (14 eyes) included in the analysis, mean/median
age was 56 (range: 33 to 83), with 5 males (62.5%). Among the 51
controls, mean age was 52 (range: 40 to 75), with 26 males (51%).
ERG findings demonstrated a functional deficit that showed a strong
correlation with structural loss.
Conclusions: Eyes with AIR show a loss of retinal tissue compared
to eyes with no known ocular pathology. The greatest loss appears to
occur in the RPE and PRL. ERG findings correlate strongly with the
loss of tissue seen in these layers. Thus, therapeutic options may be
targeted to preserve these two regions of the retina.
Commercial Relationships: Nithya Rajagopalan, None; Kathleen
E. Guinn, None; Mohammad A. Sadiq, None; Mostafa S. Hanout,
None; Salman Sarwar, None; Jose Maya, None; Liz J. Zapata,
None; Stuart G. Coupland, None; Quan Dong Nguyen, Genentech
(F), Regeneron (F); Yasir Sepah, None
Program Number: 331 Poster Board Number: C0102
Presentation Time: 8:30 AM–10:15 AM
Role of α-enolase Autoantibodies related to Rod-bipolar cell
function in Non-paraneoplastic Autoimmune Retinopathy
Stuart G. Coupland1, 2, Lulu L.C.D. Bursztyn3, 4, Jillian Belrose3,
J. Alexander Fraser5, 6, Alain A. Proulx3, 4. 1uOttawa Eye Institute,
University of Ottawa, Ottawa, ON, Canada; 2Ottawa Hospital
Research Institute, Ottawa, ON, Canada; 3Schulich School of
Medicine and Dentistry, Western University, London, ON, Canada;
4
Ivey Eye Institute, Western University, London, ON, Canada;
5
Clinical Neurological Sciences, Western University, London, ON,
Canada; 6Ophthalmology, Western University, London, ON, Canada.
Purpose: Autoimmune retinopathies are rare disorders whose
visual prognosis is typically poor. Here we report a case of
α-enolase mediated npAIR with marked ERG dysfunction which
demonstrated dramatic clinical improvement with a short course of
oral corticosteroids, and concomitant disappearance of α-enolase
autoantibodies and with unique electrophysiological presentation that
points to the role of α-enolase autoantibodies in modulating rod onbipolar cell function, distinct from cone on-bipolar cell function.
Methods: ERGs were recorded to ISCEV standards for rod,
combined rod-cone, cone and 30 Hz flicker conditions. In addition,
ERG responses were recorded to sawtooth flicker of luminance of
150 cd/m2 flickering at 8 Hz. with 100% contrast using Espion e3
(Diagnosys LLC) system with DTL microconductive fiber electrodes.
Both rapid-ON and rapid-OFF components were examined for
reduced b- to d-wave amplitude ratio indicative of ON pathway
dysfunction. Anti-retinal autoantibody testing by Western blot
analysis was also performed in the same time period as ERG.
After short course of oral corticosteroids patient showed marked
improvement in visual function and was seen 12 months later for
repeat testing.
Results: Initially, ERG was abnormal to scotopic flash, photopic flash
and 30Hz flicker stimulation with a definite negative ERG appearance
noted. On-off ERG using 8 Hz sawtooth flicker demonstrated
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
complete loss of the cone on-response with preservation of a normal
off-response, suggesting selective loss of cone depolarizing bipolar
cell (DBC) function in the inner retina. Anti-retinal autoantibody
testing by Western blot was positive for 46-kDa (α-enolase), 50kDa and 62-kDa proteins. At one year follow-up ERG showed full
recovery of rod function with no improvement in photopic cone ERG.
On-Off ERG showed only slight evidence of a cone on-response
confirming selective loss of cone DBC function in the inner retina.
Repeat anti-retinal antibody testing demonstrated the previously
identified 46kDa α-enolase reactive band was not observed.
Conclusions: Since rod ERG b-wave amplitude is entirely dependent
on the rod DBC, these findings support a pathological role for
α-enolase autoantibodies related to rod-bipolar cell function.
Identification of other cases which exhibit such improvements and
the associated autoantibody activity may enhance our understanding
of disease pathogenesis.
Commercial Relationships: Stuart G. Coupland, None; Lulu
L.C.D. Bursztyn, None; Jillian Belrose, None; J. Alexander
Fraser, None; Alain A. Proulx, None
Program Number: 332 Poster Board Number: C0103
Presentation Time: 8:30 AM–10:15 AM
Scotopic and Photopic ERG Responses in Pediatric Patients with
Usher Syndrome
Jena Tavormina1, Ronald M. Hansen1, 2, Anne Moskowitz1, 2, Heidi L.
Rehm3, 2, Margaret Kenna4, 2, Anne Fulton1, 2. 1Ophthalmology, Boston
Children’s Hospital, Boston, MA; 2Harvard Medical School, Boston,
MA; 3Brigham and Women’s Hospital, Boston, MA; 4Otolaryngology,
Boston Children’s Hospital, Boston, MA.
Purpose: To evaluate scotopic and photopic ERG responses in
pediatric patients with a genetic diagnosis of Usher Syndrome, a
recessively inherited ciliopathy characterized by hearing loss and
retinal degeneration affecting both rods and cones.
Methods: Twenty-two patients (age 2 months to 23 years) with
USH2A (n=13) or MYO7A (n=9) disease were studied. ERG
responses to a range of full-field scotopic and photopic stimuli
(including the ISCEV standard conditions) were recorded and
compared to responses in healthy controls (n=72). A model of
the activation of phototransduction was used to estimate rod
photoreceptor sensitivity (SROD) and saturated amplitude (RROD).
Post-receptor b-wave sensitivity was characterized by the stimulus
that produced a half maximum response (log σ) and saturated b-wave
amplitude (VMAX). Dark adapted thresholds were estimated using a
two-alternative, forced choice method in all patients.
Results: Responses to 30 Hz flickering stimuli were detected in all
USH2A patients (median 99, range 7 to 169 μV) and all MYO7A
patients (median 10, range 1 to 18 μV). Photopic b-wave amplitude
was within the normal range in nine of the 13 USH2A patients and
none of the nine MYO7A patients. In the seven MYO7A patients
whose photopic b-wave was detectable, the responses were less than
20% of the normal mean. Scotopic b-waves were detectable in all
USH2A patients but only six MYO7A patients; only four USH2A
patients had normal scotopic b-wave amplitudes. Responses were
sufficient for estimation of both photoreceptor and post-receptor
response parameters in nine USH2A and only two MYO7A patients.
For these 11 patients, deficits in scotopic post-receptor b-wave
sensitivity were greater than deficits in rod photoreceptor sensitivity.
Median dark adapted threshold in the 22 patients was elevated 0.9
(range 0.13-2.77) log units compared to normal and did not differ
between USH2A and MYO7A patients. Threshold elevation was
outside the 99% prediction interval for normal for 14 of the 22
patients, whereas ERG log σ values were outside the 99% prediction
interval for normal for 21 of 22.
Conclusions: The greater deficit in post-receptor than photoreceptor
sensitivity is consistent with a ciliopathy. In this sample, ERG log σ
was more often abnormal than the dark adapted threshold. Therefore,
the ERG may be a more sensitive detector of retinal dysfunction than
the dark adapted threshold in children at risk for Usher Syndrome.
Commercial Relationships: Jena Tavormina, None; Ronald M.
Hansen, None; Anne Moskowitz, None; Heidi L. Rehm, None;
Margaret Kenna, None; Anne Fulton, None
Support: NIH Grant EY010597
Program Number: 333 Poster Board Number: C0104
Presentation Time: 8:30 AM–10:15 AM
Evaluation of retinal architecture in glaucoma patients using
spectral-domain optical coherence tomography
Kathleen E. Guinn, Nithya Rajagopalan, Peter Bracha, Mohammad
A. Sadiq, Jose Maya, Mohamed Ibraheem, Sushma Rai, Vikas Gulati,
Quan Dong Nguyen, Yasir Sepah. Ocular Imaging Research and
Reading Center, Stanley M. Truhlsen Eye Institute, University of
Nebraska Medical Center, Omaha, NE.
Purpose: To examine the thickness of retinal layers in patients with
established diagnosis of glaucoma compared to a normative database.
Methods: Spectral-domain optical coherence tomography (SDOCT) images were acquired for 36 eyes (24 consecutive patients)
with known diagnosis of glaucoma and no known macular disease.
Horizontal B-scans passing through the fovea were then segmented
using Heyex v.5.2 software; retinal thickness was recorded for each
layer. Layers were measured every 0.5 mm; an average of temporal,
central, and nasal regions was taken. Various layers included: full
retinal thickness (FRT), photoreceptor layer (PRL), bipolar layer
(BPL), retinal pigment epithelium (RPE), nerve fiber layer (NFL),
ganglion cell layer and inner plexiform layer (GCL+). These
measured values were then compared to a database of 51 eyes with
no known disease.
Humphrey visual field (obtained on same day as OCT) results were
also correlated to the retinal thicknesses of each layer.
Results: Mean age of glaucoma subjects was 61 while mean age of
normals was 52. Twelve (50%) patients were male while 26 (51%)
normals were male. FRT, BP, GCL+ and NFL/GCL+ complex
showed significant thinning in all regions of the fovea. The PRL
showed significant thinning in the central and temporal zones only.
Conversely, the NFL showed significant thinning only in the central
and nasal regions. The RPE showed no significant thinning in any
region.
VFI showed a decreasing trend with the loss of both FRT and
individual retinal layers.
Conclusions: Overall, eyes with glaucoma demonstrate thinning
in various retinal layers. In particular, this study has shown for the
first time that thinning of the BPL in all foveal regions and PRL in
the central and temporal regions occurs in glaucomatous eyes. In
addition, VFI appears to correlate with retinal thickness.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
significantly greater compared with the controls (P<0.0001). The MD
at the third visit was significantly improved compared with the first
visit in the controls, whereas the PSD was significantly worsened in
the POAG eyes over the same time span. There were no significant
fluctuations in the SNR-AUCs in both the control and POAG eyes. In
the POAG eyes, the SNR-AUC CV improved as the MD (R2=0.28,
P=0.0007), PSD (R2=0.26, P=0.0013), and SNR-AUC (R2=0.35,
P=0.0001) worsened.
Conclusions: The SNR-AUC of the mfVEP showed high
reproducibility in the control eyes, whereas its CV increased in a
disease stage-dependent fashion in the POAG eyes.
Commercial Relationships: Yukako Inoue, None; Kei Kato, None;
Kumiko Ishikawa, None; Makoto Nakamura, None
Support: grant-in-aid from Japanese Goverment (No. 23592568)
Table 1. Mean thickness values for various retinal layers in central,
temporal and nasal foveal regions. p-values were from two-tailed,
homoscedastic t-tests.
Commercial Relationships: Kathleen E. Guinn, None; Nithya
Rajagopalan, None; Peter Bracha, None; Mohammad A. Sadiq,
None; Jose Maya, None; Mohamed Ibraheem, None; Sushma Rai,
None; Vikas Gulati, None; Quan Dong Nguyen, Genentech (F);
Yasir Sepah, None
Program Number: 334 Poster Board Number: C0105
Presentation Time: 8:30 AM–10:15 AM
Mid-term fluctuations in the global indices of multifocal visual
evoked potentials and Humphrey visual fields in controls and
glaucomatous eyes
Yukako Inoue, Kei Kato, Kumiko Ishikawa, Makoto Nakamura.
Ophthalmology, Kobe University, Kobe, Japan.
Purpose: We have previously reported that the degree of signalto-noise ratio (SNR) distribution overlap between a signal window
and a noise window in multifocal visual evoked potential (mfVEP)
responses, which is determined by the area under the receiveroperating characteristic curve (SNR-AUC), can quantitatively
detect glaucomatous visual functional damage (Nakamura et al. Doc
Ophthalmol, 2011). The purpose of this study was to compare midterm fluctuations in the SNR-AUCs between eyes with primary open
angle glaucoma (POAG) and control eyes.
Methods: We enrolled 30 eyes in 30 ophthalmologically normal
controls and 37 eyes in 37 POAG patients whose mean deviations
(MDs) in the Humphrey visual field (HVF) 24-2 were -15 dB or
better. MfVEPs were recorded using 2 vertical channels and 1
horizontal channel as reported. The SNR-AUCs were calculated
based on the root mean square amplitudes of the signal and noise
windows. The HVFs and mfVEPs in the same individuals were
recorded three times at different visits, and the coefficients of
variation (CV) of the MDs, pattern standard deviations (PSDs), and
SNR-AUCs were obtained. The MDs, PSDs, and SNR-AUCs from
the three visits were compared using repeated measures of analysis of
variance, and the logarithmic CVs of each parameter were compared
using t-tests between the control and POAG eyes. Linear regression
analyses were performed on the logarithmic CVs of the SNR-AUCs
against the SNR-AUCs themselves.
Results: The average (standard deviation) of the SNR-AUC and its
CV were 0.97±0.02 and 0.012±0.008, respectively, in the control eyes
and 0.87±0.09 and 0.040±0.037, respectively, in the POAG eyes. The
SNR-AUC in the POAG eyes was significantly lower and its CV was
Program Number: 335 Poster Board Number: C0106
Presentation Time: 8:30 AM–10:15 AM
Chromatic Full-field Stimulus Threshold (FST) in Proliferative
Diabetic Retinopathy
Andre Messias1, Katharina Messias1, Rafael S. Arcieri1, Fabiano
Sakamoto1, Vinicius M. Castro1, 2, Rodrigo Jorge1. 1Ophthalmology,
University of Sao Paulo, Ribeirao Preto, Brazil; 2Universitat of
Tuebingen, Tübingen, Germany.
Purpose: To describe chromatic full-field stimulus threshold (FST)
outcomes in proliferative diabetic retinopathy (PDR) and their
relationship with electroretinography (ERG).
Methods: Data from 24 patients with PDR (n=31 eyes; 56 ± 10
years of age; 50% male) were analyzed. Patients were submitted to
measurement of best-corrected visual acuity (BCVA), and full field
Electroretinography (ERG), according to ISCEV was performed
to determine a- and b-wave amplitude and implicit time for darkadapted rod (0.01 cd.s/m2), combined response (CR: 3.0 cd.s/m2)
and light-adapted cone (3.0 cd.s/m2) and 30 Hz flicker (3.0 cd.s/
m2). FST was psychophysically determined before ERG recordings,
after 25 minutes dark adaptation, using Espion E2 system with the
ColorDome LED full-field stimulator (Diagnosys LLC, Lowell,
MA), first using red (635 to 638 nm), then blue (465 to 470 nm), and
then white (6500 K) stimulus, with 5 minutes inter-session interval.
Normal subjects were evaluated to serve as controls (n=20).
Results: Mean ± SE patients’ FST was significantly higher than
(controls’) for white: -28.1 ± 1.3 dB (-42.3 ± 0.7 dB; ANOVA
P<0.001), blue: -33.9 ± 1.7 dB (-48.9 ± 0.7 dB; P<0.001); and red:
-14.6 ± 0.9 dB (-25.4 ± 0.6 dB; P<0.001). Of interest, only 7 (23%),
6 (19%) and 11 (35%) eyes were above the controls’ 5% quantile.
Mean BCVA was 0.40 ± 0.05 logMAR (range: 20/20 to 20/200),
and was not significantly correlated to FST of any color. There was
moderate (r ≥ 0.5) significant (P<0.05) correlation between white
FST and dark-adapted b-wave amplitude and implicit time.
Conclusions: These data indicate that FST is sensitive for retinal
functional changes due to proliferative diabetic retinopathy, and
might add interesting information about visual function in clinical
trials including these patients.
Commercial Relationships: Andre Messias, None; Katharina
Messias, None; Rafael S. Arcieri, None; Fabiano Sakamoto, None;
Vinicius M. Castro, None; Rodrigo Jorge, None
Support: FAPESP POST-DOCTORAL GRANT 2012/16265-0
Clinical Trial: NCT02005432
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 336 Poster Board Number: C0107
Presentation Time: 8:30 AM–10:15 AM
Pattern VER in Diabetic Patients with Good Visual Acuity
Vidhya Gunasekaran1, Byongdo Kim2, Kimberly Pham2, Jenny
Nguyen3, Evelyn Ross4, Vinna Nam2, Scott E. Brodie5, Gloria Wu6.
1
Ophthalmology, Aravind Eye Hospital, Madurai, India; 2University
of California, Berkeley, Berkeley, CA; 3University of Southern
California, Los Angeles, CA; 4University of Iowa College of Public
Health, Iowa City, IA; 5Ophthalmology, Mount Sinai School of
Medicine, New York City, NY; 6Ophthalmology, Stanford Hospital &
Clinics, Stanford, CA.
Purpose: Pattern VER to assess optic nerve function in diabetic
patients with good visual acuity. Diabetic patients are at increased
risk of glaucoma. In addition diabetic optic neuropathy can occur
with various stages of diabetic retinopathy. Pattern VER is now being
used to assess glaucoma patients.
Methods: Retrospective chart review of the database of 3500
computerized records for the ICD-9 diagnosis code of diabetic
retinopathy (362.83. 362.03. 362.02), glaucoma suspect (365.00),
glaucoma (365.11) who had the procedure code of 95930 for visual
evoked response (VER). Inclusion criteria: diabetic retinopathy,
glaucoma suspect, and glaucoma patients, Snellen visual acuity:
20/15-20/40. Exclusion criteria: optic neuritis, multiple sclerosis,
optic neuropathy from other causes (drug toxicity, pituitary disease,
CNS disease etc) or multiple diagnoses. Controls were normal
volunteers, patients with no detectable ocular pathology but had
diagnosis of non-specific headache (ICD-9 code 379.91). Visual
acuity was 20/15-20/40. The Stata statistical software program was
used. For each patient or control subject, VER data from only one eye
was used (randomization by coin toss). Using Pattern VER (LKC,
Gaithersburg MD, EM software), we measured the P100 amplitude
(P100amp), P100 implicit time (P100IT), N75 implicit time (N75IT).
Results: Total of 80 patients enrolled: Controls (C):C=20: age range
21-78 yrs, avg=53.75, sd=15.2; Diabetes (DM): n=20: age range
45-83 yrs, avg=61.9, sd=12.05. Glaucoma Suspect (GS)=20: age
range 33-78, avg=54.6, sd=13.2; POAG (G)=20: age range 43-78
yrs, avg=65.2, sd=11.3. N75 Imp time (N75IT) 2 tailed t-test: C
mean 30.9 msec vs G mean 49.4 msec, p=0.0001; C mean 30.9 msec
vs DM mean 42.3 msec, p=0.0001; G mean 49.4 msec vs GS mean
34.6 msec, p=0.0002; G mean 49.4 msec vs DM mean 42.3 msec,
p=0.0051; DM mean 42.3 msec vs GS mean 34.6 msec, p=0.03. No
significant differences were noted in N75IT between C vs GS: p=0.3.
No significant differences were noted in P100amp, P100IT: DM vs C;
GS vs C; G vs C.
Conclusions: Pattern VER has objectively quantified optic nerve
pathology in POAG patients. This small study suggests that diabetic
patients with good visual acuity may have early optic nerve pathology
as shown in the delay of N75 implicit time. Future randomized
clinical trials will be needed to further corroborate these findings.
Commercial Relationships: Vidhya Gunasekaran, None; Byongdo
Kim, None; Kimberly Pham, None; Jenny Nguyen, None; Evelyn
Ross, None; Vinna Nam, None; Scott E. Brodie, None; Gloria Wu,
None
Program Number: 337 Poster Board Number: C0108
Presentation Time: 8:30 AM–10:15 AM
Alteration of Photopic Negative Response of Multifocal
Electroretinogram elicited by seven hexagons in Patients with
Glaucoma
Muneyoshi Kaneko1, 2, Shigeki Machida1, Yuya Hoshi1, Daijiro
Kurosaka1. 1Department of Ophthalmology, Iwate Medical
University, Morioka, Japan; 2Department of Ophthalmology, Morioka
Municipal Hospital, Morioka, Japan.
Purpose: We previously reported that patients with glaucomatous
optic neuropathy (GON) have reduced photopic negative responses
(PhNRs) of full-field light-adapted flash ERG and focal ERG as well
as 5-element multifocal ERG (mfERG). Here we determine whether
data on retinal localization of glaucomatous changes can be obtained
with more detail by increasing the number of elements in mfERG
recording from 5 used in a previous study to 7.
Methods: Eight patients with GON, all with glaucomatous visual
field defect on static quantitative perimetry, were observed with
7-hexagon mfERG (center, C; temporal superior, TS; temporal
inferior, TI; nasal superior, NS; nasal inferior, NI; temporal, T; nasal,
N). The stimulus frequency was reduced to 6.25 Hz and the low- and
high-cut filters set to 3 and 30 Hz, respectively, to record the slow
waveforms.
Results: The low-frequency stimuli elicited mfERG waveforms
closely resembling those seen with full-field light-adapted flash
ERG and focal ERG, consisting of an initial negative wave and a
positive wave (N1 and P1), followed by a slow negative wave (N2,
corresponding to the PhNR).
We found no significant differences between the normal control and
glaucomatous eyes in N1 and P1 response densities across all regions.
Meanwhile, significant differences were noted in N2 response density
in the TI, C, and T regions and in N2/N1 and N2/P1 response density
ratios in the C and T regions (P < 0.05). No significant differences,
however, were noted for any mfERG components in other regions,
including TS, NS, NI, and N.
Conclusions: We previously reported that the 5-element mfERG
responses showed significant reductions of the N2 response density
and response density ratios of the N2/N1 and N2/P1 in the C
region. In the preset study, the 7-element mfERG responses showed
significant reductions of all these ERG parameters in both the C and
T regions, suggesting that the temporal regions are also susceptible
to glaucomatous damage. The results also indicate that increasing
the number of elements in mfERG recording can potentially generate
more detailed data on retinal localization of glaucomatous changes.
Commercial Relationships: Muneyoshi Kaneko, None; Shigeki
Machida, None; Yuya Hoshi, None; Daijiro Kurosaka, None
Support: Supported by a Grant (#24592639) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan(SM).
Program Number: 338 Poster Board Number: C0109
Presentation Time: 8:30 AM–10:15 AM
Multifocal Electroretinogram Amplitudes are associated with
Mean Ocular Perfusion Pressure in patients with Diabetes and
Vascular Disease
Wendy W. Harrison, Andrew Benson, Sheree Fetkin, Alicia Havens,
Elizabeth Lyon, Vladimir Yevseyenkov. Optometry, Midwestern Univ
Arizona Coll of Optometry, Glendale, AZ.
Purpose: It has been observed in patients with diabetes that mfERG
amplitudes are more variable than implicit times (IT). Sources of
amplitude variation are not fully understood and need to be better
characterized in this patient group. Mean ocular perfusion pressure
(MOPP), a function of systolic, diastolic, and intraocular pressure,
is a measure which can be altered in patients with diabetes and its
co-morbidities. It is also known to fluctuate over time. This study
evaluates the association between MOPP, systemic blood markers
and retinal function in patients with and without vascular diseases.
Methods: 10 subjects with systemic vascular disease and 25 control
subjects participated in this pilot study. Placement in the vascular
disease group required the subject to be formally diagnosed and
under treatment for both diabetes and hypertension. Each subject had
the following measured: mfERG implicit time and mfERG amplitude
(VERIS 6.3) which was averaged over the entire eye; systolic and
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
diastolic blood pressure, intraocular pressure, and an extensive blood
panel. A multivariate regression model, evaluating the association
between mfERG amplitude and other factors, was created.
Differences between groups were evaluated with T-Tests.
Results: The results indicate that mfERG amplitude is associated
with perfusion pressure as long as the age of the patient is controlled
for properly. The best multivariate model for average amplitude
included perfusion (p<0.010), mfERG IT (p<0.0001), and age
(p<0.004). It was also found that the systemic vascular disease group
was older (p<0.0001), had higher cholesterol (p<0.024), higher
triglycerides (p<0.0001), higher CRP levels (p<0.003) and reduced
amplitudes (p<0.043).
Conclusions: mfERG amplitude is a useful, but often variable,
measure of retinal function. Results of this pilot study indicate that
MOPP may be one source of mfERG Amp variation in patients with
and without vascular disease. Further studies with serial measures are
needed to learn more about the relationship of perfusion and function
in these patient groups.
Commercial Relationships: Wendy W. Harrison, None; Andrew
Benson, None; Sheree Fetkin, None; Alicia Havens, None;
Elizabeth Lyon, None; Vladimir Yevseyenkov, None
Program Number: 339 Poster Board Number: C0110
Presentation Time: 8:30 AM–10:15 AM
Cones structure and function related patterns in Usher syndrome
patients
Ieva Sliesoraityte1, Saddek Mohand-Saïd1, Dorothée Dagostinoz1,
Konstantin E. Kotliar2, Shahira Miloudi1, Jose A. Sahel1. 1INSERM,
CIC 503, Institut de la Vision, Paris, France; 2University of Applied
Sciences Biomedical Engineering, Juelich, Germany.
Purpose: Structure and function related cone measures are needed to
be standardized for efficacy assessment in gene therapy trials. We aim
to investigate cone mosaic changes in association to cone functional
patterns in Usher syndrome (USH) patients.
Methods: High-resolution macular images were obtained by en-face
adaptive optics imaging (AO, Imagine Eyes, Orsay, France) and
spectral domain optical coherence tomography (SD-OCT, Spectralis,
Heidelberg, Germany) from 15 eyes of fifteen USH patients with
confirmed gene mutations. Cone density and spatial organization of
the cone mosaic was evaluated in 50x50microns rectangles at 0.1
mm, 0.5mm and 1.0 mm from the center of the fovea. Functional
cone patterns were obtained using custom-made chromatic
pupillometer (AMTech, Dosenheim, Germany), while pupil diameter
response to the red light (640 nm) was recorded. Correlation between
cones mosaic spatial organization changes and cone function was
performed using MATLAB software. Age-matched healthy subjects’
eyes data served as a norm for comparison purposes.
Results: All patients (mean age 35 years, mean visual acuity
0.40 logarithm of the minimum angle of resolution (logMAR))
demonstrated abnormal and irregular cone mosaic patterns with
significantly decreased cone density at 0.1 mm, 0.5mm and 1.0 mm
from the center of the fovea as 30%, 20% and 20%, respectively
(p=0.001). In addition, greater decrease in cone density was related
to disruption of the photoreceptor inner segment ellipsoid band on
SD-OCT images, i.e. 30% compared to 48% for the vulnerable region
(p=0.03). The significant reduction of chromatic pupil responses were
determined in all Usher syndrome patients (15% reduction, p=0.01).
Decreased cone density was significantly associated with diminished
cones functional activity (r=0.39, p=0.015). Cone mosaic patterns
having less regular and large dark regions were related with highly
significant cones function attenuation (r=0.89, p=0.012).
Conclusions: Decreased cone density and irregularity of the cone
mosaic alongside with diminished cones function was observed in
eyes with Usher syndrome. Chromatic pupillometry and adaptive
optics imaging potentially could be implemented as objective and
sensitive tools in gene therapy trials for cones structure and function
related changes quantification in Usher syndrome.
Commercial Relationships: Ieva Sliesoraityte, None; Saddek
Mohand-Saïd, None; Dorothée Dagostinoz, None; Konstantin E.
Kotliar, None; Shahira Miloudi, None; Jose A. Sahel, None
Support: ERAREl N°58: Eur-USH, HEALTH-F2-2010-242013
Clinical Trial: NCT01954953
Program Number: 340 Poster Board Number: C0111
Presentation Time: 8:30 AM–10:15 AM
Does foveal hypoplasia lead to functional deficiencies?
Peter Heiduschka, Lea Oberfeld, Nicole Eter. Univ Eye Hosp
Muenster, Muenster, Germany.
Purpose: It is still disputed if the absence of the foveal pit, i.e.
foveal hypoplasia, leads to a functional impairment of the retina.
We analysed morphological and functional data in patients having
presented to our retina clinic.
Methods: Patients with foveal hypoplasia were selected by screening
the OCT database of our hospital. They were grouped according to
stages 1 through 4 as introduced by Thomas et al., Ophthalmology,
2011; 118:1653–1660, with stage 1 for a shallow foveal pit and stage
4 for a completely flat retina with flat retinal layers. Best corrected
visual acuity (BCVA), size of foveal avascular zone determined
by fluorescein angiography and retinal thickness determined by
OCT were correlated with the stages. Amplitudes of multifocal
electroretinography (mfERG) were compared with age-matched
control values.
Results: Among more than 1000 patients with OCT documentation,
46 patients with foveal hypoplasia were selected. 18 patients were
excluded from further consideration due to various accompanying
disorders that disturb retinal structure and/or function, such as
epiretinal gliosis, macular oedema or photoreceptor degeneration. In
normal controls, retinal thickness in the fovea was about 244 mm. In
the 25 examined patients, average retinal thickness at the site of the
fovea was 284 mm, 276 mm, 311 mm and 325 mm for stages 1 through
4, which was statistically different for stages 1, 3 and 4. BCVA
ranged between 0.1 and 1.0 with average values between 0.4 and 0.6
for each group. Large variations within the groups did allow not for
statistically significant differences, although average BCVA in group
of stage 1 was better than in the other groups. Large variations were
also found in the area sizes of foveal avascular zones, although a
clear tendency was visible that avascular zones were smaller in foveal
regions of stage 4 eyes. In almost all patients, mfERG amplitudes
obtained in the fovea were smaller than control values, and
amplitudes obtained outside the fovea were closer to control values.
There were only weak correlations between mfERG amplitudes and
BCVA, depending on the stage of foveal hypoplasia.
Conclusions: Despite the limited number of patients, our results
indicate that foveal hypoplasia leads to an impaired retinal function in
all four groups.
Commercial Relationships: Peter Heiduschka, None; Lea
Oberfeld, None; Nicole Eter, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 341 Poster Board Number: C0112
Presentation Time: 8:30 AM–10:15 AM
Electroretinographic Oscillatory Potentials and Flicker
Responses in Pediatric Patients Taking Vigabatrin
Tara L. Favazza1, Theodore Bowe1, Emily A. Swanson1, Anne
Moskowitz1, 2, Ronald M. Hansen1, 2, Aparna Raghuram1, 2, Anne
Fulton1, 2, James D. Akula1, 2. 1Department of Ophthalmology, Boston
Children’s Hospital, Boston, MA; 2Harvard Medical School, Boston,
MA.
Purpose: The cumulative dose of the antiepileptic drug vigabatrin
(VGB) has been associated with visual field (VF) loss. In
patients unable to do conventional VF tests due to immaturity or
disability, surveillance for adverse effects of VGB is done using
electroretinography (ERG). In this study, we evaluated the ERG
oscillatory potentials (OPs) and responses to flickering stimuli for
significant differences between VGB users and controls, as well as
significant correlation with dose or duration of VGB use.
Methods: Extant ERG records from VGB users (n=64) were
compared to those of healthy controls. The records included
responses of the dark-adapted eye to a range of brief, full-field blue
flashes (0.06-20 cd s/m2) of doubling intensity, and cone-isolated
responses to red flashes (0.28-35 cd s/m2) presented on a steady
white background (25.5 cd/m2). The amplitude and implicit time
of individual OPs, demonstrated by passing the records through a
5th order Butterworth filter (bandpass 70-300 Hz), were measured,
and the energy of the OPs was calculated from the area under the
Fourier-transformed record. Responses to flickering white stimuli
(2.25 cd s/m2 with a 25.5 cd/m2 background; 10, 25, 31.25 Hz )
were obtained; the amplitude and phase of the first four harmonics
in these responses were processed. Respective ANOVA were used
to detect differences in rod and cone OPs (intensity×OP×group) and
flicker (frequency×harmonic×group) parameters. ERG data were
also evaluated for significant relation to dose and to duration of VGB
using Pearson’s product moment.
Results: In VGB users, both rod and cone OP amplitudes and
flicker responses to the two higher frequencies were significantly
attenuated. In spite of this, neither OP energy nor the amplitude of the
fundamental at any of the three frequencies tested was significantly
related to VGB dose or duration of use. However, consistent with
Westall et al. (Doc Ophthalmol, 2002), inspection of the OP energy
vs. duration on VGB data suggests a possible quadratic relationship
(i.e. rising and falling).
Conclusions: Although VGB use is associated with attenuated
ERG responses, these data do not show a relationship to VGB dose
or duration. In view of these results, continued effort to develop
procedures for evaluation of the peripheral VF in VGB users is
warranted.
Commercial Relationships: Tara L. Favazza, None; Theodore
Bowe, None; Emily A. Swanson, None; Anne Moskowitz, None;
Ronald M. Hansen, None; Aparna Raghuram, None; Anne
Fulton, None; James D. Akula, None
Support: Boston Children’s Hospital Ophthalmology Foundation,
Massachusetts Lions Eye Research Fund
Program Number: 342 Poster Board Number: C0113
Presentation Time: 8:30 AM–10:15 AM
Electroretinography using a fiber electrode prototype in patients
with retinal dystrophy
Josenilson P. Pereira1, Daniel M. Rocha1, Sung E. Watanabe1, Paula
Y. Sacai1, Sergio Munoz2, Solange R. Salomao1, Adriana Berezovsky1.
1
Ophthalmology, Univ Federal de Sao Paulo, Sao Paulo, Brazil;
2
Departamento de Salud Publica, Universidad de La Frontera,
Temuco, Chile.
Purpose: To compare full-field electroretinogram (ERG) responses
recorded in patients with retinal dystrophy with monopolar DTL®
electrode to those obtained with a microfiber electrode prototype,
using the ERG standards of the International Society for the Clinical
Electrophysiology of Vision (ISCEV).
Methods: This study was approved by the Ethics Committee of the
Federal University of São Paulo (1087/08). Fifty six patients (mean
age 36.8±16.9 years, 29 females) with previously diagnosed retinal
dystrophy had full-field ERG recorded (ISCEV standard full-field
protocol) using two distinct electrodes randomly selected in two
consecutive visits in the same week. VERIS 5.1.9 system by EDI
was used for data acquisition and analysis. ERG outcomes were
analyzed by independent linear regression method by StataSE 11
statistical software. Retinal dystrophy type was classified on the basis
of standard clinical criteria as: retinitis pigmentosa, cone dystrophy,
Stargardt’s disease, Cone-rod dystrophy and others. ERG responses
were compared with normative data from our own lab.
Results: The magnitude and waveform quality obtained with the
two electrodes were similar for all ERG responses. No statistical
differences were found for amplitude and implicit time between
microfiber electrode prototype and DTL® responses. Linear
regression showed a trend line equation for rod amplitude response
(DTL=6.01+1.070 *prototype) and for cone amplitude (DTL=0.90+1.100*prototype).
Conclusions: The results showed that the ERG waveforms obtained
with the two electrode types were remarkably similar for all ERG
responses. The microfiber electrode prototype might be a choice for
low-cost alternative instrument for clinical ERG recording for retinal
function assessment.
Commercial Relationships: Josenilson P. Pereira, None; Daniel
M. Rocha, None; Sung E. Watanabe, None; Paula Y. Sacai,
None; Sergio Munoz, None; Solange R. Salomao, None; Adriana
Berezovsky, None
Support: Fapesp - Fundação de Ampara a Pesquisa do Estado de
São Paulo to AB and Capes - Coordenadoria de Aperfeiçoamento de
Pessoal de Nível Superior to JMP
Program Number: 343 Poster Board Number: C0114
Presentation Time: 8:30 AM–10:15 AM
FREQUENCY AND CAUSES OF NEGATIVE
ELECTRORETINOGRAM OVER A 10-YEAR PERIOD IN A
UNIVERSITY HOSPITAL IN BRAZIL
Daniel M. Rocha, Solange R. Salomao, Sung E. Watanabe, Josenilson
M. Pereira, Paula Y. Sacai, Adriana Berezovsky. Oftalmologia,
Universidade Federal de Sao Paulo, Sao Paulo, Brazil.
Purpose: To provide an overview of the frequency and causes of
negative electroretinograms (ERGs) over a 10-year period in a
University Hospital in Brazil.
Methods: A retrospective review was performed of all full-field
ERGs performed from March 2004 to November 2013 under ISCEV
standard conditions in the Laboratory of Clinical Electrophysiology
of Vision, Hospital Sao Paulo, Brazil. All participants had their
monocular visual acuity (VA) measured using the ETDRS chart.
Negative ERG was defined as a waveform evoked by a bright flash
under scotopic conditions with a larger a-wave than b-wave resulting
in a b/a ratio below 1.0 in at least one eye. Clinical information as
age, gender, presenting VA, history of consanguinity and diagnosis
were considered.
Results: A total of 1645 patients had both eyes tested during the
study period, with 41 (2.49%) showing negative ERG. Ages ranged
from 0.5-78 years (mean=33.8±20.7; median=32); 22 (54%) males
and 19 (46%) females. Negative ERGs were found in 14 children
(5 females and 9 males) and 27 adults (14 females and 13 males).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Frequencies of negative ERG were 1.64% for adults and 0.85%
for children (< 18 years of age). Bilateral negative ERG was
found in 27 (66%) patients and unilateral in 14 (34%). The most
common diseases associated with bilateral negative ERGs were
photoreceptor dystrophies (n=17), X-linked juvenile retinoschisis
(n=3), congenital stationary night blindness (n=1), inflammatory eye
diseases (n=1), diabetic retinopathy (n=1) and undetermined (n=4).
In unilateral cases the main causes were photoreceptor dystrophies
(n=7), inflammatory eye diseases (n=4), diabetic retinopathy (n=1)
and undetermined (n=2). History of consanguinity was found in 9
(23.1%) patients, with two with unilateral negative ERGs. Mean
VA in the better-seeing eye was 0.55 ±0.55 logMAR (20/70) and
0.88±0.69 logMAR (20/150) in the worse-seeing eye.
Conclusions: The frequency of negative ERGs over a 10-year period
from a large university public hospital in Brazil, was marginally
below the 3-5% range described in the literature. A possible factor
contributing to this result is the unavailability of ERGs under
sedation in our service decreasing the number of young children
tested. Predominantly male children showed negative ERGs. The
most likely diagnosis for both bilateral and unilateral cases was
photoreceptor dystrophies (rod-cone and cone-rod).
Commercial Relationships: Daniel M. Rocha, None; Solange R.
Salomao, None; Sung E. Watanabe, None; Josenilson M. Pereira,
None; Paula Y. Sacai, None; Adriana Berezovsky, None
Program Number: 344 Poster Board Number: C0115
Presentation Time: 8:30 AM–10:15 AM
MULTIPLE SCLEROSIS AND NEUROMYELITIS
OPTICA: ANALYSIS OF MULTIFOCAL VISUAL EVOKED
POTENTIALS
Gustavo M. Amorim1, 2, Lucas Daniel Almeida Fernandes1, 2, Lorena
Botelho Vergara1, 2, Phelipe Augusto Rabelo Paixao1, 2, Raisa L.
dos Santos1, 2, Patrick Lopes1, 2, Eliza M C B. Lacerda1, 3, Givago S.
Souza1, 3, Alexandre Rosa2, 4, Luiz Carlos L. Silveira1, 3. 1Nucleo de
Medicina Tropical, Universidade Federal do Para, Belem, Brazil;
2
Institudo de Ciencias da Saude, Universidade Federal do Para,
Belem, Brazil; 3Instituto de Ciencias Biologicas, Universidade
Federal do Para, Belem, Brazil; 4Hospital Universitario Bettina Ferro
de Souza, Universidade Federal do Para, Belem, Brazil.
Purpose: To compare the reliability indexes multifocal visual evoked
potentials (mfVEP) in patients with multiple sclerosis (MS) and
neuromyelitis optica (NMO).
Methods: Patients with multiple sclerosis (9 subjects, 18 eyes, 37 ±
9.7 years old) and NMO (12 subjects, 20 eyes, 36.1 ± 11.8 years old)
were studied by biomicroscopy, fundoscopy, visual acuity and visual
electrophysiology (mfVEP). mfVEP was recorded using Veris 6010
system with 60 sectors black and white. We estimated the signalto-noise ratio (SNR) for each form of mfVEP wave. The mfVEP
waveforms were reliable when the SNR was greater than 1.395.
Moreover, the rates of reliable forms of wave mfVEP was quantified
as the ratio between the number of waveforms confidence / total
number of sectors of six areas of the same eccentricity (from inside
the visual field R1 to R6 in exterior visual field ) and the whole visual
field. We used the binomial test, α = 0.05 as analysis.
Results: Optic neuritis (ON) was present in 5/18 eyes of patients with
multiple sclerosis and 9/ 20 patients NMO. Confidence indices of
mfVEP for the controls were higher than for the MS with or without
ON and NMO with ON in R1 - R5. In R6, the control rate was higher
than the rate for all other groups of patients. The rate of mfVEP to
reliably controls were higher than those obtained for the MS and ON
with or without NMO MS (p < 0.01) in R1 - R5. In R6, the control
rate was higher than the rate for all other groups of patients. The rate
of mfVEP reliable for NMO group was higher than the rate of set
of MS, despite the presence of NO. The rate waveform to trust the
MS group without ON was higher than for the MS group with ON
(p<0.01), and there was no difference between NMO group with and
without ON for all analyzed areas.
Conclusions: The rate of reliability mfVEP wave was the lowest
among MS patients and the presence of ON significantly decreased
the performance of patients with MS and NMO ways to generate
reliable mfVEP wave.
Commercial Relationships: Gustavo M. Amorim, None; Lucas
Daniel Almeida Fernandes, None; Lorena Botelho Vergara, None;
Phelipe Augusto Rabelo Paixao, None; Raisa L. dos Santos, None;
Patrick Lopes, None; Eliza M C B. Lacerda, None; Givago S.
Souza, None; Alexandre Rosa, None; Luiz Carlos L. Silveira,
None
Program Number: 345 Poster Board Number: C0116
Presentation Time: 8:30 AM–10:15 AM
PHYSIOLOGICAL DYSFUNCTION IN THE FELLOW EYE
OF STRABISMIC AND ANISOMETROPIC AMBLYOPIC
CHILDREN
Eric P. Andrade, Adriana Berezovsky, Paula Y. Sacai, Josenilson M.
Pereira, Daniel M. Rocha, Solange R. Salomao. Ophthalmology,
Federal University of Sao Paulo, Sao Paulo, Brazil.
Purpose: Amblyopia is a form of cerebral visual impairment in the
absence of an organic cause. Attenuated amplitudes and prolonged
latencies are common abnormalities found in pattern reversal visual
evoked potentials (PRVEP) in amblyopic eyes. However there is
scarce data on PRVEP in fellow eyes of amblyopes. The aim of this
study is to evaluate visual acuity and PRVEP in the fellow eye of
strabismic and/or anisometropic amblyopic children.
Methods: This study was approved by the Ethics Committee of the
Federal University of São Paulo (0503/08). The amblyopic group
consists of 40 children (22 girls), aged 5-14 years (mean 8.7±2.2
years), 15 anisometropic, 21 strabismic and 4 with anisometropia and
strabismus. A group of 19 healthy children (13 girls) aged 5-15 years
(8.2±2.6 years) was used as control. Visual acuity was measured
in logMAR from each eye with the best optical correction using
the ETDRS chart for distance. Grating acuity was measured from
each eye using the sweep-VEP system. Transient PRVEP recording
was obtained with checkerboard stimuli subtending 1°, 15’ and 7.5’
visual angles from both eyes in monocular stimulation condition
according to ISCEV protocol. P100 latency in milliseconds (ms), the
amplitude between the peaks of N75 and P100 in microvolts (μV)
were determined.
Results: Statistically worse visual acuity for either optotype
(0.04±0.1 logMAR; p=0.021,) or grating acuity (0.07±0.05 logMAR;
p=0.026,) were found when compared with healthy children (0.0±0.0
logMAR optotype, 0.05±0.04 logMAR grating). Significantly
prolonged P100 latency for stimulus 7.5’ in the felllow eye
(110.9±11.4) was detected when compared with controls (103.2±6.8;
p=0.01,). There were not a statiscally significant difference between
the amplitude of the control group and the fellow eye for all stimulus
(p=0.496, 0.700 and 0.422 for 1°, 15’ and 7.5’ visual angles,
respectively).
Conclusions: When compared with eyes of healthy children, fellow
eyes of amblyopic children showed worse optotype and grating
acuity, with subtle abnormalities in the PRVEP detected as prolonged
latencies for smaller size stimuli. These findings confirm previous
studies showing that the fellow eye of amblyope patients is not fully
normal.
Commercial Relationships: Eric P. Andrade, None; Adriana
Berezovsky, None; Paula Y. Sacai, None; Josenilson M. Pereira,
None; Daniel M. Rocha, None; Solange R. Salomao, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Support: CAPES - Governo Federal do Brasil
Support: Kerstan Stiftung
Program Number: 346 Poster Board Number: C0117
Presentation Time: 8:30 AM–10:15 AM
Retinal structure and function in Achromatopsia: the CNGA3
phenotype
Ditta Zobor1, Franco Stanzial2, Ulrich Kellner3, Günther
Rudolph4, Bernd Wissinger5, Susanne Kohl5, Eberhart Zrenner1.
1
Institute for Ophthalmic Research, Centre for Ophthalmology,
Tuebingen, Germany; 2Genetic Counseling, Coordinating Center
of Rare Diseases, Bolzano, Italy; 3Rare Retinal Disease Center,
Augenzentrum Siegburg, Siegburg, Germany; 4Department of
Ophthalmology, University of Munich, Munich, Germany; 5Institute
for Ophthalmic Research, Molecular Genetics Laboratory, Tuebingen,
Germany.
Purpose: To evaluate retinal function, and to characterize the retinal
changes in patients with achromatopsia (ACHM) due to mutations
in the CNGA3 gene by using spectral-domain optical coherence
tomography (SD-OCT) and fundus autofluorescence (FAF)
Methods: ACHM patients with known mutations in the CNGA3
gene were examined. A complete ophthalmological examination
was performed by the same investigator in every patient including
psychophysical tests (ETDRS visual acuity, color vision tests, visual
field, microperimetry and dark adaptation thresholds) and extended
electrophysiology (Ganzfeld and multifocal ERG). The appearance
and thickness of all retinal layers were evaluated by SD-OCT and
FAF.
Results: Thirty patients (mean age 33.7 years, range: 7 to 56 years)
were included. Mean BCVA was 20/140 in the complete forms
(cACHM, 28 cases) and 20/60 in the incomplete cases (iACHM, 2
cases). The Rayleigh anomaloscope matches were consistent with a
rod-dominated function in every cACHM patient. Microperimetry
indicated an overall lower retinal sensitivity within 20° of visual
field. In the electrophysiological examinations, photopic responses
were non-detectable in cACHM patients, but residual cone responses
could be observed in the iACHM patients. ISCEV rod threshold
amplitudes and implicit times were within normal limits, while
Vmax was significantly below normal values (p<0.05). In contrast,
slope (n) and semisaturation intensity (k) were found to be within
normal limits. On the morphological level, SD-OCT examination
showed no specific changes in 22.2%, IS disruption at the fovea
in 36.2%, absent IS in 22.2%, an “intraretinal bubble“ in 13.8%
and outer retinal atrophy including RPE loss was seen in 5.6% of
all cases. Foveal hypoplasia was found in 21 patients (70%), but
surprisingly, no correlation with BCVA could be observed. The
severity of morphological and functional changes lacked a robust
association with age. A specific correlation to the genotype could not
be observed.
Conclusions: Thirty CNGA3-related ACHM patients were examined
with identical functional and morphological methods. In preparation
to a therapeutic gene therapy trial, high-resolution techniques were
used to assess photoreceptor structure and function in patients
with ACHM. These findings will be useful for the identification of
patients concerning future therapeutic trials. The data imply that the
therapeutic window seems to be wider than previously indicated.
Commercial Relationships: Ditta Zobor, None; Franco Stanzial,
None; Ulrich Kellner, None; Günther Rudolph, None; Bernd
Wissinger, None; Susanne Kohl, None; Eberhart Zrenner,
Neurotech USA (C), Pfizer USA (C), QLT Inc. (C), Retina Implant
AG Germany (C), Retina Implant AG Germany (F), Retina Implant
AG Germany (I), Retina Implant AG Germany (P), Servier Paris (C),
Steinbeis GmbH Stuttgart Germany (C), Steinbeis GmbH Stuttgart
Germany (I)
Program Number: 347 Poster Board Number: C0118
Presentation Time: 8:30 AM–10:15 AM
The effect of cataract surgery on blue-yellow and standard
pattern visual evoked potentials
Eva Koch, Niklas Plange, Sara Jamali, Gernot Roessler, Peter Walter,
Babac Mazinani, Matthias Fuest. Department of Ophthalmology,
RWTH Aachen University, 52074 Aachen, Germany.
Purpose: Blue-yellow visual evoked potentials (BY-VEPs) may be
used for diagnostics of functional ganglion cell damage in glaucoma
and other ocular diseases.
In this study we investigated the impact of lenticular opacities on BYVEPs by examining patients before and after cataract surgery.
Methods: 18 patients with moderate cataract were included in a
prospective study. Transient on/off isoluminant blue-yellow 2° checks
were used for short-wavelength stimulation (BY-VEP), transient
large 1° (M1) and small 0.25° (M2) black-white checks for standard
pattern reversal VEPs. VEPs were acquired before (24 ±30 days) and
after cataract surgery (14 ±16 days). The contralateral eye was used
as a control.
Results: Amplitude and latency of M1 and M2 peaks did not change
significantly from before to after surgery. The amplitude of the BYVEPs did not change significantly after cataract surgery (pre-surgery:
-7.42 ±3.43mV, post surgery: -7.93 ±3.65mV, p=0.42), yet the latency
of the main negative peak showed a significant decrease (pre-surgery:
143.9 ±12.9ms, post-surgery: 133.2 ±7.7ms, p=0.0006). The BCVA
improvement was significant from before to after cataract surgery
(pre-surgery: 0.344 ±0.125 LogMAR, post-surgery: 0.224 ±0.179
LogMAR, p=0.013) yet not correlated to the absolute decrease in
latency of the BY-VEP after surgery (r=0.309, p=0.22). No significant
changes were found in the contralateral eye.
Conclusions: The BY-VEP is sensitive to lenticular opacities of
the human lens, presumably due to the increased short wavelength
absorption in the aging eye. This fact should be considered when
applying BY-VEPs for diagnostics.
Commercial Relationships: Eva Koch, None; Niklas Plange,
None; Sara Jamali, None; Gernot Roessler, None; Peter Walter,
None; Babac Mazinani, None; Matthias Fuest, None
Program Number: 348 Poster Board Number: C0119
Presentation Time: 8:30 AM–10:15 AM
The Effects of Non-Dilated and Dilated Pupil at Different
Eccentricity on Multifocal Electroretinogram
Muhamad-Syukri Mohamad-Rafiuddin, Saiful Azlan Rosli, Ai-Hong
Chen, Wan-Nurdiana Wan-Hamat. Optometry Department, Universiti
Teknologi MARA (UiTM), Puncak Alam, Malaysia.
Purpose: The objective of the study is to investigate the effects
of non-dilated pupil and dilated pupil on different eccentricities of
multifocal electroretinogram (mfERG).
Methods: Seventeen emmetropic subjects aged 20 to 23 years
old were recruited in this study. Multifocal electroretinogram was
performed to conform to ISCEV 2012 standard. The stimulus array
of 61 hexagonal elements with 60 degree was presented during
recording with frame frequency of 75 Hz. The mfERG recording was
done in the non-dilated eye first, followed by the dilated eye. Pupil
diameter measurement was performed using manual pupilometer
prior to multifocal electroretinography. The eye was anesthetized
with a drop of Alcaine 0.5% prior to DTL electrode insertion and
then was fully dilated using a drop each of Mydriacyl 1.0% and
Mydfrin 2.5%, and the maximum pupil dilation was measured after
60 minutes. N1 and P1 implicit time and amplitudes were recorded
across 5 different eccentricities from centrally located Ring 1 to the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
most peripheral Ring 5. Statistical analysis was performed using
Statistical Package for Social Sciences version 20.
Results: The mean pupil diameter for non-dilated eye was
3.8mm±0.41 while the mean pupil diameter for dilated eye was
8.13mm±0.35. There was no significant difference found in N1 and
P1 implicit time between dilated and non-dilated eye except for Ring
5 P1 implicit time, t(16) = -2.82, p = 0.01. N1 and P1 amplitudes for
Ring 1 and Ring 2 in dilated and non-dilated eye had no significant
difference while Ring 3 N1 [t(16) = -2.60, p = 0.02], Ring 4 N1 [t(16)
= -3.41, p = 0.004], Ring 5 N1 [t(16) = -4.26, p = 0.001], Ring 3 P1
[t(16) = 3.33, p = 0.004], Ring 4 P1 [t(16) = 4.18, p = 0.001] and
Ring 5 P1 [t(16), p < 0.001] had significant differences.
Conclusions: Our findings suggested that the multifocal
electroretinogram could be performed without dilation in clinical
investigation for central retina only.
Commercial Relationships: Muhamad-Syukri MohamadRafiuddin, None; Saiful Azlan Rosli, None; Ai-Hong Chen, None;
Wan-Nurdiana Wan-Hamat, None
Support: Fundamental Research Grant Scheme Reference No. [600RMI/ST/FRGS 5/3/Fst (45/2011)] & Exploratory Research Grant
Scheme Reference No. [600-RMI/ERGS 5/3 (59/2011)], Ministry of
Education, Malaysia
Program Number: 349 Poster Board Number: C0120
Presentation Time: 8:30 AM–10:15 AM
Intact extrastriate maps following V1 quarterfield lesion
Hiroshi Horiguchi1, Yaping J. Liao2, Brian A. Wandell3, Jonathan
Winawer4. 1Ophthalmology, Jikei University, School of Medicine,
Tokyo, Japan; 2Ophthalmology, Stanford University Medical Center,
Stanford, CA; 3Psychology, Stanford University, Stanford, CA;
4
Psychology, NYU, New York, NY.
Purpose: Primary visual cortex (V1) is a key cortical area in
distributing signals across visual cortex. There are other pathways
that bypass V1 which convey signals from the eyes to extrastriate
visual areas. Here we report a case study of a 68 year old man with a
lesion to one-quarter of V1 (right ventral). Our principal goal was to
characterize the portions of the extrastriate maps that do not receive
input from V1.
Methods: Visual function was assessed with Humphrey and
Goldmann visual field testing. T1- and T2-weighted MRI scans (3T)
were used to identify the lesion extent. Population Receptive Fields
(pRFs) were measured using functional MRI with a moving bar
stimulus paradigm (Dumoulin and Wandell 2008). The pRF method
was used to identify retinotopic maps and measure the visual field
coverage within the maps.
Results: The lesion resulted in a complete loss of ventral visual field
maps bilaterally (hV4 and VO-1/2) and the ventral (but not dorsal)
portion of right V1/V2/V3, representing the left upper visual field.
Consistent with the V1/V2/V3 lesion, visual field testing showed a
complete homonymous quadrantanopia in the left upper visual field.
Visual field coverage was assessed by superimposing the pRFs
within a map (Winawer et al. 2010). Right V1 covered only the lower
left quarterfield, as expected due to the lesion. In contrast, several
extrastriate map clusters in the right hemisphere - V3-A/B, temporaloccipital (TO-1/2), and lateral occipital (LO-1/2) - each covered the
complete left hemifield. PRF size was quantified as a function of
eccentricity separately for upper and lower visual field. Within each
map cluster, in both upper and lower visual field, pRF size increased
with eccentricity. PRF size also varied systematically between
clusters, increasing from V1 to LO-1/2 to V3-A/B to TO-1/2. The
sizes did not differ systematically between upper visual field and
lower visual field within any of the maps.
Conclusions: One quarter of the visual field was not represented in
V1/V2/V3, but nonetheless was intact in several extrastriate visual
field map clusters. Surprisingly, pRF measures for the quarterfield in
which the patient cannot see were quantitatively similar to measures
for the regions in which the patient can see. Hence, pathways which
bypass V1/V2/V3 do not enable the patient to see but do support
intact extrastriate visual field maps.
Commercial Relationships: Hiroshi Horiguchi, None; Yaping J.
Liao, None; Brian A. Wandell, None; Jonathan Winawer, None
Support: NEI grant RO1-EY03164 (BW), NEI grant K99-EY022116
(JW)
Program Number: 350 Poster Board Number: C0121
Presentation Time: 8:30 AM–10:15 AM
Visual evoked potentials (VEPs) to lateralized stimuli to measure
the interhemispheric transfer time (IHTT)
Ilie Cretu1, 4, Solange Milazzo1, 2, Pierre Betermiez1, Michel Petitjean3.
1
Department of Ophthalmology, University Hospital of Amiens,
Amiens, France; 2University of Picardy Jules Vernes, Amiens,
France; 3Service de Physiologie - Explorations Fonctionnelles,
Hôpital Ambroise Paré, Boulogne-Billancourt, France; 4Department
of Ophthalmology, Hospital of Abbeville, Abbeville, France.
Purpose: To evaluate in a population of normal subjects the VEPs
to lateralized stimuli to measure the interhemispheric transfer time
(IHTT), and methodological variability factors.
Methods: We recorded in 36 young right-handed women evoked
responses of visual occipital regions right (O2) and left (O1) in
response to visual stimulation achieved through a screen with
checkerboards alternants either in open fields, or half-fields when
the line of sight coincides with a central focus. The test is performed
in monocular eye test is chosen by lot with balancing choices, not
to have any effect of precession. The atmosphere is scotopic total.
The reversal of checkerboard is carried out at a frequency of 1.7Hz.
Depending on the distance, the size of the checkerboard is 1 °.Were
recorded average of 100 traces for each lead, during two successive
series.
Results: The results are presented according to three factors
analyzed: the eye, the conditions (reproducibility and effect number),
the observer. For stimulation in the full field, there is no effect
or observer eye for the latencies of waves N75 and P100, and no
effects eye or averaging effect for the latency of the N135 wave.
For the half-full stimulation, there is no eye effect, or observer
reproducibility, but there is a half-full field effect in the left eye. For
TTIH, there is no significant difference between half-full fields, or
effect or observer reproducibility, but there is an effect eye. Linear
regression was highly significant between the P100 and TTIH.
Conclusions: The VEPs to lateralized stimuli seem a good method
to calculate the IHTT, with good reproducibility and a minimal
observer-effect. Further work on patient disease would be interesting
to screen for a prolonged IHTT in inflammatory demyelinating CNS.
Commercial Relationships: Ilie Cretu, None; Solange Milazzo,
None; Pierre Betermiez, None; Michel Petitjean, None
Program Number: 351 Poster Board Number: C0122
Presentation Time: 8:30 AM–10:15 AM
Tachistoscope and Visual Working Memory in Sport-related
Concussion
Jeffrey Bennett1, Scott Doberstein2, Dennis Siemsen1, Logan Galezio2.
1
Opthalmology, Mayo Clinic, Rochester, MN; 2University of
Wisconsin-La Crosse, La Crosse, WI.
Purpose: It is estimated that between 300,000 and 3.8 million US
athletes sustain sports-related concussions each year. Using an iPad
tachistoscope application test we developed, concussed collegiate
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
football players were tested on visual working memory within the
acute phase of a concussion. This study sought to determine if a
measurable decline in visual working memory occurs in acutely
concussed football players compared to preseason baseline test
scores.
Methods: The test consists of 12 flash cards, each shown for
approximately 0.60 seconds. The visual stimuli used are known, rote
images unique to the subject’s occupation or athletic team. After
the flash card is shown, 4 descriptions of the flash card’s image are
shown to the subject. The subject has 4 seconds to choose the correct
answer using the touch screen. The order of the 12 flash cards and the
4 answer choices are randomized for each test. A prior study assessed
the reliability of this iPad app on 58 normal subjects. The test/retest
correlation coefficient was 0.72, indicating substantial agreement. 57
returning players for the 2013 UW-La Crosse football team consented
to participate in the study. A test was created for 3 sets of player
positions: Defense; QB WR; OL, TE, RB. The tests were presented
and demonstrated to the team by the PI at a preseason team meeting.
Following the meeting, each of the 57 subjects took a baseline test.
During the course of practices and 10 games, players suspected of
being concussed by coaches or training staff were removed from play.
Each of these players was tested with the iPad test on the sideline by
a training staff member. For each player suspect of being concussed,
an uninjured player of the same position was tested as a normal
comparison.
Results: During the course of the 2013 season, 7 concussed and 7
uninjured players were retested. Comparing the baseline and retest
scores found no statistical significance for concussed players (p=0.45)
and uninjured players (p=0.60). A comparison of change of baseline
to retest scores between the concussed and uninjured players was also
not statistically significant (p=0.64).
Conclusions: In our small sample size of concussed players, a
measurable decline in visual working memory did not occur in
acutely concussed football players when tested with a tachistoscope.
Continued testing with more subjects is needed to confirm a
relationship between concussion and visual working memory.
Commercial Relationships: Jeffrey Bennett, None; Scott
Doberstein, None; Dennis Siemsen, None; Logan Galezio, None
202 ipRGCs
Monday, May 05, 2014 8:30 AM–10:15 AM
S 210DE Paper Session
Program #/Board # Range: 1230–1236
Organizing Section: Visual Neuroscience
Program Number: 1230
Presentation Time: 8:30 AM–8:45 AM
Genetic dissection of retinal circuits underlying the pupillary
light reflex
Alan C. Rupp1, Samer Hattar1, 2. 1Biology, Johns Hopkins University,
Baltimore, MD; 2Neuroscience, Johns Hopkins University, Baltimore,
MD.
Purpose: The pupillary light reflex (PLR) is crucial for proper visual
function by regulating the amount of light entering the eye. The PLR
begins with light detection in the retina; however, the retinal circuitry
underlying this reflex is poorly understood. We therefore sought to
identify the cell types and circuits within the retina that mediate the
PLR.
Methods: To investigate the cells and retinal circuits underlying the
PLR, we used a genetic silencing approach to remove the function of
specific cell types within the retina. We evaluated the dark-adapted
PLR in each of these mutant mouse lines in response to broadspectrum white light similar to that in the environment.
Results: Contrary to predictions from previous work, mutant
mice lacking rod function displayed a substantial reduction in
PLR sensitivity, lacking pupil constriction until the light intensity
reached photopic levels. In contrast, cone mutant mice displayed no
observable defects in PLR sensitivity or kinetics. Additionally, we
have found that mice containing rods as the only photoreceptors have
a normal PLR at all light intensities, including photopic. However,
animals with cones as the only photoreceptors have only a brief,
transient PLR and only at the brightest light intensities. Lastly,
though the PLR requires rods, we found that the PLR persists in
animals lacking the primary and secondary rod circuits.
Conclusions: We have uncovered a primary involvement of rods in
driving the PLR across a wide range of light intensities. In addition,
we have uncovered evidence of a novel rod pathway in the retina for
the PLR that is distinct from the conventional rod circuits.
Commercial Relationships: Alan C. Rupp, None; Samer Hattar,
None
Support: NIH grant GM076430
Program Number: 1231
Presentation Time: 8:45 AM–9:00 AM
A retinal projection to the iris mediates pupil constriction
Tiffany M. Schmidt1, Alan C. Rupp1, Kylie S. Chew1, Benjamin
Yungher3, Yinghong Cui4, Jurgen Wess4, Kevin Park3, Samer Hattar1, 2.
1
Biology, Johns Hopkins University, Baltimore, MD; 2Neuroscience,
Johns Hopkins University, Baltimore, MD; 3Miami Project to
Cure Paralysis, University of Miami Miller School of Medicine,
Miami, FL; 4Molecular Signaling Section, Laboratory of Bioorganic
Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases,, Bethesda, MD.
Purpose: The pupillary light reflex (PLR) is critical for proper visual
function, regulating the amount of light entering the eye. It has been
thought that the melanopsin-expressing, intrinsically photosensitive
retinal ganglion cells (ipRGCs) drive the PLR via activation of brain
circuits involving the olivary pretectal nucleus (OPN) and ultimate
release of acetylcholine from parasympathetic fibers in the iris
muscle. However, it was recently reported that melanopsin is capable
of mediating the PLR in isolation from the brain. We therefore
investigated the relative contributions of these two mechanisms in
driving the PLR.
Methods: We utilized neuronal injury models, anatomical tracing of
neuronal projections, and genetic and pharmacological silencing of
cholinergic signaling to examine the relative contribution of retinal,
central, and direct inputs to the PLR.
Results: We identified a new pathway by which ipRGCs drive the
PLR via a direct projection to the iris. Using genetic tracing methods,
we identified ipRGC axons innervating both the ciliary body and
iris muscle. To determine whether this projection is functional, we
then performed optic nerve injury to completely isolate the eye from
brain circuitry. In the initial days following injury, we observed
constriction of the pupil that was independent of brain circuitry.
Interestingly, this constriction was completely absent 5 weeks
following optic nerve injury, coincident with the death of ganglion
cells in this injury paradigm, and indicating the RGCs are required
for this brain-independent PLR. We further show that cholinergic
signaling is required for this reflex, because silencing cholinergic
signaling pharmacologically or genetically abolishes the PLR. We
next examined the ipRGC subtypes involved in this reflex. Ablation
of the M1 subtype of ipRGC results in loss of both consensual
and ipsilateral PLR, indicating that ipRGCs are in fact required
for this reflex. Interestingly, mice lacking ipRGCs that project to
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
the OPN, but retaining a subset of M1 ipRGCs, lack a consensual
PLR, but retain normal ipsilateral PLR. This indicates that the
centrally-mediated and brain-independent PLR are driven by distinct
populations of ipRGC.
Conclusions: We have identified a novel mechanism by which
ipRGCs drive the PLR via a direct projection to the iris muscle. This
pathway combines with the centrally-mediated PLR to drive the
ipsilateral PLR through modulation of cholinergic signaling.
Commercial Relationships: Tiffany M. Schmidt, None; Alan C.
Rupp, None; Kylie S. Chew, None; Benjamin Yungher, None;
Yinghong Cui, None; Jurgen Wess, None; Kevin Park, None;
Samer Hattar, None
Support: Intramural Research Program of the National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK), NIH to JW;
EY022543 to TS, GM076430 to SH
Program Number: 1232
Presentation Time: 9:00 AM–9:15 AM
Circuit properties of an S-ON/M-OFF chromatic signal in the M5
melanopsin ganglion cell
Maureen E. Estevez, Lauren E. Quattrochi, Inkyu Kim, David M.
Berson. Neuroscience, Brown University, Providence, RI.
Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs)
express melanopsin, detect light directly, and are further influenced
by rods and cones. There are five subtypes of ipRGCs in mice
(M1-M5), with different forms, responses, and projections. The M1
subtype is critical for non-image-forming functions, such as circadian
photoentrainment. Other subtypes (e.g., M4 & M5) project to brain
targets important for pattern vision, but their roles are unclear.
We recently discovered that the M5 type carries an S-ON/M-OFF
synaptically-driven chromatic signal. However, the circuitry and
spatial properties of this signal are unknown. Because M5 cells
stratify only in the ON IPL, we speculated that the long-wavelength
OFF signal would be conferred by sign-inverting amacrine cells.
Methods: We performed whole-cell voltage clamp recordings of M5
cells from adult mice of the Opn4cre/+;Z/EG line, in which all known
ipRGC subtypes express GFP. We targeted M5 cells by their small,
round somas. We delivered light stimuli (1s steps; 360 or 520 nm)
using small spots that fit within the cells’ receptive field center and
larger ones that extended well beyond it. We recorded light-evoked
currents while holding at 0, -64, and -69 mV, then applied L-AP4 to
block the ON channel.
Results: M5 ipRGCs, distinguishable from other ipRGC subtypes
by their morphology, also prove to have unique synaptically-driven
physiology. In response to center-only stimuli, M5 cells exhibited
inward currents in response to both 360 and 520 nm light. However,
when both center and surround were stimulated, M5 cells exhibited
outward currents in response to 520 nm light, but inward currents at
360 nm light. The outward current triggered by long-wavelengths
was abolished when holding at the reversal potential for chloride.
All synaptically-driven currents were abolished by L-AP4. Thus,
the OFF-channel makes no obvious contribution to the response of
these cells, and the long-wavelength inhibition is presumably driven
by ON-type GABAergic or glycinergic amacrine cells fed at least
partly by M cones. We did not observe chromatic opponency in other
ipRGC subtypes.
Conclusions: The M5 type of ipRGCs constructs an S-ON center and
M-OFF surround using circuitry reminiscent of that reported for blue
OFF-center ganglion cells in the ground squirrel (but with inverted
chromatic preference). This suggests a role for the M5 ipRGC in
spatial and chromatic vision.
Commercial Relationships: Maureen E. Estevez, None; Lauren E.
Quattrochi, None; Inkyu Kim, None; David M. Berson, None
Support: NIH Grants F32-EY021994 and R01-EY012793
Program Number: 1233
Presentation Time: 9:15 AM–9:30 AM
Action potential-dependent calcium influx into ganglion cell
photoreceptors mediates retrograde signal transmission to
dopaminergic amacrine neurons
Cameron Atkinson, Dao-Qi Zhang. Eye Research Institute, Oakland
University, Rochester, MI.
Purpose: We have reported that intrinsically-photosensitive retinal
ganglion cells (ipRGCs) appear to drive a subset of dopaminergic
amacrine (DA) neurons, providing a novel retrograde signaling
pathway that is likely involved in mediating retinal light adaptation
(Zhang et al., 2008, 2012; Atkinson et al., 2013). IpRGCs respond
to light with a membrane depolarization and an increased action
potential (AP) frequency. Here we sought to determine whether the
depolarization, increased APs, or both mediate synaptic transmission
to DA neurons.
Methods: Whole-cell voltage-clamp recordings were made from
ipRGCs genetically labeled by GFP and from RFP-labeled DA
neurons in whole-mount mouse retinas. Retrograde signaling from
ipRGCs to DA neurons was isolated by blocking rod/cone input with
L-AP4 in wild-type retinas or by using retinal degeneration 1 (rd1)
retinas in which rods and cones have degenerated.
Results: Light-evoked inward currents of DA neurons in rd1
retinas were completely eliminated by the sodium channel blocker
tetrodotoxin (TTX; n=4). The same results were observed in 4 DA
neurons from wild-type retinas with L-AP4, indicating that TTX
appears to block excitatory synaptic transmission from ipRGCs to
DA neurons. Interestingly, TTX had no effect on the light-induced
inward currents of ipRGCs but eliminated all spontaneous and lightevoked APs (n=2). These results suggest that the TTX blockade of
synaptic transmission to DA neurons results from the loss of APs in
ipRGCs. Further data suggest that this excitatory signal transmission
is primarily calcium dependent because ipRGC signals to DA neurons
were almost undetectable in the presence of the non-selective calcium
channel blocker cadmium (n=4).
Conclusions: Dendrodendritic synapses (via graded potentials) and
recurrent axon collaterals of ipRGCs (via APs) have been proposed
as two potential routes of signal transmission from ipRGCs to DA
neurons (Zhang et al., 2012). Our physiological data support the
latter route in which APs generated in ipRGCs are propagated along
recurrent axon collaterals, triggering calcium influx into the axon
collateral terminals that may directly synapse onto DA neurons.
Commercial Relationships: Cameron Atkinson, None; Dao-Qi
Zhang, None
Support: Oakland University Provost’s Graduate Student Research
Award
Program Number: 1234
Presentation Time: 9:30 AM–9:45 AM
A circadian clock in intrinsically photosensitive retinal ganglion
cells is required for normal visual function
Christophe Ribelayga1, Zhijing Zhang1, Alexia Vidal1, Rachel
Zimmerman2. 1Ophthalmology & Visual Science, University of Texas
Medical School at Houston, Houston, TX; 2Undergraduate Program,
Rice University, Houston, TX.
Purpose: Melanopsin-expressing intrinsically photosensitive retinal
ganglion cells (ipRGCs) play a key role in various non-image
forming functions, such as circadian photoentrainment and acute
masking of locomotor activity by light. In addition, ipRGCs mediate
retrograde visual signaling in the retina. We recently demonstrated
in the mouse that ipRGCs express all of the core components of the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
mammalian circadian clock. Here we tested the functionality of the
ipRGC clock in an ipRGC-specific circadian-clock-deficient mouse
model.
Methods: An ipRGC-specific circadian-clock-deficient mouse line
was generated by conditionally knocking out the essential circadian
clock component Bmal1 in ipRGCs using the Cre-loxP system.
Specifically, we crossed Bmal1f/f mice with Opn4Cre/Cre;Z/G mice.
Immunohistochemistry was used to label cell markers and assay the
expression of BMAL1 in retinal cells. Light entrainment and negative
masking of the voluntary locomotor activity rhythm was tested
using activity monitoring with wheeled cages. We measured spatial
frequency threshold (i.e. acuity) and contrast sensitivity of freely
moving mice by observing their optomotor responses to moving sinewave gratings, using the Optomotry system.
Results: In the Bmal1f/f;Opn4Cre/+;Z/G retina, BMAL1 expression
pattern was normal among retinal cells but below detection threshold
in ipRGCs. We did not find any obvious morphological defects in
the retinal tissue in this line, at least at 2 months of age. However,
we were unable to detect the somatas of ipRGCs in Bmal1f/
f;Opn4Cre/+;Z/G retinas using an antibody against melanopsin. Yet,
ipRGC cells showed normal levels of GFP expression, indicating
that ipRGCs are present and melanopsin expression is considerably
reduced in these animals. In addition, the locomotor activity rhythm
of Bmal1f/f;Opn4Cre/+;Z/G animals was normally entrained by
light but showed abnormal increased activity during the light phase,
thus suggesting a decrease in the negative masking effect of light on
locomotor behavior. Finally, Bmal1f/f;Opn4Cre/+;Z/G mice showed
an altered circadian rhythm in contrast sensitivity, with a decrease
during the day. Acuity was normal in Bmal1f/f;Opn4Cre/+;Z/G mice.
Conclusions: Our data provide evidence that a functional circadian
clock in ipRGCs is required for normal function of both the imageforming and the non-image-forming visual systems.
Commercial Relationships: Christophe Ribelayga, None; Zhijing
Zhang, None; Alexia Vidal, None; Rachel Zimmerman, None
Support: This work was supported by the National Institutes of
Health (EY018640, EY010608, OD010768), the Hermann Eye Fund
and a Challenge Grant to The University of Texas Medical School at
Houston from Research to Prevent Blindness.
Program Number: 1235
Presentation Time: 9:45 AM–10:00 AM
High Photosensitivity of the Human Circadian System
Kwoon Y. Wong. 1Ophthalmology and Visual Sciences, University of
Michigan, Ann Arbor, MI; 2Molecular, Cellular and Developmental
Biology, University of Michigan, Ann Arbor, MI.
Purpose: The light/dark cycle is the predominant synchronizer of
circadian rhythms in humans. The clock-controlled secretion of
melatonin from the pineal gland is widely used as a circadian marker.
Photic suppression of nocturnal melatonin release in humans has been
shown to be relatively insensitive, with a threshold of about 10 lux or
13 log photons cm-2 s-1. However, all previous studies were performed
under light-adapted conditions, which could reduce photosensitivity.
I aimed to reexamine the threshold intensity using fully dark-adapted
subjects.
Methods: Subjects were kept in a room that was completely dark
except during stimulus light presentation. The stimulus was a 468nm
blue LED whose intensity was adjusted using neutral density filters,
and it was presented continuously for 1 hr in each trial through a
Ganzfeld dome. Saliva was collected periodically from each subject
and analyzed by the Bühlmann melatonin radioimmunoassay.
Results: In darkness, melatonin level in all subjects rose
monotonically for 2 - 3 hours at early subjective night before
reaching a plateau that lasted several hours. All prior studies tested
for melatonin suppression during the plateau, and initially I also
used this period. But similar to previous researchers, I found that
melatonin level fluctuated greatly during the plateau, making it
difficult to detect small changes in melatonin secretion. Thus, I did all
subsequent testing during the early-night rising phase, as the nearly
linear increase in melatonin improved the signal-to-noise ratio in
the measurements. One-hour exposure to 10.3 log photons cm-2 s-1
suppressed melatonin level by nearly 20% (p-value < 0.001). The
suppression induced by 1-hr 9.2 log photons cm-2 s-1 was smaller
(~7%) but still significant (p-value = 0.03). The color of the 9.2 log
photons cm-2 s-1 light was barely discernable, suggesting this intensity
was near the S-cone threshold.
Conclusions: I have found the human circadian system to be at least
four orders of magnitude more sensitive to light than previously
demonstrated, and the threshold intensity reported here is within
about 2 log units of that for intracellularly recorded primate
melanopsin ganglion cells (Dacey et al., Nature 2005). Light at
night can disrupt sleep, cause depression, impair learning, and even
increase cancer risks. The high photosensitivity of the circadian
system suggests that at night, even the dim light from a television or
street lights could be harmful.
Commercial Relationships: Kwoon Y. Wong, None
Support: NIH Grants EY18863, EY23660 and EY07003, and
Research to Prevent Blindness Scientific Career Development Award
Program Number: 1236
Presentation Time: 10:00 AM–10:15 AM
Melanopsin Retinal Ganglion Cells are Spared in Wolfram
Syndrome
Fred N. Ross-Cisneros1, Chiara La Morgia2, 3, Billy X. Pan1, Jens
Hannibal4, Neil Miller5, Valerio Carelli2, 3, Alfredo A. Sadun1.
1
Neuro-Ophthalmology, Keck School of Medicine, University of
Southern California, Los Angeles, CA; 2Biomedical and Neuromotor
Sciences, University of Bologna, Bologna, Italy; 3IRCC Instituto
delle ScienzeNeurologiche di Bologna, Bellaria Hospital, Bologna,
Italy; 4Clinical Biochemistry, Bispebjerg Hospital and Rigshopitalet,
Copenhagen, Denmark; 5Wilmer Eye Institute, Johns Hopkins School
of Medicine, Baltimore, MD.
Purpose: To examine if melanopsin retinal ganglion cells (mRGCs)
are spared in Wolfram Syndrome (WS), an autosomal-recessive
disease partly characterized by optic atrophy resulting from the loss
of RGCs. The pattern of retinal nerve fiber loss in the optic nerves
of WS has been shown to be similar to that seen in Leber hereditary
optic neuropathy (LHON), a condition in which mRGCs are known
to be spared.
Methods: Right eyes were obtained at autopsy from a 25-year-old
male WS patient and a 24-year-old healthy male control. Eyes were
fixed in formalin within 12 hours, dissected at the horizontal meridian
at the level of the optic nerve, processed, embedded into paraffin,
and serially cut at 5 mm. Every fifth section was immunostained for
melanopsin using an indirect immunoperoxidase staining method
with an HRP-bound secondary. Diaminobenzadine was used as the
chromogen and tissues were counterstained with hematoxylin. The
retinal sections were examined for number, location, and morphology
of mRGCs.
Results: The control retina showed a normal cellular architecture.
The WS retina demonstrated widespread loss of regular RGCs,
especially in the papillomacular bundle region. The average number
of mRGCs found in 10 sections from the control patient was 6.0
cells per section. The average number of mRGCs in 10 sections
from the WS patient was 5.3 cells per section. For both the control
and WS patient, mRGCs and their dendritic arbors were distributed
throughout the temporal and nasal retina. The mRGCs found in the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
macula were approximately equal. They were also found slightly
more often in the retinal inner nuclear layer (INL) at 52% and 55%
respectively. The morphology of these cells appeared similar in
both patients and was characterized by densely stained somas and
dendrites. Dendritic arbors from mRGCs in the GCL were found to
project to both the inner and outer sublamina of the inner plexiform
layer (IPL). Cells in the INL were found to project their dendrites to
the outermost sublamina of the IPL.
Conclusions: Our assessment of mRGCs in a WS patient compared
with a control demonstrates significant loss of regular RGCs but
relative sparing of mRGCs. Remarkably, in the WS patient, we found
a higher number of mRGCs in the heavily degenerated temporal zone
that includes the papillomacular bundle. This finding supports the
existence of an anatomical substrate which would allow for both the
pupillary reflex and circadian photoentrainment in WS.
Commercial Relationships: Fred N. Ross-Cisneros, Edison
Pharmaceutical, Inc. (F); Chiara La Morgia, None; Billy X. Pan,
None; Jens Hannibal, None; Neil Miller, None; Valerio Carelli,
Edison Pharmaceuticals, Inc. (F), Sigma-Tau, Inc. (F); Alfredo A.
Sadun, Edison Pharmaceuticals, Inc. (F), Stealth Peptides, Inc. (F)
Support: Research to Prevent Blindness, International Foundation
for Optic Nerve Diseases (IFOND), Struggling Within Leber’s,
Eierman Foundation, The Poincenot Family and NIH Grant #
EY03040.
226 Disease Models
Monday, May 05, 2014 11:00 AM–12:45 PM
S 210DE Paper Session
Program #/Board # Range: 1638–1644
Organizing Section: Visual Neuroscience
Program Number: 1638
Presentation Time: 11:00 AM–11:15 AM
Targeted ablation of Crb2 in photoreceptor cells induces Retinitis
Pigmentosa
Celso H. Alves1, Lucie P. Pellissier1, Marina Garcia Garrido2,
Vithiyanjali Sothilingam2, Christina Seide2, Susanne C. Beck2, John
G. Flannery3, Mathias W. Seeliger2, Jan Wijnholds1. 1Neuromedical
genetics, Netherlands Institute for Neurosciences, Amsterdam,
Netherlands; 2Ocular Neurodegeneration, Institute for Ophthalmic
Research, Centre for Ophthalmology, Eberhard Karls University of
Tübingen, Tubingen, Germany; 3Molecular and Cellular Biology,
The Helen Wills Neuroscience Institute, University of California,
Berkeley, CA.
Purpose: In humans, the Crumbs homologue-1 (CRB1) gene
is mutated in progressive types of autosomal recessive retinitis
pigmentosa and Leber congenital amaurosis. In mammals, the
Crumbs family is composed of: CRB1, CRB2, CRB3A and
CRB3B. Recently, we showed that removal of mouse Crb2 from
retinal progenitor cells, and consequent removal from Müller glia
and photoreceptor cells, results in severe and progressive retinal
degeneration with concomitant loss of retinal function that mimics
retinitis pigmentosa due to mutations in the CRB1 gene. We studied
the effects of cell-type specific loss of CRB2 from the developing
and from the adult mouse retina using targeted conditional deletion of
Crb2 in photoreceptors or Müller cells.
Methods: The Crb2 conditional knockout mice were crossed with
Crx-Cre or Pdgfrα-Cre transgenic mice, in order to ablate CRB2
during retinal development, from photoreceptor or Müller cells,
respectively. Retinas from animals with different ages were analyzed.
In vivo analyses of retinal function were performed using optical
coherence tomography (OCT), scanning laser ophthalmoscopy (SLO)
and electroretinography (ERG). To ablate CRB2, in the adult mouse
retina, we generated adeno-associated viral (AAV) vectors encoding
Cre recombinase and short hairpin RNA against Crb2.
Results: In vivo analyses by OCT and SLO on retinas lacking CRB2
in photoreceptors showed progressive thinning of the photoreceptor
layer and cellular mislocalization. ERG under scotopic conditions
showed severe attenuation of the a-wave, suggesting degeneration
of photoreceptors resulting in severe retinitis pigmentosa. Retinas
lacking CRB2 in developing photoreceptors showed early onset of
abnormal lamination. Retinas lacking CRB2 in developing Müller
cells showed late onset retinal disorganization, resulting in mild
retinitis pigmentosa. Short-term removal of CRB2 from the adult
retina did not result in major abnormalities in retinal structure.
Conclusions: Our data show that CRB2 has a redundant function
in mouse Müller glia cells, however in photoreceptor cells CRB2
is crucial for proper retinal development. Removal of CRB2 from
photoreceptors leads to severe and progressive degeneration with
a concomitant loss of retinal function. Furthermore, under normal
physiological conditions, CRB2 is not essential for maintenance of
adhesion between adult photoreceptors or Müller glia cells.
Commercial Relationships: Celso H. Alves, None; Lucie P.
Pellissier, None; Marina Garcia Garrido, None; Vithiyanjali
Sothilingam, None; Christina Seide, None; Susanne C. Beck,
None; John G. Flannery, None; Mathias W. Seeliger, None; Jan
Wijnholds, None
Support: Rotterdamse Vereniging Blindenbelangen, Landelijke
St. voor Blinden en Slechtzienden, St. Blindenhulp, St. Oogfonds
Nederland, St. Retina Nederland, Netherlands Institute for
Neuroscience, Foundation Fighting Blindness [TA-GT-0811-0540NIN and TA-GT-0313-0607-NIN], and The Netherlands Organisation
for Health Research and Development [ZonMw grant 43200004],
European Union [HEALTH-F2-2008-200234].
Program Number: 1639
Presentation Time: 11:15 AM–11:30 AM
Leprdb/db mouse models of Type 2 diabetes exhibit rapid
defects in RPE electrophysiology that correlate with systemic
hyperglycemia
Ivy S. Samuels1, 2, Brent A. Bell2, Neal S. Peachey1, 2. 1Research
Service, Louis Stokes VA Medical Center, Cleveland, OH;
2
Ophthalmic Research, Cole Eye Institute, Cleveland Clinic,
Cleveland, OH.
Purpose: To determine the effects of Type 2 diabetes on function of
the outer retina as measured by electroretinography.
Methods: Leprdb/db mice were bred on BKS and B6.BKS background
strains to establish two models of Type 2 diabetes. Leprdb/db mice and
control littermates (Lepr+/db ;Lepr+/+) underwent standard strobe flash
electroretinography and DC-ERG testing beginning at 4 weeks
of age. Blood glucose and plasma insulin levels were measured.
Histological analysis and in vivo imaging was performed to assess
structure of the outer retina.
Results: In comparison to control littermates, Leprdb/db mice on the
BKS background displayed elevated blood glucose levels at 4 weeks,
and became overtly hyperglycemic at 8 weeks of age, which was
sustained. DC-ERG testing of these mice demonstrated reductions
in the c-wave, revealing disrupted RPE function as early as 4 weeks
of age. While b-wave amplitude was reduced at 8 weeks of age,
a-wave amplitude was normal at both time points. Leprdb/db mice on
the B6.BKS background were severely hyperglycemic by 4 weeks
of age; however, blood glucose levels fell to normoglycemic levels
by 12 weeks and were only slightly elevated compared to control
littermates until 24 weeks, when hyperglycemia returned. These
mice displayed hyperinsulinemia and obesity for the duration of their
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
lives. Reductions in c-wave amplitude for the B6.BKS mice were
also identified as early as 4 weeks; reductions in other measures
of RPE function were observed at both 8 and 12 weeks. B-wave
amplitude was also reduced at 8 weeks in the B6.BKS Leprdb/db
mice, which remained low through 16 weeks of age. At 12 weeks,
a-wave amplitudes were also reduced. At 24 weeks, when mice were
morbidly obese and hyperglycemic, measures of RPE function, both
a- and b- wave amplitudes, as well as the light adapted response
was reduced in B6.BKS mice compared to littermate controls. No
structural damage was observed by in vivo imaging or histological
analysis in either strain of Leprdb/db mice at any age.
Conclusions: The Leprdb/db mouse model of Type 2 diabetes
displays altered outer retina function at times which correlate
with hyperglycemia and precede the development of vascular
damage, oxidative stress, or anatomical abnormality. These findings
demonstrate that outer retina function is markedly affected by
elevated glucose levels.
Commercial Relationships: Ivy S. Samuels, None; Brent A. Bell,
None; Neal S. Peachey, None
Support: Department of Veterans Affairs (Career Development
Award to ISS); The Foundation Fighting Blindness; Research to
Prevent Blindness
Program Number: 1640
Presentation Time: 11:30 AM–11:45 AM
A mouse model to assess gene therapy for RLBP1-associated
retinal dystrophy
Chad E. Bigelow1, Shawn M. Hanks1, Joanna Vrouvlianis1, Oliver
Turner2, Gregory Argentieri2, George Bounoutas1, Vivian W. Choi1,
Thaddeus P. Dryja1, Seshidhar Reddy Police1, Bruce D. Jaffee1.
1
Ophthalmology, Novartis Institutes for BioMedical Research,
Cambridge, MA; 2Preclinical Safety, Novartis Institutes for
BioMedical Research, East Hanover, NJ.
Purpose: To develop a mouse model to test recombinant adenoassociated viral (rAAV) vectors for RLBP1 gene delivery to the eye.
Methods: Retinal morphology was evaluated in RLBP1+/+ and
RLBP1-/- mice of 4, 10, and 16 months of age. Assessments were
made by viewing sections stained with hematoxylin and eosin or by
using image processing on a subset of the sections to determine retina
and outer nuclear layer thickness.
Dark-adapted electroretinograms (ERGs) were used to assess
photoreceptor function in RLBP1+/+, RLBP1+/-, and RLBP1-/mice. Dark-adapted photoreceptor function was compared between
genotypes using a 15-step intensity response series (-1.0 to 3.7 log
scot cd s m-2). The rate of dark adaptation was assessed by monitoring
ERG a-wave amplitude recovery after a photobleach (16 xenon
flashes: 3.7 log scot cd s m-2).
In order to test the ability of gene delivery to restore visual function
to RLBP1-/- mice, 3.6x108 vg/eye of AAV-pRLBP1-hRLBP1 vectors
(NVS1 or NVS2) with different serotypes were injected subretinally.
12 weeks post-injection, recovery of a-wave amplitude (dark
adaptation) 4 hours post-bleach was measured.
Results: There were no notable differences in retinal morphology
with age or between genotypes based on light microscopy
examination or image processing. Dark-adapted photoreceptor
function was also indistinguishable between genotypes. However,
slow dark adaptation was observed in RLBP1-/- mice compared
to wild-type or heterozygous controls. RLBP1+/+, RLBP1+/-, and
RLBP1-/- groups at ~3 hours post-bleach exhibited a-wave recoveries
of 89%, 97%, and 14%, respectively.
Subretinal delivery of NVS1 or NVS2 increased the rate of dark
adaptation in RLBP1-/- mice. Four hours post-photobleach, naïve
eyes recovered 13% of a-wave amplitude compared to 38% (p<0.01)
and 87% (p<0.001) for those receiving NVS1 or NVS2, respectively.
Serotype-related efficacy differences were significant: eyes that
received NVS2 exhibited a >2-fold increase in a-wave amplitude 4
hours post-bleach vs. those receiving NVS1 (p<0.001).
Conclusions: RLBP1-/- mice exhibit slow dark adaptation that is
similar to the deficit observed in patients with RLBP1-associated
retinal dystrophy. ERG-based assessments in this model indicate that
the rate of dark adaptation can be substantially improved with viral
vector-mediated gene delivery and that the assay possesses sufficient
sensitivity to differentiate viral vectors with different properties.
Commercial Relationships: Chad E. Bigelow, Novartis Institutes
for BioMedical Research (E); Shawn M. Hanks, Novartis Institutes
for BioMedical Research (E); Joanna Vrouvlianis, Novartis
Institutes for BioMedical Research (E); Oliver Turner, Novartis
Institutes for BioMedical Research (E); Gregory Argentieri,
Novartis Institutes for BioMedical Research (E); George Bounoutas,
Novartis Institutes for BioMedical Research (E); Vivian W. Choi,
Novartis Institutes for BioMedical Research (E); Thaddeus P. Dryja,
Novartis Institutes for BioMedical Research (E); Seshidhar Reddy
Police, Novartis Institutes for BioMedical Research (E); Bruce D.
Jaffee, Novartis Institutes for BioMedical Research (E)
Program Number: 1641
Presentation Time: 11:45 AM–12:00 PM
A Missense Mutation in Canine CNGA3 Eliminates Retinal Cone
Function: A Novel Model for Achromatopsia
Emily V. Dutrow1, Naoto Tanaka2, Keiko Miyadera1, William R.
Crumley1, Shelby L. Reinstein1, Margret L. Casal1, Jacqueline C.
Tanaka2, Gustavo D. Aguirre1, Karina E. Guziewicz1. 1Clinical
Studies-Philadelphia, University of Pennsylvania, Philadelphia, PA;
2
Department of Biology, Temple University, Philadelphia, PA.
Purpose: Achromatopsia is an autosomal recessive retinal disorder
causing day-blindness, poor visual acuity and monochromacy
as a result of impaired signal transduction in cone photoreceptor
cells. Transmission of light-evoked visual signals requires cyclic
nucleotide-gated (CNG) channels on cone outer segment membranes.
We identified a one-year-old German shepherd dog with the clinical
manifestations of day-blindness. The aim of this study was to identify
and characterize the genetic basis, visual phenotype and molecular
pathogenesis of the disease.
Methods: ERG recordings under light and dark-adapted conditions
were evoked for the day-blind dog and compared to normal ERGs
recorded under identical conditions. Cone phototransduction cascade
genes CNGB3, CNGA3, GNAT2, PDE6C, and PDE6H were selected
for mutation screening. For expression studies, cDNA was ligated
into a YFP expression vector and the CNGA3-R424W plasmid was
generated by site-directed mutagenesis. HEK cells were transfected
with either WT or CNGA3-R424W-YFP construct. Excised patch
clamping was performed to monitor cAMP and cGMP activation
at -60mV to +60mV. Cellular localization of YFP-tagged CNGA3
protein was visualized using fluorescence microscopy. The amino
acid topology of the canine CNGA3 transmembrane segments (S1S6) was analyzed in silico.
Results: Behavioral study of the affected German shepherd
indicated complete day-blindness and ERG recordings confirmed
loss of cone function. Phenotype-directed candidate gene analysis
revealed a R424W (C1270T) missense mutation in exon 7 of canine
CNGA3. HEK cells expressing CNGA3-R424W-YFP showed no
cyclic nucleotide-activated current with patch-clamp recordings in
comparison to WT. Furthermore, mutant homomeric channels showed
abnormal cellular localization indicating that the mutation alters
subunit trafficking; the nature of this mislocalization is still under
investigation. According to amino acid alignment with Kv1.2/2.1,
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
the R424 residue is located in the C-terminal end of S6. Homology
modeling in silico is underway to predict noncovalent interactions of
this residue.
Conclusions: We report the first canine model for CNGA3achromatopsia and characterize its molecular mechanism. Based on
our findings, we propose the R424W as a valuable model for studying
CNG channel malfunction and potentially developing therapies for
cone cell signal transduction diseases caused by CNGA3 mutations.
Commercial Relationships: Emily V. Dutrow, None; Naoto
Tanaka, None; Keiko Miyadera, None; William R. Crumley,
None; Shelby L. Reinstein, None; Margret L. Casal, None;
Jacqueline C. Tanaka, None; Gustavo D. Aguirre, None; Karina
E. Guziewicz, None
Support: NEI/NIH EY-06855, -17549, -019304, -018241,
Foundation Fighting Blindness, Macula Vision Research Foundation,
Hope for Vision, Penn Vision Research Center, Van Sloun Fund for
Canine Genetic Research
Program Number: 1642
Presentation Time: 12:00 PM–12:15 PM
A novel mouse model for complete Congenital Stationary Night
Blindness (cCSNB)
Marion Neuillé1, Said El Shamieh1, Elise Orhan1, Christelle
Michiels1, Kinga M. Bujakowska1, 2, Olivier Poch3, Jose A. Sahel4,
5
, Isabelle Audo6, 5, Christina Zeitz1. 1Institut de la Vision Univ
Pierre et Marie Curie Paris 6; INSERM, UMR_S968; CNRS,
UMR_7210, Paris, France; 2Massachusetts Eye and Ear Infirmary,
Ocular Genomics Institute, Boston, MA; 3Integrative Bioinformatics
and Genomics Laboratory ICube; CNRS, UMR_7357, Strasbourg,
France; 4Univ Pierre et Marie Curie Paris 6; INSERM, UMR_S968;
CNRS, UMR_7210; CHNO, INSERM-DHOS CIC 503; Fondation
Ophtalmologique Adolphe de Rothschild; Académie des SciencesInstitut de France, Paris, France; 5UCL-Institute of Ophthalmology,
London, United Kingdom; 6Univ Pierre et Marie Curie Paris 6;
INSERM, UMR_S968; CNRS, UMR_7210; CHNO, INSERMDHOS CIC 503, Paris, France.
Purpose: Despite that mutations in LRIT3 lead to autosomal
recessive cCSNB, the exact role of the corresponding protein in
the ON-bipolar cell signaling cascade remains to be elucidated. To
develop a tool to study the function and pathogenic mechanism of
LRIT3, we wanted to identify the full length mouse Lrit3 cDNA and
genetically and functionally characterize a commercially available
Lrit3 knock-out (ko) mouse.
Methods: Mouse retinal mRNA was extracted and full length coding
Lrit3 cDNA obtained by RT-PCR. Genomic DNA was isolated and
genotyped for the Lrit3 ko allele, common mutations in laboratory
mouse strains and known genes underlying cCSNB. The ko model
was confirmed on cDNA level. Visual function was measured by
Ganzfeld electroretinography. Retinal structure was investigated
by fundus auto-fluorescence, histology and spectral domain optical
coherence tomography (SD-OCT). The functional characterization
was performed at 6 weeks and 6 months.
Results: In contrast to publicly available databases, the mouse Lrit3
cDNA codes for a protein with 681 amino acids instead of 560.
We confirmed on DNA and RNA level that with the insertion of a
selection cassette a premature stop codon is introduced. This would
code for a presumably non-functional short 206 amino acid protein.
The mouse line does not harbor other mutations present in common
laboratory mouse strains, nor in other cCSNB genes. Lrit3-/- mice
exhibit a so called no b-wave (nob) phenotype with an abnormal
scotopic electroretinogram (ERG), which lacks the b-wave, whereas
the a-wave amplitude and implicit time are normal. The photopic
ERG is also altered with severely decreased b-wave amplitude and
delayed implicit times for both a- and b-waves. No obvious fundus or
histology abnormalities are observed. However, SD-OCT data reveal
minor differences with thinned inner nuclear layer and ganglion cell
complex. This nob phenotype is noted at 6 weeks and 6 months.
Wild-type and heterozygous mice have a normal phenotype at the two
time-points.
Conclusions: The stationary nob phenotype of mice lacking Lrit3,
which we named Lrit3nob6, confirms the findings previously reported
in patients carrying LRIT3 mutations and is similar to other cCSNB
mouse models. This study describes a novel mouse model, which will
be useful to investigate the pathogenic mechanism associated with
LRIT3 mutations and clarify the role of LRIT3 in the ON-bipolar cell
signaling cascade.
Commercial Relationships: Marion Neuillé, None; Said El
Shamieh, None; Elise Orhan, None; Christelle Michiels, None;
Kinga M. Bujakowska, None; Olivier Poch, None; Jose A.
Sahel, Second Sight (F), UPMC/Essilor (P); Isabelle Audo, None;
Christina Zeitz, None
Support: “Agence Nationale de la Recherche” [ANR-12BSVS1-0012-01_GPR179] (CZ), Foundation Voir et Entendre (CZ),
Prix Dalloz for “la recherche en ophtalmologie” (CZ), Ville de Paris
and Region Ile de France, LABEX LIFESENSES [reference ANR10-LABX-65] supported by French state funds managed by the ANR
within the Investissements d
Program Number: 1643
Presentation Time: 12:15 PM–12:30 PM
Retinal inhibitory signaling is compromised in diabetes
Johnnie Moore-Dotson1, Reece Mazade1, Adam Bernstein1, 2, Melissa
Romero-Aleshire1, Heddwen Brooks1, Erika Eggers1, 2. 1Physiology,
University of Arizona, Tucson, AZ; 2Biomedical Engineering,
University of Arizona, Tucson, AZ.
Purpose: Diabetic retinopathy causes severe retinal damage that
ultimately leads to blindness. It was previously thought that diabetic
retinal injury was solely a result of vascular damage, but recent
studies have shown changes in retinal signaling that suggest altered
inhibitory GABAergic signaling prior to vascular changes. We
previously found that spontaneous GABAergic amacrine cell input to
rod bipolar cells is increased without changes in morphology in early
diabetes. The purpose of this study is to determine whether lightevoked inhibitory signaling in the inner retina is compromised in a
mouse model of diabetes.
Methods: Diabetes was induced in C57BL/6J mice at 5 weeks of age
by i.p. injections of streptozotocin (STZ) at a dose of 75 mg/kg over
3 days. Diabetes was confirmed by blood glucose levels >200 mg/
dL. Six weeks post injections, whole-cell voltage clamp recordings
of light-evoked (L) inhibitory (IPSCs) and excitatory postsynaptic
currents (EPSCs) were made from rod bipolar cells (RBCs) in dark
adapted retinal slices. RBCs were held at the reversal potential for
cations or Cl- ions to isolate L-IPSCs or EPSCs, respectively. Light
responses were elicited by a 30 ms full field LED stimulus. GABAA
and GABAC receptor (R) inputs were pharmacologically isolated.
The peak amplitude and charge transfer (Q) were measured.
Results: The L-EPSCs of RBCs that represent rod photoreceptor
inputs were not different from control in STZ mice. The peak
amplitude of L-IPSCs was significantly reduced in STZ mice (n = 17
cells) at multiple light intensities compared to control (n = 16 cells, p
< 0.05). The Q from STZ mice was reduced at a rod dominant light
intensity. The GABACR mediated response was on average reduced,
but this was not significant. However, GABAAR mediated responses
(p < 0.05) were attenuated at multiple intensities in STZ mice.
Conclusions: These results show that light-evoked inhibition to
RBCs is decreased in early diabetes. The decrease in light-evoked
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
inhibition of RBCs from STZ mice suggests that amacrine cells are
less sensitive to light activation in diabetes. Reduced amacrine cell
input is not due to decreased RBC activation because RBC L-EPSCs
are not different in diabetic mice. These results are consistent with
ERG data that indicates altered inhibitory signaling and likely
contribute to the changes in retinal electrical signaling that occurs in
diabetic retinopathy.
Commercial Relationships: Johnnie Moore-Dotson, None; Reece
Mazade, None; Adam Bernstein, None; Melissa Romero-Aleshire,
None; Heddwen Brooks, None; Erika Eggers, None
Support: NIH Institutional Training Grant 2T32HL7249-36A1;
JDRF Innovation Grant
Program Number: 1644
Presentation Time: 12:30 PM–12:45 PM
Evidence that Huntington’s Disease Affects Retinal Structure and
Function
Mary A. Johnson1, Harald Gelderblom2, Klaus Rüther2, Josef Priller2,
Steven L. Bernstein1, 3. 1Ophthal and Vis Science, Univ of Maryland
Sch of Medicine, Baltimore, MD; 2Experimental Neurology, ChariteUniversitätsmedizin, Berlin, Germany; 3Anatomy & Neurobiology,
Univ of Maryland Sch of Medicine, Baltimore, MD.
Purpose: Huntington’s disease (HD) is a fatal autosomal dominant
genetic disease, characterized by progressive neurologic degeneration
affecting muscle coordination, cognition and mood/psychiatric
health. HD is caused by a trinucleotide expansion repeat mutation
in the Huntingtin (HTT) gene. Disease symptoms typically appear
between the 2nd and 4th decades of life, based on the extent of the
expansion-mutation. Numerous areas in the affected brain contain
HTT-inclusion bodies and protein aggregates produced by the mutant
HTT protein. While HTT expansion repeat transgenics have been
shown to exhibit retinal photoreceptor degeneration, these models
were hampered by artificially long expansions that may cause ‘off
effects’. We wanted to evaluate retinal function and structure in an
HTT model that more closely resembles the human disease.
Methods: Transgenic knock-in Huntington’s rats containing
the human HTT gene with a 60-75 expansion repeat were used.
Both Wild type (WT) and mutant pairs from the same litters were
evaluated, and at different ages. Following at least 12 hours of
dark-adaptation, we measured electroretinograms (ERGs) in pairs of
HTT rats and their WT littermates, using the ISCEV protocol and an
additional paradigm we developed to expose changes in presumed
horizontal cell function. This test measured disinhibition of the
photopic response when recorded on a dim vs. bright background.
Rats were euthanized shortly thereafter and retinas immunostained
for HTT, calbindin (stains horizontal cells), Choline acetyl transferase
(ChAT) (stains a subset of amacrine cells), and evaluated by confocal
microscopy.
Results: No differences were seen in the ERG a- and b-waves of the
HTT and normal rats. However, HTT rats showed reduced oscillatory
potential amplitudes and disinhibition of the photopic response.
Immunohistochemistry showed HTT accumulation in the horizontal
cells and suggested a loss of ChAT (+)-amacrine cells.
Conclusions: Huntington’s disease appears to selectively affect
retinal interneurons, causing an unusual inner retinal degeneration,
the first such described. Since the disinhibition test appears to
selectively target horizontal cell function, this test may be a potential
biomarker for future treatment studies.
Commercial Relationships: Mary A. Johnson, None; Harald
Gelderblom, None; Klaus Rüther, None; Josef Priller, None;
Steven L. Bernstein, None
278 Photoreceptors and their synapses
Monday, May 05, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 2361–2371/B0060–B0070
Organizing Section: Visual Neuroscience
Program Number: 2361 Poster Board Number: B0060
Presentation Time: 3:45 PM–5:30 PM
Estimating photopigment excitations from color sensor outputs of
a personal light exposure device
Dingcai Cao, Pablo A. Barrionuevo. Ophthalmology and Visual
Sciences, University of Illinois at Chicago, Chicago, IL.
Purpose: The human circadian clock is regulated by light exposure.
To fully understand how daily light exposure contributes to circadian
rhythm, it will be helpful to know the relative photopigment
(rod pigment, cone pigment and melanopsin) excitations of light
exposures. The ActiWatch Spectrum (Phillips Respironics) is a
commercially available device to record personal exposure to light.
The purpose of the current study was to estimate photopigment
excitation from the color-sensor outputs.
Methods: The spectral sensitivity functions of the red (R), green
(G), blue (B) sensors were obtained from a previous published work
(Price et al, Light Research and Technology, 44:17-26, 2012). The
outputs of the R, G, B sensors were computed under 64 illuminants
(27 daylight illuminants, 27 fluorescent lights, 5 high pressure
lights, and 5 LED-based lights; correlated color temperature CCT:
2000K-10000K). The excitations of rod and cone (S-, M-, and L-)
photoreceptors and melanopsin were computed for each illuminant
using human corneal spectral sensitivity functions. The predictability
of photopigment excitations and CCT from R, G, B outputs was
assessed using a regression method.
Results: For all of the illuminants, a linear combination of R, G,
B outputs could predict the excitations well for S-cones (R2 =
97.8%), rods (R2 = 97.9%) and melanopsin (R2 = 96.5%). The
predictability from R, G, B outputs became relatively poor for
L-cones (R2 = 89.1%), M-cones (R2 = 84.5%) or luminance (R2
= 88.1%), particularly for three-band fluorescent lights due to low
ActiWatch R and G sensor sensitivities between 570nm -590nm
and spikes in the fluorescent light spectrum. A separate function for
three-band fluorescent lights was needed to improve the fits of L- or
M- cone excitations. Meanwhile, a second order polynomial function
combining R, G, B outputs predicted correlated color temperature
satisfactorily for all illuminant types (R2 = 96.8%). Finally, R, G,
B outputs could classify illuminant types (fluorescent vs. daylight
illuminants) satisfactorily (sensitivity = 85%, specificity = 84%).
Conclusions: The R, G, B outputs produce a reasonable estimate
of photopigment (S-cone, M-cone, L-cone, rod and melanopsin)
excitations, but separate weightings are required for disparate
illuminant types. R, G, B outputs may also be useful for classifying
illuminant types.
Commercial Relationships: Dingcai Cao, None; Pablo A.
Barrionuevo, None
Support: NIH NEI grant R01 EY019651 (D. Cao), UIC core grant
for vision research P30-EY01792, Unrestricted Departmental Grant
from the Research to Prevent Blindness, IBRO John G. Nicholls
Research Fellowship (P. Barrionuevo)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2362 Poster Board Number: B0061
Presentation Time: 3:45 PM–5:30 PM
Visual phenotyping in mutant mice deleted for the taurinetransporter gene
Wahiba HADJ SAÏD1, Nicolas G. Froger1, Ivana Ivkovic1, Manuel
Simonutti1, Nathalie Neveux5, 6, Takashi Ito4, Junichi Azuma4, JoséAlain Sahel1, 3, Serge A. Picaud1, 2. 1Transmission de l’information
visuelle,pharmacotoxicité rétinienne et neuroprotection, Centre
de Recherche INSTITUT DE LA VISION UMR S 968 Inserm
UPMC / CHNO des Quinze-Vingts, Paris, France; 2Fondation
Adolphe de Rothschild, Paris, France; 3Centre Hospitalier National
d’Ophtalmologie des Quize-Vingts, Paris, France; 4Department
of Pharmacy, School of Pharmacy, Hyogo University of Health
Sciences, Kobe, Japan; 5Service de Biochimie, Groupe Hospitalier
Cochin – Hôtel-Dieu, Assistance Publique – Hôpitaux de Paris,
Paris, France; 6Laboratoire de Nutrition, EA 4466, Université Paris
Descartes, Faculté de Pharmacie, Paris, France.
Purpose: In the 70s, taurine was shown to induce photoreceptor
degeneration. This rod and cone degeneration was also shown is in
knockout mice for the taurine transporter (Tau-T KO), which takes
up taurine into tissues and cells. More recently, by investigating the
retinal phototoxicity of vigabatrin, we found that this antiepileptic
drug induces a taurine depletion. It led us to discover that both retinal
ganglion cells and cone photoreceptors appear as the primary sites
of damage in albino adult taurine-depleted animals. We have here
further investigated the visual phenotype of the Tau-T KO mouse.
Methods: Visual acuity was evaluated by optokinetics tests, retinal
cell function was measured by electroretinogram (ERG) recordings
on heterozygous (HT) and homozygous (HO) Tau-T KO mice as
compared to wild-type C57BL/6J mice (WT). Cell loss was examined
in vivo, using optic coherence tomography (OCT). Histological
examinations were then performed on both retinal sections and retinal
flatmounts to quantify photoreceptors and retinal ganglion cells
(RGC).
Results: The deletion of gene encoding for Tau-T caused a
marked depletion of plasma taurine concentrations In HO mice
(371±6 mM, 771±176 mM and 732±40 mM for HO, HT and WT
mice, respectively). Interestingly, the mutation induced severe
impairment of visual function since the optokinetic score was
reduced in HO mice compared to WT mice, while the HT mice
were unaffected. Such visual impairments were correlated to
changes in electroretinograms. Indeed, retinal function in the rod
and cone pathways was rapidly abolished in HO mice as indicated
by both scotopic and photopic ERG measurements. OCT imaging
indicated a reduction of the total retinal thickness with a loss of the
photoreceptors layers in HO mice. Histology showed a cone loss,
whereas RGC density appeared unaffected in 9-week HO mice.
Conclusions: These data showed that the absence of Tau-T leads
to a profound retinal degeneration, with a primary photoreceptor
disruption, due to the taurine depletion. Because the RGC loss
was only found in taurine-depleted albino animals, the absence of
RGC loss in the pigmented tau-T KO mice is consistent with the
notion of RGC phototoxicity. Further studies will investigate the
light phototoxicity to RGCs in tau-T KO mice. These data further
demonstrate the crucial role of the Tau-T for maintaining the visual
function.
Commercial Relationships: Wahiba HADJ SAÏD, None; Nicolas
G. Froger, None; Ivana Ivkovic, None; Manuel Simonutti, None;
Nathalie Neveux, None; Takashi Ito, None; Junichi Azuma, None;
José-Alain Sahel, Genesignal (C), GenSight Biologics (C), Pixium
Vision (C), Sanofi-Fovea (C); Serge A. Picaud, None
Support: Institut National de la Santé et de la Recherche
Médicale (INSERM), Pierre et Marie Curie University (UPMC),
Centre National de la Recherche Scientifique (CNRS), Fondation
Ophtalmologique A. de Rothschild (Paris), Agence Nationale pour la
Recherche (ANR: GLAUCOME), the European Community contract
TREATRUSH (n° HEALTH-F2- 2010-242013),Fondation Voir et
Entendre, Fondation pour la Recherche Médicale, Fondation Roland
Bailly, the Fédération des Aveugles de France, IRRP, the city of Paris,
the Regional Council of Ile-de-France, and the French State program
“Investissements d
Program Number: 2363 Poster Board Number: B0062
Presentation Time: 3:45 PM–5:30 PM
Silent Substitution Stimuli silence Cones Light Responses but not
their Output
Sizar Kamar1, 2, Marcus Howlett1, Maarten Kamermans1. 1Retinal
Signal Processing, Netherlands Institute for Neuroscience,
Amsterdam, Netherlands; 2Ophthalmology, Leiden Univ Medical
Center, Leiden, Netherlands.
Purpose: The silent substitution concept was suggested by
Rushton et al. as a way of investigating the contribution of specific
photoreceptor types to vision. A silent substitution stimulus consists
of two alternating stimuli, of which wavelengths and radiances are
chosen such that they present steady excitation in one cone type,
while others are modulated. In principle this procedure prevents
modulation of the phototransduction cascade in the silenced cone
type. Since, the output of cones is determined by both the modulation
of the phototransduction cascade and the feedback signal from
horizontal cells, we asked the question how the output of the cones is
affected by the silent substitution stimulus.
Methods: Responses of cones from isolated goldfish retina were
measured by whole cell voltage clamp. The retina was stimulated
with either silent substitution or cone isolating stimulus, specifically
tailored for each cone type. Direct light responses were measured
outside the activation range of the Ca2+-current of cones (-70 mV).
And the cone output, i.e. the modulation of the Ca2+-current of
cones, was measured at the half activation potential of the Ca2+current (about -40 mV).
Results: For the cone isolating stimuli, light responses were only
seen when stimuli were matched to the cone type being recorded
indicating that the spectral composition of our stimuli were well
matched to each cone type spectral sensitivity. No direct light
responses were found in any of the cones when the appropriate
silent substitution stimuli were used. However, the silent substitution
stimuli for the specific cone types modulated the cone output.
Conclusions: Although silent substitution stimuli may prevent direct
light responses in cones, they do not silences the cone output. This
modulation of the cone output is generated via negative feedback
from horizontal cells to cones. These results indicate that caution is
needed by using silent substitution and cone isolating stimuli to study
the wiring of the visual system.
Commercial Relationships: Sizar Kamar, None; Marcus Howlett,
None; Maarten Kamermans, None
Support: Mosaic grant, NWO
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2364 Poster Board Number: B0063
Presentation Time: 3:45 PM–5:30 PM
Opsin gene identification and evaluation of color vision in an
albino capuchin monkey (Sapajus/Cebus sp)
Leonardo D. Henriques1, Paulo R. Goulart3, Julio C. Oliveira3,
Daniela M. Bonci1, 2, Givago S. Souza4, 5, Luiz Carlos L. Silveira4,
5
, Olavo F. Galvão3, Dora F. Ventura1, 2. 1Experimental Psychology,
University of São Paulo, São Paulo, Brazil; 2Israelite Study and
Research Institute, Albert Einstein Israelite Hospital, São Paulo,
Brazil; 3Experimental School of Primates, Federal University of Pará,
Belém, Brazil; 4Biological Science Institute, Federal University of
Pará, Belém, Brazil; 5Tropical Medicine Center, Federal University of
Pará, Belém, Brazil.
Purpose: Albinism is characterized by a deficit of melanin
production, leading to reduced or absent pigmentation of some
organs. Anatomic differences described for an Old World Monkey
include lack of fovea, high concentration of ganglion cells on the
expected foveal area and higher crossing of visual pathways, but
it was not clear how those differences would affect vision and
specifically color vision. We investigated color discrimination in
an albino capuchin monkey (Sapajus sp, formerly Cebus) using a
modified version of the Cambridge Colour Test and compared it with
healthy Sapajus data. Behavioral phenotypic characterization was
compared with opsin gene genotype.
Methods: The animal was trained via positive reinforcement (190mg banana pellet) to touch over an approximately square target
(5cm2) on a pseudoisochromatic display, independently of hue and
position and then exposed to the test condition. For the test target
chromaticity varied along 20 equidistant vectors, with background
chromaticity fixed at the achromatic point of the CIE 1976 diagram
(u’= 0.1977, v’= 0.4689). Target chromaticity varied between
1100x10-4 and 20x10-4 u’v’ units along each vector, following a
staircase procedure. The orientation of the best-fit ellipse generated
for the 20 threshold points guided phenotypic characterization (Fig.
1). Exons 3 and 5 of the X-linked opsin genes were analysed in order
to infer the spectral sensitivity of M/L cones.
Results: The albino subject successfully learned the visual
discrimination task, and produced color discrimination thresholds
typical of deuteranopy (Fig. 2). The genetic analysis showed an SYT
allele expressing an opsin with λ peak at 560-563nm, consistent with
results described for other non-albino deuteranope Sapajus (Goulart
et al., 2013).
Conclusions: There were no color vision or opsin gene differences
between the albino variant and healthy deuteranopic subjects of
Sapajus. Behavioral and genetic data obtained in this rare specimen
will be complemented by ophthalmological and electrophysiological
measures for a complete characterization of the animal’s visual
capabilities.
Figure 1. Ellipses for the discrimination thresholds of two
deuteranope Sapajus, the albino subject from this work and a normal
subject tested by Goulart et al. (2013).
Figure 2. Color discrimination thresholds from albino subject and
deuteranope monkeys of the Sapajus genera.
Commercial Relationships: Leonardo D. Henriques, None; Paulo
R. Goulart, None; Julio C. Oliveira, None; Daniela M. Bonci,
None; Givago S. Souza, None; Luiz Carlos L. Silveira, None;
Olavo F. Galvão, None; Dora F. Ventura, None
Support: Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP). processo n0 2011/05059-8, Programa Nacional
de Cooperação Acadêmica (CAPES/ PROCAD) Bolsa - processo
n0182/2007, Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) processo n0484228/2011-0
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2365 Poster Board Number: B0064
Presentation Time: 3:45 PM–5:30 PM
Physiological role of M2-group cone visual pigments on
photoresponse in chicken green knock-in mice
Keisuke Sakurai1, Akishi Onishi2, Hiroo Imai3, Osamu Chisaka3,
Takahiro Yamashita3, Kei Nakatani1, Yoshinori Shichida1. 1University
of Tuskuba, Tsukuba, Japan; 2CDB, Riken, Kobe, Japan; 3Kyoto
University, Kyoto, Japan.
Purpose: Rod and cone photoreceptors exhibit photoresponses
different from each other and contain similar visual pigments proteins
with distinctive molecular properties. Phylogenic analyses on visual
pigments indicate that the rhodopsin group diverged from one of
the cone visual pigment groups, referred to as M2 (RH2) group.
To elucidate physiological significance of the divergence of visual
pigments, we have created knock-in mice, where rhodopsin were
replaced with chicken green-sensitive cone opsin as a representative
of M2 group, and carried out electrophysiological recordings of the
photoreceptors.
Methods: Knock-in mice, in which chicken green cDNA was
introduced into mouse rhodopsin loci, were generated with
homologous recombination on mouse ES cells. Using a suction
electrode, we recorded membrane currents from singe rod
photoreceptors of knock-in and wild-type mice.
Results: With an immunohistochemistry of mice retina, chicken
green cone pigments ectopically expressed by homologous
recombination were found to be properly localized in rod outer
segments. Electrophysiological analysis of suction recordings
showed that photoisomerization of visual pigments required for
the half-saturating response was 16 R* and 42 R* in wild-type and
homozygote, respectively, indicating that the sensitivity as a function
of photoisomerization is 0.4-fold lower in homozygote than that
in wild-type. The mean amplitude of single-photon response (pA)
calculated with an ensemble variance-to-mean ratio was 0.53 in wildtype and 0.22 in homozygote, which is in a good agreement with the
result of photoisomerization-response relation. Moreover, the kinetics
of dim flash response of homozygote was significantly accelerated as
compared with that of wild-type. Whereas time-to-peak was 159 ms
in wild-type and 145 ms in homozygote, integration time was 300 ms
in wild-type and 189 ms in homozygote.
Conclusions: The expression pattern of chicken green opsin from
recombinant allele is apparently under the endogenous regulatory
control. The physiological results suggest that the dim-flash kinetics
as well as response amplitude may be affected by property of visual
pigments.
Commercial Relationships: Keisuke Sakurai, None; Akishi
Onishi, None; Hiroo Imai, None; Osamu Chisaka, None; Takahiro
Yamashita, None; Kei Nakatani, None; Yoshinori Shichida, None
Program Number: 2366 Poster Board Number: B0065
Presentation Time: 3:45 PM–5:30 PM
The wrb gene encodes a novel protein required for ribbon
synapse function
Lauren L. Daniele1, Farida Emran2, Brian D. Perkins1. 1Ophthalmic
Research, Cole Eye Institute, Cleveland Clinic Foundation,
Cleveland, OH; 2Centre for Research in Neuroscience, McGill
University, Montreal, QC, Canada.
Purpose: Ribbon synapses of sensory neurons tonically release
glutamate to signal graded changes in stimulus intensity over a large
operating range. The ribbon structure is found at photoreceptor, hair
cell, and bipolar cell synapses where it tethers synaptic vesicles
in close proximity to the presynaptic active zone. The zebrafish
hi1482 mutant was isolated in a screen for abnormal visual system
development and results from a retroviral insertion in the gene
encoding tryptophan rich basic protein (Wrb). The goal of this
study was to gain insight into the role of this novel protein in ribbon
synapse structure and function.
Methods: Electroretinography (ERG), optokinetic response
measurements (OKR), Immunohistochemistry (IHC) and electron
microscopy (EM) analyses were employed to assess ribbon synapse
function, protein expression, and ultrastructure. Real-time q RTPCR was used for relative quantification of wrb expression. As
homozygous mutants do not survive to adulthood, all fish were
analyzed at 5 days post fertilization, when rods are not active.
Results: The hi1482 retroviral insertion represents a hypomorphic
mutation, with expression of wrb reduced to less than 1% of WT.
Mutant OKR was minimal, with saccade frequency reduced to 15%
of WT and gain of OKR slow phase negligible at the highest intensity
and contrast. Mutants have severely diminished b-wave responses,
with maximal b-wave amplitudes only ~20% of WT. ERG a-waves
had comparable amplitudes in mutant vs. WT. Since photoreceptor
number appears normal in mutant fish, ERG and OKR results point to
a specific defect in cone photoreceptor synaptic transmission. Mutant
cone photoreceptor synapses had a greater number of misaligned,
floating ribbons than WT and a partial disruption in SV2 and ribeye
localization. More severe disruptions of ribbon architecture were
encountered in mechanosensory hair cells of mutants, where ribeyepositive presynaptic ribbons were scarce. Despite disrupted signaling,
the post-synaptic contacts of bipolar cells at cone synaptic terminals
were structurally normal in wrb mutants.
Conclusions: The attenuated synaptic transmission at ribbon
synapses and the mislocalization of key presynaptic components in
the wrb mutant suggest that wrb is important for the assembly or
maintenance of ribbon synapses.
Commercial Relationships: Lauren L. Daniele, None; Farida
Emran, None; Brian D. Perkins, None
Support: NIH Grant EY021865
Program Number: 2367 Poster Board Number: B0066
Presentation Time: 3:45 PM–5:30 PM
Microanatomy of the postsynaptic triads at the ribbon synapse of
rod spherules in the mouse retina
Hong Lim Kim1, 2, Eun Jeong Kim1, Ji Hyun Jeon1, Sun-Sook Paik1,
Stephen C. Massey3, In-Beom Kim1, 4. 1Department of Anatomy, The
Catholic University of Korea, Seoul, Republic of Korea; 2Integrative
Research Support Center, The Catholic University of Korea, Seoul,
Republic of Korea; 3Ophthalmology and Visual Sciences, The
University of Texas-Medical School at Houston, Houston, TX;
4
Catholic Neuroscience Institute, The Catholic University of Korea,
Seoul, Republic of Korea.
Purpose: The ribbon synapse at the photoreceptor terminal is the
first synapse in the retina. Recent studies have revealed the molecular
structure of the synaptic ribbon but there are few studies concerning
the postsynaptic elements at ribbon synapses. Therefore, we
examined the microanatomy of the postsynaptic triad invaginating the
rod spherule by Focused Ion Beam Scanning Electron Microscopy
(FIB-SEM) combined with 3D image analysis.
Methods: C57BL/6J mouse retinas were dissected and fixed in 2%
parformaldehyde and 2.5% glutaraldehyde. The tissues were stained
en bloc and embedded in Epon. Retinal pieces were cut horizontally
through the outer plexiform layer with 30-nm thickness and we
automatically scanned the cutting surface by FIB-SEM. The scanned
images were added one by one and reconstructed with Mimics
software.
Results: Synaptic triads: The postsynaptic structure was composed
of one or two bipolar dendrites as the lower central element with
two horizontal cell axon terminals providing lateral elements closest
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
to the synaptic ribbon. All the processes entered the rod spherule
together as a thin bundle at one side of the synaptic ribbon plate.
Afterwards, the two horizontal elements ran parallel on either side of
the ribbon in an arc, giving a ‘hook’ or ‘question mark’ appearance
when viewed from the side. When one bipolar dendrite formed the
central element, it expanded horizontally like a fan with a concave
top opposed to the vertical synaptic ribbon. However, when two
bipolar dendrites entered the rod spherule, they expanded vertically
and parallel to each other.
Conclusions: These results demonstrate that the organization of
postsynaptic triads at rod ribbon synapses is variable with one or
two low but central bipolar elements flanked by two horizontal cell
processes in close apposition to the synaptic ribbon.
Commercial Relationships: Hong Lim Kim, None; Eun Jeong
Kim, None; Ji Hyun Jeon, None; Sun-Sook Paik, None; Stephen
C. Massey, None; In-Beom Kim, None
Support: Basic Science Research Program
(2013R1A2A2A01014070) through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science, and Technology
Program Number: 2368 Poster Board Number: B0067
Presentation Time: 3:45 PM–5:30 PM
Electron tomography reveals 3D architecture of the cone ribbon
synapse
Jun Zhang1, Alioscka Sousa2, Richard D. Leapman2, Jeffrey
Diamond1. 1Synaptic Physiology Section, NINDS/NIH, Bethesda,
MD; 2Laboratory of Cellular Imaging and Macromolecular
Biophysics, NIBIB/NIH, Bethesda, MD.
Purpose: Previous studies suggested that the readily releasable pool
(RRP) at ribbon synapses (RSs) in cone consists of 20 (salamander)
~36 (primate) synaptic vesicles (SVs) that can be released rapidly
in response to membrane depolarization. To better understand the
physical arrangement of the RRP at cone terminals, we used scanning
transmission electron tomography (ET) to quantify and visualize the
SVs tethered to the ribbon, the subset of those SVs that are docked at
the base of the ribbon, and also synaptic ribbon morphology.
Methods: ET of cone RSs was performed on routine EM-embedded
retinas from P18 Sprague-Dawley rats. Thick sections were collected
on Formvar-coated slot grids, counterstained with heavy metals,
covered with evaporated carbon, and coated with 10 or 20 nm gold
particles. Dual-axis tilt series of selected RSs were acquired using an
FEI Tecnai TF30 TEM (at 300-kV beam voltage) from 0.2 mm thick
sections with a tilt range of +65° to −65° in 2° angular increments,
and from 1.0-1.2 mm thick sections with a tilt range of +55° to −55°
in 1.5° angular increments. Images were acquired with pixel sizes of
either 0.75 or 1.4 nm. Tilt series were aligned and reconstructed by
means of IMOD software, and were then rendered, segmented, and
analyzed using Amira software.
Results: Seven entire cone RSs were analyzed. The ribbon at each
synapse exhibited plate-like, rectangular morphology, with average
reconstructed dimensions of 310±94.1 nm (length; mean±SD) x
236±45.7 nm (height) x 40.4±0.8 nm (width). Each ribbon was
tethered to 84±27.2 SVs by thin filaments; 37±2.7% (30±9.3) of those
SVs either touched or were tethered to the presynaptic membrane
and constituted the RRP. Most SVs in the RRP were at the base of
the ribbon, but a few SVs in the middle of the ribbon formed tethers
with the presynaptic membrane. SVs had diameters of 38.5±6.5
nm (n=772) and were tethered to the ribbon by several filaments
(27.1±5.2 nm in length, n=62) or to the presynaptic membrane by
short filaments (11.2±5.1 nm, n=62).
Conclusions: This study imaged, for the first time, the full 3D
architecture of mammalian cone RSs, in which regular docked and
tethered SVs, as well as plate-like, rectangular-shaped synaptic
ribbon were visualized. Quantitative analysis revealed an RRP size
that is consistent with previous results and indicates a morphological
correlate of the functionally defined RRP.
Commercial Relationships: Jun Zhang, None; Alioscka Sousa,
None; Richard D. Leapman, None; Jeffrey Diamond, None
Support: NINDS Intramural Research Program
Program Number: 2369 Poster Board Number: B0068
Presentation Time: 3:45 PM–5:30 PM
Zebrafish Transgenic Reports Mushashi1 (Msi1) in Retinal
Neurons
Ralph F. Nelson1, Reena R. Abraham1, Sara Patterson1, Jennifer L.
Strykowski2, Lin Li3, Harold A. Burgess2, Victoria P. Connaughton4.
1
Basic Neurosciences Program, NINDS NIH, Bethesda, MD;
2
Behavioral Neurogenetics, NICHD, NIH, Bethesda, MD;
3
Ophthalmic Molecular Genetics, NEI, NIH, Rockville, MD;
4
Biology, American University, Washington, DC.
Purpose: Gal4 insertion in the line Et(SCP1:Gal4ff)y245 (y245) is
localized to the promoter of msi1, making y245 a candidate reporter
for msi1. Here we compare immunoreactivity (IR) of an Msi1
antibody to y245;UAS: kaede fluorescence.
Methods: The 14h1 rat Msi1 antibody (MBL), selected because
zebrafish Msi1 contains a sequence similar to the 14h1 antigenic
peptide, revealed a single 41kDa band on Western blots of 6dpf
larval heads. For IR, 6dpf heads were fixed in 4% PFA in PBS,
impregnated with 30% sucrose, embedded in OCT, and cryosectioned
at 10 mm. After incubation in blocking buffer (2% fetal bovine
serum or BSA, 0.2% Triton X-100, 1% DMSO in PBS), sections
were incubated overnight (4C) in 14h1 (1:200) in blocking buffer.
Goat-anti-rat Alexafluor594 (Abcam, 1:200) secondary antibody and
confocal microscopy revealed IR patterns. ERG b2 and PIII waves
were isolated from 6dpf eyes perfused with 50mM CNQX or 20mM
Na Aspartate (respectively) in 95%O2/5%CO2-saturated MEM
(Invitrogen), and recorded with glass microelectrodes (WPI DAM80
amplifier) using a spectral stimulation protocol (Nelson & Singla,
2009).
Results: Western blots of 6dpf heads showed no change of Msi1
expression in mutants (y245+/+). In mutants, hets, and WT eyes,
14h1-IR occurred in perinuclear cytoplasm of ganglion and amacrine
cells, inner plexiform layer (IPL), cone synaptic pedicles, and a high
band of cone inner segments. Descending cone axons crossed an
unreactive dark band between inner segments and pedicles. Bipolar
somata labeled faintly. Kaede was brightest in y245;UAS:kaede
mutants and localization was identical to 14h1-IR, though in mutants,
Müller cells and the dark photoreceptor band were also labeled.
Kaede stained cones were previously shown to be red and green
types (Cohen et al, 2013). WT retinas displayed regular layering, but
y245 mutants often revealed thin and irregular IPL (33% reduction in
mean thickness). Mutant 6dpf b2 and PIII spectral sensitivities were
depressed.
Conclusions: y245 gene products are found in the same retinal cell
types and layers as 14h1-IR suggesting y245 is a complete msi1
reporter. Localization of Msi1, an RNA binding protein, within
synaptic layers is of interest for function. y245 mutants show changes
in PIII and b2 responsiveness, and altered retinal histology, but
no gross reduction in protein expression. This suggests that y245
interferes with the regulation of Msi1 expression.
Commercial Relationships: Ralph F. Nelson, None; Reena R.
Abraham, None; Sara Patterson, None; Jennifer L. Strykowski,
None; Lin Li, None; Harold A. Burgess, None; Victoria P.
Connaughton, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2370 Poster Board Number: B0069
Presentation Time: 3:45 PM–5:30 PM
Forskolin modulates photoresponses in mouse rods but not as
strongly as in amphibian photoreceptors
Teemu Turunen1, Michael L. Firsov2, Ari O. Koskelainen1.
1
Department of Biomedical Engineering and Computational Science,
Aalto University School of Science, Espoo, Finland; 2I.M. Sechenov
Institute of Evolutionary Physiology and Biochemistry, Russian
Academy of Sciences, 194223 Saint Petersburg, Russian Federation.
Purpose: Intracellular [cAMP] is regulated by circadian rhythm.
The level of cAMP peaks at night and decreases during the daytime
(Chaurasia et al., J. Neurochem., 2006). The increase in [cAMP]
can be mimicked by introducing the adenylate cyclase activator,
forskolin, which accelerates the synthesis of cAMP. Photoreceptor
sensitivity and kinetics have recently been shown to be regulated
by cAMP via multiple targets in the phototransduction cascade in
amphibian rods (Astakhova et al., J. Gen. Physiol., 2012). Single cell
recordings on Rana ridibunda rods suggest that forskolin increases
intracellular Ca2+ and decreases basal PDE activity. This is seen
as delayed deactivation kinetics and over twofold increase in rod
sensitivity. The object of this study was to find out whether similar
kinds of effects by forskolin are present also in mice rods.
Methods: Dark-adapted ERG responses to green LED light
flashes were recorded from WT and GCAP-/- mice (C57BL/6J).
Isolated retina was perfused with modified Ringer’s solution at
37±1°C containing BaCl2, DL-AP4 and/or aspartate to isolate rod
photoresponses. 5 or 10 mM forskolin dissolved in DMSO was
introduced in the perfusion solution for 20 minutes before solution
was changed back to normal. Saturated and small stimulus responses
were followed throughout the experiment. ERG responses were
simultaneously recorded transretinally (TERG) and across the outer
segment layer with microelectrodes (LERG).
Results: Forskolin increased the saturated photoresponse amplitude
by 40-50 % in CGAP-/- mice, while only a minor increase was
observed in WT mice. The maximal effect was achieved within 10
minutes after introducing forskolin. With TERG fractional small
stimulus responses of WT and CGAP-/- rods grew 30 % and 50 %
respectively, while 10 % and 20 % increases were seen in LERG
recordings. Also in CGAP-/- mice the time constant of small response
recovery (τrec) as well as the time-to-peak increased by 10-20 % in
LERG recordings.
Conclusions: Forskolin caused notable effects in CGAP-/- mouse
rod sensitivity and time-to-peak which seemed to be a result from
a delayed deactivation of responses. The effects were considerably
smaller than in amphibian photoreceptors. The growth in the
saturation amplitude observed in CGAP-/- mice rods can be due to the
lack of Ca2+ feedback to guanylate cyclase activity.
Commercial Relationships: Teemu Turunen, None; Michael L.
Firsov, None; Ari O. Koskelainen, None
Support: Academy of Finland (Grant 269747), International
Graduate School in Biomedical Engineering and Medical Physics
Program Number: 2371 Poster Board Number: B0070
Presentation Time: 3:45 PM–5:30 PM
Mislocalization of Cone Nuclei Impairs Cone Dark Adaptation in
Mice
Yunlu Xue1, 2, David S. Razafsky1, Didier M. Hodzic1, Vladimir J.
Kefalov1. 1Ophthal & Visual Sciences, Washington Univ in St Louis,
St Louis, MO; 2Neuroscience Program, Division of Biology &
Biomedical Sci., Washington Univ in St. Louis, St. Louis, MO.
Purpose: The nuclei of rod and cone photoreceptors form the
outer nuclear layer (ONL) of the retina, with cone nuclei localized
specifically at the apical side. The expression of EGFP-Kash2 in
cones disrupts this organization and results in the mislocalization
of the cone nuclei to the basal side of the ONL. We examined how
the localization of cone nuclei affects the function of cones by using
transgenic EGFP-Kash2 mice.
Methods: EGFP-Kash2 transgenic mice were crossed with
HGRP-Cre mice to induce cone-specific expression of EGFPKash2 and immunostaining was performed to confirm cone nuclei
mispositioning and synaptic defects. To facilitate cone recordings,
all mice were in the Gnat1-/- (transducin α-subunit knockout)
background and were dark adapted overnight prior to the recordings.
Cone photoresponses were obtained from transretinal recordings, and
cone b-wave responses were obtained from in-vivo ERG recordings.
We determined the functional properties of cones in dark-adapted
conditions and their recovery from exposure to bright bleaching light.
Results: EGFP-Kash2 expression in cones resulted in the
mislocalization of >95% of cone nuclei to the basal side of the
ONL as well as the OPL. While this mislocalization did not affect
the inner and outer segments in 6 month-old mice, the structural
organization of cone pedicles was severely disrupted. We carried
out electrophysiological experiments to determine the functional
consequences of these observations. In dark-adapted conditions, the
mislocalization of cone nuclei did not affect the kinetics or maximal
amplitude of cone flash responses, but decreased the sensitivity (I1/2)
by 2.8-fold. In addition, the maximal cone b-wave amplitude was
decreased by 1.6-fold in EGFP-Kash2/HGRP-Cre mice. Notably,
the level of cone b-wave sensitivity recovery 50 minutes following a
bright bleach in-vivo was 2.6-fold lower. The level of cone sensitivity
recovery 7 minutes after a bleach in isolated retina was also
suppressed by 1.5-fold.
Conclusions: The mislocalization of cone nuclei reduced the cone
sensitivity and compromised the synaptic transmission between
cones and bipolar cells in EGFP-Kash2/HGRP-Cre mice. Notably,
cone dark adaptation was impaired both in vivo and in isolated
retina suggesting that the positioning of cone nuclei is important
in maintaining the chromophore supply to cones through both the
pigment epithelium and the retina visual cycles.
Commercial Relationships: Yunlu Xue, None; David S. Razafsky,
None; Didier M. Hodzic, None; Vladimir J. Kefalov, None
Support: EY019312, EY021126, EY002687, EY022632 to the
Department of Ophthalmology and Visual Sciences at Washington
University, Research to Prevent Blindness
279 Circadian, adaptation, and modulation
Monday, May 05, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 2372–2376/B0071–B0075
Organizing Section: Visual Neuroscience
Program Number: 2372 Poster Board Number: B0071
Presentation Time: 3:45 PM–5:30 PM
Experimental analysis of variance adaptation in the horseshoe
crab eye
Tchoudomira Valtcheva, Christopher L. Passaglia. Chemical and
Biomedical Engineering, University of South Florida, Tampa, FL.
Purpose: To characterize contrast-dependent gain changes in the
crab eye as a function of mean illumination level and investigate their
physiological origins.
Methods: A visual stimulation system was built to drive single or
multiple ommatidial receptors of the horseshoe crab eye, consisting
of an optical coupler that focused light from a computer monitor onto
a 150um or larger optical fiber. The monitor output was modulated
according to random binary sequences having different mean
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
luminance and luminance variance. Single optic nerve fiber responses
to the white noise stimulus were then recorded from live male
horseshoe crabs and saved to computer. The spike train records were
analyzed in terms of a linear-nonlinear model. In other experiments,
electroretinograms (ERGs) were recorded from the eye stimulated by
a linearized LED modulated by the same random binary sequences.
ERG records were also evaluated by white noise analysis.
Results: Optic nerve recordings from the horseshoe crab eye
demonstrate existence of variance adaptation. The sensitivity of the
adaptive process to stimulus contrast was the same across mean light
levels ranging from 0.6 to 60 cd/m2. Single ommatidial and whole
eye illumination gave similar results, indicating that this process
does reside in lateral inhibitory synapses of the retinal network. ERG
recordings did not show evidence of variance adaptation, implying
that contrast-dependent gain changes originate in post-receptoral
mechanisms.
Conclusions: Variance adaptation does not depend on mean light
level over the photopic to mesopic range. Its origin appears to lie in
eccentric cells, which encode photoreceptor signals in optic nerve
spike trains. Possible mechanisms include spike generation, selfinhibition, or a heretofore unknown process.
Commercial Relationships: Tchoudomira Valtcheva, None;
Christopher L. Passaglia, None
Program Number: 2373 Poster Board Number: B0072
Presentation Time: 3:45 PM–5:30 PM
Trafficking and turnover of Cx36 in HeLa cells studied by
fluorescent pulse-chase labeling
Yanran Wang1, 2, John O’Brien1, 2, Cheryl K. Mitchell1. 1Vision and
Ophthalmology, University of Texas Health Science Center Houston,
Houston, TX; 2Neuroscience, The University of Texas Graduate
School of Biomedical Science, Houston, TX.
Purpose: Electrical synapses formed of the gap junction (GJ)
protein Cx36 show a great deal of functional plasticity in the retina,
much dependent on changes in phosphorylation of the connexin.
However, GJ turnover may also be important for regulating cell-cell
communication, and turnover rates of Cx36 have not been studied.
Methods: We utilized HaloTag technology to perform pulse-chase
analysis of Cx36 turnover in transiently transfected HeLa cells. The
HaloTag protein forms irreversible covalent bonds with chloroalkane
ligands, allowing specific protein labeling. The HaloTag open reading
frame was inserted into an internal site in the C-terminus of Cx36
designed not to disrupt phosphorylation sites or C-terminal proteinprotein interactions. HeLa cells were pulse labeled with Oregon
Green (OG) HaloTag ligand and chase labeled at various times with
tetramethylrhodamine (TMR) ligand. Cells were fixed 30 minutes
after initiation of chase labeling.
Results: Cx36-Halo formed large plaques at sites of contact
between transfected HeLa cells and was also contained in many
intracellular vesicles. Pulse labeled Cx36 was gradually replaced by
newly synthesized Cx36 labeled with the chase ligand (TMR). The
half-life of Cx36 in junctional plaques was 2.8 hours. Disruption
of the Golgi apparatus with brefeldin A prevented the addition of
new connexins to junctional plaques. Two classes of intracellular
vesicles were observed. Small chase-labeled vesicles were interpreted
to be trafficking Cx36 for exocytosis; large ring-shaped vesicles
containing pulse label and occasionally chase label were interpreted
to be endocytic and degradation vesicles. Newly synthesized vesicles
were added to existing GJs throughout the GJ, not just at edges. Old
GJ was removed from the center as well as the ends of the existing
plaques. Both classes of vesicle were associated with actin filaments
labeled with phalloidin leading to the GJ. Thick actin bundles
connected all edges of GJ plaques, but actin filaments were rare
within any plaque.
Conclusions: Two-color fluorescent pulse-chase labeling allows
discrimination of exocytic and endocytic vesicles and revealed
unique aspects of connexin trafficking to and from gap junctions.
Turnover of Cx36 could contribute to long-term changes in coupling
by changing the number of available channels in a gap junction. This
can complement short-term regulation of channel opening by Cx36
phosphorylation.
Commercial Relationships: Yanran Wang, None; John O’Brien,
None; Cheryl K. Mitchell, None
Program Number: 2374 Poster Board Number: B0073
Presentation Time: 3:45 PM–5:30 PM
Retinal photoreception modulates brain serotonin function
Chad R. Jackson, Noah H. Green, Douglas McMahon. Biological
Sciences, Vanderbilt University, Nashville, TN.
Purpose: Clinical and animal studies show that seasonal light
signals, transduced and transmitted by the retina, are involved in
mood regulation. For example, people who suffer from Seasonal
Affective Disorder display alterations in retinal responses that
correlate with winter time decreases in mood. The neurotransmitter
serotonin is known to be important for mood regulation and the midbrain Raphe nuclei are the sole site of serotonin production in the
brain. We hypothesized that decreased retinal function would impact
serotonergic function in the Raphe Nucleus. To test this hypothesis
we assayed dorsal raphe 5-HT neuron activity and gene expression
in strains of mice with degeneration of rod and cone photoreceptors
(C3H, rd1/rd1), and loss of melanopsin photo transduction (Opn4-/-).
Methods: In vitro physiology: Dorsal raphe nuclei were placed on
perforated multi-electrode arrays and recorded for serotonergic cell
baseline firing rates in the presence of 40mM tryptophan and 3mM
phenylepherine. Next, serotonin was perfused over the slice for 5
minutes at a concentration of 40mM to observe autoinhibition, which
identifies 5-HT neurons.
qRT-PCR: Mid-brains were removed, total RNA extracted, and
reverse-transcribed (~250ng) into cDNA. qRT-PCR reactions were
performed with 2μL cDNA, 12.5μL of SYBR Green Supermix, 8.5μL
water and 1μL of 300nM forward and reverse primers in a Bio-Rad
CFX96 Real-Time System. Each sample was assayed in duplicate.
H&E staining: Mouse eyes were removed and dropped fixed
overnight in Davidson’s Fixation. Next, eyes were dehydrated in
alcohol washes, placed in Xylene, and then in Paraplast Xtra. The
blocks were cut at 6 μM sections, dried overnight, and stained with
hematoxylin and eosin.
Results: Mice with substantial reduction of rod and cone
photoreceptors (C3H, rd1/rd1), or elimination of melanopsin from
retinal ganglion cells (Opn4-/-) showed significant reductions in
5-HT neuron firing rates in vitro. Also, Opn4-/- mice displayed
reductions in the expression of Tph2, and Sert, key genes regulating
the synthesis and bioavailability of 5-HT, and Pet-1, which is required
for the serotonergic neuron phenotype.
Conclusions: We found that mice with decreases in photoreception
display lower serotonergic physiological function and genetic
markers. These findings suggest that the visual system functionally
impacts the serotongeric system and implicates it as a potential site to
modulate seasonal mood disorders.
Commercial Relationships: Chad R. Jackson, None; Noah H.
Green, None; Douglas McMahon, None
Support: NIH Grant EY015815; Vanderbilt Vision Research Center
Grant EY008126; Vanderbilt Silvio Conte Pilot Grant MH096972
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2375 Poster Board Number: B0074
Presentation Time: 3:45 PM–5:30 PM
Using Change Blindness to Study the Effect of Visual Attention in
Visual Area V4
Daniel J. Foster, Fabrice Arcizet, Koorosh Mirpour, James W. Bisley.
Neurobiology, University of California Los Angeles, Los Angeles,
CA.
Purpose: Visual attention is necessary to perceive and react to our
world; when we do not attend something, we are often completely
unaware of it. This lack of perception is exemplified in change
blindness tasks in which we are unable to detect a difference between
two scenes of objects separated by a blank screen, even though the
change may be large. Past studies have suggested that behavioral
effects of attention are due to modulation of neural activity in
the visual cortex. Here we test this hypothesis by asking whether
attentional modulation in visual area V4 can explain performance in a
change detection task.
Methods: Two animals were trained in a change detection task
comprised of one, two, four, or eight orientated bars on a screen. The
bars were shown to the animals twice for 500 ms, with a 100 ms gap
between presentations. They had to spread their attention to all the
bars and determine if one of had rotated between the presentations.
In a control condition, one location was loaded with a high reward,
which biased the animal’s attention towards that location.
Results: The animals’ performance decreased as the number of
stimuli increased. Given the results of past studies, we expected
to see the neural activity of V4 correlate with the degree to which
attention was spread; high activity correlating with less spread of
attention and lower activity with more spread of attention. Using
extracellular electrodes to measure spiking activity from single
neurons, we found that the neurons in V4 did not vary as a function
of the number of stimuli. When the animals were tested on the high
reward task, attentional modulation in V4 was seen concurrent with
improved performance at that location.
Conclusions: Since the V4 activity did not vary as a function of the
number of stimuli, but the behavior did, these results suggest that the
attentional modulation seen in past studies is not responsible for the
decreased performance seen with the greater set sizes. We believe the
change in performance is due to a second physiological mechanism,
which limits the information that can be passed forward to cognitive
processing areas.
Commercial Relationships: Daniel J. Foster, None; Fabrice
Arcizet, None; Koorosh Mirpour, None; James W. Bisley, None
Support: National Eye Institute EY019273, and the McKnight
Foundation
Program Number: 2376 Poster Board Number: B0075
Presentation Time: 3:45 PM–5:30 PM
Cell type-specific distribution of dopamine D1a receptors in
retina revealed in the Drd1a-tdTomato BAC transgenic mouse
Bozena Fyk-Kolodziej1, Paul D. Walker1, Tomomi Ichinose1, 2.
1
Anatomy and Cell Biology, Wayne State University, Detroit, MI;
2
Ophthalmology, Wayne State University, Detroit, MI.
Purpose: Light-induced dopamine release by amacrine cells
regulates transition from rod to cone driven signal flow in the
retina. Dopamine acts at different levels through modulation of
photoreceptor light responses, voltage-gated channels, and gap
junctions. Although dopamine D1 receptors (D1Rs) are involved, it
has been difficult to localize D1Rs to specific subtypes of neurons
due to inadequate whole-cell staining with specific D1R antibodies.
We have overcome this limitation using the BAC transgenic mouse,
which shows strong tdTomato fluorescence in whole DR1a receptorexpressing neurons.
Methods: Drd1a-tdTomato BAC transgenic mice were used which
express tdTomato under the control of DR1a promoter. Specific types
of neurons were labeled by markers. Bipolar cells: type1 - NK3R;
type 2 and 6 - Syt 2b; type 3 - HCN4; type 4 - Csen; type 5 - HCN1;
RBC - PKC α. Type 9 was revealed by contact with genuine S-opsin
cone. Horizontal cells: Calbindin. In addition, individual neurons
were injected with neurobiotin for subsequent staining with Alexa
conjugated streptavidin. tdTomato expression was enhanced by Dsred
antibody.
Results: Strong tdTomato fluorescence was observed in entire cells,
allowing for morphological subtype identification. The number of
cells positive for DR1a within the INL and the GCL was distributed
uniformly throughout the retina. Horizontal cells were positively
labeled for DR1a. Many, but not all subtypes of bipolar cells
(BCs) were positive for DR1a. For OFF BCs, DR1a was positive
in subtypes -1 and -4, but was negative in subtypes -2 and -3. In
ON BCs, subtypes -5 (partly), -6, and -9 BCs were DR1a positive.
Interestingly, terminals of DR1a expressing type-5 BCs were widefield, located adjacent to the ON ChAT band, and were positive for
HCN1. Another subset of subtype -5 with umbrella like terminals
were negative for DR1a. Rod BCs did not express DR1a; however,
strong fluorescence was seen in close proximity to axon terminals,
suggesting the presence of DR1a in AII or A17 amacrine cell (AC).
Because A17 ACs were negative for Tomato fluorescence, the AII
ACs probably express DR1a.
Conclusions: We have localized DR1a to many subtypes of cone
BCs. Since detailed dopaminergic effects on BCs mediated by DR1a
have not been studied, these results could lay the groundwork for
further physiological experiments to elucidate dopamine action in
cone circuitry.
Commercial Relationships: Bozena Fyk-Kolodziej, None; Paul D.
Walker, None; Tomomi Ichinose, None
Support: Midwest Eye Banks, RPB, NIH Grant EY020533
280 Retinal ganglion cells
Monday, May 05, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 2377–2395/B0076–B0094
Organizing Section: Visual Neuroscience
Contributing Section(s): Retina
Program Number: 2377 Poster Board Number: B0076
Presentation Time: 3:45 PM–5:30 PM
Morphological and Functional Characterization of Ex Vivo
Retinal Explants from Adult Mice
Soile Nymark1, 2, Symantas Ragauskas3, 4, Virpi Savolainen1, 2,
Andrea Holme5, Jari A. Hyttinen1, 2, Giedrius Kalesnykas3, 6. 1Dept
of Electronics&Communications Engin, Tampere University of
Technology, Tampere, Finland; 2BioMediTech, Tampere, Finland;
3
Department of Ophthalmology, University of Eastern Finland,
Kuopio, Finland; 4Institute of Innovative Medicine, Vilnius,
Lithuania; 5Flow Cytometry Core, University of Alberta, Edmonton,
AB, Canada; 6Experimentica Ltd., Kuopio, Finland.
Purpose: In order to facilitate the development of new therapies, in
vitro model systems are needed to investigate the effects, benefits
and risks of the treatments to the retinal tissue. In this study, we
characterized a retinal ex vivo culture system from adult mice using
immunohistochemistry, stereology and electrophysiology.
Methods: Retinae of young C57Bl/6J mice (age under 8 weeks) were
isolated and cultured under ex vivo conditions up to 17 days in serum
free medium supplemented with B27 and N2. Electrophysiological
characterization of the retinae was performed by microelectrodearray
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
(MEA) technique. Spontaneous activity of ganglion cells as well
as their responses to flashes and steps of light was recorded during
the culturing period. Viability of the tissue was also followed by
immunohistochemistry and stereology.
Results: Our immunohistochemical analysis shows that cultured
retinal explants preserve retinal morphology for at least 17 days,
although significant thinning of the outer nuclear layer was observed.
Retinal functionality is retained substantially even though it is
gradually lost during the whole culturing period. Spontaneous
electrical activity of retinal ganglion cells was recorded up to 14 days
in culture. The responsiveness of ganglion cells to light stimulation
was lost faster, during the first week in culture.
Conclusions: Based on our electrophysiological and
immunohistochemical characterization, retinal explants from adult
mice retain their morphology and functionality for a remarkably long
time period in optimal culturing conditions. This culture system thus
represents a valuable tool for screening neuroprotective compounds
using morphological and functional correlates ex vivo.
Commercial Relationships: Soile Nymark, None; Symantas
Ragauskas, None; Virpi Savolainen, None; Andrea Holme, None;
Jari A. Hyttinen, None; Giedrius Kalesnykas, Experimentica Ltd
(E)
Support: Academy of Finland grant number 260375
Program Number: 2378 Poster Board Number: B0077
Presentation Time: 3:45 PM–5:30 PM
Indirect activation elicits strong correlations between light and
electrical responses in ON but not OFF ganglion cells
Maesoon Im1, Shelley I. Fried1, 2. 1Neurosurgery, Massachusetts
General Hosp/HMS, Boston, MA; 2Boston VA Healthcare System,
Boston, MA.
Purpose: For improved quality of vision elicited by retinal
prosthetics, it is important to understand the relationship between
the parameters of stimulation and the resulting neural activity within
the retina. Moreover, the prosthetic must be able to simultaneously
create appropriate spiking patterns, e.g. those that match the light
responses for each type of ganglion cell. Surprisingly, the similarities
between light- and electrically-elicited response patterns have not
been well studied. Here, we measured the response patterns elicited
by epiretinal electric stimulation in RGCs and explored correlations
between the electrically- and the light-elicited spikes within each
type.
Methods: Cell-attached patch clamp was used to record spikes
from RGCs in the isolated rabbit retina. RGCs were classified as
ON or OFF cells by their response to stationary flashes. ON cells
were further subdivided based on the presence of doublets or triplets
in their spontaneous responses. After cell type classification, a
monophasic half-sinusoidal wave (duration of 4ms, corresponds to
half period of single 125Hz sine wave, amplitude range of -100mA
to 100mA with 10mA increment) was presented typically 7 times to
targeted RGCs. We recorded the spiking activity in 35 ON cells and
46 OFF cells.
Results: The electric response patterns of RGCs had clear
distinctions across cell types. ON BT cells (transient light responses
with doublets in the spontaneous activity) always (n=18/18)
contained three or more bursts of spikes while ON BS cells typically
(n=13/17) responded with two bursts. ON cells generally showed
strong correlations between electrically- and light-elicited responses
in terms of strength and timing. In contrast, the electric response
patterns from OFF cells did not neatly differentiate into two distinct
groups. Also, the OFF cells had worse correlations to light responses
than did ON cells. There were further differences between ON
and OFF cells when the responses to repetitive stimulation were
examined: each new stimulus strongly suppressed responses to
previous stimuli in ON cells. This unique behavior of ON cells
created differences between ON and OFF cells in response to
repetitive stimulation with various inter-stimulus intervals.
Conclusions: Our results indicate that electric stimulation elicits
responses that more closely match physiological responses in ON
cells than in OFF cells.
Commercial Relationships: Maesoon Im, None; Shelley I. Fried,
None
Support: 1I01RX000350-01A1 (VA Merit Review) & 1R01
EY019967-01 (NIH)
Program Number: 2379 Poster Board Number: B0078
Presentation Time: 3:45 PM–5:30 PM
Human retina as an in-vitro model system
Mutter Marion, Katja Reinhard, Thomas A. Muench. Centre for
Integrative Neuroscience, Tuebingen, Germany.
Purpose: Several approaches to treat blindness are being tested,
ranging from virus-vector mediated optogenetic therapy (Busskamp
et al 2010) to implantable chips to electrically stimulate surviving
retinal neurons (Zrenner 2002). To further develop and improve these
methods, in particular for translational aspects (clinical treatment
of patients), it is desirable to examine the treatment success directly
on human retina. Thus, our goal was to establish an in-vitro testing
system for the evaluation of treatment approaches in human retina.
This includes: (1) evaluating if post mortem human retina is suited
for this purpose, in particular (2) a detailed characterization of
ischemic influences on the retina and (3) the establishment of
organotypic tissue culture conditions for the human retina.
Methods: We systematically investigated the influence of duration of
ischemia on the health status of post-mortem pig retina. Human retina
was obtained from enucleations (ex-vivo) and cornea donations (postmortem) from the University Eye Hospital of Tübingen. For both pig
and human retina, the viability was assessed by electrophysiological
recordings (MEA) and histologically. Furthermore, we set up a tissue
culturing procedure to maintain the retina in healthy conditions.
Results: Despite the current opinion that retinal ganglion cell death
during ischemia begins after 2 hours, and is completed after 4 hours
(Hayreh & Zimmerman 2005), we were able to record ganglion cell
spiking activity in pig retina after up to 14 hours ischemia time.
Ganglion cells of post mortem human retina, were still activity when
the tissue was obtained 15, 25 (2x) and even 27.5 hours post mortem.
In culture, ganglion cells of the 25h-old post mortem human retina
maintained activity for at least 96 hours. Additionally, we were able
to maintain light responsiveness in cultured retina (obtained ex vivo)
for at least 48 (mouse), 72 (pig), and 24 (human) hours.
Conclusions: Our results demonstrate that retinal ganglion cells can
survive ischemia considerably longer than expected. With such tissue,
we have successfully established culture conditions to maintain
human retina in an active physiological state for several days, so that
it remains accessible for testing treatment approaches. In addition,
our results may be relevant for assessing the time frame within which
the treatment of retinal central artery occlusions is indicated.
Commercial Relationships: Mutter Marion, None; Katja
Reinhard, None; Thomas A. Muench, None
Support: Werner Reichardt Centre for Integrative Neuroscience
Tübingen (DFG EXC 307); Bernstein Center for Computational
Neuroscience Tübingen (BMBF FKZ 01GQ1002)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2380 Poster Board Number: B0079
Presentation Time: 3:45 PM–5:30 PM
Insights into visual processing of human retina in-vitro
Katja Reinhard, Thomas A. Muench. University of Tübingen,
Tübingen, Germany.
Purpose: Retinal information processing has been characterized in
many animal models. However, except for a short communication by
Weinstein et al. in 1971, no physiological in-vitro data from human
retina has been published. It is often alleged that sophisticated image
processing might be absent in human retina. Further, with respect to
novel treatment options against visual impairment, we believe that it
is important to gain more knowledge about the targeted tissue – the
human retina. We thus aimed at studying human retina function on
cell and system levels in-vitro.
Methods: Pieces of human retina – donated by patients who had to
undergo a medically indicated enucleation – were placed ganglion
cell side-down on a 60-electrode multi-electrode array (MEA),
and stimulated with various light stimuli. In addition, we recently
implemented a high-density MEA with 11011 electrodes (Frey et al.
2009), and performed a first successful measurement with human
retina.
Results: During stimulation, we recorded spiking activity from
retinal ganglion cells (RGCs), and in 8 out of 15 donated retinas we
could measure abundant light responses. The recorded cells showed
diverse properties: ON-, OFF- and ON-OFF-responses, different
response latencies and transiencies, and different speed tuning.
Interestingly, human RGCs are tuned to higher speeds and higher
temporal frequencies compared to mouse RGCs.
Conclusions: The observed differences in speed tuning might be
explained by the higher angular velocities to which human RGCs are
exposed in the bigger human eye. Further, differences in temporal
frequency tuning suggest species specific kinetic differences of
synaptic processing within retinal circuitry. Taken together, we show
that it is possible to record light responses from human retina invitro, and we provide a glimpse into the diversity of its information
processing. In the future, the use of high-density MEAs will allow
further comprehensive characterization of human RGC types.
Commercial Relationships: Katja Reinhard, None; Thomas A.
Muench, None
Support: DFG EXC 307; BMBF FKZ 01GQ1002; PhD Stipend by
Pro Retina Foundation Germany
Program Number: 2381 Poster Board Number: B0080
Presentation Time: 3:45 PM–5:30 PM
The differential role of dopamine on the response characteristics
of retinal ganglion cell subtypes.
David Sprinzen, Michael L. Risner, Douglas McMahon. Vanderbilt
University, Nashville, TN.
Purpose: We investigated the effect of dopamine on spatial and
temporal response dynamics of retinal ganglion cells (RGCs) by
comparing wild type and retinal tyrosine hydroxylase knockout
mice (rThKO). Previous studies have shown significant effects of
dopamine in the retina in light adaption, however it is unclear how
dopamine may affect the different channels of light encoding. We
used a multi-electrode array (MEA) and recorded from RGCs while
showing various patterns of light stimulation to both dark adapted
and light adapted retinas.
Methods: Mouse retinas were dissected in dim red light, placed
ganglion cell side down on perforated MEAs,and perfused with
oxygenated Ames’. Stimulation protocols were delivered through a
monochrome microLED monitor. In a marching square protocol a
120 x 120 mm square (32 cd/m2) was indexed through a 12 x 16 grid
covering the MEA and the induced spike frequency was correlated
to the stimulus position to create a receptive field map. Cells were
defined as ON, OFF, or ON/OFF and sustained or transient based
on the response at the receptive field center. A white noise protocol
consisted of a binary randomly flickering checkerboard updated at
75 Hz. The receptive field and response latency were defined through
retro-correlating the spike triggered average stimulus for each cell.
Directionally selectivity was assessed with a full-field moving bar in
eight directions, from 0 to 315 degrees in 45-degree intervals.
Results: Dopamine knock out in the retina caused divergent
functional changes in different retinal ganglion cell types. In OFFcenter cells, exhibited a significant decrease in the response duration
under dark-adapted conditions but an increase in the response time
under light-adapted conditions. ON-center transient cells and ONOFF directionally selective cells, showed a significant decreases in
the receptive field size in the dark adapted state but a slight increase
in the light adapted state, representing an overall blunting of the
effect of light in manipulating the adapted state of the retina.
Conclusions: The heterogeneous effects of dopamine knock out on
RGCs may represent divergent roles of these information channels
in relation to light adaption. In the future, we plan on using D1 and
D2 receptor agonists to assess the relative contribution of signaling
pathways to the functional changes we found.
Commercial Relationships: David Sprinzen, None; Michael L.
Risner, None; Douglas McMahon, None
Support: NIH Grants RO1-EY09256, FEY023163A, and P30EY008126
Program Number: 2382 Poster Board Number: B0081
Presentation Time: 3:45 PM–5:30 PM
Characteristic morphology of T-dominant ganglion cells in rat
retina
Caiping Hu1, 4, Peng Chen2, 1, Xuemin Jin3, Malcolm M. Slaughter4.
1
Zhengzhou Eye Institute, Zhengzhou, China; 2Zhengzhou Second
Hospital, Zhengzhou, China; 3First Affiliated Hospital of Zhengzhou
University, Zhengzhou, China; 4University at Buffalo, Buffalo, NY.
Purpose: To explore the special morphology of retinal ganglion cells
with dominant T-type Ca2+ currents, which include some intrinsically
photosensitive retinal ganglion cells (ipRGCs).
Methods: The whole cell patch clamp and confocal imaging were
used to show the characteristic morphology of retinal ganglion
cells with dominant T-type Ca2+ currents. Their light responses
and dendritic stratification in the inner plexiform layer (IPL) were
analyzed. The melanopsin-expressed ganglion photoreceptors with
dominant T-type Ca2+ currents were compared with the conventional
T-dominant ganglion cells in morphology and electrophysiology.
Results: The 3D structure of T-dominant ganglion cells in the inner
plexiform layer(IPL) have strictly stratification, including ON,
OFF, ON-OFF type and non-ON/OFF types. Their dendritic system
had fewer branches in the largest distribution area. Their dendritic
system morphologically occupied the specific sublayers which were
perpendicular to the conventional photoreceptor-bipolar complex. As
a special example, T-dominant ganglion photoreceptors exemplified
the common characteristic morphology of T-dominant ganglion cells.
The morphology-function relationship suggested T-burst signals
could sense such temporal dimension as circadian cycle in visual
system.
Conclusions: The morphology of T-dominant ganglion cells fitted
their physiological functions. The holistic integrity of spatial
information can be carried out by T-burst signals and Na+ spike
signals in these large retinal ganglion cells. With specific anatomic
design, the conventional photoreceptor-bipolar complex and
melanopsin-dependent photoreceptors can exploit common signaling
tools for encoding the space-time coordinates.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Commercial Relationships: Caiping Hu, None; Peng Chen, None;
Xuemin Jin, None; Malcolm M. Slaughter, None
Support: VA Center for the Prevention and Treatement of Visual
Loss (C6810-C) and VA Carrer Development Award (MMH))
Program Number: 2383 Poster Board Number: B0082
Presentation Time: 3:45 PM–5:30 PM
Time-dependent changes in spontaneous and light-evoked retinal
ganglion cell activity in a mouse model of blast-induced traumatic
brain injury
Laura Dutca1, 2, Frederick R. Blodi1, 3, Adam Hedberg-Buenz1, 4,
Malini Shankar4, 3, Michael G. Anderson1, 4, Randy H. Kardon1,
2
, Steven F. Stasheff1, 3, Matthew M. Harper1, 2. 1Research, Dept
of Veterans Affairs, Iowa City, IA; 2Ophthalmology & Visual
Sci, University of Iowa Children’s Hospital & Carver College of
Medicine, Iowa City, IA; 3Pediatrics (Neurology), University of
Iowa Children’s Hospital & Carver College of Medicine, Iowa City,
IA; 4Department of Molecular Physiology and Biophysics, The
University of Iowa, Iowa City, IA.
Purpose: To analyse the in vitro function of retinal ganglion cells
(RGCs) after exposure to blast.
Methods: Mice were exposed to an overpressure wave (20 PSI)
directed to the head using a custom-built blast chamber. Individual
RGCs (~ 250 cells for each time point) from freshly dissected wholemounted retinas were monitored using a multielectrode array at 7
days, 5 weeks and 4 months after exposure to blast. Spontaneous
activity and the light evoked responses (to full field flashes) for each
RGC were measured and compared with retinas from control mice
and across time-points. Statistical analysis was performed using the
Mann-Whitney test.
Results: Seven days after blast exposure, the spontaneous activity of
RGCs was slightly increased compared to controls. The fraction of
cells that responded to the onset of light decreased significantly, while
the median response amplitude (spikes/sec) and response duration
were similar to those of control RGCs. The median amplitude of
responses to light OFF-set increased significantly, while response
duration decreased.
Five weeks post-blast, spontaneous activity was strikingly increased
compared to both controls and 7d recordings. The fraction of cells
responding to the onset of light was similar to that at 7d, while
median response amplitude and response duration were significantly
increased compared to both control and 7d post-blast. OFF response
amplitude was significantly increased, while response duration was
similar to control levels.
By four months post-blast, all these measures of RGC physiology had
recovered to near-normal values, similar to those at 7d post-blast.
Conclusions: Exposure to blast induces dramatic alterations in
physiological activity of RGCs after an initial period of relatively
normal function. These latent effects may indicate an optimal
time interval during which such dysfunction may be prevented or
ameliorated. The return to more normal RGC function by later time
points may reflect the physiology of subpopulations that survive
long-term, after a substantial proportion of RGCs die. The differential
effects of blast exposure on ON and OFF responses may indicate
differential susceptibility of particular RGC types to this injury.
Better understanding of the RGC physiology after blast exposure will
help in the development of improved clinical testing and treatment of
those suffering from TBI.
Commercial Relationships: Laura Dutca, None; Frederick R.
Blodi, None; Adam Hedberg-Buenz, None; Malini Shankar,
None; Michael G. Anderson, None; Randy H. Kardon, Acorda (C),
Dept. of Veterans Affairs Research Foundation Iowa City Iowa City
(S), Fight for Sight Inc (S), Novartis (C); Steven F. Stasheff, None;
Matthew M. Harper, None
Program Number: 2384 Poster Board Number: B0083
Presentation Time: 3:45 PM–5:30 PM
Light-Evoked Retinal Ganglion Cell Synaptic Responses and
Microglial Morphology are Modulated by P2X7 Receptor
Activation and Bacterial Lipopolysaccharide (LPS)
Seetal Chavda1, Philip J. Luthert2, 3, Thomas E. Salt1. 1Visual
Neuroscience, UCL Institute of Ophthalmology, London, United
Kingdom; 2Ocular Biology and Therapeutics, UCL Institute of
Ophthalmology, London, United Kingdom; 3NIHR Biomedical
Research Centre in Ophthalmology, London, United Kingdom.
Purpose: ATP-gated P2X7 receptors (P2X7Rs) have shown to act
as conduits for retinal ganglion cell (RGC) damage, a consequence
of various neurodegenerative conditions in the visual pathway.
P2X7Rs are also associated with the induction of early microglial
activation, and the release of neuroinflammatory mediators, following
neuronal injury. Using a dark-adapted, ex vivo mouse retinal
wholemount preparation, the present study aimed to characterise
the effect of P2X7R activation, and lipopolysaccharide (LPS; a
microglial activator), on light-evoked NMDA receptor (NMDAR)mediated RGC synaptic responses. The effect of LPS on microglial
morphology was also investigated, under similar conditions.
Methods: ON and OFF field excitatory post-synaptic potentials
(fEPSPs) were recorded from the ganglion cell layer of acutely
isolated retinal wholemounts (adult, male C57BL/6 mice) in
response to a light stimulus (1s-duration peak wavelength 562nm
flash, repeated every 3s). The NMDAR-mediated component of the
responses was pharmacologically isolated with a Mg2+-free Krebs
medium containing NBQX, picrotoxin, strychnine and tetrodotoxin.
Additional compounds were applied to the bathing medium. Retinae
were stained for microglia (α-IBA-1/IgG-Alexa488) and visualised
with confocal microscopy.
Results: BzATP (300mM), a P2X receptor agonist, depressed the ON
(78±2% of control; P<0.0001, n=21) but not the OFF RGC fEPSP
peak amplitude. The effect of BzATP on the ON RGC fEPSP was
reduced by the selective P2X7R antagonist, A438079 (10mM; 31±2%
reduction; P<0.05, n=6). Furthermore, bath-applied LPS (10 μg/ml)
depressed the ON fEPSP peak amplitude (86±3% of control; P<0.05,
n=7), an effect which was reduced in the presence of A438079.
Under similar conditions, microglial process area was significantly
reduced with LPS treatment (74±3% of control, P<0.0001, n=71
cells, 4 retinae), suggesting a transition to an early stage of microglial
activation.
Conclusions: In summary, LPS-mediated early microglial activation
induces a depression of ON-retinal ganglion cell responses, possibly
through a mechanism involving P2X7R activation. Since changes
in neurotransmission and microglial function are early indicators
of neuropathology, these results contribute to the understanding of
neural-immune interactions in retinal disease.
Commercial Relationships: Seetal Chavda, None; Philip J.
Luthert, None; Thomas E. Salt, None
Support: Lundbeck
Program Number: 2385 Poster Board Number: B0084
Presentation Time: 3:45 PM–5:30 PM
Pharmacological blockade of HCN channels changes evoked and
spontaneous activity of retinal ganglion cells
Sebastian Bemme, Michael Weick, Tim Gollisch. Department of
Ophthalmology, University Medical Center Göttingen, Göttingen,
Germany.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Purpose: Phosphene perception is a characteristic side effect,
occurring in patients treated with ivabradine. This drug is a selective
bradycardic agent which reduces the heart rate by blocking HCN4channels in the sinoatrial node. The induction of phosphenes by
ivabradine is ascribed to the blockade of hyperpolarization-activated
and cyclic nucleotide-gated (HCN) channels in retinal photoreceptors.
In the retina, HCN channels play an important role in efficient
encoding of stimuli at high-frequency by shaping the photoreceptor
membrane potential. Previous studies showed that blocking HCN
channels by genetic knockout or pharmacology results in prolonged
hyperpolarization of photoreceptor inner segments. Complementary
to these studies, we here examined the effect of pharmacological
HCN channel blockade on the encoding of visual signals by retinal
ganglion cells (RGCs).
Methods: We recorded spikes from individual ganglion cells of
isolated mouse retina mounted on a multielectrode array. Responses
to light stimuli as well as spontaneous spiking of ganglion cells were
measured with and without administration of 3 mM ivabradine or 3
mM cilobradine, an alternative HCN channel blocker.
Results: By analyzing the cells’ temporal filter properties, we
found that administration of ivabradine or cilobradine prolonged
the response delay and reduced responses to stimuli of high
temporal frequencies, in agreement with previous studies of
electroretinograms. Furthermore, we found that ON-type RGCs
primarily showed increasing and OFF-type RGCs decreasing
spontaneous spike rates during drug administration. OFF-type RGCs
moreover changed their firing patterns to more burst-like spiking.
Conclusions: Our data suggest that reduced band-pass filtering under
pharmacological HCN-channel blockade underlies the measured
imbalance of spontaneous activity between ON-type and OFF-type
cells, which may contribute to the reported phosphene perception in
treated patients.
Commercial Relationships: Sebastian Bemme, None; Michael
Weick, None; Tim Gollisch, Novartis (basic research grant) (F)
Support: International Human Frontier Science Program
Organization; Deutsche Forschungsgemeinschaft (DFG-SFB 889)
Program Number: 2386 Poster Board Number: B0085
Presentation Time: 3:45 PM–5:30 PM
Distinct contribution of kainate and NMDA receptors to OFFpathway circuitry for midget, parasol and small bistratified
ganglion cells in the macaque monkey retina
Dennis M. Dacey, Joanna D. Crook, Lauren Anderson, Beth B.
Peterson, Orin S. Packer. Biological Structure, University of
Washington, Seattle, WA.
Purpose: In primate the midget, parasol and small bistratified
ganglion cell populations together provide the major contribution to
the primary visual pathway and thus to the perception of form, color
and motion. Receptive field properties of these pathways have been
well characterized but less is known about the underlying circuits and
synaptic mechanisms utilized by each pathway. The purpose of this
study was to characterize the contributions of AMPA, kainate and
NMDA type glutamate receptors to the midget and parasol OFFcenter types and the “yellow-OFF” pathway of the small bistratified
cell.
Methods: Ganglion cell types were identified in the macaque
monkey retina in vitro and were voltage clamped using standard
methods. Light-evoked postsynaptic currents were measured
with spots restricted to the receptive field center that selectively
modulated the long and middle wavelength-sensitive cones (L+M,
50% contrast). Conductance analysis was used to resolve excitatory
NMDA, non-NMDA and inhibitory conductances (Crook et al., Vis
Neurosci, 2013; first view, 1-28).
Results: NMDA receptors contributed about 20% to the total
excitatory conductance in OFF-parasol cells at physiological
membrane potentials (-55 mV). By contrast, for both the OFF-midget
and the L+M-OFF response of the small bistratified cells, NMDA
receptors mediated a surprisingly large fraction, from 50-70%, of
the excitatory conductance. Unexpectedly, after application of the
AMPA/kainate receptor antagonist NBQX (10-50 mM), the NMDAmediated conductance was preserved with little or no attenuation in
all three ganglion cell types. Further addition of UBP 310 (1-5 mM),
a selective kainate receptor antagonist, abolished all light evoked
synaptic currents, suggesting that in midget, parasol and small
bistratified cell circuits the transmission from cones to OFF-bipolar
cells is mediated largely by an NBQX-resistant kainate receptor.
Conclusions: The NMDA receptor-mediated component of
OFF-pathway excitation in midget and small bistratified cells
is surprisingly large, comprising over 50% of the excitatory
conductance at physiological membrane potentials suggesting a role
for NMDA receptors in color-opponent circuitry. Unexpectedly, in all
three major pathways, kainate receptors appear to mediate the major
fraction of synaptic transmission from cones to OFF-bipolar cells.
Commercial Relationships: Dennis M. Dacey, None; Joanna D.
Crook, None; Lauren Anderson, None; Beth B. Peterson, None;
Orin S. Packer, None
Support: NIH Grants RR00166, EY06678 and a Vision Training
Grant, EY07031
Program Number: 2387 Poster Board Number: B0086
Presentation Time: 3:45 PM–5:30 PM
Set-β regulates retinal ganglion cells’ neurite growth and optic
nerve regeneration
Karan Patel1, 2, Ephraim Trakhtenberg1, 2, Yan Wang3, Melina I.
Morkin3, Stephanie Fernandez1, Gregory Mlacker1, 2, Kapil Gupta1, 2,
Susan Dombrowski4, Xiongfei Liu1, 2, Jeffrey L. Goldberg1, 3. 1Bascom
Palmer Eye Institute, Miami, FL; 2University of Miami Miller
School of Medicine, Miami, FL; 3Shiley Eye Center, University of
California, San Diego, FL; 4Genomatix Software, Inc., Ann Arbor,
MI.
Purpose: The failure of the adult mammalian central nervous system
(CNS) neurons to regenerate axons is a major clinical problem
associated with traumatic and ischemic optic neuropathies and
glaucoma. Transcriptional regulators like Set-β are well-positioned
to regulate intrinsic axon regeneration ability, which declines
developmentally in maturing CNS neurons. Set-β also functions
at cellular membranes and its subcellular localization is already
known to be disrupted in Alzheimer’s disease, but many of its other
biological mechanisms have not yet been explored in neurons. Here,
we investigated Set-β’s role in retinal ganglion cells (RGC) neurite
growth and axon regeneration.
Methods: Embryonic and postnatal rat retinal sections
were immunostained against RGC marker Brn3b and Set-β.
Immunofluorescence intensity was analyzed with AxioVision (Zeiss).
Set-β mRNA expression in RGCs was analyzed with qRT-PCR at
E19, P8, and P21. Set-β, myristoylated (myr)-Set-β, Set-β mutants,
PP2A-A, and mCherry were overexpressed in purified P3-4 RGCs,
and Neurite length was quantified using ImageJ Plugin Neurite
Tracer. For in vivo, myr-Set-β or GFP were delivered intravitreally
through AAV2 into anesthetized animals prior to the optic nerve
crush. Analysis with ANOVA and post hoc test (SPSS).
Results: We found that Set-β was upregulated postnatally in RGCs,
and was primarily localized to the nucleus but was also detected
adjacent to the plasma membrane. Remarkably, nuclear Set-β
suppressed whereas cytoplasmic membrane Set-β promoted neurite
growth in rodent RGC and hippocampal neurons. Mimicking serine
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
9 phosphorylation delayed nuclear import and furthermore blocked
the ability of nuclear Set-β to suppress neurite growth. We also
showed that nuclear Set-β regulates transcription of multiple genes
in RGCs, identified Set-β’s cytoplasmic binding partner PP2A-A
which suppressed neurite growth, and detected a novel isoform
of Set-β transcript lacking the nuclear localization signal. Finally,
we demonstrated that increasing recruitment of Set-β to cellular
membranes promoted optic nerve axon regeneration after injury in
vivo in adult rats.
Conclusions: Set-β differentially regulates axon growth
and regeneration depending on subcellular localization and
phosphorylation, and myr-Set-β gene therapy could be relevant for
developing treatments for optic neuropathies.
Commercial Relationships: Karan Patel, None; Ephraim
Trakhtenberg, None; Yan Wang, None; Melina I. Morkin, None;
Stephanie Fernandez, None; Gregory Mlacker, None; Kapil
Gupta, None; Susan Dombrowski, None; Xiongfei Liu, None;
Jeffrey L. Goldberg, None
Support: NEI EY020913, EY022589, EY014801; NINDS T32
NS007492; AHA 11PRE7310069
Program Number: 2388 Poster Board Number: B0087
Presentation Time: 3:45 PM–5:30 PM
Physiological and morphological characterization of ganglion
cells in the salamander retina
Jing Wang, Samuel Wu. Ophthalmology, Baylor College of Medicine,
Houston, TX.
Purpose: To evaluate physiological and morphological properties of
ganglion cells (GCs) in dark-adapted salamander retina by measuring
light responses via patch-clamp recording and visualizing threedimensional morphology via confocal imaging.
Methods: 1. A mixture of gap-junction-impermeable (Lucifer yellow,
LY) and -permeable dye (Neurobiotin, NB) was applied to optic
nerve stump of the salamander for retrograde labelling of GCs and
cells coupled with GCs. 2. Light-evoked current responses were
recorded from 41 GCs in dark-adapted salamander flat-mounted
retinas by voltage-clamp recording and the cell morphology was
revealed by intracellular LY loading under a confocal microscope.
Results: 1. In the GC layer, retrograde-identified GCs with both
LY and NB labelling constituted 78±5% of total neurons; 8±1% of
neurons with NB signal but no LY signal; and 14±1% with neither LY
nor NB labelling. 2. LY loading experiments showed that majority
of GCs (93%) have symmetrically-distributed dendritic fields.
Narrow-field cells (< 200um) account for 19% of GCs; mediate-field
(200 to 300um) 51%; and wide field (> 300um) 30%. 64% GCs
receive mixed rod/cone inputs; 26% receive rod-dominated input;
and 10% receive cone-dominated input. Based on physiological and
morphological characteristics, seven types of GCs were identified.
(1) Transient ON-OFF alpha GCs: large somas and dendritic fields
and exhibit transient ON-OFF response to a light step. (2) ON alpha
GCs: large somas and dendritic fields and exhibit ON responses. (3)
OFF alpha GCs exhibit OFF light responses. (4) Transient ON-OFF
beta GCs: smaller somas, dense dendritic field and exhibit ON-OFF
responses. (5) Transient ON-OFF gamma GCs: sparsely branching
dendrites and exhibit transient ON-OFF responses. (6) Transient ONOFF theta GCs: small soma, densely branching dendrites and exhibit
transient ON-OFF responses. (7) Asymmetrical unilateral GCs: >90%
dendrites distributed in one side of the soma and exhibit ON response
to 500 nm light and ON-OFF response to 700nm light.
Conclusions: By using both morphological and physiological
criteria, we identified seven types of GCs in the GC layer of
salamander retina. Analysis of response sensitivity, polarity and
waveform suggests that some GCs receive segregated bipolar cell
inputs where others receive mixed bipolar cell inputs. About 1/5 of
neurons in salamander retinal GC layer are displaced amacrine cells.
Commercial Relationships: Jing Wang, None; Samuel Wu, None
Support: NEI EY04446, EY019908, 02520, Texas Retinal Research
Foundation, Research to Prevent Blindness, International Retinal
Research Foundation
Program Number: 2389 Poster Board Number: B0088
Presentation Time: 3:45 PM–5:30 PM
A novel classification system for rat RGCs in retinal degeneration
James R. Tribble1, Paulina Samsel1, Stephen D. Cross1, Frank
Sengpiel2, James E. Morgan1, 3. 1School of Optometry and Vision
Science, Cardiff University, Cardiff, United Kingdom; 2Cardiff
School of Biosciences, Cardiff University, Cardiff, United Kingdom;
3
University Hospital of Wales, Cardiff Eye Unit, Cardiff, United
Kingdom.
Purpose: Retinal ganglion cells (RGC) types are typically classified
based on the morphology of their dendritic arbour. This can be
problematic when quantifying the dendritic remodeling that occurs
in rodent models of retinal degenerative diseases where labeling
bias among RGC types could be an important potential confounder.
To control for this effect we developed a novel quantitative RGC
classification based on proximal dendritic features that are usually
resistant to early degeneration.
Methods: Retinas from 20 adult Brown Norway Rats were flat
mounted and labelled ballistically (Biorad Helios gene gun) by
delivery of DiO and DiI coated tungsten coated particles. RGC
dendritic arbours were imaged with a Zeiss LSM 510 confocal
microscope. RGCs were classified according to Sun et al. (2002)
using soma diameter, dendritic field diameter and stratification depth.
Primary and secondary dendrite features were quantified and assessed
via principle component analysis (PCA) to generate novel condensed
variables or components. Discriminant analysis was then used to
assess their value in the classification of RGC types. A hold out
sample (n=16, taken from RGC traces in Sun et al. 2002) provided
validation of the model.
Results: 140 RGCs were imaged; according to standard classification
(Sun et al. 2002) 26% (n=37) were RGCA, 29% (n=40) RGCB, 31%
(n=43) RGCC and 14% (n=20) RGCD.PCA gave a 3 component
solution, separating RGCs based on descriptors of cell soma and
dendritic field size, dendritic tree asymmetry and branching density.
RGCA and RGCC were separated from the smaller RGCB and RGCD
based on descriptors of cell soma and dendritic tree size. RGCA and
RGCC were separated based on branching density, while RGCB and
RGCD were separated by a combination of all 3 factors. Discriminant
analysis showed that the new variables correctly classified 64.3%
(n=90) of RGCs and 62.5% (n=10) of the hold-out sample indicating
an effective model.
Conclusions: Primary and secondary dendrite characteristics provide
quantitative data for a classification system that is relatively resistant
to early degeneration. By measuring only the proximal dendritic
field the atrophy of distal dendrites and reduction in overall dendritic
field size does not confound the classification. This quantitative
classification system can be used to control for sample bias in the
assessment of RGC degeneration in experimental retinal disease.
Commercial Relationships: James R. Tribble, None; Paulina
Samsel, None; Stephen D. Cross, None; Frank Sengpiel, None;
James E. Morgan, None
Support: BBSRC grant BB/F016352/1
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 2390 Poster Board Number: B0089
Presentation Time: 3:45 PM–5:30 PM
Excitotoxic and Ischemic Conditions Change the Expression of
Gap Junction Connexins in the Inner Retina
Abram Akopian1, 2, Tamas Atlasz2, Stewart A. Bloomfield1, 2.
1
Biological and Vision Sciences, State University of New York
College of Optometry, New York, NY; 2NYU School of Medicine,
New York, NY.
Purpose: Secondary cell death via gap junctions plays a critical role
in the cell loss associated with neurodegenerative diseases (Belousov
and Fontes 2013). In the retina we reported that secondary cell death
accounts for most cell loss that occurs under excitotoxic and ischemic
conditions (ARVO, 2011). Secondary cell death in CNS may be
connexin specific and connexins may be up- or down-regulated
under different pathological conditions (Rouach et al., 2002). Here
we examined whether secondary cell death of retinal ganglion cells
(RGCs) under excitotoxic and ischemic conditions is connexinspecific and whether the expression of Cx36 and Cx45 in inner retina
is differentially effected.
Methods: Excitotoxicity was induced in vitro by incubation of mouse
retinas in NMDA. Transient retinal ischemia was induced in vivo by
elevation of IOP. Levels of cell death were assayed histologically and
antibodies against Cx36 and Cx45 were used to assess their levels in
the IPL.
Results: Consistent with our earlier work, we found that excitotoxic
and ischemic conditions produced a significant loss of RGCs.
Ablation of Cx36 in the Cx36 knockout (KO) mouse retina resulted
in an ~70% decrease in RGC loss under excitotoxic conditions,
whereas RGC loss in the Cx45 KO retina was not statistically
different than that seen in WT mice. In contrast, RGC loss with
ischemia was significantly reduced in Cx45 KO retinas, whereas the
loss in Cx36 KO retinas was similar to that in the WT. In WT retinas
the expression of Cx36 and Cx45 in the IPL followed a punctuate
pattern typical for gap junctions. Under excitotoxic conditions the
expression of Cx45 was down-regulated, whereas there were no
detectable changes in Cx36 expression. In contrast, induction of
ischemic conditions produced a dramatic change in Cx36 expression,
which appeared as dense clusters around nuclei rather than as puncta.
We found no change in the control punctate labeling pattern of Cx45
expression in ischemic retinas.
Conclusions: Secondary cell death of RGCs is connexin specific
where Cx36 gap junctions play a role under excitotoxic conditions
and Cx45 gap junctions play a role during ischemia. These results are
consistent with changes in connexin expression seen under these two
conditions. These results suggest that targeting of specific connexins
can be a novel therapeutic strategy for reducing RGC loss under
different pathological conditions.
Commercial Relationships: Abram Akopian, None; Tamas Atlasz,
None; Stewart A. Bloomfield, None
Support: NIH Grant EY007360
Program Number: 2391 Poster Board Number: B0090
Presentation Time: 3:45 PM–5:30 PM
Ranibizumab (Lucentis®) suppresses the autocrine VEGFelicited survival of purified retinal ganglion cells
Nicolas G. Froger1, Valérie Forster1, Ivana Ivkovic1, Frederic
Matonti1, 3, José-Alain Sahel1, 2, Serge A. Picaud1. 1INSERM U968
/ UPMC Univ Paris-06 / CNRS-7210, Institut de la Vision, Paris,
France; 2Centre Hospitalier National d’Ophtalmologie des QuinzeVingts, Paris, France; 3UMR 7289, CNRS - Aix Marseille Université,
Institut de Neurosciences de la Timone, Marseille, France.
Purpose: Vitreous injections of ranibuzumab, an antibody fragment
against the vascular endogenous growth factor type A (VEGF-A), are
largely used against neovascularisation and edema occurring both in
age-related macular degeneration and diabetic retinopathy. VEGF-A
was recently reported to exert a neuroprotective effect on RGCs in
different pathological models. We here report that purified retinal
ganglion cells (RGCs) can produce VEGF-A in culture to promote
their own survival. We further show that VEGF antibodies, including
lucentis, can suppress this VEGF autocrine effect on purified RGCs
Methods: RGCs from the adult rat retina were purified by
immunopanning and seeded in a 96-well at different cell densities
(from 8 000 to 50 000 cells/well) and cultured in a serum deprived
medium. After 6 days in vitro (DIV), culture media were harvested
in order to measure the amounts of VEGF-A164 by ELISA. After
their calceinAM labeling, viable RGCs were counted on automated
platform d to quantify RGC survival.
Results: After 6 DIV the medium used for RGC culture was found
to contain detectable amounts of VEGF-A164. Interestingly, the
VEGF-A concentration was correlated to the RGC initial seeding
density. Concentrations of VEGF-A were even increasing in an
exponential manner with the number of surviving RGC cells. To
assess if VEGF was contributing to the RGC survival, the rabbit
polyclonal antibody against murine VEGF-A164 was applied in
the RGC culture medium (2 mg/ml for 6 DIV). As a consequence,
measured VEGF-A164 concentrations were significantly reduced
and the RGC survival was decreased significantly by 52%. Similarly,
application of different concentrations of Lucentis (2, 50, 150, 500
and 1000 mg/ml) on RGC cultures reduced the VEGF-A amounts
into culture medium. This effect was associated to a dose-dependent
decrease of the RGC survival.
Conclusions: These data showed that RGCs are able to produce
VEGF-A in culture in order to maintain their own survival. This
autocrine neurotrophic effect was neutralized by VEGF-A antibodies,
including ranibuzumab,. These results raise questions on anti-VEGF
strategies.
Commercial Relationships: Nicolas G. Froger, None; Valérie
Forster, None; Ivana Ivkovic, None; Frederic Matonti, None;
José-Alain Sahel, Genesignal (C), GenSight Biologics (C), Pixium
Vision (C), Sanofi-fovea (C); Serge A. Picaud, None
Support: Institut National de la Santé et de la Recherche
Médicale (INSERM), Pierre et Marie Curie University (UPMC),
Centre National de la Recherche Scientifique (CNRS), Fondation
Ophtalmologique A. de Rothschild (Paris), Fondation pour le
recherche Médicale, Fondation Roland Bailly, Agence Nationale
pour la Recherche (ANR: GLAUCOME), the European Community
contract TREATRUSH (n° HEALTH-F2- 2010-242013), the
Fédération des Aveugles de France, IRRP, the city of Paris, the
Regional Council of Ile-de-France, and the French State program
“Investissements d
Program Number: 2392 Poster Board Number: B0091
Presentation Time: 3:45 PM–5:30 PM
The RNA binding protein RBPMS is a specific marker of
ganglion cells in the mammalian retina
Allen Rodriguez1, Luis Pérez de Sevilla Müller1, Nicholas Brecha1, 2.
1
Neurobiology, UCLA, Los Angeles, CA; 2Jules Stein Eye Institute,
UCLA, Los Angeles, CA.
Purpose: Transcription factors and RNA-binding proteins that are
specific to retinal ganglion cells (RGCs) have been reported in the
mammalian retina. We report the development and characterization
of a new set of antibodies against RNA-binding protein with multiple
splicing (RBPMS) that specifically labels the entire RGC population.
Methods: Affinity purified polyclonal guinea pig and rabbit
antibodies were generated to the N-terminus of RBPMS. The
RBPMS polypeptide sequence used for immunization is identical
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
in mouse, rat, monkey and human, and 95% similar for guinea
pig. Mouse and rat retinal extracts were evaluated using western
blotting. Mouse, rat, guinea pig, rabbit and monkey retinal sections
and whole-mounts were evaluated using single and double labeling
immunohistochemistry. RBPMS-immunoreactive cells were
measured for somatic size and density. Unilateral optic nerve
transection or crush was performed in rats and mice.
Results: The guinea pig and rabbit antibodies detected a single band
of ~24 kDa on western blots of cell lysates from HEK293T cells
transfected with human RBPMS cDNA. No bands were detected in
non-transfected lysates. A prominent single band at ~24 kDa was
also detected in mouse and rat retinal extracts. Specific RBPMS
immunoreactivity was mainly localized to medium to large diameter
somata in the ganglion cell layer and to a few medium and large
somata at the border of the inner plexiform layer and inner nuclear
layer. The size and density of RBPMS immunoreactive cells in mouse
and rat retina were comparable to earlier semi-quantitative estimates
of RGCs. All Brn3a, SMI-32 and melanopsin immunoreactive RGCs
also express RBPMS immunoreactivity. RBPMS immunoreactivity
is not expressed in syntaxin (HPC-1) immunoreactive cells in the
INL and GCL, consistent with the specificity of RBPMS as a RGC
marker. 86% or 98% of RBPMS immunoreactive cells are lost
following optic nerve crush or transection at three weeks post injury
in mouse and rat, respectively. These findings are consistent with a
very weak immunostained band at ~24 kDa in a rat retinal extract
collected 56 days after optic nerve transection.
Conclusions: RBPMS antibodies are robust reagents for the
identification and quantification of RGCs in multiple mammalian
species, and they will be particularly useful for tracking RGCs in
chronic disease or acute injury models.
Rat RGCs labeled by a RBPMS antibody. Scale = 50mm.
Commercial Relationships: Allen Rodriguez, None; Luis Pérez de
Sevilla Müller, None; Nicholas Brecha, None
Support: DOD / TATRC Contract Number: W81XWH-10-2-0077,
NIH EY04067, NIDDDK P30 DK41301 (UCLA Cure Center Core)
and a VA Merit Review (NCB). NCB is a VA Career Research
Scientist.
Program Number: 2393 Poster Board Number: B0092
Presentation Time: 3:45 PM–5:30 PM
GABA Inhibition Controls the Threshold Sensitivity of Retinal
Ganglion Cells Independent of Dopaminergic Circuitry
Feng Pan, Abram Akopian, Stewart A. Bloomfield. Biological and
Vision Sciences, State University of New York College of Optometry,
New York, NY.
Purpose: Dark-adapted retinal ganglion cells (RGCs) in the
mouse retina can be differentiated based on their extensive range
of threshold sensitivities covering 3 log units (Volgyi et al., 2004).
We previously showed that blockade of GABA circuits shifted
the thresholds of RGCs suggesting that inhibition controls their
sensitivity. Here we used selective GABA receptor blockers to
determine the location of the inhibitory circuits and examined
whether they interact with dopaminergic pathways that also affect
neuronal sensitivity (Li & Dowling, 2000; Hermann et al., 2011).
Methods: The light-evoked responses of RGCs in C57BL mouse
retinas were obtained using tungsten microelectrode or multielectrode
array recordings. Intensity-response functions and threshold
sensitivities were then computed from responses to varying intensity,
full-field light stimulation.
Results: Application of the nonselective GABA blocker PTX
(100 μM) produced a leftward shift of most RGC I-R functions
indicating an increase in sensitivity. Application of the selective
GABAA receptor blocker SR-95531 (20 μM) had minimal effect
on the thresholds of RGCs. However, application of the selective
GABAC receptor blocker TPMPA (100 μM) increased the sensitivity
of most RGCs by 1-3 log units, comparable to the effects of PTX
alone. Application of the dopamine D2 receptor blocker eticlopride
(25 μM) decreased the thresholds of most RGCs by 0.25 -1.00 log
unit, but subsequent application of PTX still produced an increase
in sensitivity. Consistent with this finding, the increase in sensitivity
of RGCs produced by PTX was never reversed by subsequent
application of eticlopride. Application of the D1 receptor blocker
SCH-23390 (10 μM) decreased the thresholds of most RGCs by
0.25-0.50 log units, but subsequent application of PTX increased the
thresholds of RGCs. Likewise, the increase in sensitivity produced by
PTX was not reversed by subsequent application of SCH-23390.
Conclusions: Our results indicate that inhibition derived mainly by
activation of GABAC receptors controls the threshold sensitivity of
RGCs. Dopaminergic circuits also control the sensitivity of RGCs,
but this action is relatively small compared to that of the GABAergic
inhibition. Moreover, the GABAergic control of RGC sensitivity
appears independent of that provided by dopaminergic circuitry.
Commercial Relationships: Feng Pan, None; Abram Akopian,
None; Stewart A. Bloomfield, None
Support: NIH Grant EY007360
Program Number: 2394 Poster Board Number: B0093
Presentation Time: 3:45 PM–5:30 PM
Loss and recovery of retinal ganglion cell function after distal
injury of the retino-collicular pathway
Tsung-Han Chou, Mario J. Rojas, Ning Wang, Yihui Chen, Rong Wen,
Vittorio Porciatti. Bascom Palmer Eye Inst, Univ of Miami, Miller
Sch of Med, Miami, FL.
Purpose: Intrinsic survival mechanisms of injured neurons are
difficult to study because of concurrent destructive mechanisms
leading to cell death. Here we investigate adaptive plasticity of retinal
ganglion cells (RGCs) following superior colliculus (SC) injury in
mice, a model that does not cause RGC death over several months
(Yang et al, IOVS 2013).
Methods: RGC function (pattern ERG, PERG), outer retina function
(photopic ERG, FERG) and inner/outer retina thickness (SD-OCT)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
were serially assessed in anesthetized C57BL/6J mice (n=14) at
closely-spaced time points before and after surgical ablation of the
superficial layers of the right SC. Integrity of optic nerve axons
and transport was established by means of intravitreal injections of
cholera toxin B (CTB). At endpoint, confocal microscopic analysis
of RGCs was performed on TUJ1-labeled retinal flat mounts. BDNF
expression was quantified in Western blots of retina homogenates.
Results: After right SC lesion, the PERG amplitude did not change
in the right eye but rapidly decreased in the left eye to 44% of
baseline, recovered slowly after one week to reach 68% of baseline
at one month and then remained stable over three months (ANOVA,
P<0.001). CTB-labeled optic nerves and tracts were similar in the
two eyes. After SC lesions, OCT-determined inner retina thickness
(ILM+RNFL+GCL+IPL) remained normal in the left eye for about
one week and then significantly thinned by about 4 mm (P<0.01),
remaining at reduced thickness thereafter. At the three-month
endpoint, the mean RGC soma size was reduced in the left eye while
RGC density was normal, and BDNF expression was increased
(P<0.01). The FERG was unchanged.
Conclusions: After SC injury, RGC function undergoes three
distinct phases—sudden loss, slow recovery, stabilization at a
subnormal level. The PERG plateau level is associated with inner
retina thinning but not cell death, normal axonal transport and BDNF
overexpression. These structural-functional-molecular changes
indicate that RGCs have an intrinsic ability to reorganize and repair
themselves to regain function after injury. The SC-lesion model is
useful to investigate intrinsic survival mechanisms of RGCs.
Commercial Relationships: Tsung-Han Chou, None; Mario J.
Rojas, None; Ning Wang, None; Yihui Chen, None; Rong Wen,
None; Vittorio Porciatti, None
Support: NIH Grant R01EY019077, NIH center grant P30EY14801, Research to Prevent Blindness
Program Number: 2395 Poster Board Number: B0094
Presentation Time: 3:45 PM–5:30 PM
Injury-dependent retinal ganglion cell plasticity: effect of
exogenous trophic factors
Mario J. Rojas, Tsung-Han Chou, Adam S. Rosner, Rong Wen,
Vittorio Porciatti. Bascom Palmer Eye Institute, University of Miami,
Miller School of Medicine, Miami, FL.
Purpose: Distal injury of the retino-collicular pathway in mice causes
reduction of retinal ganglion cell (RGC) electrical responsiveness
associated with RGC shrinkage, BDNF overexpression, but not cell
death (Yang et al, IOVS 2013). Here we investigated whether these
adaptive changes are modifiable with exogenous trophic factors.
Methods: RGC function (pattern ERG, PERG), outer retina function
(Photopic ERG, FERG) and inner/outer retina thickness (SD-OCT)
were serially assessed in anesthetized C57BL/6J mice (n=24) before
and 7, 14, 30, 60, 90 days after receiving surgical ablation of the
superficial layers of the superior colliculus (SC). Two microliters
of either BDNF (2mg/ml, n=10) or CNTF (1.5 mg/ml, n=10) were
intravitreally injected in the left eye two weeks after right SC
injury. Control mice (n=14) received SC injury but did not receive
intravitreal injections.
Results: In all mice groups, two weeks after SC injury the
inner retina (ILM+NFL+RGCL+IPL) became significantly
(P<0.01) thinner by 4.5 mm on average whereas the outer retina
(INL+OPL+ONL+PRL) did not change. PERG amplitude dropped
by 50% on average (P<0.01). Intravitreal injections of either BDNF
or CNTF temporarily restored normal inner retina thickness for two
weeks (P<0.01), which then returned to the post-injury thinned state.
After either BDNF or CNTF injection, the PERG slowly recovered
to reach a slightly subnormal plateau level between one month and
three months (-25% of baseline on average, P=0.05). SC-injured
controls that did not receive treatment displayed a slow recovery of
PERG amplitude to a subnormal plateau as BDNF/CNTF-injected
mice (two-way ANOVA: effect of treatment, N.S). For all the above
conditions, the FERG was unchanged.
Conclusions: Early loss of RGC function after SC injury undergoes
a spontaneous partial recovery while inner retina becomes thinner.
Intravitreal injections of either BDNF or CNTF temporarily
restore normal inner retina thickness but do not significantly alter
spontaneous recovery of RGC function. These plastic changes are
a conspicuous phenomenon that can be studied in an in vivo mouse
model using translational tools such as OCT and PERG. Results have
implications for better understanding, monitoring, and treatment of
glaucoma and optic nerve disorders.
Commercial Relationships: Mario J. Rojas, None; Tsung-Han
Chou, None; Adam S. Rosner, None; Rong Wen, None; Vittorio
Porciatti, None
Support: NIH Grant R01EY019077, NIH center grant P30EY14801, Research to Prevent Blindness
301 Bipolar and amacrine cells
Tuesday, May 06, 2014 8:30 AM–10:15 AM
S 210DE Paper Session
Program #/Board # Range: 2637–2643
Organizing Section: Visual Neuroscience
Program Number: 2637
Presentation Time: 8:30 AM–8:45 AM
The Atrx Chromatin Remodeler is Required in Retinal Bipolar
Cells for Amacrine and Horizontal Cell Survival
Pamela S. Lagali1, 2, Chantal Medina1, 2, Keqin Yan1, Adam Baker1,
Stuart G. Coupland3, 4, Valerie A. Wallace1, 2, David Picketts1,
2 1
. Regenerative Medicine, Ottawa Hospital Research Institute,
Ottawa, ON, Canada; 2Biochemistry, Microbiology and Immunology,
University of Ottawa, Ottawa, ON, Canada; 3Cellular and
Molecular Medicine, University of Ottawa, Ottawa, ON, Canada;
4
Ophthalmology, University of Ottawa Eye Institute, Ottawa, ON,
Canada.
Purpose: The success of photoreceptor repair or replacement
strategies for the treatment of retinal degenerative diseases critically
depends on the continued survival and function of the inner retinal
neurons. Morphological defects and death of these neurons are
known to occur in later stages of retinal degeneration and may limit
the effectiveness of long-term therapies for vision restoration. We
have shown that the chromatin remodeling protein Atrx is important
for the maintenance and function of amacrine and horizontal cells.
We aim to understand the mechanism by which Atrx activity mediates
retinal inhibitory interneuron survival.
Methods: Atrx was deleted in different retinal cell populations by
generating cell type-specific conditional knockout mice via Cre
recombinase-mediated genetic excision using germline transgenic
and in vivo electroporation approaches. Morphological, functional,
and genetic analysis of the Atrx-deleted retinas was performed
using immunohistochemistry and fluorescence microscopy,
electroretinography, and quantitative RT-PCR respectively.
Results: Amacrine and horizontal cell disorganization and loss occurs
when Atrx is deleted in multipotent progenitor cells during embryonic
retinal development, but not when the gene is inactivated in lineagerestricted, post-mitotic amacrine and horizontal precursor cells.
Selective genetic ablation of Atrx postnatally in retinal bipolar cells
recapitulates the effects of early pan-retinal gene deletion, indicating
that Atrx activity in these neurons is responsible for the function
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
and survival of the retinal inhibitory interneurons. Further genetic
and immunohistochemical analysis of the mutant mice reveals
dysregulation of bipolar cell marker genes, misexpression of bipolar
subtype-specific proteins, and alterations in neuronal morphology that
may underlie defects in inner retinal circuitry.
Conclusions: The loss of amacrine and horizontal cells from Atrxdeleted retinas appears to occur through a non-cell autonomous
mechanism. Our analyses implicate a role for bipolar cells in
retinal inhibitory interneuron survival and function. Atrx-mediated
chromatin remodeling may be important for the regulation of
specific genes that are involved in retinal neuron synaptic activity,
connectivity, and homeostasis. These genes may represent potential
targets of neuroprotective strategies for retinal degenerative disease
therapies.
Commercial Relationships: Pamela S. Lagali, None; Chantal
Medina, None; Keqin Yan, None; Adam Baker, None; Stuart G.
Coupland, None; Valerie A. Wallace, None; David Picketts, None
Support: Foundation Fighting Blindness-Canada
Program Number: 2638
Presentation Time: 8:45 AM–9:00 AM
AAV-Mediated Expression Targeting of Retinal Rod Bipolar Cells
with An Optimized mGluR6 Promoter
Zhuo-Hua Pan1, 2, Qi Lu2, Tushar Ganjawala2, JrGang Cheng3.
1
Ophthalmology, Wayne State Univ Sch of Med, Detroit, MI;
2
Anatomy & Cell Biology, Wayne State University, Detroit, MI;
3
Neuroscience Center, University of North Carolina, Chapel Hill, NC.
Purpose: AAV-mediated expression of microbial rhodopsins in
surviving inner retinal neurons is a promising approach to restoring
vision after retinal degeneration. Targeting ChR2 to specific bipolar
cells is particularly appealing; but AAV-mediated targeted gene
expression to specific bipolar cells, especially administered by
intravitreal injection, poses a challenge. The use of a 200 bp enhancer
of mGluR6 promoter and a basal SV40 promoter has been shown to
be able to target ChR2 to ON bipolar cells by electroporation (Lagali
et al., 2008). However, AAV-mediated delivery of ChR2 to ON
bipolar cells with the same construct was achieved only via subretinal
injection (Doroudchi et al., 2011). In this study, we aimed to develop
mGluR6 promoter constructs for AAV-mediated gene delivery to
specific retinal bipolar cell type(s) via intravitreal injection.
Methods: A series of AAV2 expression cassettes were constructed
with the combination of various sequences of mGluR6 promoter,
the 200 bp mGluR6 enhancer, and intron sequences of mGluR6
carrying a transgene of mCherry or ChR2-GFP. AAV vectors were
produced by packaging the expression cassettes into AAV serotype 2
with an Y444F capsid mutation and were injected intravitreally into
the eyes of C57BL/6J mice at age of approximately one month. The
expression was examined one month after viral injection.
Results: The constructs containing the 200 bp mGluR6 enhancer and
a ~1 kb mGluR6 promoter sequence resulted in selective targeting
of the transgenes to bipolar cells, predominantly rod bipolar cells
(RBCs). The expression of the transgenes in RBCs was significantly
enhanced with the use of a shortened mGluR6 promoter sequence and
the inclusion of mGluR6 intron 3 and 4. The transduction efficiency
was viral concentration dependent. At the concentration of ~1 x 1013
vg/ml, the expression of the transgenes in RBCs was observed across
the entire retina with the highest density in the peripheral regions,
where the majority of RBCs could be transfected.
Conclusions: We developed optimized mGluR6 promoter
constructs that can achieve AAV-mediated targeted gene expression
predominantly in RBCs administered by intravitreal injection.
The ability of AAV-mediated targeted gene delivery to RBCs via
intravitreal injection could facilitate the development of optogenetic-
based therapies for vision restoration as well as genetic manipulation
in RBCs in vitro and in vivo.
Commercial Relationships: Zhuo-Hua Pan, RetroSense (C),
Wayne State University (P); Qi Lu, None; Tushar Ganjawala,
None; JrGang Cheng, None
Support: NIH grant EY17130, core grant EY04068 to Department
of Anatomy and Cell Biology at Wayne State University, the Ligon
Research Center of Vision, and Research to Prevent Blindness to
Department of Ophthalmology at Wayne State University.
Program Number: 2639
Presentation Time: 9:00 AM–9:15 AM
A Synaptic Basis for Small World Network Design in the ON
Inner Plexiform Layer of the Rabbit Retina
J Scott Lauritzen, Noah T. Nelson, Crystal L. Sigulinsky, Nathan
Sherbotie, John Hoang, Rebecca L. Pfeiffer, James R. Anderson, Carl
B. Watt, Bryan W. Jones, Robert E. Marc. Ophthalmology & Visual
Sciences, University of Utah, Salt Lake City, UT.
Purpose: Converging evidence suggests that large- and intermediatescale neural networks throughout the nervous system exhibit “small
world” design, characterized by high local clustering of connections
yet short path length between neuronal modules (Watts & Strogatz,
1998 Nature; Sporns et al., 2004 Trends in Cog Sci). It is suspected
that this organizing principle scales to local networks (Ganmor et al.,
2011 J Neurosci; Sporns, 2006 BioSystems), but direct observation
of synapses and local network topologies mediating small world
design has not been achieved in any neuronal tissue. We sought direct
evidence for synaptic and topological substrates that instantiate small
world network architectures in the ON inner plexiform layer (IPL)
of the rabbit retina. To test this, we mined ≈ 200 ON cone bipolar
cells (BCs), and ≈ 500 inhibitory amacrine cell (AC) processes in the
ultrastructural rabbit retinal connectome (RC1).
Methods: BC networks in RC1 were annotated with the Viking
viewer, and explored via graph visualization of connectivity and 3D
rendering (Anderson et al., 2011 J Microscopy). Small molecule
signals embedded in RC1, e.g. GABA, glycine, and L-glutamate,
combined with morphological reconstruction and connectivity
analysis allow for robust cell classification. MacNeil et al. (2004 J
Comp Neurol) BC classification scheme used for clarity.
Results: Homocellular BC coupling (CBb3::CBb3, CBb4::CBb4,
CBb5::CBb5) and within-class BC inhibitory networks (CBb3 Ç
AC —| CBb3, CBb4 Ç AC —| CBb4, CBb5 Ç AC —| CBb5) in
each ON IPL strata form laminar-specific functional sheets with high
clustering coefficients. Heterocellular BC coupling (CBb3::CBb4,
CBb4::CBb5, CBb3::CBb5) and cross-class BC inhibitory networks
(CBb3 Ç AC —| CBb4, CBb4 Ç AC —| CBb3, CBb4 Ç AC —|
CBb5, CBb5 Ç AC —| CBb4, CBb3 Ç AC —| CBb5, CBb5 Ç AC
—| CBb3) establish short synaptic path lengths across all ON IPL
laminae.
Conclusions: The retina contains a greater than expected number
of synaptic hubs that multiplex parallel channels presynaptic to
ganglion cells. The results validate a synaptic basis (ie. direct
synaptic connectivity) and local network topology for the small world
architecture indicated at larger scales, providing neuroanatomical
plausibility of this organization for local networks, and are consistent
with small world design as a fundamental organizing principle of
neural networks on multiple spatial scales.
Commercial Relationships: J Scott Lauritzen, None; Noah T.
Nelson, None; Crystal L. Sigulinsky, None; Nathan Sherbotie,
None; John Hoang, None; Rebecca L. Pfeiffer, None; James R.
Anderson, None; Carl B. Watt, None; Bryan W. Jones, None;
Robert E. Marc, Signature Immunologics, Inc. (E)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Support: NIH EY02576 (RM), NIH EY015128 (RM), NSF 0941717
(RM), NIH EY014800 Vision Core (RM), RPB award to Moran Eye
Center, RPB Career Development Award (BWJ), Thome Foundation
grant for AMD Research (BWJ).
Program Number: 2640
Presentation Time: 9:15 AM–9:30 AM
A non-spiking, wide-field amacrine cell that rapidly integrates
visual signals over long distances in the primate
Michael B. Manookin1, Christian Puller1, Fred Rieke2, Maureen
Neitz1, Jay Neitz1. 1Ophthalmology, University of Washington,
Seattle, WA; 2Physiology and Biophysics and HHMI, University of
Washington, Seattle, WA.
Purpose: Stimulation outside of the classical receptive field produces
clear and significant effects on visual processing, but little is known
about the mechanisms mediating these long-range effects. Wide-field
amacrine cells are a likely culprit, but, with a few exceptions, the
function of these cells remains uncertain. Particularly little is known
about wide-field amacrine cells in the primate. Here, we studied the
physiology of a class of primate amacrine cells whose extensive
dendritic arbor is well suited for integrating visual signals over broad
regions of space.
Methods: We recorded from a class of displaced amacrine cells in an
in vitro, whole-mount preparation of macaque retina. Responses were
recorded to various light stimuli including spatio-temporal noise,
which was used to characterize a cell’s receptive field (Chichilnisky,
2001 Network). We performed recordings in current-clamp with a
K-based pipette solution that included lucifer yellow so we could
recover the cells’ morphology after recording. Recordings were
performed at an eccentricity of ~4-8 mm from the fovea.
Results: Recorded amacrine cells appeared to constitute a single
class based on their light responses, cellular morphology, and
stratification pattern. Cells showed on average nine straight, smooth
dendrites that extended 0.8-1.2 mm from the soma. These dendrites
exhibited putative synaptic varicosities along their full length but
lacked spines and axonal processes. This class of amacrine cells
comprised OFF and ON types, stratifying in the outer and inner
sublamina of the inner plexiform layer, respectively. The OFF type
depolarized at light offset and the ON type depolarized at light onset;
neither type exhibited a classical center-surround receptive field.
Action potentials were not observed to either light stimulation or
current injection despite depolarizations of >20 mV. Nonetheless,
visual inputs located >0.5 mm from the cell body were effective in
producing changes in somatic voltage.
Conclusions: The morphology of the wide-field amacrine cells
characterized here is consistent with the wiry-type amacrine cells
described by Mariani (1990, J Comp Neuro) in macaque retina. These
cells appear well suited for rapidly integrating and communicating
over distances.
Commercial Relationships: Michael B. Manookin, None;
Christian Puller, None; Fred Rieke, None; Maureen Neitz, None;
Jay Neitz, None
Support: Helen Hay Whitney Foundation, NEI R01EY09303, NEI
R01EY11850, Research to Prevent Blindness, Core Grant for Vision
Research (P30EY01730), Howard Hughes Medical Institute
Program Number: 2641
Presentation Time: 9:30 AM–9:45 AM
Cocaine- and amphetamine-regulated transcript (CART): a novel
retinal neuropeptide
S. Anna Sargsyan, P. Michael Iuvone. Ophthalmology, Emory
University, Atlanta, GA.
Purpose: Dopamine modulates multiple dimensions of lightadapted vision, including daytime contrast sensitivity, visual acuity,
and light-adapted electroretinographic responses (Jackson et al., J
Neurosci 2012). CART is highly expressed in the brain, where it
functions as a neuropeptide and a modulator of dopamine signaling
(Rogge et al., Nature Reviews 2008). The mRNA for CART, Cartpt,
is highly expressed in the retina (Douglass et al., J Neurosci 1995),
and CART was detected in on-off direction-selective retinal ganglion
cells [RGCs (Kay et al., J Neurosci 2011)], other bistratified RGCs
(Ivanova et al., JCN 2013), and dopaminergic amacrine cells [DaACs
(Gustincich et al., PNAS 2004)]. Yet its function in the retina has not
been explored. Our study aimed to characterize the CART-expressing
retinal cells and elucidate whether dopamine can affect CART levels.
Methods: Immunofluorescence staining for CART and tyrosine
hydroxylase was used to identify CART-expressing cells and DaACs,
respectively, in retinas of wild type (WT), ThloxP/loxP and Chx10CreThloxP/loxP [retina-specific dopamine deficiency (Jackson et
al., J Neurosci 2012)] mice. ImageJ image analysis software was
used to quantify CART levels. Treatment with 10 mg/kg of L-3-4dihydroxyphenylalanine (L-DOPA) of Chx10-CreThloxP/loxP mice
was used to study the effect of restoring retinal dopamine on CART
levels. qRT-PCR was used to examine the expression of Cartpt in
retinas of WT and dopamine D4 receptor-deficient (Drd4-/-) mice.
Results: CART-like immunoreactivity was detected in DaACs,
in another subset of amacrine cells, in previously characterized
RGCs, and in the outer segments of photoreceptors. The cells
with the highest expression of CART were DaACs. CART levels
were significantly reduced in the processes of DaACs of Chx10CreThloxP/loxP mice when compared to those of ThloxP/loxP
control mice. Daily L-DOPA treatment for one week increased CART
levels in the processes of DaACs of Chx10-CreThloxP/loxP mice.
Retinal Cartpt expression showed diurnal rhythmicity in WT mice,
which was significantly altered in Drd4-/- mice.
Conclusions: CART was widely expressed in many cell types in
the retina, with its highest expression seen in DaACs. Retinal CART
expression was modulated by dopamine. Cartpt expression had
diurnal rhythmicity in WT mice, suggesting that its expression was
under the control of circadian clock transcription factors.
Commercial Relationships: S. Anna Sargsyan, None; P. Michael
Iuvone, None
Support: NIH grants R01EY004864, P30EY006360, T32EY007092,
and support from Research to Prevent Blindness
Program Number: 2642
Presentation Time: 9:45 AM–10:00 AM
Cellular localization and rhythmic expression of melatonin
receptor 1 in the rat retina
Shi-Jun Weng, Wen-Long Sheng, Xiong-Li Yang, Yong-Mei Zhong.
Fudan University, Shanghai, China.
Purpose: Retinal melatonin modulates various visual functions via
two subtypes of specific receptors, namely MT1 and MT2 receptors.
The expression of these receptors is reported to be highly speciesand neuron subtype-dependent, and exhibits daily fluctuation. We
sought to (1) explore the cellular localization of MT1 receptor in the
rat retina and (2) determine whether and how the retinal expression
levels of this receptor are regulated by circadian clock and/or light.
Methods: Localization of MT1 receptor was studied in vertical
retinal sections by immunofluorescence double staining, using a
rabbit anti-MT1 antibody, in combination with various markers for
major retinal neuron types. Diurnal and circadian variations in MT1
receptor expression levels of whole retina were assessed by Western
blotting, using retinas harvested from rats entrained to a 12: 12 h LD
cycle and kept in DD, respectively. To examine photic regulation of
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
MT1 levels during the dark period, rats were given a 2-h light pulse
just before retinas were collected.
Results: Immunostaining for MT1 receptor was strong in both the
INL and GCL, whereas weaker labeling was observed in the IPL
and ONL. In the outer retina, horizontal cells and bipolar cells were
all MT1-positive. In the inner retina, labeling for MT1 receptor
was localized to glycinergic amacrine cells, including AII amacrine
cells. GABAergic amacrine cells were also immunoreactive to
MT1 receptor. Most cholinergic amacrine cells and a subset of
dopaminergic amacrine cells were immunolabeled for MT1.
Additionally, MT1 receptor immunoreactivity was seen in almost
all Brn3a-labeled ganglion cells, as well as in Müller glial cells.
The expression levels of retinal MT1 receptors showed evident
diurnal variations, being significantly elevated during the light
period. However, the free-running retinal MT1 receptor levels under
DD condition seemed not to display obvious circadian changes.
Furthermore, 2-h light exposure applied during the dark period
acutely up-regulated retinal MT1 receptor levels.
Conclusions: MT1 receptor is expressed in multiple types of rat
retinal neurons, suggesting that melatonin may modulate the activity
of these cells. Melatonin-induced modulation may exhibit diurnal
changes due to daily fluctuation of the expression levels of MT1
receptor in the retina.
Commercial Relationships: Shi-Jun Weng, None; Wen-Long
Sheng, None; Xiong-Li Yang, None; Yong-Mei Zhong, None
Support: The Ministry of Science and Technology of China
(2011CB504602); The National Natural Science Foundation of China
(30930034, 31070967, 31171055, 31121061, 31100796); ARVO/
Pfizer Collaborative Research Fellowship to Shi-Jun Weng
Program Number: 2643
Presentation Time: 10:00 AM–10:15 AM
The influence of dopamine on contrast sensitivity in
physiologically defined retinal ganglion cells
Michael L. Risner, David Sprinzen, Douglas McMahon. Biological
Sciences, Vanderbilt University, Nashville, TN.
Purpose: Previously, our lab has found that light-mediated vision
is altered in mice that lack retinal dopamine. Specifically, we found
that the light-adapted ERG b-wave is blunted and behavioral contrast
sensitivity is reduced in retinal tyrosine hydroxylase knockout mice
(rThKO). The purpose of the present study was to further investigate
the retinal mechanism(s) that control light-adapted vision using the
rThKO mouse model. Here we examined light-adapted contrast
sensitivity of retinal ganglion cells (RGCs) in control and rThKO
mice using multi-electrode array (MEA) electrophysiology.
Methods: Retinas were harvested and flat mounted on 6 x 10 or
8 x 8 MEAs. To measure contrast sensitivity, drifting sinusoidal
gratings were projected onto the retinal surface using a small lightemitting diode (LED) monitor that was attached to the epifluorescent
port of an upright microscope. The image was focused onto the
photoreceptor layer of the retina using a 10X objective. The mean
luminance of the gratings was 0.12 mW/cm2. Contrast ranged from
3 to 30%. Sinusoidal gratings from 1.0 to 28.5 cycles/mm were
used to measure contrast sensitivity. Gratings were presented at four
different angles: 0, 90, 180, and 270 degrees. Stimuli were presented
in random order and each contrast-spatial frequency combination
was presented at least 16 times. Contrast-response functions were
obtained for each spatial frequency by measuring the average number
of spikes. Contrast threshold was defined as just above overall mean
spike rate of all stimuli.
Results: To initially assess contrast sensitivity we combined all RGC
types. Thus far we have obtained recordings from 21 RGCs from
control animals and 9 RGCs from rThKO animals. Our preliminary
results indicate reduced contrast sensitivity in rThKO RGCs. Contrast
sensitivity is reduced at low, middle, and high spatial frequencies.
Conclusions: These results corroborate our previous psychophysical
findings. We will further investigate the influence of dopamine on
contrast sensitivity in physiologically defined RGCs and rescue
contrast sensitivity using dopamine agonists.
Commercial Relationships: Michael L. Risner, None; David
Sprinzen, None; Douglas McMahon, None
Support: NIH Grant F32EY023163, NIH Grant RO1EY09256
348 ERG and VEP: human studies
Tuesday, May 06, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 3493–3511/D0093–D0111
Organizing Section: Visual Neuroscience
Program Number: 3493 Poster Board Number: D0093
Presentation Time: 11:00 AM–12:45 PM
Adaptation recovery of the photopic multi-focal ERG in early
and intermediate age-related macular degeneration
Athanasios Panorgias1, Megan Tillman1, Erich E. Sutter2, John
S. Werner1, 3. 1Ophthalmology and Vision Science, University of
California Davis, Sacramento, CA; 2Electro-Diagnostic Imaging,
Inc., Redwood City, CA; 3Neurobiology, Physiology and Behavior,
University of California Davis, Davis, CA.
Purpose: To test whether age-related macular degeneration (AMD)
affects retinal recovery after fast light adaptation using the photopic
multi-focal m-sequence technique.
Methods: Ten subjects with early (n=6) and intermediate (n=4)
AMD [76.4 ± 8.1 years old (mean ± 1 S.D.), range: 61-86 years]
were tested using the multi-focal ERG (mfERG). AMD classification
was based on a clinical exam and fundus image review by a retinal
specialist. ETDRS best-corrected visual acuity (BCVA) was between
20/16 and 20/32. Ten age-matched normal subjects (77.5 ± 8.1 years
old, range: 61-88 years) without any ocular or retinal pathologies
and ETDRS BCVA of 20/25 or better underwent the same mfERG
testing. mfERGs were recorded using the VerisPro software (EDI)
that supports extraction of retinal responses at different inter-stimulus
intervals (ISIs) and different flash combinations. An m-sequence
of 16, resulting in ~14-min recordings, was used. The stimulus
consisted of 103 hexagons having a peak luminance of 2.66 cd•s/
m2. The responses were grouped into macular and peripheral retinal
areas. In the macular region, the stimulus subtended a ~10° radial
area centered on the fovea. The peripheral stimulus formed a ring
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
with inner and outer radii ~10° and 20°, respectively. Single-flash
responses preceded by a double flash were extracted from the signal
at varying ISIs and the single flash amplitude was plotted as a
function of ISI (called the recovery function). The rate of recovery
was defined by the slope of the linear regression equation fitted
through the linear phase of the recovery function.
Results: The AMD subjects showed, as expected, lower mfERG
responses in the macular area. However, no statistically significant
difference was found in the rates of recovery between AMD and
normal subjects for both the macular and peripheral areas (two-way
ANOVA, p=0.104, α=5%).
Conclusions: AMD is known to affect predominantly outer retinal
structures (photoreceptors and RPE) and this is manifested on the
retinal electrophysiological responses, especially in the macular
area. The results, however, suggest that the disease does not affect
the recovery rate of the remaining functioning cones after fast light
adaptation.
Commercial Relationships: Athanasios Panorgias, None; Megan
Tillman, None; Erich E. Sutter, Electro-Diagnostic Imaging,
Inc. (E), Electro-Diagnostic Imaging, Inc. (I), Electro-Diagnostic
Imaging, Inc. (P); John S. Werner, None
Support: NIH (AG 04058), Research to Prevent Blindness
Program Number: 3494 Poster Board Number: D0094
Presentation Time: 11:00 AM–12:45 PM
Sleep Quality in Age-Related Macular Degeneration (AMD)
Robert M. Purbrick1, 2, Jovi C. Wong2, Rukhsana Safa2, Iona
Alexander2, Rupal Morjaria1, 2, Katharina Wulff2, Russell G. Foster2,
Susan M. Downes1, 2. 1Oxford Eye Hospital, Oxford University
Hospitals, Oxford, United Kingdom; 2Nuffield Laboratory of
Ophthalmology, Nuffield Department of Clinical Neurosciences,
Oxford University, Oxford, United Kingdom.
Purpose: To assess the effect of AMD on sleep quality.
Photosensitive retinal ganglion cells (pRGCs) relay the light signal to
entrain the body’s circadian clock. The effect of AMD on pRGCs, and
the non-visual responses to light that pRGCs mediate, is unknown.
Methods: Patients attending medical retina clinics with AMD and no
other significant ocular comorbidity completed the Pittsburgh Sleep
Quality Index (PSQI). A global PSQI score (0-21) is calculated from
seven components: sleep quality; latency; duration; habitual sleep
efficiency; sleep disturbance; use of sleep medication; and daytime
dysfunction. A global score of >5 reflects disturbed sleep. PSQI
scores were assessed in relation to best visual acuity (VA) across both
eyes and stage of AMD (early vs late).
Results: 81 patients participated in this study (four patients were
excluded due to existence of ocular comorbidities). Thus data from
77 eligible patients were analysed with a mean age of 78 years. Mean
global PSQI was 5 (range 1-14) and 31 patients (40%) had a score
suggestive of a sleep problem (i.e. PSQI >5), though this did not
correlate with logMAR visual acuity in AMD patients (r = -0.005).
There was no significant difference in global PSQI between groups of
patients according to stage of AMD (p = 0.222).
Conclusions: No relationship was found between global PSQI
score and best VA across both eyes, or disease stage, in our study
population of AMD patients. This implies that sleep is not affected
by age-related macular degeneration. Although 31 patients (40%)
had a global PSQI of >5 and mean global PSQI across the group was
5, suggesting prevalent sleep disturbance, this cannot be attributed
to AMD. Indeed, sleep disturbance in healthy older people has been
previously reported. pRGCs are distributed in a reticular fashion
over the entire retina and receive input from rods and cones. Thus,
compensatory mechanisms due to rod and cone survival, as well as
peripheral pRGC survival, may allow a normal sleep phenotype to
persist despite AMD. Our data support the idea that central retinal
degeneration is unlikely to affect sleep and circadian entrainment.
Commercial Relationships: Robert M. Purbrick, None; Jovi C.
Wong, None; Rukhsana Safa, None; Iona Alexander, None; Rupal
Morjaria, None; Katharina Wulff, None; Russell G. Foster, None;
Susan M. Downes, None
Support: Wellcome Trust Programme Grant 090684/Z/09/Z
Program Number: 3495 Poster Board Number: D0095
Presentation Time: 11:00 AM–12:45 PM
To investigate the impact of diabetic retinopathy of varying
severity on sleep.
Rupal Morjaria1, 2, Iona Alexander2, Obaid Kousha1, Rukhsana Safa2,
Robert M. Purbrick1, 2, Victor Chong1, 2, Katharina Wulff2, Russell
G. Foster2, Susan M. Downes1, 2. 1Ophthalmology, Oxford Hospitals
NHS Trust, Oxford, United Kingdom; 2Nuffield Department of
Ophthalmology, Nuffield Department of Clinical Neurosciences,
Oxford, United Kingdom.
Purpose: Sleep is essential for life and the body’s metabolic
systems require sleep of good quantity and quality for their proper
functioning. Glucose metabolism can be affected adversely by several
sleep disorders. Retinal ganglion cells have been reported to be
affected by diabetes. Melanopsin-retinal ganglion cells (mpRGCs)
are integral to the entrainment of 24-hour circadian cycle with rods
and cones also involved. The purpose of our study is to investigate
the impact of diabetic retinopathy on sleep in patients with varying
severity of retinopathy.
Methods: Patients attending diabetic retinopathy clinics with no
significant ocular co-morbidities completed the self-rated Pittsburgh
Sleep Quality Index (PSQI) to assess subjective sleep quality and a
Hospital Anxiety and Depression scale (HADS). The PSQI scores
seven different sleep components (scale of 0 to 3), combined to
produce a global sleep score of 0 to 21. A PSQI score ≥6 indicates
poor sleep. The HADS scale consists of 14 questions, 7 items for
depression (HADS-D) and 7 for anxiety (HADS-A) combined
to produce a score of 0-21.Using ETDRS grading, patients were
allocated to 3 groups depending on severity of retinopathy: no/
mild 10-35, moderate 35-53, severe >61. Statistical analysis was
performed using SPSS.
Results: 327 patients participated, (110 were excluded due to
ineligibility/incomplete data). 217 completed questionnaires were
analysed. The mean PSQI score across the three diabetic retinopathy
severity groups did not reveal a statistically significant difference
(mild =5.10, moderate =5.18, severe =5.45 p >0.05). Spearman’s
correlation was significant with global PSQI and HADS-A, r = 0.254
(p <0.001) and HADS-D, r = 0.424 (p <0.001) but not with EDTRS
score, r = 0.053 (p =0.427) and r = -0.010 (p=0.878).
Conclusions: Our study showed that no significant differences in the
PSQI and HADS with increasing stages of diabetic retinopathy. The
HADS did however correlate to global sleep quality suggesting that
anxiety and depression score increases with decrease in sleep quality.
Presumably therefore there are enough functioning melanopsin
cells and or rods and cones for entrainment to be unaffected even in
moderate and severe stages of diabetic retinopathy.
Commercial Relationships: Rupal Morjaria, None; Iona
Alexander, None; Obaid Kousha, None; Rukhsana Safa, None;
Robert M. Purbrick, None; Victor Chong, None; Katharina
Wulff, None; Russell G. Foster, None; Susan M. Downes, None
Support: WTPG grant – 090684/Z/09/Z Melanopsin signalling:
phototransduction, behavioural regulation and clinical relevance.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 3496 Poster Board Number: D0096
Presentation Time: 11:00 AM–12:45 PM
Visual consequences of mild traumatic brain injury in veterans
Chrystyna Rakoczy1, Radouil T. Tzekov2, 3. 1James A Haley Veterans’
Hospital, Tampa, FL; 2The Roskamp Institute, Sarasota, FL;
3
University of South Florida, Tampa, FL.
Purpose: Traumatic brain injury (TBI), a major cause of death
and disability worldwide can lead to vision damage. The extent
of subjective visual dysfunction present in combat veterans with
mild TBI is still unknown. The purpose of this study is to evaluate
subjective vision-related complaints, visual field deficit and
retinal nerve fiber layer (RNFL) thickness in Servicemembers
with documented mild TBI due to blast and/or blunt trauma. It is
hypothesized that visual/ocular symptomology and measures will be
similar between the groups.
Methods: 90 patients with documented mild TBI were evaluated.
All patients underwent symptom review and detailed ophthalmic
examination, including visual acuity (VA). 87 patients had visual
field testing (HVF) in both eyes, 75 had SD-OCT in at least one eye
and 49 had contrast sensitivity (CS) tested in both eyes. Based on
the history of head trauma, patients were divided into three groups:
Group 1 (Gr1) blast only, Group 2 (Gr2) blunt only and Group 3
(Gr3) blast and blunt trauma.
Results: Patients were categorized: 39 as Gr1, 18 as Gr2 and 33 as
Gr3. The average age was 33.9, 38.4 and 35.1 years, respectively.
Blurred vision (BV) was reported in (Gr1, Gr2, Gr3) 90%, 89% and
73%, photosensitivity (PT) in 85%, 73% and 79%, perception of
field deficit (PVFD) in 23%, 44% and 27%. The average values for
VA were: -0.063, -0.058 and -0.038 log MAR, for CS: 1.93, 1.75 and
1.69 log CS, for HVF mean deviation: -4.6, -4.4 and -4.0 dB, for total
RNFL thickness: 99.7, 92.4 and 100.7 μm. There was no significant
difference between the mean values of the three groups in any of
the above measures. By quadrant analysis, mean temporal RNFL
thickness was lower in all of the groups compared to a normal mean
(p<0.0001), while Gr2 mean was lower compared to Gr1 (p<0.05).
Conclusions: A large number of veterans with mild TBI reported
subjective visual complaints such as BV and PT. Whether blunt,
blast or both, the mechanism of injury did not affect the frequency of
reported subjective symptoms. Similarly, VA, CS, HVF and RNFL
thickness did not vary significantly between the trauma groups.
However, measurements of temporal RNFL were lower than normal
in all groups and significantly thinner in the blunt group. This leads
us to believe that the mechanism of injury may determine damaging
effects on already normally thin temporal RNFL. Chronology of
multiple mechanism trauma may have an effect on RNFL damage as
well.
Commercial Relationships: Chrystyna Rakoczy, None; Radouil T.
Tzekov, None
Program Number: 3497 Poster Board Number: D0097
Presentation Time: 11:00 AM–12:45 PM
Effect of Luminance on the Visual Evoked Potential (VEP) in
Visually-Normal and Mild Traumatic Brain Injury (mTBI)
Populations
Vanessa Fimreite, Naveen K. Yadav, Kenneth J. Ciuffreda. Biological
and Vision Sciences, SUNY College of Optometry, New York, NY.
Purpose: To assess the effect of luminance on VEP amplitude and
latency in the visually-normal (VN) and in the mild traumatic brain
injury (mTBI) adult populations; the findings are equivocal in the
former, and have never been tested in the latter, populations.
Methods: VN individuals (n=20, mean = 23 years) and those
with mTBI (n=12, mean = 27 years; 2 months to 10 years postinjury) were tested. Pattern VEP testing was employed using the
DIOPSYSTM NOVA–TR system (17 H x 15 V degree field size, 20’
check size, 74 cd/m2 luminance, 1 Hz temporal frequency, 20 second
trials, 1 meter distance, binocular viewing with spectacle correction),
which served as the baseline condition. Luminance levels were then
reduced with five different neutral density filters (ND) (0.5, 1.0,
1.5, 2.0, and 2.5), presented in a counterbalanced manner, with all
compared to baseline. Luminance levels were reduced to 23.6, 7.4,
2.2, 0.5, and 0.2 cd/m2, respectively, with the ND filters. Four trials
were averaged for each test condition.
Results: In both groups, the mean VEP amplitude significantly
reduced beyond 2.2 cd/m2 (1.5 ND) (p<0.05). At each luminance
level, the mean VEP amplitude was significantly lower in mTBI than
in VN (p<0.05). In contrast, in both groups, the mean VEP latency
increased progressively and significantly with luminance decrease
(p<0.05). At each luminance level, however the mean VEP latency
was significantly higher in mTBI than in VN (p<0.05). Mean latency
variability was significantly higher (~30-300%) in mTBI than in VN
(p<0.05).
Conclusions: The VEP amplitude was robust to large reductions
in luminance in both groups. In contrast, the VEP latency was
more sensitive to luminance reduction in both groups. However,
the differential latency increase and its related increased variability
with luminance reduction, in the mTBI population as compared to
VN, suggests a magnocellular pathway deficit, perhaps reflecting
increased neural noise consequent to the brain injury.
Commercial Relationships: Vanessa Fimreite, None; Naveen K.
Yadav, None; Kenneth J. Ciuffreda, None
Support: T35 NIH/NEI Research Grant 5-T35-EY020481
Program Number: 3498 Poster Board Number: D0098
Presentation Time: 11:00 AM–12:45 PM
Effect of Binasal Occlusion and Base-In Prisms on the VisualEvoked Potential (VEP) in the Visually-Normal and Mild
Traumatic Brain Injury Populations
Naveen K. Yadav, Kenneth J. Ciuffreda. Biological and Vision
Sciences, SUNY, College of Optometry, New York, NY.
Purpose: To assess the effect, and relative contribution, of binasal
occlusion (BNO) and base-in prisms (BI) on the visually-evoked
potential (VEP) amplitude and latency in the visually-normal
(VN) and in the mild traumatic brain injury (mTBI) populations.
Clinically, BNO, at times in conjunction with BI prisms, is added to
the spectacle prescription of individuals with mTBI to reduce their
abnormally-increased visual motion sensitivity (VMS).
Methods: Subjects were comprised of VN adults (n=20, mean age
25 years), and adults having mTBI (n=11, mean age 34 years, 1-10
years post-insult) and abnormally-increased VMS. There were 4 test
conditions: 1) pattern VEP (64 x 64 checkerboard pattern, 17H x 15V
degree field size, 1 Hz temporal frequency, 85% contrast, 74 cd/meter
square, 20 second trial duration, 1 meter test distance, binocular
viewing with spectacle correction), which served as the baseline
comparison condition; 2) pattern VEP with BNO; 3) pattern VEP
with 2 pd base-in (BI) prisms before each eye; and 4) pattern VEP
with the combination of the above BNO and BI prisms, with the last
3 conditions counterbalanced. Four trials were averaged for each test
condition. Figure 1.
Results: In VN, the mean VEP amplitude decreased significantly
(p<0.05) (~3 mV) with BNO in all subjects, as well as with the
combination of BNO and BI prisms. There was no effect of BI
prisms only. In contrast, in mTBI, the mean VEP amplitude increased
significantly (p<0.05) (~3 mV) in all subjects with BNO only. In both
groups, latency remained normal.
Conclusions: Only BNO alone demonstrated significant, but
opposite, directional effects on the VEP amplitude, in both groups.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Therefore, BNO alone can be used clinically for the objective
differential diagnosis of suspected mTBI with VMS. We speculate
that mTBI patients habitually attempt to suppress visual information
in the near retinal periphery to reduce their abnormal VMS. With
addition of the BNO in mTBI, the attempted motion suppression
is now rendered unnecessary. This leads to the spread of reduced
inhibition, thus producing enhanced central visual field responsivity.
In contrast, in VN, it may reflect reduction of normal excitation
over the same spatial regions, thus reducing central visual field
responsivity.
Figure 1: Binasal occluders
Commercial Relationships: Naveen K. Yadav, None; Kenneth J.
Ciuffreda, None
Program Number: 3499 Poster Board Number: D0099
Presentation Time: 11:00 AM–12:45 PM
Oculomotor Vision Rehabilitation in Mild Traumatic Brain
Injury: Effect on the Visual Evoked Potential (VEP) and Visual
Attentional (VAT) Responsivity
Kenneth J. Ciuffreda, Naveen K. Yadav. Biological and Vision
Sciences, SUNY College of Optometry, New York, NY.
Purpose: To assess the effect of oculomotor vision rehabilitation
(OVR) on the VEP amplitude and latency, as well as subjective
and objective visual attention (VAT), in mild traumatic brain injury
(mTBI).
Methods: Young adults with mTBI (n=7, mean = 29 years, 1-6
years post-insult) having oculomotor and VAT deficits were trained
and tested. OVR included training (6 weeks, 9 hours total, 3 hours
each system) of the three oculomotor systems (version, vergence,
and accommodation). Pattern-VEP testing was performed before
and after the successful OVR using the DIOPSYSTM system (17H x
15V degree field size, 20’ check size, 85% contrast, 74 candelas per
square meter, 1 Hz temporal frequency, 20 second trials, 1 meter test
distance, binocular viewing with spectacle correction). Subjective
(VSAT percentile) and objective [VEP; eyes-closed alpha (8-13
Hz) power ÷ eyes-open alpha power, the attenuation ratio (AR)]
(Willeford et al., 2013) VAT were also assessed before and after
the OVR. Three VEP trials were averaged for each of the 2 test
conditions in each subject at each test session.
Results: There was a significant increase in the group mean VEP
amplitude (17.10 to 19.15 mV), and a significant decrease in its
variability (1.89 to 1.03 mV), following OVR (p ≤ 0.05). Group mean
VEP latency and its variability did not change significantly before
and after OVR (p ≥ 0.05). There was a significant increase in VSAT
percentile score (40.25 to 59.5 percentile) (p ≤ 0.05), as well as in the
alpha AR for both the full alpha band (8-13 Hz), and selected subbands (10, 11, and 13 Hz), following OVR (p ≤ 0.05). The significant
increases in VEP amplitude and the AR ratios were found in all
subjects following the OVR.
Conclusions: These findings demonstrate for the first time that
OVR improves neuro-cortical activity in mTBI patients. The VEP
amplitude increased with reduced variability, and VAT improved
both subjectively and objectively, with this latter finding being
consistent with earlier subjective results (e.g., Solan et al., 2003),
thus suggesting that embedded in OVR is attentional training/
enhancement. Furthermore, these results demonstrate that the VEP
could be used reliably and objectively to assess the effect of OVR in
mTBI.
Commercial Relationships: Kenneth J. Ciuffreda, None; Naveen
K. Yadav, None
Program Number: 3500 Poster Board Number: D0100
Presentation Time: 11:00 AM–12:45 PM
A Retrospective Analysis of Photosensitivity in Mild Traumatic
Brain-Injury (mTBI)
James Q. Truong, Kenneth J. Ciuffreda, Esther Han, Irwin Suchoff.
Biological and Vision Sciences, SUNY College of Optometry, New
York, NY.
Purpose: To determine whether photosensitivity (PS) changes
over time, and if so, what factors may be related with the change;
furthermore, to determine whether tint density changes over time, all
in mTBI.
Methods: A retrospective analysis of 100 electronic patient records
(ages 18-40 years) with mTBI and PS was conducted. All charts were
from the clinics of SUNY/Optometry from the years 2004 to 2011.
An initial computer query using the ICD-9 diagnostic code of 368.13
was used (i.e., “visual discomfort” including PS). Only charts with
documented PS were selected for analysis. Numerous factors were
assessed, with key findings reported here.
Results: 50% of the patients reduced in PS over time, with most
occurring after year 1 post-injury (43%); 2% increased, and the
remainder did not change. 33% of patients who wore tinted lenses
decreased in PS, in contrast to 73% for those who did not. 77% of
patients who used tint maintained the same tint density over time,
while 23% reduced, and none increased. 82% of patients who wore
contact lenses (non-tinted) decreased in PS, in contrast to 40% for
those who did not. Factors associated with retarding PS reduction
were: presence of migraines (25%), loss of consciousness at the
time of trauma (29%), and having multiple brain injuries (32%).
Factors with equal distribution in PS reduction (~50%) were gender,
refractive error, medications, visual field defect type, and type of
illumination.
Conclusions: The findings suggest that neural adaptation to PS is
a long-term process, with most occurring after year one. The use of
spectacle lens tint inhibited this adaptive process; this may change
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
the way PS is treated clinically (i.e., use of low density tints or no
tints). Use of non-tinted contact lenses fostered this adaptive process
resulting in decreased PS. This too may change the way PS is treated
clinically (i.e., use of non-tinted contact lenses). These findings are
promising, as they provide prognostic indicators and initial guidance
in the management and treatment of photosensitivity in the clinic
population.
Commercial Relationships: James Q. Truong, None; Kenneth J.
Ciuffreda, None; Esther Han, None; Irwin Suchoff, None
Program Number: 3501 Poster Board Number: D0101
Presentation Time: 11:00 AM–12:45 PM
The Effect of Induced Meridional Refractive Defocus on the
Amplitude and Implicit Time of Multifocal Electroretinogram
(mfERG)
Saiful Azlan Rosli, Ai-Hong Chen, Nur-Fadzilah Che Alwi,
Muhamad-Syukri Mohamad-Rafiuddin. Optometry Department,
Universiti Teknologi MARA (UiTM), Puncak Alam, Malaysia.
Purpose: The aim of the study was to investigate the effect of
horizontal and vertical meridional refractive defocus on amplitude
and implicit time of P1 in multifocal electroretinogram (mfERG) at
three different areas of the retina.
Methods: Six combinations of astigmatism were induced by soft
toric contact lenses, C1 (plano/-1.00 × 180), C2 (plano /-2.00 × 180),
C3 (plano /-3.00 × 180), C4 (plano /-1.00 × 90), C5 (plano /-2.00 ×
90), C6 (plano /-3.00 × 90), in the fully dilated pupil condition on
ten young healthy emmetropic subjects. The amplitude and implicit
time of P1 were evaluated at three difference locations of the retina,
namely area A (1 hexagon: 19.8 deg2 at 0° viewing angle), area B
(20 hexagons: 42.9 deg2 at 17.2° of viewing angle) and area C (20
hexagons: 47.9 deg2 at 15.4° viewing angle). The horizontal defocus
was gathered from C1, C2 and C3 whereas C4, C5 and C6 simulated
the vertical defocus. The effects of horizontal and vertical defocus
among the same cylindrical refractive power were measured in the
areas of A, B and C.
Results: From the comparison, C1 and C4, C2 and C5 as well as C3
and C6 showed insignificant value between horizontal and vertical
defocus of the focal lines for the same cylindrical power in the three
difference retinal areas, where p > 0.05.
Conclusions: The effects of horizontal defocus and vertical defocus
below or equal to -3.00 DC did not influence the reading of P1
amplitude and implicit time of mfERG.
Commercial Relationships: Saiful Azlan Rosli, None; Ai-Hong
Chen, None; Nur-Fadzilah Che Alwi, None; Muhamad-Syukri
Mohamad-Rafiuddin, None
Support: Exploratory Research Grant Scheme (ERGS), 600-RMI/
ERGS 5/3 59/2011 and Fundamental Research Grant Scheme
(FRGS), 600-RMI/ST/FRGS 5/3/Fst 45/2011, Ministry of Education,
Malaysia.
Program Number: 3502 Poster Board Number: D0102
Presentation Time: 11:00 AM–12:45 PM
Pattern electroretinogram in pre-perimetric and hemifield loss
glaucoma eyes and its correlation with Fourier Domain OCT
macular thickness measurements
Andre C. Kreuz, Maria K. Oyamada, Mario L. Monteiro.
Ophthalmology, University of São Paulo, São Paulo, Brazil.
Purpose: To investigate the ability of transient and steady-state
pattern electroretinogram (PERG) to detect amplitude response
reduction in pre-perimetric and hemifield loss glaucoma patients.
To verify the correlation between PERG and fourier-domain optical
coherence tomography (FD-OCT) macular thickness measurements.
Methods: Fourteen eyes from 9 patients with optic nerve
glaucomatous damage (group 1) and 23 eyes from 20 healthy subjects
(group 2) were submitted to ophthalmic examination, including
standard automated perimetry (SAP – Humphrey 24-2 SITA Standard
test) and FD-OCT (3DOCT-1000; Topcon, Inc). Transient and
steady state PERG were recorded according to the ISCEV with the
RETiscan System (Roland Consult, Wiesbaden, Germany, 2006).
The stimulus was binocularly generated with black-and-white checks
(measuring 0.24 minutes of arc) with a mean luminance of 80 cd/
m2 and a contrast of 97%. Reversal rate was either 3 Hz (transient
response) or 10 Hz (steady state response). Amplitudes and peak
times for P50 and N95 were measured. OCT-measured full-thickness
macular measurements were registered according to an overlaid
OCT generated checkboard with 36 checks. Macular thickness
measurements were averaged globally and for two half (superior
or inferior) of that area. Data from the two groups were compared.
Correlation between PERG and FD-OCT was investigated.
Results: Transient PERG P50 and N95 amplitude measurements
(mean ± SD) were 0.82 ± 0.46 and 1.33 ± 0.71 in group 1 and 1.43
± 0.79 and 2.42 ± 1.39 in group 2 (p=0.004 and 0.004, respectively).
Steady-state P1 amplitude was 1.23 ± 0.5 (group 1) and 2.50 ±
1.32 (group 2, p<0.001). Average, superior half and inferior half
macular thickness measurements were 242.8 ± 20.5, 250.7 ± 27.4 and
234.8 ± 22.5 for group 1 and 258.2 ± 16.0, 258.3 ± 11.1 and 258.1
± 26.2 for group 2 (p= 0.014; 0.159 and 0.012, respectively). No
significant correlation was found between PERG responses and OCT
measurements.
Conclusions: Although PERG amplitudes (P1, P50 and N95) and
OCT measurements (average and inferior hemifield) were significant
lower in the glaucoma group compared to controls, no significant
correlation was observed between such measurements. Our study
suggests that PERG and OCT quantify neural loss differently, but
both technologies are useful in detecting abnormalities in patients
with glaucoma.
Commercial Relationships: Andre C. Kreuz, None; Maria K.
Oyamada, None; Mario L. Monteiro, None
Program Number: 3503 Poster Board Number: D0103
Presentation Time: 11:00 AM–12:45 PM
Evaluation of the Unaffected Fellow Eye of Unilateral Exfoliation
Syndrome and Exfoliation Glaucoma Eyes using Short Duration
Transient Visual Evoked Potentials (SD-tVEP)
Lam Lu1, Peter H. Derr2, Jessica V. Jasien1, Alberto O. Gonzalez
Garcia2, Celso Tello1, Jeffrey M. Liebmann1, 3, Robert Ritch1, 4.
1
Einhorn Clinical Research Center, New York Eye and Ear Infirmary,
New York, NY; 2Diopsys Inc., Pine Brook, NY; 3NYU School of
Medicine, New York, NY; 4New York Medical College, Valhalla, NY.
Purpose: To evaluate the fellow eye of unilateral Exfoliation
Syndrome (XFS) and Exfoliation Glaucoma (XFG) patients using
SD-tVEP.
Methods: The study population was divided into three age-matched
groups: 1) 15 randomly selected eyes of 15 healthy subjects
(70.2±5.4 yr); 2) 30 eyes of 15 unilateral XFS patients (73.9±6.0
yr); and 3) 26 eyes of 13 unilateral XFG patients (70.5±9.1 yr). SDtVEP’s were recorded using the Diopsys NOVA System (Diopsys,
Inc. Pine Brook, NJ). An area under the curve (AUC) analysis of the
SD-tVEP parameters was performed comparing the XFS/XFG eyes
and the healthy eyes. 1-way ANOVA was performed on the SD-tVEP
parameters to determine if significant differences existed between the
XFS/XFG eye and the unaffected fellow eyes; between the XFS and
XFG eyes; between the XFS/XFG fellow eye and the healthy eyes;
and between the XFS/XFG and the healthy eyes.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Results: No significant difference was found between the XFS/
XFG and fellow eye (p=0.43, p=0.21 respectively) under SD-tVEP
parameters, nor was one found between the XFS and XFG eyes
(p=0.39). In addition, the differences between both XFS/XFG
fellow eyes and the healthy eyes approached but were not significant
(p=0.054, p=0.06). However, significant differences were found
between both XFS/XFG eyes and the healthy eyes (p=0.01, p=0.03
respectively). Comparing the healthy eyes to the XFS/XFG eyes, the
AUC for the XFS eyes was Hc P100 amplitude 0.77 (0.58 - 0.96);
Hc P100 latency 0.75 (0.56 - 0.93); Lc P100 amplitude 0.71 (0.50 –
0.91); and Lc latency 0.74 (0.56 – 0.93) and the AUC for the XFG
eyes was Hc P100 amplitude 0.78 (0.59 - 0.97); Hc P100 latency 0.84
(0.67 - 1.00); Lc P100 amplitude 0.76 (0.57 – 0.95); and Lc latency
0.75 (0.55 – 0.95).
Conclusions: Short-duration transient VEP may be capable of
detecting subtle alterations in the uninvolved fellow eye of unilateral
XFS/XFG eyes prior to other abnormalities. Further study with a
larger number of patients is needed to confirm these preliminary
findings.
Commercial Relationships: Lam Lu, None; Peter H. Derr,
Diopsys (E); Jessica V. Jasien, None; Alberto O. Gonzalez Garcia,
Diopsys (E); Celso Tello, Diopsys (R); Jeffrey M. Liebmann,
Diopsys (R); Robert Ritch, Diopsys (R)
Program Number: 3504 Poster Board Number: D0104
Presentation Time: 11:00 AM–12:45 PM
Macular function measured with mfERG in prematurely-born
children at school-age
Hanna M. Akerblom1, Gerd Holmstrom1, Sten Andreasson2.
1
Neurosience/Ophthalmolgy, Uppsala University, Uppsala, Sweden;
2
Lund University, Lund, Sweden.
Purpose: Children born preterm have affected visual functions
compared to children born at term, including decreased visual
acuity and reduced contrast vision. We have previously shown
morphological changes, measured with optical coherent tomography
(OCT), of the macula of prematurely-born children. The purpose of
this study is to evaluate the function of the macula with multifocal
electroretinogram (mfERG) and investigate correlations between
macular function and visual acuity (VA), gestational age (GA), birth
weight (BW) and central macular thickness measured with OCT.
Methods: A preterm group of nine children, 9-13 years old, born
before 32 weeks of gestation, were evaluated with multifocal mfERG
(VERIS, EDI) and OCT (CIRRUS, Carl Zeiss, Meditec). Their mean
gestational age at birth was 29 weeks and mean birth weight was
1259 g. Four children had no retinopathy of prematurity (ROP) and
five had mild ROP in the neonatal period. A group of 11 full-term
children, 8-19 years old, with normal visual acuity, were used as
controls. A program with 103 hexagones stimulation was used and
the amplitudes and implicit times of the first positive peak, P1, were
presented in five concentric rings and a “sum of all groups”, ring 1
being the most central ring.
Results: The amplitudes of the P1 response of ring 1 and 2 were
significantly reduced in the preterm group compared to controls
(p=0.05) Ring 3-5 and “sum of all groups” were borderline. The
implicit times of the P1 response showed no difference between the
two groups. There was no correlation between P1 amplitude and
implicit times with VA, GA, BW or central macular thickness.
Conclusions: The central macular function, measured with mfERG,
is reduced in school-aged, prematurely born children compare to
children born at term. These results could be one reason why children
born preterm have affected visual functions even if they had no or
only mild ROP in the neonatal period. A possible explanation is that
preterm birth per se affects the development of the macula leading to
permanent morphological as well as functional changes.
Commercial Relationships: Hanna M. Akerblom, None; Gerd
Holmstrom, None; Sten Andreasson, None
Program Number: 3505 Poster Board Number: D0105
Presentation Time: 11:00 AM–12:45 PM
Retinal Remodeling in Retinopathy of Prematurity
James D. Akula1, 2, Anca Mocofanescu1, R D. Ferguson3, 1, Mircea
Mujat3, Jena Tavormina1, Tara L. Favazza1, Emily A. Swanson1, Anne
Moskowitz1, 2, Ronald M. Hansen1, 2, Anne Fulton1, 2. 1Ophthalmology,
Boston Children’s Hospital, Boston, MA; 2Ophthalmology, Harvard
Medical School, Boston, MA; 3Biomedical Imaging Group, Physical
Sciences, Inc., Andover, MA.
Purpose: The sensitivity of the electroretinographic (ERG) a-wave,
which originates in photoreceptors, is low in mature eyes with a
history of retinopathy of prematurity (ROP). However, the sensitivity
of the ERG b-wave, which originates in postreceptor retina, often
becomes normal with age (Harris et al., 2011, Doc Opthalmol
122(1):19). Furthermore, near the ‘rod ring,’ teenaged patients with
a history of ROP have fairly normal retinal sensitivity but larger
areas for complete scotopic spatial summation than do either preterm
individuals with no history of ROP or term born controls (Tavormina
et al., 2013, ARVO Abs. 5850). Collectively, these data suggest
reorganization of the peripheral, postreceptor circuitry that trades
spatial resolution for increased sensitivity. Thus, the retinal layers
near the rod ring were studied by adaptive-optics optical coherence
tomography (OCT) in this preliminary study.
Methods: Spectral-domain OCTs of a 1° slice located 18° nasal of
the fovea were obtained in 12 term-born controls, 4 preterm (≤32
weeks gestation) subjects and 3 subjects with a history of ROP.
Flattened, aligned and averaged frames were manually marked in
ImageJ to delineate the following layers: 1) nerve fiber, 2) inner
plexiform and ganglion cell, 3) inner nuclear, 4) outer plexiform,
5) outer nuclear, 6 and 7) respective mitochondria sparse and
mitochondria dense portions of the inner segment, 8) outer segment,
and 9) retinal pigment epithelium and choriocapillaris. Mean layer
thickness was plotted for each group and inspected for patterns.
The total thickness of the aggregate ‘postreceptor’ layers (1-4) and
‘photoreceptor’ layers (5-9) were tested for significant differences by
two-factor (group, depth) repeated measures ANOVA, followed by
Tukey’s HSD post-hoc test.
Results: Inspection of the means revealed that, relative to term,
ROP layers 1-4 (postreceptor) were notably thicker, layers 5-8
(photoreceptor) were thinner, and 9 was almost unchanged.
Correspondingly, there was no main effect of group (P=0.914) but
there was a highly significant group×depth interaction (P<0.001) and
the HSD test revealed significantly thicker postreceptor retina and
thinner photoreceptor retina in ROP than in term subjects; in preterm
subjects, neither depth was distinguishable from ROP or term.
Conclusions: There is anatomic evidence for postreceptor
remodeling compensatory to loss of photoreceptor input in the
peripheral ROP retina.
Commercial Relationships: James D. Akula, None; Anca
Mocofanescu, None; R D. Ferguson, Physical Sciences, Inc. (E),
Physical Sciences, Inc. (P); Mircea Mujat, Physical Sciences, Inc.
(E); Jena Tavormina, None; Tara L. Favazza, None; Emily A.
Swanson, None; Anne Moskowitz, None; Ronald M. Hansen,
None; Anne Fulton, None
Support: NIH Grant EY010957, Children’s Hospital Ophthalmology
Foundation
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 3506 Poster Board Number: D0106
Presentation Time: 11:00 AM–12:45 PM
Infantile nystagmus syndrome in childhood: Isolation of the
visual cortical signal
John P. Kelly1, 2, Felix Darvis2, James O. Phillips1, 2, Avery H.
Weiss1, 2. 1Ophthalmology OA.6.293, Seattle Children, Seattle, WA;
2
University of Washington, Seattle, WA.
Purpose: Visual cortical responses are selectively reduced to
checkerboard reversal stimulation when recorded from patients with
infantile nystagmus syndrome (INS). This study examined if visual
evoked potential (VEP) responses could be improved by signal
processing and if these improvements were related to presumed
foveation periods.
Methods: Subjects were 12 children (0.4 – 14 yrs age) with INS
(normal retina without albinism or optic nerve hypoplasia). VEP
amplitude, latency, and signal-to-noise ratios (SNR) were recorded
to contrast-reversing (1.4 Hz) checkerboards of 163 arc minutes
and compared to brief onset of horizontal gratings that reduced
retinal image motion (0.5 cycle/degrees, 150/500 ms on/off period;
constant mean luminance). Individual VEP epochs underwent 1)
latency correction using phase cross correlation, or 2) selection of
epochs based on phase consistency at 13 frequencies (5.5-21.9 Hz)
in the Fourier domain. The probability of foveation in 9 subjects was
estimated by the percentage of time eye velocity was ≤ 3 degrees/
second from video-oculography.
Results: VEP amplitude and SNR was significantly correlated with
the probability of foveation for check reversal only (amplitude, r =
0.69; p = 0.026; SNR, r = 0.76; p = 0.011). VEP amplitude increased
up to 2.5 fold after latency correction (p < 0.0001) and increased up
to 5.4 fold after selective averaging based on phase consistency (p
< 0.0001). Improvement in amplitude was always greater for check
reversal compared to pattern-onset. The changes in VEP amplitude
were confirmed by similar improvements in SNR (p < 0.0001). Phase
correction had no effect on latency with either stimulus condition (p
≥ 0.06). Improvements in VEP amplitude were accounted for by a
significant relationship between SNR and the probability of foveation
(r = 0.66; p = 0.038).
Conclusions: The reduction in visual cortical signal in patients with
INS is significantly related to a reduction in SNR due to reduced
foveation periods. This reduction in VEP is associated with loss of
VEP coherency or an absence of cortical phase-locking. Our findings
were specific to stimuli undergoing retinal image motion, since there
was no significant relationship to transient horizontally oriented
stimuli. Our VEP analysis likely extracts brief epochs in the visual
cortical signal in which the retinal image motion is minimal thus
allowing for better visual sampling.
Commercial Relationships: John P. Kelly, None; Felix Darvis,
None; James O. Phillips, None; Avery H. Weiss, None
Support: Peter LeHaye, Barbara Anderson, and William O. Rogers
Endowment Funds
Program Number: 3507 Poster Board Number: D0107
Presentation Time: 11:00 AM–12:45 PM
Visual evoked cortical potential kernels elicited by m-sequences:
a principal component analysis
Carolina S. Araujo1, Givago S. Souza1, 2, Luiz Carlos L. Silveira1,
2 1
. Instituto de Ciencias Biologicas, Universidade Federal do Para,
Belem, Brazil; 2Nucleo de Medicina Tropical, Universidade Federal
do Para, Belem, Brazil.
Purpose: To evaluate the contribution of principal components of the
second order kernels of pseudo-random VECP waveform in different
combinations of achromatic contrast and spatial frequency.
Methods: Nine normal subjects were tested (24.1±6.2 yo).
Achromatic sinusoidal gratings (8° visual angle) were temporally
controlled by an m-sequence in order to simulate pattern reversal
presentation mode. Six levels of Michelson contrast (3% to 99%)
and seven spatial frequencies (0.4 cpd to 10 cpd) were used. The
Veris system was used for visual stimulation, electrophysiological
recording, and extraction of the first and second slice of the second
order kernel (K2.1 and K2.2, respectively). Principal component
analysis was applied in the K2.1 and K2.2 waveforms using singular
value decomposition. We estimated the gain of the RMS amplitude
of the principal components from K2.1 and K2.2 as a function of
contrast and compared with data from retinal ganglion cells published
by Kaplan & Shapley (1986).
Results: Two principal components (PC) explained most of the
variance from each slice of the second order kernel. The variance
explained by the PC extracted from the K2.1 was 49±5% for the first
PC and 20±3% for the second PC, whereas the variance explained by
the PC extracted from K2.2 was 44±3% for the first PC and 22±2%
for the second PC. The K2.1 first PC was very similar to the original
K2.1, with the same N1, P1, and P2 components. The K2.1 second
PC showed lower amplitudes and were similar to K2.1 first PC. The
waveforms of the two PC extracted from K2.2 were dominated by
negative polarity waveforms. The contrast-response function of the
K2.1 first PC showed higher gain than K2.1 second PC, and both
PCs from K2.2, mainly at low and intermediate spatial frequencies.
The mean RMS amplitude function versus contrast of K2.1 first PC
co-varied well with M cell responses from Kaplan & Shapley (1986),
while contrast-response functions of K2.1 second PC and both PCs
from K2.2 co-varied well with P cell responses from Kaplan &
Shapley (1986).
Conclusions: K2.1 first principal component seems to be generated
by the activity of a high contrast sensitivity pathway, while the
other major K2.1 and K2.2 components seem to be generated by
a mechanism of low contrast sensitivity. Visual system M and P
parallel pathways are natural candidates to represent the generating
mechanisms of the second order kernels principal components.
Commercial Relationships: Carolina S. Araujo, None; Givago S.
Souza, None; Luiz Carlos L. Silveira, None
Support: BRAVO ALLERGAN Grant
Program Number: 3508 Poster Board Number: D0108
Presentation Time: 11:00 AM–12:45 PM
The influence of stimulus size on L- and M-cone driven
electroretinograms
Mellina M. Jacob1, Bruno D. Gomes1, Givago S. Souza1, 2, Manoel da
Silva1, Declan J. Mckeefry3, Neil R. Parry4, Luiz Carlos L. Silveira1, 2,
Jan Kremers5. 1Instituto de Ciências Biológicas, Universidade Federal
do Pará, Belém, Brazil; 2Núcleo de Medicina Tropical, Universidade
Federal do Pará, Belém, Brazil; 3Bradford School of Optometry and
Vision Science, University of Bradford, Bradford, United Kingdom;
4
Vision Science Centre, Manchester Royal Eye Hospital, Manchester,
United Kingdom; 5Department of Ophthalmology, University
Hospital Erlangen, Erlangen, Germany.
Purpose: To determine the ERG responses to isolated L- and M-cone
stimuli as a function of stimuli size and temporal frequency, and
correlate them with post-receptoral pathways properties.
Methods: Flicker ERGs were recorded monocularly from four
trichromatic subjects using corneal fiber electrodes. Sinusoidal
stimuli were presented in a Ganzfeld bowl, containing four colored
light-emitting diodes (LED). The mean luminance of the white
background was 284 cd/m2. A triple “silent substitution” method was
applied, resulting in either L-cone or M-cone isolating stimuli each at
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
10% cone contrast. Flicker ERG measurements were repeated at five
temporal frequencies (8, 12, 30, 36 and 48 Hz), in 14 different spatial
stimulus configurations implemented by black cardboard field stops:
one full-field stimulus, seven circular stimuli varying in size between
10° and 70° in 10° steps, and six annular stimuli with 70° outer
diameter and inner diameters between 10° and 60° varying in 10°
steps. Fast Fourier Transform (FFT) was used to extract amplitude
and phase from the fundamental component. L/M ratio and phase
difference were estimated from the averaged responses.
Results: At 8 and 12 Hz, the averaged amplitudes were constant for
all stimuli configurations, and the L/M ratio was close to unity. In
30, 36 and 48 Hz, ERG amplitude increased with increasing stimulus
size. L-cone driven ERGs were slightly larger than M-cone driven
ERGs. The L/M ratio varied with stimuli size and was particularly
large for full field stimuli. The L/M ratio differed for 30, 36 or 48 Hz
frequencies.
Conclusions: The data confirm results that we presented at last
year’s ARVO using reddish backgrounds and suggest that ERG
responses to low and high temporal frequencies stimuli are processed
by different post-receptorals mechanisms, probably parvocellular
and magnocellular pathways. In addition, the L/M ratio for the
high frequency mechanism depends upon stimulus size but also on
temporal frequency.
Commercial Relationships: Mellina M. Jacob, None; Bruno D.
Gomes, None; Givago S. Souza, None; Manoel da Silva, None;
Declan J. Mckeefry, None; Neil R. Parry, None; Luiz Carlos L.
Silveira, None; Jan Kremers, None
Support: A “Science without borders” stipend from CNPq, DFG
grant KR 1317/13-1. LCLS is a CNPq research fellow
Program Number: 3509 Poster Board Number: D0109
Presentation Time: 11:00 AM–12:45 PM
Rod- and cone-isolated flicker electroretinograms and their
response summation characteristics
J Jason McAnany, Jason C. Park, Dingcai Cao. Ophthalmology and
Visual Sciences, University of Illinois at Chicago, Chicago, IL.
Purpose: To define the amplitude and phase characteristics of rodand cone-isolated flicker electroretinograms (ERGs) and to determine
how rod and cone responses summate to generate the non-receptorisolated flicker ERG.
Methods: Following 30 minutes of dark-adaptation, full-field ERGs
were obtained from four normally-sighted subjects (ages 25 to 36
years) using a 4-primary LED-based ganzfeld photostimulator and
standard ERG recording techniques. The 4 primaries were either
modulated sinusoidally in phase to achieve simultaneous rod and
cone activation (ERGROD+CONE; non-receptor-isolated) or in different
phases to achieve rod-isolated (ERGROD) and cone-isolated (ERGCONE)
responses by means of triple silent substitution. ERGs were measured
at two mean luminance levels (2.4 cd/m2 and 24 cd/m2) and four
temporal frequencies (2, 4, 8, and 15 Hz). The amplitude and phase
of the fundamental response component for each condition were
derived by Fourier analysis.
Results: At both luminance levels, response phase decreased
approximately linearly as stimulus temporal frequency increased
for all three paradigms, with the ERGROD and ERGCONE phases
differing by approximately 180 deg at all frequencies. The
relationship between response amplitude and stimulus frequency was
complex and depended on the paradigm and mean luminance level.
Specifically, ERGROD amplitude decreased as stimulus frequency
increased for both luminance levels, whereas ERGCONE amplitude
depended on mean luminance: at 2.4 cd/m2, ERGCONE amplitude
decreased as stimulus frequency increased; at 24 cd/m2, ERGCONE
amplitude decreased from 2 to 8 Hz and increased from 8 to 15 Hz.
There were substantial differences under the ERGROD+CONE paradigm
at the two luminance levels: the relationship between response
amplitude and stimulus frequency was weakly band-pass at 2.4 cd/
m2 and was U shaped at 24 cd/m2, due to ERGROD and ERGCONE
responses having similar amplitudes and opposite phases at 4 and 8
Hz.
Conclusions: The pattern of responses for the combined rod and
cone paradigm at both luminance levels can be accounted for by
the summation of signals arising from the rod and cone pathways
that have opposite response phase. Destructive interference between
the rod and cone pathway signals was greatest for conditions
that generated similar rod and cone ERG amplitudes, resulting in
decreased amplitude under the combined rod and cone condition.
Commercial Relationships: J Jason McAnany, None; Jason C.
Park, None; Dingcai Cao, None
Support: NIH research grant R00EY019510 (JM), R01EY019651
(DC), NIH core grant P30EY001792, and an unrestricted
departmental grant from Research to Prevent Blindness.
Program Number: 3510 Poster Board Number: D0110
Presentation Time: 11:00 AM–12:45 PM
Contribution of oscillatory potentials to the ON- and OFFphotopic electroretinogram (ERG) in human
Jonathan Gotzmann1, Ioannis Dimopoulos2, Yves Sauve2, 1.
1
Physiology, University of Alberta, Edmonton, AB, Canada;
2
Ophthamology, University of Alberta, Edmonton, AB, Canada.
Purpose: To describe changes in the time, power and frequency
domain of the ON (b-wave) and OFF (d-wave) oscillatory potential
(OP) components of the human ERG.
Methods: Full-field ERG ON- and OFF- responses were recorded
from the eyes of 9 healthy subjects (aged 20-49 years) with
dilated pupils using DTL fiber electrodes and an Espion e2 system
(Diagnosys LLC; 0.3-300Hz bandpass), with 30 cd.m-2 background
adaptation and stimulus intensity of 2.75 log cd.m-2. Light stimulus
duration was increased in a stepwise fashion from 10ms to 800ms
in eight steps. A total of 20 traces were averaged at each step.
Oscillatory potentials of the b- and d-waves were analyzed with
trough-to-peak measurement and also Fast-Fourier-Transform (FFT)
to determine the dominant power (mV2) and frequency (Hz).
Results: Four distinct OP peaks were consistently phase-locked to
the ON response (ON-OPs). Power of the ON-OPs peaked at shorter
duration stimuli (<20ms) followed by an exponential decay with
longer durations. The dominant frequency remained ~140Hz for all
durations. The ON-OPs (OP1-4) timing also remained constant for
all stimulus durations (peaks at t=19ms, t=25ms, t=32ms and t=40ms
after stimulus onset) with amplitudes remaining constant after a
stimuli durations >20ms. For the OFF phase, two distinct OPs were
distinguished. The OFF-OPs (OP1-2) timing remained constant for
stimuli durations >100ms (peaks at t=19ms and t=26ms after stimulus
offset). The dominant power of the OFF-OPs increased with stimulus
duration, reaching a plateau after a 500ms duration. The dominant
frequency remained ~120Hz for all durations At a short duration
stimulus of 10ms OFF-OP1 significantly contributed to ON-OP3,
while with a stimulus duration of 20ms, OFF-OP1 contributed
significantly to ON-OP4. The OP peaks measured from a filtered
trace precede the peaks found in the raw trace by 1-2ms for both the
ON and OFF response.
Conclusions: Our results imply that OPs to short duration stimuli,
as used in the clinic, represent a mixed contribution from both the
ON and OFF retina circuitry. Selective testing of these two distinct
circuits would be optimally achieved using longer duration stimuli
such as 200 msec, which allows a clear separation of both response
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
types without the discomfort of subject exposure to longer duration
stimuli.
Commercial Relationships: Jonathan Gotzmann, None; Ioannis
Dimopoulos, None; Yves Sauve, None
Support: CIHR MOP 125873
Program Number: 3511 Poster Board Number: D0111
Presentation Time: 11:00 AM–12:45 PM
Statistical Decomposition of the Electroretinogram into its
Components
Ye Chen1, Jessica Tang2, Marc Sarossy2, 1. 1The Royal Victorian Eye
and Ear Hospital, East Melbourne, VIC, Australia; 2Centre for Eye
Research Australia, East Melbourne, VIC, Australia.
Purpose: In the early 20th Century, Piper, Granit and others
decomposed the electroretinogram (ERG) into components using
pharmacological techniques. In this study, we decompose the ERG
into early, mid and late components using statistical curve fitting
techniques in R.
Methods: This study tests the reliability of a statistical
decomposition of the photopic full-field ERG into a combination
of Gaussian curves. 39 subjects with ‘normal’ functional eyes were
recruited. Each subject underwent 2 sessions of bilateral ERG tests at
five different brightness levels. Raw data obtained from the tests was
recorded in the Espion software. This was exported to the R statistical
program. A parametric function consisting of a linear combination
of three Gaussian curves was fitted to the data using non-linear least
squares.
Results: Rapid convergence and successful curve fitting was
achieved for 446 out of 457 traces. Reconstructed waveforms showed
good correlation with the original raw data for a- and b-waves
(r=0.945 for a-wave and r=0.972 for b-wave). Bland Altman plots
also showed good agreement between modelled waveforms and raw
data.
Conclusions: Statistical decomposition of the electroretinogram into
early, mid and late components is possible and reliable. The analysis
of individual components may prove useful in the study of particular
diseases.
Commercial Relationships: Ye Chen, None; Jessica Tang, None;
Marc Sarossy, None
349 Retinal Development
Tuesday, May 06, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 3512–3520/D0112–D0120
Organizing Section: Visual Neuroscience
Program Number: 3512 Poster Board Number: D0112
Presentation Time: 11:00 AM–12:45 PM
Dopamine D2 receptor regulates the functional development of
retina
Ning Tian1, Hongping Xu2, Ping Wang1. 1Ophthalmology & Visual
Science, University of Utah, Salt Lake City, UT; 2Neurobiology, Yale
University, New Haven, CT.
Purpose: Dopamine receptors play important roles in the activity
dependent synaptic plasticity in CNS and multiple subtypes of
dopamine receptors are expressed by retinal neurons. Our recent
study showed that D1 dopamine receptor regulates the developmental
enhancement of ERG b-wave and the light response gain between
bipolar and ganglion/amacrine cells. In this study, we further
determined whether D2 dopamine receptor plays important roles in
the activity-dependent development of mouse retina.
Methods: Mouse retinal light responses were recorded using ERG
measurements. Young and adult wild type (WT) mice and mice
lacking dopamine D2 receptor were used to evaluate the roles of
dopamine D2 receptor in the development of retina. ERGs were also
recorded from mice reared in constant darkness to determine whether
dopamine D2 receptor plays critical roles in the activity-dependent
development of moue retina. The amplitudes, kinetics and response
gains of ERG waveforms are quantitatively analyzed.
Results: 1) Opposite to the mutation of D1 dopamine receptor, the
amplitude of inner retinal light responses measured as ERG OPs
is selectively enhanced in D2-/- mice. 2) Unlike the D1 dopamine
receptor mutation which preferentially affects the response gain
between b-wave and OPs at the scotopic range, dopamine D2
receptor mutation preferentially enhance the relative strength of
OPs evoked by high light intensities (photopic) through selective
enhancement of the response gain between b-wave and OPs. 3)
The amplitudes of ERG of D2-/- mice are not different from that
of age-matched WT mice before eye opening. 4) The amplitude
enhancement of inner retinal light responses of D2-/- mice is
completely eliminated by light deprivation.
Conclusions: 1) Deletion of dopamine D2 receptor has opposite
effect on the inner retinal light response in comparison with D1
dopamine receptor mutation. 2) D2 dopamine receptor preferentially
regulates the retinal light responses after eye opening. 3) Effects
induced by mutation of D2 dopamine receptor on ERG are lightsensitive.
Commercial Relationships: Ning Tian, None; Hongping Xu,
None; Ping Wang, None
Support: NIH Grant EY012345
Program Number: 3513 Poster Board Number: D0113
Presentation Time: 11:00 AM–12:45 PM
Influence of ON and OFF Pathways on Visual Function
Development
Moe H. Aung1, Hanna Park1, Curran S. Sidhu1, P M. Iuvone1,
2
, Machelle T. Pardue1, 3. 1Neuroscience/Ophthalmology, Emory
University, Atlanta, GA; 2Pharmacology, Emory University, Atlanta,
GA; 3Rehab R&D Center of Excellence, Atlanta VA Medical Center,
Atlanta, GA.
Purpose: ON and OFF pathways are essential components in visual
discrimination. In this study, we evaluated the roles of ON and OFF
pathways in mediating visual function during development using
pathway-specific mutant mice.
Methods: Nob mice have a null mutation in the Nyx gene that
encodes for the protein nyctalopin; this mutation results in lack of
visual signal transmission in the retinal ON pathways. Alternatively,
Vsx1 knockout (KO) mice have a null mutation in the visual system
homeobox gene 1 (Vsx1), which leads to defects in the differentiation
and function of most cone OFF bipolar cells and consequently
defective OFF pathway signaling. With their respective wildtype controls (WT), C57BL/6 for nob and 129Sv for Vsx1 KO,
we followed the development of visual acuity and peak contrast
sensitivity weekly from 2 to 8 weeks of age using the virtual
optokinetic system.
Results: Both nob and Vsx1 KO mice had significantly lower visual
acuity levels than their WT counterparts early in development
(p<0.05), with nob mice having more severe deficits than Vsx1
KO mice. Both nob and Vsx1 KO mice showed improvements in
visual acuity thresholds that plateaued at similar thresholds as WT.
However, nob mice did not reach WT levels until postnatal week 7,
while Vsx1 KO mice were indistinguishable from WT starting at 3
weeks of age. Both nob and Vsx1 KO mice had consistently lower
contrast sensitivities than their WT counterparts throughout the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
study period (p<0.001). Moreover, the spatial frequency that elicited
maximal contrast sensitivity differed between the two WT strains
(C57BL/6 vs. 129Sv).
Conclusions: Collectively, our results suggest that having a
functional ON or OFF pathway is sufficient for normal visual acuity
development by adulthood, but both pathways are necessary for
proper contrast sensitivity perception. It would be interesting to
determine if the differential effects of ON or OFF pathway defect on
visual acuity and contrast sensitivity are due to differences in retinal
dopamine content, a key modulator of the optokinetic response.
Commercial Relationships: Moe H. Aung, None; Hanna Park,
None; Curran S. Sidhu, None; P M. Iuvone, None; Machelle T.
Pardue, None
Support: NIH EY016435 (MTP), NIH P30 EY006360, Research to
Prevent Blindness, and the Department of Veterans Affairs
Program Number: 3514 Poster Board Number: D0114
Presentation Time: 11:00 AM–12:45 PM
Double olfm1a and olfm1b knockout in zebrafish causes moderate
abnormality in retinal development and function
Naoki Nakaya1, Tohei Yokogawa2, Haohua Qian3, Fumihito Ono4,
Harold A. Burgess2, Stanislav I. Tomarev1. 1Section of Retinal
Ganglion Cell Biology, National Eye Institute/NIH, Bethesda, MD;
2
Section of Vertebrate Organogenesis, National Institute of Child
Health and Human Development/NIH, Bethesda, MD; 3Visual
Function Core, National Eye Institute/NIH, Bethesda, MD; 4Section
of Model Synaptic Systems, National Institute on Alcohol Abuse and
Alcoholism/NIH, Rockville, MD.
Purpose: The olfm1 gene encodes a secreted glycoprotein highly
conserved in vertebrates. There are two olfm1 genes in zebrafish,
olfm1a and olfm1b. Both of these genes are expressed in the brain
and retina starting from 16 h post fertilization to adults. We generated
a null mutant of both olfm1a and olfm1b genes and analyzed its
retinal structure and visual function.
Methods: Olfm1a and olfm1b mutant alleles with nonsense point
mutations were obtained from the Wellcome Trust Sanger Institute.
Olfm1a and olfm1b null mutants were bred to generate double null
mutant (olfm1a/b null). Spontaneous movement, optokinetic and
optomotor responses, behavioral responses to light increments and
decrements and electroretinogram (ERG) to increased (ON) and
decreased (OFF) illumination were compared between olfm1a/b
null and wild-type larvae 7 days post fertilization (dpf). The retinal
morphology was examined by immunostaining of frozen sections.
Total RNA was isolated from 3 and 7 dpf larvae for RNA sequencing
analysis.
Results: Body shape, behavior and fertility of adult olfm1a/b null fish
appeared to be normal. At 7 dpf, the thickness of the retinal ganglion
cell and inner plexiform layers was reduced while the outer nuclear
layer was thicker in olfm1a/b null retina as compared with wildtype. The size of other retinal layers was similar in olfm1a/b null and
wild-type larvae. The optomotor response of olfm1a/b null larva to
OFF stimulation was normal. However, the optokinetic response and
behavioral responses to light increments was significantly reduced in
olfm1a/b null as compared with wild-type larvae. The ERG response
of olfm1a/b null larvae was slightly reduced in both ON and OFF
stimulation conditions as compared with wild-type. RNAseq analysis
of 3 dpf larvae showed reductions in expression of genes encoding
some transcription factors (pax6), calcium channels (cacna1a),
AMPA receptors, the MAP kinase pathway, and genes involved in
axon growth (neurotrophic factor receptors and neurofilaments)
for olfm1a/b null larvae compared with wild-type. Many of these
changes in the gene expression levels were recovered to the normal
levels at 7 dpf. Nevertheless, down-regulation of indicated genes at
early developmental stages may contribute to the observed defects in
olfm1a/b null retina.
Conclusions: Olfm1a and olfm1b genes are involved in retinal
development and visual functions in zebrafish.
Commercial Relationships: Naoki Nakaya, None; Tohei
Yokogawa, None; Haohua Qian, None; Fumihito Ono, None;
Harold A. Burgess, None; Stanislav I. Tomarev, None
Support: The Intramural Research Programs of the National
Eye Institute, National Institutes of Child Health and Human
Development and National Institute on Alcohol Abuse and
Alcoholism in National Institute of Health
Program Number: 3515 Poster Board Number: D0115
Presentation Time: 11:00 AM–12:45 PM
MMP-2 and MT1-MMP as axonal outgrowth-promoting
molecules in the neuroretina
Lieve K. Moons1, Tom Buyens1, Kim Lemmens1, Manuel SalinasNavarro1, Niels Behrendt2, Inge Van Hove1, Djoere Gaublomme1,
Lies De Groef1. 1Biology Department, Zoological Inst, University of
Leuven (KU Leuven), Leuven, Belgium; 2The Finsen Laboratory,
University of Copenhagen, Copenhagen, Denmark.
Purpose: Intensive research efforts focus on elucidating mechanisms
that can enhance a regenerative capacity in the adult CNS. Over the
past years considerable knowledge was obtained from studying optic
nerve regeneration in adult animals. As matrix metalloproteinases
(MMPs) are upregulated during CNS repair, reduce glial scar
formation and potentially promote axonal regrowth, MMPs or their
underlying molecules likely form potent regenerative molecules.
Here, we investigate the possible role of specific MMPs in axonal
outgrowth of injured retinal ganglion cells (RGCs).
Methods: RGC neurite outgrowth was analysed using ex vivo
culturing of retinal explants from neonatal mice. Adult zebrafish
were subjected to optic nerve crush (ONC) and axonal regeneration
was followed using biocytin tracing. Immunohistochemistry (IHC),
Western blotting (WB) and gel zymography were used to investigate
MMP expression. MMP function was studied by using MMP
deficient animals or MMP-inhibiting compounds.
Results: Broad-spectrum MMP inhibition reduces neurite extension
of RGCs from retinal explants, implicating MMPs as beneficial
factors in axonal regeneration. Additional studies, using more specific
inhibitors and MMP deficient mice, disclosed that MMP-2 and MT1MMP, but not MMP-9, are involved in this process. Furthermore,
administration of a novel antibody to MT1-MMP that selectively
blocks proMMP-2 activation, revealed a functional co-involvement
of these proteinases in determining RGC outgrowth. Subsequent
immunostainings showed expression of both MMPs in/on RGC axons
and glial cells and gel zymography revealed the presence of active
MMP-2 in retinal explants.
In the retina of adult zebrafish subjected to ONC, WB and IHC
confirmed a restricted time-dependent upregulated MMP-2 and MT1MMP expression in both Müller glia and RGC axons during axonal
regrowth. Furthermore, broad-spectrum MMP inhibition in the retina
greatly reduced the regenerative response after ONC. Currently,
more specific MMP inhibition/knockdown is being applied in vivo
in the zebrafish eye to determine their effects on RGC survival, glial
reactivity, axonal regeneration and tectal reinnervation.
Conclusions: Overall, our results suggest that MT1-MMP activates
proMMP-2 at the axolemma to exert its axonal outgrowth-promoting
function. These observations are currently being validated in a mouse
ONC model, characterized by partial regeneration of RGC axons.
Commercial Relationships: Lieve K. Moons, None; Tom Buyens,
None; Kim Lemmens, None; Manuel Salinas-Navarro, None; Niels
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Behrendt, None; Inge Van Hove, None; Djoere Gaublomme, None;
Lies De Groef, None
Support: This work was supported by national grants from the
Research Council of KU Leuven (KU Leuven BOF-OT/10/033),
the Research Foundation Flanders (FWO) (FWO G05311.10), the
Danish Cancer Society, the Lundbeck Foundation, and the European
Community’s Seventh Framework Programme FP7/2007-2011 under
grant agreement n°201279. Manuel Salinas-Navarro is a PD fellow
of the Research Foundation Flanders (FWO), Belgium. Tom Buyens,
Kim Lemmens and Lies De Groef are fellows of the Flemish Institute
for the promotion of scientific research (IWT), Belgium.
Program Number: 3516 Poster Board Number: D0116
Presentation Time: 11:00 AM–12:45 PM
The Transcription Factor Math5 Is Not Required For
Specification Of A Subset Of Retinal Ganglion Cells
Justin Brodie-Kommit1, Tiffany M. Schmidt1, Samer Hattar1, 2.
1
Biology, Johns Hopkins University, Baltimore, MD; 2Neuroscience,
Johns Hopkins University, Baltimore, MD.
Purpose: Retinal ganglion cells (RGCs) are the sole conduits
of light information to the brain for both image and non-image
forming functions. During embryonic development, RGCs arise
from retinal progenitors via expression of transcription factors at
discrete developmental timepoints. One such transcription factor,
Math5 (Atoh7), is a basic helix loop helix transcription factor that
was thought to be required for RGC specification. In support of
this, Math5 mutant mice lack an optic nerve and chiasm, and have
greater than 80% reduction in RGC number. Interestingly, we have
previously reported that the melanopsin-expressing, intrinsically
photosensitive (ip)RGCs continue to be generated in the retina after
Math5 is downregulated (through embryonic day 18.5). This led us
to hypothesize that a subset of ipRGCs might arise independently of
Math5.
Methods: We utilized Math5Cre and Bax-/- mouse lines as well as Credependent reporter lines to analyze RGC development and lineage.
Results: Lineage tracing indicated that, surprisingly, only half
of melanopsin immunopositive cells express Math5 during
development. However, Math5-/- animals lack 90% of ipRGCs. To
resolve this apparent inconsistency, we sought to determine whether
Math5 negative ipRGCs die secondarily in Math5 mutant animals.
To test this, we generated double mutant animals that lack Math5
as well as the proapoptotic factor Bax (Bax-/-). In double mutant
animals (Math5-/-; Bax-/-), we observe a 6-fold greater proportion of
ipRGCs (~60%) remaining relative to Math5-/- animals. We next
examined whether conventional RGCs that express the transcription
factor Brn3a (expressed in 70% of RGCs) also die secondarily in
Math5-/- animals. We found a substantial rescue of Brn3a positive
RGCs in Math5-/-; Bax-/- retinas. Neurobiotin labeling confirmed these
surviving Brn3a positive cells project axons to the optic disc.
Conclusions: These results indicate that a subset of both ipRGCs
and conventional RGCs do not require Math5 for specification. The
greater loss of ipRGCs and RGCs in Math5-/- animals compared to
Math5-/-; Bax-/- animals, suggests that Math5 negative RGC survival
is dependent on Math5 positive RGCs. This secondary cell death
could be due to lack of trophic support since there is a severely
retarded optic nerve in Math5 mutant animals.
Commercial Relationships: Justin Brodie-Kommit, None; Tiffany
M. Schmidt, None; Samer Hattar, None
Support: NIH Grant GM076430-09
Program Number: 3517 Poster Board Number: D0117
Presentation Time: 11:00 AM–12:45 PM
The Role of NMDA Receptor Activity in Retinal Ganglion Cell
Dendrite Development
Eerik Elias1, 2, Ping Wang2, Ning Tian2, 1. 1Interdepartmental Program
in Neuroscience, University of Utah, Salt Lake City, UT; 2Department
of Ophthalmology and Visual Science, University of Utah School of
Medicine, Salt Lake City, UT.
Purpose: To elucidate mechanisms underlying the dendrite
developmental plasticity of retinal ganglion cells, we examined
the role of glutamate receptors on retinal ganglion cell dendrite
elongation and filopodia elimination.
Methods: We used the JamB genetically labeled subtype of RGCs as
our working model. JamB-CreER:YFP ganglion cell dendritic arbors
were imaged in whole mount retina using confocal microscopy.
Dendrite length, area, branching, and filopodia number were traced
and measured using Neurolucida. Visual inputs were blocked by
dark-rearing pups after P5. Glutamatergic activity was blocked using
daily intraocular injections of AP5 and CNQX from P9 to P13 or
genetic ablation of the NMDA receptor in these RGCs.
Results: To test the role of visual inputs on dendrite development,
we dark-reared mice from P5 to P30 and found a modest effect on
filopodia elimination in JamB RGCs. Anticipating that spontaneous
glutamatergic activity in the retina may also contribute to RGC
filopodia elimination, we blocked spontaneous glutamatergic
activity by daily intraocular injections of AP5 and CNQX from P9
to P13. This led to an increase in filopodia density due to decreased
dendrite length but no change in filopodia number. We confirmed
this result by examining NMDAR knockout JamB cells (JamBCreER:YFP:Grin1-/-). As expected, Grin1-/- JamB RGCs have
decreased dendrite outgrowth like the pharmacologic blockade.
However, filopodia elimination in these cells was significantly
decreased as well, suggesting that NMDA and non-NMDA glutamate
receptors might regulate the RGC dendritic development in a
differential manner. This effect was dramatic at P13. To test if this
effect persists into adulthood, we examined Grin1-/- JamB RGCs at
P30 and found that they are indistinguishable from wild-type JamB
RGCs, suggesting that a compensatory mechanism exists to drive
dendrite elongation and filopodia elimination in the absence of the
NMDA receptor.
Conclusions: Our study demonstrated that ganglion cell dendrite
outgrowth and pruning of filopodia require glutamatergic activity and
visual input that act via NMDA and possibly non-NMDA glutamate
receptors.
Commercial Relationships: Eerik Elias, None; Ping Wang, None;
Ning Tian, None
Support: NIH NEI 2R01EY012345-12A2
Program Number: 3518 Poster Board Number: D0118
Presentation Time: 11:00 AM–12:45 PM
A role for HCN channels in coordinating activity during
glutamatergic retinal waves
Marla Feller1, 2, Alana Firl2, 1. 1Molecular and Cell Biology,
University of California, Berkeley, Berkeley, CA; 2Vision Science
Graduate Group, University of California, Berkeley, Berkeley, CA.
Purpose: During the second postnatal week, transient glutamatergic
circuits give rise to retinal waves, which are characterized by
spontaneous depolarizations that propagate laterally across the retina.
Previously, we showed that while retinal waves are accompanied by
a large transient increase in extrasynaptic glutamate, only a subset
of neurons in the ganglion cell layer (GCL) and inner nuclear layer
(INL) participate in retinal waves. Inhibition is thought to play a role
in determining which cells participate in waves, but the mechanism
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
by which inhibition limits depolarization during waves remains
unknown. Here we explore the relative role of excitation provided by
bipolar cells and inhibition provided by amacrine in regulating the
complex depolarization patterns of glutamatergic waves.
Methods: Spontaneous calcium transients of individual neurons in
the INL and GCL were recorded with 2-photon calcium imaging.
Short applications of glutamate (1mM) were delivered to the INL
through a glass electrode using a PV820 pneumatic PicoPump.
Participation of AII amacrine cells was monitored using the
FBXO32 mouse, which expressed GFP in AII amacrine cells. We
also examined the role of coordinated inhibition on the patterns
of activation in both the INL and GCL with application of the
hyperpolarization-activated cyclic nucleotide-gated (HCN) channel
blocker ZD7288 (50 mM).
Results: First, we found that short applications of exogenous
glutamate did not initiate waves, indicating that excitatory input
from bipolar cells is insufficient for initiating a wave. Second, using
two-photon calcium imaging in FBXO32 mice, we determined that
AII amacrine cells participate in glutamatergic waves. Third, we
tested the role of HCN channels, which play a critical role in network
entrainment in other neural circuits. Bath application of ZD7288
increased wave frequency and uncorrelated cell activity between
waves in the GCL and INL. AII amacrine cells were desynchronized
by ZD7288. Note, the effects of HCN blockade are different from
the effects of inhibition blockade on waves, where the frequency
of waves increases but there is no evidence of an increase in
uncorrelated firing.
Conclusions: Our data indicate wave initiation and propagation
is orchestrated by a coordinated circuit involving inhibitory
interneurons. We propose a model in which glutamate release from
bipolar cells is modulated by a network of inhibitory amacrine cells.
Commercial Relationships: Marla Feller, None; Alana Firl, None
Support: NIH RO1-EY-013528
Program Number: 3519 Poster Board Number: D0119
Presentation Time: 11:00 AM–12:45 PM
RGC receptive field diameters are smaller in dark reared mice
Nikolay Akimov, Rene C. Renteria. Physiology, Univ of Texas Health
Science Center at SA, San Antonio, TX.
Purpose: The mouse visual system matures during a critical period
lasting for approximately two weeks after eye opening. Data show
that form vision during this period can influence development of each
stage of the visual system. Because the maturation of cortical and
subcortical visual areas may be affected by experience-dependent
changes to their retinal inputs during development, we asked whether
visual experience alters the development of receptive field (RF)
diameters of ON and OFF retinal ganglion cells (RGCs).
Methods: C57Bl6 mice were reared either in control conditions
consisting of a 12/12-hr light/dark cycle (normally reared, NR)
or in complete darkness (dark-reared, DR) from before birth to
postnatal day (P)30-39. Light-evoked spiking responses of RGCs
were recorded in vitro using a multi-electrode array (MultiChannel
Systems, Inc.; 60 electrodes with 100 mm separation). RFs were
mapped with a Gaussian white-noise checkerboard presented at 25
Hz and 60 mm check size followed by spike-triggered averaging
(STA). RF diameter was considered to be the average diameter at 1
standard deviation of the 2D Gaussian fit at the peak of the STA.
Results: In both ON and OFF RGC populations of DR mice, RGC
RF diameters were significantly smaller than in NR mice. Values
were (mean ± sem) NR, ON = 206.7 ± 1.5 mm and OFF = 220.6 ±
4.8 mm; DR, ON = 190.1 ± 2.4 mm and OFF = 206.0 ± 4.0 mm. These
represent decreases of 15% and 13% in RF area for ON and OFF
RGCs, respectively.
Conclusions: Dark rearing from birth to the end of the critical period
modifies spatial RF properties of ON and OFF RGCs in the mouse
retina. These findings suggest that early visual experience is critical
for normal refinement of retinal circuits. Experience-dependent
maturation of the earliest stage of the visual system may thus affect
development of functional properties of neurons in higher visual
centers.
Commercial Relationships: Nikolay Akimov, None; Rene C.
Renteria, None
Support: National Center for Research Resources grant (CTSA)
UL1RR025767 to UTHSCSA
Program Number: 3520 Poster Board Number: D0120
Presentation Time: 11:00 AM–12:45 PM
Cone Photoreceptor Afferents and Dendritic Development of
S-cone Bipolar Cells in the Mouse Retina
Li Jia, Wei Li. National Eye Institute, Bethesda, MD.
Purpose: To gain insight into guidance mechanisms that regulate
the formation of specific connections between true S cones and S
cone bipolar cells (SCBCs) in the retina, we ask whether altering the
number and type of cone afferents affects the dendritic development
and synapse formation of the postsynaptic SCBCs. To identify factors
that distinguish true S cones from M cones for specific synaptic
targeting, we set out to purify S and M cones and compare their gene
expression profiles.
Methods: SCBCs are labeled in a transgenic mouse line expressing
Clomeleon (Clm) driven by thy1 promoter. Thrb2-/-, Sop-/- and
Clm1 mice were crossed to generate Clm;Thrb2+/+, Clm;Thrb2-/-,
Clm;Sop-/- and Clm;Thrb2-/-;Sop-/- mice. Whole mount retinae were
immunolabeled with GFP, cone arrestin, Sopsin and then imaged on
confocal. The dendritic morphology of SCBCs was analyzed and
compared. True S cones were FACS sorted from a BAC transgenic
mouse line, Sop-Venus, where Venus (a variant of GFP) is inserted
in the endogenous Opn1sw locus on the BAC clone. M cones were
sorted from Mop-cre/ZEG mice in which M cones are labeled with
EGFP.
Results: Two mouse models with altered densities and types of
cones were used. In Thrb2-/- mice, which lack thyroid hormone
receptor β2 (TRβ2), M-opsin expression is abolished and all cones
become S-cones. In Sopsin-/- mice, the Sopsin gene is knocked
out. We obtained Clm;Thrb2+/+, Clm;Thrb2-/-, Clm;Sopsin-/- and
Clm;Thrb2-/- ;Sopsin-/- (DKO) mice and compared the dendritic
morphology of SCBCs in these mice. We found that the numbers of
SCBCs in Thrb2-/-, Sopsin-/- and DKO mice are comparable to that
in wildtype. Morphologically, SCBCs in Thrb2-/- and Sopsin-/- mice
are indistinguishable from those in wildtype in terms of dendritic
length, number of dendritic branches and number of cone contacts.
To obtain a pure population of true S cones, we focused on the dorsal
third of the Sop-Venus retina where individual cones express either
Sopsin or Mopsin exclusively. We have sorted true S cones and M
cones using FACS and their expression profiles are being compared
using RNAseq.
Conclusions: Our results show that dendritic development of SCBCs
does not depend on the expression of Mopsin or Sopsin. Specific
connections between true S cones and SCBCs appear to depend on
factors other than cone opsin, which we are exploring by comparing
gene expression patterns of true S and M cones.
Commercial Relationships: Li Jia, None; Wei Li, None
Support: NEI Intramural Research Program
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
364 Leading Edge — New Directions in the Analysis of Retinal
Image Motion - Minisymposium
Tuesday, May 06, 2014 3:45 PM–5:30 PM
S 210DE Minisymposium
Program #/Board # Range: 3521–3525
Organizing Section: Visual Neuroscience
Program Number: 3521
Presentation Time: 3:45 PM–4:06 PM
An anatomical basis of direction-selectivity in the mouse retina
Kevin L. Briggman. NINDS, NIH, Bethesda, MD.
Presentation Description: We have used large-scale electron
microscopy coupled with two-photon calcium imaging in the mouse
retina to study wiring specificity in the direction-selectivity circuit.
Commercial Relationships: Kevin L. Briggman, None
Support: NIH Intramural Program
Program Number: 3522
Presentation Time: 4:06 PM–4:27 PM
Role of the starburst network in direction selectivity and beyond
Jimmy Zhou. Yale University, New Haven, CT.
Presentation Description: The talk will review recent results on
the synaptic function of the starburst network in direction selectivity
and the functional properties of cholinergic and GABAergic
neurotransmission in the inner plexiform layer of the mammalian
retina.
Commercial Relationships: Jimmy Zhou, None
Support: NIH grants EY017353 and EY10894
Program Number: 3523
Presentation Time: 4:27 PM–4:48 PM
Electrical signalling in superior coding ON OFF DSGC
Gautam Awatramani. Biology, University of Victoria, Victoria, BC,
Canada.
Presentation Description: ON-OFF DSGCs preferentially respond
to stimuli moving in one of four orthogonal directions through
well-defined chemical synaptic interactions. Previously, it has been
observed that a single subset of ON-OFF DSGCs is electrically
coupled. However, which type or the functional implications on
coupling have not been well characterized. Here, we first present
anatomical and electrophysiological evidence that indicates that
the superior coding population of DSGCs, genetically labelled in
Hb9::eGFP mouse retina, is the only type that is electrically coupled.
Moreover, identification of a genetically labelled coupled class of
DSGCs presented the opportunity for studying roles of gap junctions
during specific neural computations. Our experiments address how
weak gap junction conductances on distal dendrites can interact with
chemical synapses and provide superior coding DSGCs with unique
processing capabilities compared to their uncoupled counterparts.
Commercial Relationships: Gautam Awatramani, None
Support: CIHR
Program Number: 3524
Presentation Time: 4:48 PM–5:09 PM
Adaptation in direction selective responses
Marla Feller. Molecular and Cell Biology and Helen Wills
Neuroscience Institute, University of California, Berkeley, Berkeley,
CA.
Presentation Description: Direction selectivity in the retina requires
asymmetric inhibitory inputs from starburst amacrine cells. Yet,
short visual stimulation with drifting gratings induces a long-lasting
reversal of direction preference of direction-selective retinal ganglion
cells (DSGCs). Here, I will present recent progress in characterizing
adaptation of starburst responses to this same stimulation and present
a model as to how this adaptation underlies the reversal of directional
preference in DSGCs.
Other Authors — Michal Rivlin - Etzion (Weitzmann Institute), Anna
Vlasits (UC Berkeley)
Commercial Relationships: Marla Feller, None
Support: NIH EY019498
Program Number: 3525
Presentation Time: 5:09 PM–5:30 PM
Cell types and trans-synaptic circuits for processing directional
motion
Andrew Huberman. University of California, San Diego, San Diego,
CA.
Presentation Description: I will describe recent work from our lab
on the structure and function of neural circuits for sensing directional
motion in the visual system.
Using anatomy, rabies virus circuit mapping, physiology and
behavior, we discovered a dedicated set of set of visual pathways
for sensing directed self motion and object motion. These circuits
are segregated from the very first synapse in the brain and extract
direction information from physiologically and molecularly distinct
cohorts of cells. The implications of this work for visual perception in
primates, including humans, will also be discussed.
Commercial Relationships: Andrew Huberman, None
Support: NIH/NEI and the Glaucoma Research Foundation
415 Bipolar and amacrine cells
Wednesday, May 07, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 4164–4175/A0169–A0180
Organizing Section: Visual Neuroscience
Contributing Section(s): Retinal Cell Biology
Program Number: 4164 Poster Board Number: A0169
Presentation Time: 8:30 AM–10:15 AM
Changes in dopaminergic cells during progression of retinal
degeneration
Elena Ivanova1, 2, Botir Sagdullaev1, 2. 1Ophthalmology, Weill Medical
College of Cornell University, New York, NY; 2Burke Medical
Research Institute, White Plains, NY.
Purpose: In the retina, dopamine is released by a single type of
interplexiform neuron, dopaminergic amacrine cells (DACs). In
the healthy retina, dopamine release is increased during light onset
and triggers multiple changes associated with light adaptation,
including modulation of gap junctions. Progressive photoreceptor
cell loss during retinal degeneration (RD) leads to altered lightdriven input to DACs. However, structural and functional changes
in DACs during RD remain unclear. The aim of this study was to
determine anatomical changes in DACs during the course of retinal
photoreceptor degeneration in murine models of retinitis pigmentosa.
Methods: The retinas of rd1, rd10, and wt mice at P30, P60, and
P120 were fixed and processed for immunohistochemistry and
confocal microscopy. The dopaminergic cells were indirectly labeled
for tyrosine hydroxylase (the key enzyme in dopamine production)
and vesicular monoamine transporter 2 (responsible for dopamine
uptake into synaptic vesicles). The densities and processes of DACs,
and the distribution of the dopamine transporter, were quantified at
identified retinal poles and eccentricities across different age groups.
Results: In advanced stages of retinal degeneration, the number
of retinal DACs was similar between wt and rd10 mice but was
significantly decreased in rd1 mice. The densities of the dopaminergic
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
processes in the inner plexiform layer (IPL) were highest in wt
retinas, followed by rd10, and were significantly lower in rd1 mice.
The number of the characteristic varicosities formed by dopaminergic
processes in the IPL, which express dopamine transporter, was also
diminished in RD retinas.
Conclusions: The densities and fine anatomical features of DACs
are affected during the course of retinal degeneration. These changes
might correlate with the function of these amacrine cells and
indicate modified dopamine release/uptake in the degenerated retina.
Alterations in the dopaminergic system can cause changes in retinal
light adaption status and lead to abnormal coupling of the neurons in
the degenerated retina, contributing to dystrophic neuronal activity.
Commercial Relationships: Elena Ivanova, None; Botir
Sagdullaev, None
Support: EY020535
Program Number: 4165 Poster Board Number: A0170
Presentation Time: 8:30 AM–10:15 AM
A Genetic Approach to Elucidate Visual Processing in the
Zebrafish Retina
Stella Glasauer, Matthias Gesemann, Stephan C. Neuhauss.
Institute of Molecular Life Sciences, University of Zurich, Zurich,
Switzerland.
Purpose: Each photoreceptor is contacted by several bipolar cell
types, marked by distinct morphologies and responses to visual
inputs. Transgenic zebrafish lines provide powerful tools to study
connectivity and function of cells. The goal of this study is to
transgenically label subtypes of retinal bipolar cells and study their
functions. As we aim to use regulatory regions of genes specifically
expressed in bipolar cells to drive transgene expression, it is
necessary to identify new markers of bipolar cells. One such gene
encodes a Ca2+ channel subunit mutated in patients suffering from
progressive cone dystrophy. We have characterized the orthologous
genes (cacna2d4a and b) in zebrafish.
Methods: Candidate genes have been identified by RNA in-situ
hybridization on developing and adult zebrafish retinae. In order to
isolate the regulatory elements of these candidate genes, we cloned
the more compact regulatory regions of the puffer fish. Those
regulatory regions were used to drive expression of membrane-tagged
reporters in zebrafish, which visualize cell shapes and target cells
for electrophysiology.To address functions of cacna2d4a and b, we
used Morpholino-mediated knockdown followed by histological
examination and electroretinographiy.
Results: We found that genes encoding Calcium binding proteins
(cabp2a, 2b, 5a and 5b) show strong and restricted, but distinct
expression patterns in bipolar cells. Similar to mice, genes encoding
accessory Calcium channel subunits α2δ (cacna2d4a and b) are
expressed in bipolar cells and additionally in photoreceptors.
Knockdown of either cacna2d4a or b does not change morphology
of 5 day old zebrafish retinae and also resulted in no alterations in
electroretinography.
Conclusions: We were able to find promising candidate genes to
use in our transgenic approach. The restricted expression of cabps
to few cell types reflects their specialized roles, probably in visual
processing. The fact that knockdown of the zebrafish cacna2d4 genes
does not show a phenotype with the methods employed may be due
to the progressive nature of the related cone dystrophy.
Commercial Relationships: Stella Glasauer, None; Matthias
Gesemann, None; Stephan C. Neuhauss, None
Support: Swiss National Science Foundation
Program Number: 4166 Poster Board Number: A0171
Presentation Time: 8:30 AM–10:15 AM
A Pannexin-Mediated Purinergic Pathway in the Vertebrate
Retina
Wen Shen1, Yufei Liu1, Richard L. Chappell2, Harris Ripps2.
1
Biomedical Science, Florida Atlantic University, Boca Raton, FL;
2
Marine Biological Laboratory, Woods Hole, MA.
Purpose: Previously, we demonstrated that pannexin 1 channels,
known to be ATP release sites in many tissues, are expressed
predominately on cone-dominant ON-bipolar cells. The purpose
of this study was to demonstrate the importance of the pannexinmediated purinergic pathway that transmits signals from conedominant bipolar cells to rod-dominant bipolar cells in the inner
retina.
Methods: Whole cell patch-clamp recordings were obtained from
bipolar, amacrine and ganglion cells in tiger salamander retinal slices.
These cells were used to determine the effects of P2X3 receptors
and the selective antagonist A317491 on spontaneous EPSCs,
light-evoked EPSCs and voltage-gated Ca2+ channels in response
to ATP and its analogs. ATP release from pannexin 1 channels was
detected in luciferase-luciferin ATP assay, which could be blocked by
application of a specific inhibitor of pannexin 1 channels, 10Panx-1.
Results: We find that increasing extracellular ATP levels - by
applying 200microM ATP - suppresses dim light-evoked EPSCs
in ganglion cells. Moreover, it inhibits spontaneous release of
glutamate from bipolar cells in the dark. Both effects were reversed
by A317491, a P2X3 receptor antagonist. However, ATP had a
limited effect on cone-dominated light responses in ganglion cells,
suggesting that the rod pathway is selectively affected by local ATP
levels in the inner plexiform layer. Moreover, exogenous ATP tends
to increase IPSCs in ganglion cells, indicating that ATP may increase
GABA or glycine release from amacrine cells that initiate IPSCs
in ganglion cells. Further evidence shows that ATP and its analogs
enhance voltage-gated Ca2+ channels in a group of amacrine cells.
Conclusions: Our results indicate that ATP release via pannexin
1 channels in cone-dominated ON-bipolar cells directly activates
P2X3 receptors in amacrine cells. This provides a negative feedback
signal to rod-dominated bipolar cells, and results in the suppression
of EPSCs in ganglion cells. In addition, this study reveals that the
purinergic system by which the inner retina conveys signals from
the cone-pathway to the rod pathway is inhibited in the light-adapted
retina.
Commercial Relationships: Wen Shen, None; Yufei Liu, None;
Richard L. Chappell, None; Harris Ripps, None
Support: NSF - IOS 1021646
Program Number: 4167 Poster Board Number: A0172
Presentation Time: 8:30 AM–10:15 AM
Light driven S-nitrosylation in the retina
Ryan Tooker, Jozsef Vigh. Biomedical Sciences, Colorado State
University, Fort Collins, CO.
Purpose: Nitric oxide (NO) synthesis in the retina is triggered
by light stimulation. NO has been shown to be a neuromodulator
influencing light adaptation at various levels of visual signal
processing, primarily by activation of the NO receptor soluble
guanylate cyclase, consequent cGMP elevation and protein kinase
G action. Recently, we reported that in goldfish retina endogenous
or exogenous NO selectively enhanced Mb-type bipolar cell
input/output ratio for dim scotopic inputs (≤2.4x108 photons/
cm2/s) independent of cGMP and protein kinase G, and in fact,
the NO effect was mediated through S-nitrosylation, in which NO
covalently binds to the thiol group of a protein’s cysteine residue
(Tooker et al, 2013). The purpose of this study was to examine
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
light-evoked S-nitrosylation in the goldfish and mouse retina by
immunohistochemical methods.
Methods: Standard immunohistochemical procedures were utilized
for detection of S-nitrosylated proteins on 20 μm thick vertical
sections of goldfish and mouse retinas. Retinas were exposed to
various illumination protocols in order to produce distinct light
adaptation states before paraformaldehyde fixation.
Results: Dark adapted fish retinal sections were devoid of S-nitroso
immunolabeling (SNI). In light adapted retinas, all retinal layers
were strongly labeled, with particularly strong labeling in the inner
plexiform (IPL) and ganglion cell layer (GCL). Goldfish retinas that
received a light stimulation (1010 photons/cm2/s, 505 nm, 5-10 sec)
capable of potentiating rod-mediated responses in Mb terminals
(Tooker et al, 2013) showed relatively weak SNI in the ON sublamina
of the IPL, often outlining Mb terminals. N-Ethylmaleimide, a potent
inhibitor of S-nitrosylation reactions, eliminated SNI independent of
illumination protocol. The SNI pattern in mouse retinas was similar
to that seen in goldfish under matching illumination protocols.
Further, there was a drastic reduction in overall S-nitrosylated protein
immunoreactivity in light adapted retinas from mice lacking neuronal
nitric oxide synthase.
Conclusions: Our results suggest that the extent of S-nitrosylation in
goldfish and mouse retina depends on the intensity of illumination,
consistent with intensity dependent production/release of NO in the
retina. Our data also suggest that S-nitrosylation might be influencing
retinal signal processing at multiple stages during light adaptation.
Commercial Relationships: Ryan Tooker, None; Jozsef Vigh,
None
Support: NEI EY019051
Program Number: 4168 Poster Board Number: A0173
Presentation Time: 8:30 AM–10:15 AM
Sensitivity and kinetics of GPCR signaling in rod to ON-bipolar
synaptic transmission differentially control visually guided
behavior
Ignacio Sarria, Yan Cao, Kirill A. Martemyanov. Neuroscience, The
Scripps Research Institute, Jupiter, FL.
Purpose: Transmission of the light signal from rods to downstream
ON-bipolar cells (ON-BC) is essential for dim vision and
dysfunctions in components of this signaling pathway have been
shown to cause night blindness in humans. In ON-BC the signaling
is mediated by the G protein signal transduction, where the mGluR6
receptor constantly stimulated in the dark by glutamate activates
Go and closes the TRPM1 channel. We have shown that RGS7 and
RGS11 control Go deactivation and their complete knockout in mice
eliminates ON-BC responses to light-flashes. Here we study the
effect of progressive RGS loss to the light- responses of ON-BC cells
and visual performance in mice.
Methods: We generated a novel inducible RGS7 knockout by
crossing RGS7flx/flx mice on RGS11 KO background (RGS11-/:RGS7 flx/flx) with the driver line ubiquitously expressing tamoxifeninducible Cre-ERT2 recombinase to produce RGS11-/-:RGS7 flx/
flx:CAG CreERT2 (cDKO mice). RGS7 elimination was induced
tamoxifen gavage. Protein expression, quantification, and localization
where analyzed by western blotting and immunohistochemistry
respectively. Light-evoked responses were studied by ERG. Mouse
visual behavior was assessed using a water maze task with a visible
escape platform.
Results: Gradual elimination of RGS in ON-BC results in a new
and progressively deteriorating b-wave phenotype. Decreases in
RGS7 concentration reduced the amplitude, sensitivity and slowed
onset kinetics. Additionally, scotopic visual performance worsens as
RGS7 abundance drops, ending in complete nigh-blindness. Based
on quantification of RGS7 levels we correlated major parameters
of the b-wave with performance in behavioral task. We observed
amplitude, sensitivity and onset kinetics were differentially impacted
by RGS concentration loss. The b-wave amplitude and onset kinetics
were more readily affected, decreasing with smaller reductions in
RGS concentration. In contrast, changes in response sensitivity
required greater reduction in RGS7 levels. Interestingly behavioral
performance was strictly correlated with changes in response
sensitivity rather than amplitude or kinetics.
Conclusions: We conclude that different parameters of ON-BC cell
response are tuned to different concentration ranges of RGS proteins.
However, ultimately visual behavior requires high sensitivity of the
synaptic transmission at the first visual synapse.
Commercial Relationships: Ignacio Sarria, None; Yan Cao, None;
Kirill A. Martemyanov, None
Support: EY018139
Program Number: 4169 Poster Board Number: A0174
Presentation Time: 8:30 AM–10:15 AM
Differential Function of Gγ13 in Rod Bipolar and ON Cone
Bipolar Cells
Hari Ramakrishnan, Anuradha Dhingra, Marie E. Fina, Arkady
Lyubarsky, Sergei Nikonov, Noga Vardi. Neuroscience, University of
Pennsylvania, Philadelphia, PA.
Purpose: Glutamate released from photoreceptors in the dark
activates the mGluR6 receptor in ON bipolar cells; this leads
to activation of Go, closure of TRPM1 channel, and cell’s
hyperpolarization. A flash of light decreases glutamate in the cleft;
this inactivates Go, a process that is facilitated by GTPase activating
proteins. It is now known that Go is comprised of Gαo1 (with minor
contribution from Gαo2) and Gβ3, but the Gγ subunit was not
identified. Localization studies suggest that Gγ13 is the partner of
Gβ3, but no functional data are present.
Methods: To test the function of Gγ13, we generated a Gng13-null
mouse and performed ERG recordings and immunocytochemical
staining.
Results: We found that the amplitude of the scotopic ERG b-wave
in the Gng13-null mice was about half of that in the wild type. The
implicit time of the scotopic ERG b-wave was increased, especially
in response to low intensities. Furthermore, examination of ERG
b-wave at different age groups showed a progressive decline in
the amplitude of the scotopic ERG b-wave relative to the WT. In
contrast, the photopic ERG b-wave was hardly affected at any
age. Immunostaining for the GTPase Activating Proteins RGS11,
R9AP and Gβ5 showed a significant two-fold reduction in staining
intensity in the dendritic tips of rod bipolar cells and no effect on
dendritic tips of ON cone bipolar cells. Similarly, staining for Gβ3
was reduced more profoundly in rod bipolar cells than in cone
bipolar cells. Staining for Gαo, mGluR6, TRPM1, and PKC-α were
only slightly reduced in the Gng13-null mouse. Analysis of ON
bipolar cDNA library showed that the mRNAs for Gγ5, Gγ10 and
Gγ11 are expressed. Quantitative RT-PCR of retinal cDNA showed
greater values for these transcripts in the Gng13-null retinas, but the
difference was not significant.
Conclusions: These data suggest that Gγ13 dimerizes with Gβ3,
but it contributes to light signaling in rod bipolar cells more than to
signaling in ON cone bipolar cells. Furthermore, Gγ13 contributes
less than Gβ3 since deletion of Gβ3 dramatically reduced the light
response in both rod and ON cone bipolar cells. Although it is
possible that other Gγ subunits contribute to mediating mGluR6
coupling in the ON bipolar cascade, our findings suggest that the Gβγ
dimer contributes to signaling indirectly via regulating expression of
other cascade elements.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Commercial Relationships: Hari Ramakrishnan, None;
Anuradha Dhingra, None; Marie E. Fina, None; Arkady
Lyubarsky, None; Sergei Nikonov, None; Noga Vardi, None
Program Number: 4170 Poster Board Number: A0175
Presentation Time: 8:30 AM–10:15 AM
Distribution of goldfish mixed-input ON bipolar cells during
retinal growth
Christina Joselevitch1, 2, Flávio T. da Silva1, Amanda B. Garcia1,
Vitor H. Corredor1, Marcela Y. Ohanian1, Dora F. Ventura1, 2.
1
Experimental Psychology, Universidade de São Paulo, São Paulo,
Brazil; 2Núcleo de Neurociências e Comportamento, Universidade de
São Paulo, São Paulo, Brazil.
Purpose: Fish grow throughout life. As the fish retina grows, new
cells are continuously added. Even though new rods originate from
precursors present throughout the retina and are homogeneously
distributed, rod-driven cells are added at the retinal margin and
little is known about their distribution. The scope of this study was
to investigate whether goldfish mixed-input ON bipolar cells (ON
mBCs), which communicate with both rods and cones, follow the
distribution and density of rods during retinal growth.
Methods: ON mBCs of 57 goldfish ranging from 32 to 185 mm in
standard length were immunostained for PKC and the cell density
and distribution of labeled neurons was studied on whole-mounted
retinas. The values obtained were correlated with morphometrical
data for the same animals, and the maximal acuity of the ON mBC
mosaic was calculated for each case.
Results: The distribution of ON mBCs is homogeneous. Even though
the absolute number of cells increases 6.4 times with growth, mean
density decreases 4.17 times and intercellular spacing increases
1.84 times when one compares values from the smallest and largest
animals. Because the eye lens grows in proportion to other eye
structures (lens diameter = 3.33 times; eye length = 3.04 times;
eye diameter = 2.91 times), so does the image projected onto the
retina. Thereby, the maximal acuity of the ON mBC mosaic actually
increases from one cycle/degree in small animals to two cycles/
degree in large animals.
Conclusions: Although ON mBCs are predominantly rod-driven,
their proliferation and density do not follow those of rods during
growth, since retinal stretch predominates over neurogenesis for
this neuronal population. Nonetheless, the changes suffered by the
retinal magnification factor during growth overcompensate for the
concomitant decay in cell density. As a result, the maximal acuity of
the goldfish ON mBC mosaic increases as the goldfish grows.
Commercial Relationships: Christina Joselevitch, None; Flávio
T. da Silva, None; Amanda B. Garcia, None; Vitor H. Corredor,
None; Marcela Y. Ohanian, None; Dora F. Ventura, None
Support: CNPq (472150/2010–3 and 136249/2011-6) and FAPESP
(2010/16469-0 and 2008/58731-2)
Program Number: 4171 Poster Board Number: A0176
Presentation Time: 8:30 AM–10:15 AM
Bipolar Cells Restructure Dendrites After Selective Ablation of
Photoreceptors
Corinne Beier1, Jennifer Kung3, Philip Huie3, 4, Roopa Dalal3, Daniel
V. Palanker3, 4, Alexander Sher2. 1Electrical Engineering, University
of California - Santa Cruz, Santa Cruz, CA; 2Santa Cruz Institute for
Particle Physics, University of California - Santa Cruz, Santa Cruz,
CA; 3Ophthalmology, Stanford University, Stanford, CA; 4Hansen
Experimental Physics Laboratory, Stanford University, Stanford, CA.
Purpose: In the rabbit retina there is evidence of constructive
retinal plasticity in response to focal ablation of a small patch of the
photoreceptor layer by laser photocoagulation. Over a few months,
healthy photoreceptors migrate inwards filling the damaged area
and restoring visual sensitivity to the lesion site. We investigated the
changes in the morphology of the bipolar cells beneath the lesion
during the healing process in order to understand how the migrating
photoreceptors connect to the deafferented bipolar cells.
Methods: Line-shaped photocoagulation lesions of Barely Visible
clinical grade were produced in rabbits with a 532-nm laser, using
beam diameter 100 μm, scanned along 1.5mm of superior retina.
Retinal ganglion cell responses to spatio-temporal white noise
stimulus were recorded on a 512-electrode array. Functional healing
was characterized as a return of visual sensitivity over the lesion
site. Photoreceptor migration and changes in rod bipolar cells were
visually assessed using immunohistochemistry (PKCα) with confocal
microscopy.
Results: The lesioned areas of the retina, after a two-month healing
period, regain almost complete visual sensitivity. Immunostaining
shows no damage to the inner nuclear layer at 2 days after the
procedure with rod bipolar cells showing overall normal dendritic
structure. However, the rod bipolar cells within the 1-month and
2-month old lesion sites show structural changes in their dendrites.
Thinner dendrites are pruned and in many cells are replaced by a
single thick process reaching towards the edges of the lesion. This
structural change becomes less pronounced in 2-month old lesions.
In contrast, we did not observe significant changes in the dendritic
morphology of the bipolar cells bordering the lesions. The rod bipolar
cells maintain normal axon morphology and correct axon termination
sites in the ON lamina of the IPL throughout the healing period.
Conclusions: The deafferented rod bipolar cells appear to be seeking
viable pre-synaptic partners after the lesioning procedure. At the same
time, the dendrites of the surrounding bipolar cells are not following
the migrating receptors. These results suggest that the healthy
photoreceptors migrate inside the damaged area, abandon their old
post-synaptic partners and establish new functional connections with
the deafferented bipolar cells.
Commercial Relationships: Corinne Beier, None; Jennifer Kung,
None; Philip Huie, None; Roopa Dalal, None; Daniel V. Palanker,
None; Alexander Sher, None
Support: Burroughs Wellcome Fund Career Award at the Scientific
Interface, Pew Charitable Trusts Scholarship in the Biomedical
Sciences, NIH EY023020 – 01 (AS).
Program Number: 4172 Poster Board Number: A0177
Presentation Time: 8:30 AM–10:15 AM
Degeneration of retinal ON bipolar cells induced by serum
including autoantibody against TRPM1 in mouse model of
paraneoplastic retinopathy
Shinji Ueno, Koji M. Nishiguchi, Hiroko Terasaki. Ophthalmology,
Nagoya Univ School of Med, Nagoya, Japan.
Purpose: Evidence has been obtained that the transient receptor
potential melastatin 1 (TRPM1) protein was the antigen for the
autoantibody against ON bipolar cells in PR patients. The purpose of
this study was to determine whether the serum of a PR patient with
the TRPM1 antibody will cause a degeneration of ON bipolar cells.
Methods: Seventy C57BL/6 mice at 7-10 weeks-old-age were used.
Sera were collected from one PR patient with TRPM1 antibody
and one visually normal male subject. Mice were anesthetized with
ether, and 1 mL of the serum of the patient was injected intravitreally
into one of the eyes and serum from the control into the other eye
of C57BL/6 mice. Electroretinogram (ERG) and histopathological
analysis including immuhohistochemical analysis and transmission
electron microscopic examination were performed.
Results: The electroretinograms (ERGs) of the mice were altered
acutely after the injection, and the shape of the ERGs resembled that
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
of the patient with PR. Histological analysis showed that some of
the bipolar cells were apoptotic by 5 hours after the injection, and
degenerated bipolar cells were found 3 days later to be engulfed by
macrophages. At 3 months, the inner nuclear layer was thinner and
the amplitudes of the ERGs were still reduced.
Conclusions: Our results indicate that the serum of a patient with PR
contained an antibody against TRPM1 that led to an acute death of
retinal ON bipolar cells.
Commercial Relationships: Shinji Ueno, None; Koji M.
Nishiguchi, None; Hiroko Terasaki, None
Support: Ministry of Education, Culture, Sports, Science and
Technology Japan 23791977
Program Number: 4173 Poster Board Number: A0178
Presentation Time: 8:30 AM–10:15 AM
EAAT5 and EAAT6 in the Zebrafish outer Retina
Stephanie Niklaus, Simon Früh, Matthias Gesemann, Stella
Glasauer, Stephan C. Neuhauss. Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland.
Purpose: Excitatory amino acid transporters (EAATs) play a crucial
role in retinal signaling. EAATs are capable of removing glutamate
from the synaptic cleft, thus contributing to precise signal termination
and avoidance of excitotoxicity. Furthermore, transport induces an
uncoupled Cl- conductance. Here, we analyze the expression and
function of the EAAT5 and EAAT6 paralogs in the zebrafish retina.
In addition, we examine a potential wave-length dependency of the
different glutamate signaling pathways.
Methods: Expression of eaat5a, 5b, 6a and 6b was assessed
with in situ hybridization and localization of the proteins with
immunohistochemistry on whole mount larvae and adult retinal
sections using custom made peptide antibodies. Functional analysis
is being performed by morpholino mediated gene knock-down and
subsequent electroretinogram (ERG) measurements.
Results: In the adult zebrafish retina, all four genes are expressed
in photoreceptors. Eaat5a and eaat5b are additionally expressed in
the inner nuclear layer, and eaat5a in ganglion cells. Two different
splice variants of eaat5b are found, that probably differ in their Clconductance. Protein expression reveals localization of EAAT5a, 5b
and 6a in the outer plexiform layer, while EAAT6b is found at the
base of the photoreceptor outer segments. EAAT5b localizes to all
cone to ON-bipolar cell synapses but not to synapses between rods
and ON-bipolar cells.
Conclusions: The obtained results indicate an involvement of
EAAT5b in photopic but not in scotopic vision. This would be in line
with studies that suggest that the scotopic ON-response is mediated
by the mGluR6 pathway while EAATs seem to be important in the
photopic ON-response. An involvement of EAAT5b in the cone
ON-response remains to be assessed by ERG recordings along with
the examination of wavelength-dependent differences in the activated
glutamate signaling pathways.
Commercial Relationships: Stephanie Niklaus, None; Simon
Früh, None; Matthias Gesemann, None; Stella Glasauer, None;
Stephan C. Neuhauss, None
Support: Swiss National Science Foundation
Program Number: 4174 Poster Board Number: A0179
Presentation Time: 8:30 AM–10:15 AM
VIP-expressing amacrine cells in mouse retina: Multiple subtypes
with heterogeneous properties
Nicholas Brecha1, 2, Alex Solomon1, Allen Rodriguez1, Helen Vuong1,
3
, Luis Pérez de Sevilla Müller1, Belinda Wong1, Steven A. Barnes1,
4 1
. Neurobiology, Univ of California-Los Angeles, Los Angeles, CA;
2
Veterans Administration Greater Los Angeles Healthcare System,
Los Angeles, CA; 3Molecular, Cellular, and Integrative Physiology,
UCLA, Los Angeles, CA; 4Departments of Physiology & Biophysics
and Ophthalmology & Visual Sciences, Dalhousie University,
Halifax, NS, Canada.
Purpose: Amacrine cells form a large and heterogeneous group
of inhibitory interneurons with specific roles in distinct retinal
microcircuits. Our goal is to define the function of the amacrine
cell subclasses expressing vasoactive intestinal peptide (VIP) by
understanding the morphological and electrophysiological properties
of this cell population in the mouse retina.
Methods: VIP-tdTomato and VIP-Brainbow mouse lines were
generated by crossing a VIP-Cre transgenic mouse line (JAX #10908)
with a Cre-dependent tdTomato (Ai9, JAX #7909) or Brainbow2.1
(JAX #13731) reporter mouse line. Retinal sections and wholemounts were evaluated using immunohistochemistry and intracellular
tracer injection. VIP-tdTomato cells were recorded with whole cell
patch clamp techniques in retinal slices.
Results: VIP-tdTomato fluorescent cell bodies in the inner nuclear
layer (INL) and their processes were distributed to laminae 1, 3, 4
and 5 of the inner plexiform layer (IPL). Brainbow fluorescence was
confined to individual cells with well-defined processes and these
formed multiple types based on the ramification of their processes
in the IPL. There were also occasional VIP-tdTomato cell bodies
in the ganglion cell layer (GCL). Neurobiotin injection of VIPtdTomato cells in the INL revealed coupling to numerous other
amacrine and ganglion cells. VIP-tdTomato cells in the GCL showed
no tracer coupling. VIP-tdTomato cells in the INL were found in
all retinal regions, while those in the GCL were found mainly in
superior-temporal retina. Cell density in the INL was ~610 cells/
mm2 and the highest cell density in the GCL was ~28 cells/mm2.
All tdTomato fluorescing cells contained VIP immunoreactivity,
and all VIP immunoreactive cells contained tdTomato fluorescence.
Every VIP-tdTomato cell also contained GABA immunoreactivity
and none contained glycine immunoreactivity. No VIP-tdTomato
cells expressed the ganglion cell marker RBPMS. Voltage clamp of
VIP-tdTomato cells revealed differential expression of Na and BK
channels between morphological subtypes. Under current clamp,
action potentials were absent but spikelets were frequently observed.
Conclusions: We have identified a novel amacrine cell population
consisting of several subtypes that are characterized by VIP
expression. These findings provide the foundation for functional
studies to define the roles of these amacrine cells in visual
information processing.
Commercial Relationships: Nicholas Brecha, None; Alex
Solomon, None; Allen Rodriguez, None; Helen Vuong, None; Luis
Pérez de Sevilla Müller, None; Belinda Wong, None; Steven A.
Barnes, None
Support: NIH RO1 EY04067. NCB is a VA Career Research
Scientist.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 4175 Poster Board Number: A0180
Presentation Time: 8:30 AM–10:15 AM
The neurocircuitry of A8 bistratified amacrine cells in the mouse
retina
Sammy Chi Sam Lee1, 2, Arndt Meyer3, Karin Dedek3, Silke
Haverkamp2. 1Save Sight Institute, University of Sydney, Sydney,
NSW, Australia; 2Neuroanatomy, Max Planck Institute for Brain
Resesarch, Frankfurt a.M., Germany; 3University of Oldenburg,
Oldenburg, Germany.
Purpose: There are at least 20 different types of amacrine cells
in the mammalian retina and here we describe the morphology,
connectivity, and the development of a glycinergic amacrine cell, the
A8 amacrine cell, in the mouse retina.
Methods: A8 amacrine cells were identified in a transgenic mouse
line where green fluorescent protein is expressed under the thy1
promoter. The distinct bistratified morphology first appears at
postnatal day 8 and matures to an adult morphology through to
postnatal day 15. Adult retinas were triple labeled with bipolar
and synaptic markers to determine the synaptic connectivity of A8
amacrine cells. Antibodies to C-terminal binding protein (CtBP2)
were used to identify ribbon synapses; antibodies against glycine
receptor α (1-4) subunits were used to identity potential postsynaptic
partners. In addition, we injected single A8 cells with neurobiotin to
determine cell coupling.
Results: We found an average of 276±65 CtBP2 puncta per cell for
the ON plexus and 367±84 puncta for the OFF plexus (n=10 cells).
49% of the CtBP2 puncta in the OFF plexus were from t2 cone
bipolar axons (n=7 cells). 39% of the CtBP2 puncta in the ON plexus
were from t6 bipolar cell axons (n=4 cells) and only few were from
rod bipolar axons (7%; n=4 cells). We also found all glycine receptor
α subunits associated with A8 amacrine cells in varying numbers
with GlyRα1 being the highest in the OFF-plexus. In addition, A8
amacrine cells were coupled to ON cone bipolar cells and provided
putative inhibitory feedback via GlyRα1 to OFF cone bipolar cells.
Furthermore, we found evidence that A8 cells provide glycinergic
output via GlyRα1 onto sustained A-type ganglion cells.
Conclusions: We predict the A8 amacrine cell functions as an ONOFF crossover-inhibiting cell with an increase in excitation from ONbipolar cells leading to an increase in inhibition of OFF-bipolar cells.
We also predict the A8 cell modulates sustained responses of A-type
ganglion cells to enhance the dynamic range through “push-pull” of
excitatory and inhibitory inputs.
Commercial Relationships: Sammy Chi Sam Lee, None; Arndt
Meyer, None; Karin Dedek, None; Silke Haverkamp, None
Support: Deutsche Forschungsgemeinschaft (DFG) Grant number:
HA 5277/2-2, WE 849/16-1/2
427 Synaptic mechanisms
Wednesday, May 07, 2014 11:00 AM–12:45 PM
S 210DE Paper Session
Program #/Board # Range: 4526–4531
Organizing Section: Visual Neuroscience
Program Number: 4526
Presentation Time: 11:00 AM–11:15 AM
Fast fusion kinetics of primed vesicles at a mammalian cone
photoreceptor synapse
Chad Grabner, Steven H. DeVries. Ophthalmology, Northwestern
University, Chicago, IL.
Purpose: Cone photoreceptor ribbon synapses signal the end of a
light step by releasing a burst of transmitter into the synaptic cleft.
The timing and synchronicity of release determine the peak cleft
glutamate concentration and hence influence the amplitude of the
postsynaptic response; however, postsynaptic responses are subject
to non-linearities. Membrane capacitance measurements provide a
direct readout of vesicle fusion, and such measurements have not
been obtained at the mammalian cone synapse.
Methods: Recordings were obtained from ground squirrel (Ictidomys
tridecemlineatus) retina. Membrane capacitance (Cm) was measured
in whole-cell voltage clamp using the ‘sine+dc’ routine and a HEKA
EPC-10 amplifier. Cells were held at -70 mV, excepted during
stimulation. Patch electrodes contained a CsCl based solution with 10
mM EGTA. Slices were bathed in a bicarbonate buffered saline (5%
CO2) supplemented with TBOA (200 mM) and CsCl (5 mM).
Results: Release kinetics were examined by varying stimulus
duration over a range of 1-30 ms and stepped to -10 mV. The change
in Cm at 1 and 30 ms differed by only 2-fold (11.4 ± 1.7 vs. 22.6
± 1.8 fF; n = 5 cells), and the plot of Cm over pulse duration was
well-fit by a double exponential with time constants of 0.9 ms (16 fF
~290 vesicles) and 10 ms (7 fF ~127 vesicles). From a separate set
of cells the relationship between Cm and membrane voltage levels
was explored with a family of steps between -50 and 30 mV, given
for 1 or 30 ms. Release reached a maximum at -10 mV for both short
and long steps, the release function was bell-shaped, which suggests
the profile of Ca2+ entry greatly influences the apparent vesicle pool
size. HEPES (15 mM), which blocks inhibitory proton feedback onto
cone voltage-dependent Ca2+ channels, was able to shift release to
the first kinetic phase.
Conclusions: The combined size of the two fast pools equals or
slightly exceeds the size of the vesicle pool that is estimated to be
membrane docked at a cone’s ~20 ribbons. The bulk of transmitter is
released at a very high rate upon depolarization, but there also seems
to be a slower component that may be a consequence of the retarding
effect of proton feedback.
Commercial Relationships: Chad Grabner, None; Steven H.
DeVries, None
Support: EY012141
Program Number: 4527
Presentation Time: 11:15 AM–11:30 AM
Kainate Receptors Mediate Signaling in Both Transient and
Sustained OFF Bipolar Cell Pathways in the Mouse Retina
Bart G. Borghuis1, 2, Loren L. Looger5, Susumu Tomita3, 4, Jonathan
B. Demb2. 1Anatomical Science and Neurobiology, University of
Louisville, Louisville, KY; 2Ophthalmology and Visual Science, Yale
University, New Haven, CT; 3Department of Cellular and Molecular
Physiology, Yale University, New Haven, CT; 4Program in Cellular
Neuroscience, Neurodegeneration and Repair, Yale University,
New Haven, CT; 5Janelia Farm Research Campus, Howard Hughes
Medical Institute, Ashburn, VA.
Purpose: A fundamental question in sensory neuroscience is how
parallel processing is implemented at the level of molecular and
circuit mechanisms. In the retina, it has been proposed that distinct
OFF cone bipolar cell types generate fast/transient and slow/sustained
pathways by the differential expression of AMPA- and kainate-type
receptors, respectively. However, the functional significance of these
receptors in the intact circuit during light stimulation remains unclear.
Here, we evaluated the contribution of AMPA and kainate receptors
to light-evoked responses of OFF bipolar cells in the whole-mount
mouse retina.
Methods: We measured light-evoked (λmax 395 nm) glutamate
release from bipolar cells in the mouse retina in vitro, by two-photon
fluorescence imaging of a glutamate sensor (iGluSnFR) expressed
on postsynaptic amacrine and ganglion cell dendrites. We perturbed
AMPA and kainate receptor function using non-selective (DNQX:
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
100 μM) or selective blockers (AMPA: 100 μM GYKI 52466, 100
μM GYKI 53655; kainate: 50 μM UBP310, 1 μM ACET); L-AP4 (20
μM) was used to block the ON pathway. To validate and complement
imaging experiments, excitatory currents were recorded from
ganglion and bipolar cells with targeted whole-cell recordings.
Results: Light-evoked glutamate release persisted at all OFF levels
of the inner plexiform layer in the presence of DNQX but was
abolished by subsequent application of L-AP4, indicating that crossover inhibition from DNQX-resistant ON pathways can drive release
from bipolar terminals throughout the OFF layers. In subsequent
recordings we first applied L-AP4 to isolate OFF responses mediated
by the coneÇOFF bipolar cell synapse. In both transient and
sustained OFF layers, cone-driven glutamate release from bipolar
cells was blocked by antagonists to kainate receptors, but not AMPA
receptors. Electrophysiological recordings from bipolar and ganglion
cells confirmed the essential role of kainate receptors for signaling
in both transient and sustained OFF pathways. Kainate receptors
mediated contrast responses at temporal frequencies up to 20 Hz,
exceeding the limits implied by the time constant for recovery from
desensitization measured previously (0.5 – 1.5 s).
Conclusions: Light-evoked responses in all mouse OFF bipolar
pathways depend on kainate, not AMPA, receptors.
Commercial Relationships: Bart G. Borghuis, Borghuis
Instruments (I); Loren L. Looger, None; Susumu Tomita, None;
Jonathan B. Demb, None
Support: This research was funded by NIH/NEI grants R01
EY014454 (JBD) and R21 EY023038 (BGB, JBD), NIH MH085080
(ST) and the Howard Hughes Medical Institute (LLL).
Program Number: 4528
Presentation Time: 11:30 AM–11:45 AM
Kainate Receptors Mediate Synaptic Input to Transient and
Sustained OFF Pathways in the Primate Retina
Theresa Puthussery1, Kumiko Percival2, 3, Sowmya Venkataramani1,
Jacqueline Gayet1, Ulrike Grunert2, 3, William R. Taylor1. 1Department
of Ophthalmology - Casey Eye Institute, Oregon Health & Science
University, Portland, OR; 2Department of Ophthalmology - Save
Sight Institute, The University of Sydney, Sydney, NSW, Australia;
3
Australian Research Council Centre of Excellence in Vision Science,
The University of Sydney, Sydney, NSW, Australia.
Purpose: In the OFF retinal pathway, parallel temporal channels
are thought to arise from the selective expression of AMPA or
kainate type glutamate receptors in the dendrites of different OFF
bipolar cell types. AMPA receptors are thought to transmit high
temporal frequency signals, whereas kainate receptors are presumed
to encode lower temporal frequencies. We tested this hypothesis in
the macaque retina, where the low (midget/parvocellular) and high
frequency (parasol/magnocellular) temporal pathways have been well
characterized.
Methods: Retinas from adult rhesus macaques were obtained from
the Tissue Distribution Program at the Oregon National Primate
Research Center from animals used for unrelated experiments.
Whole-cell voltage-clamp recordings were made from bipolar cells
in light-adapted retinal slices. Light-evoked spikes and synaptic
currents were recorded from ganglion cells in retinal wholemounts.
The localization of the GluK1 kainate receptor subunit was examined
using immunohistochemistry.
Results: We recorded from five OFF bipolar cells types: flat midget
bipolar (FMB; n = 33) and the diffuse bipolar (DB) cell types DB1
(n = 14), DB2 (n = 24), DB3a (n = 11) and DB3b (n = 20). We
found that glutamate-evoked responses in all types were strongly
suppressed (>90%) by application of the kainate receptor selective
antagonist, ACET (0.1-1 μM), but not by the AMPA receptor
selective antagonist, GYKI 53655 (10 μM). Control recordings from
horizontal cells showed that glutamate-evoked currents were blocked
by GYKI 53655 (n = 4), but not ACET (n = 6). OFF bipolar types
showed evidence of kainate receptor heterogeneity, with differences
in: response kinetics to agonist application, sensitivity to ACET, and
immunohistochemical expression of the GluK1 receptor subunit.
Finally, we found that ACET reversibly blocked light-evoked spiking
in OFF midget and OFF parasol ganglion cells (n = 5 cells each), but
had no effect on the corresponding ON ganglion cell types (n = 5 ON
parasol, n = 3 ON midget). Voltage-clamp recordings revealed that
ACET blocked light-evoked excitatory input to OFF ganglion cells
without altering ON-pathway mediated crossover inhibition (n = 4
OFF parasol, n = 4 OFF midget).
Conclusions: The results demonstrate that kainate receptors mediate
synaptic transmission from cones to all OFF cone bipolar cell types
in the macaque retina.
Commercial Relationships: Theresa Puthussery, None; Kumiko
Percival, None; Sowmya Venkataramani, None; Jacqueline Gayet,
None; Ulrike Grunert, None; William R. Taylor, None
Support: NIH Grant: EY014888 (W.R.T.), Collins Medical Trust
(T.P.), Research to Prevent Blindness, Lew R. Wasserman Award
(W.R.T.)
Program Number: 4529
Presentation Time: 11:45 AM–12:00 PM
Molecular determinants for synaptic targeting of mGluR6 in
retinal ON-bipolar neurons
Yan Cao, Kirill A. Martemyanov. Neuroscience, Scripps Research
Institute, Jupiter, FL.
Purpose: G protein-coupled receptor mGluR6 is the key molecule
mediating the synaptic transmission in ON-bipolar cells (ON-BC)
at the first visual synapse with photoreceptors. The localization of
mGluR6 in ON-BC is restricted to the postsynaptic compartment
at the dendritic tips. However, when expressed in transfected cells,
mGluR6 is localized throughout the entire plasma membrane. This
suggests that mGluR6 localization in ON-BC is determined by an
active targeting mechanism. Furthermore, elimination of mGluR6
not only abolishes depolarizing response of ON-BC, but also impairs
postsynaptic accumulation of other signaling components including
RGS proteins and an effector channel TRPM1. While the spatial
restriction of signaling at synapse is thought to be an important
contributor to the timely responses of ON-BC, virtually nothing
is known about how this organization is achieved. In this study,
we investigated molecular determinants in mGluR6 that ensure its
selective postsynaptic delivery by analyzing subcellular localization
of various mGluR6 mutant constructs following their in vivo
expression in mouse retinas.
Methods: Mouse retinas were electroporated with GFP tagged
constructs driven by minimal mGluR6/SV40 hybrid promoter after
subretinal microinjections at P0 and harvested at P21. The subcellular
localization of the constructs was analyzed by confocal microscopy
following their immunohistochemical detection.
Results: We first investigated whether mGluR6 has unique targeting
elements not present in other mGluRs. We ectopically expressed
GFP tagged mGluR8 that shares substantial amino acid similarity
and G protein coupling specificity with mGluR6. In other neurons,
mGluR8 is documented to be localized at axonal terminals. However,
when expressed in ON-BC mGluR8 accumulated at the dendritic
tips, similarly to mGluR6. We further performed mutagenesis studies
with mGluR6. Deletion of the C terminus didn’t impair postsynaptic
targeting of mGluR6. However, deletion of ligand binding or
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
cysteine-rich domains resulted in marked destabilization of mGluR6
complicating examination of their subcellular localization.
Conclusions: mGluR6 and mGluR8, two different metabotropic
glutamate receptors are both targeted to ON-BC dendritic tips. This
points to the absence of a unique postsynaptic targeting determinant
specific to mGluR6. Instead targeting of mGluRs in ON-BC may rely
on recognition of common structural features.
Commercial Relationships: Yan Cao, None; Kirill A.
Martemyanov, None
Support: NIH Grant EY018139 (KAM)
Program Number: 4530
Presentation Time: 12:00 PM–12:15 PM
Clustering of syntaxin-4 and cation-chloride transporters
beneath S-cones suggests a synaptic specialization for color vision
exclusive to human and non-human primates
Christian Puller, Michael B. Manookin, Maureen Neitz, Jay Neitz.
Ophthalmology, University of Washington, Seattle, WA.
Purpose: Recently, we found elevated expression levels of the
SNARE protein syntaxin-4 in horizontal cells beneath short
wavelength sensitive (S-)cones of macaque retina. This phenomenon
was not observed beneath S-cones of mouse and ground squirrel
retinas (Puller et al., 2012, ARVO 53:6323). Here, we have
surveyed the expression patterns of syntaxin-4 and cation-chloride
cotransporters in the outer retinas of additional primate species,
including representatives of human, Old World and New World
monkeys.
Methods: Immunohistochemical labeling of syntaxin-4, the sodiumpotassium-chloride cotransporter NKCC and the potassium-chloride
cotransporter KCC2 was performed on vertical sections and wholemount preparations of human, macaque, baboon, and marmoset
retinas. The labeling was combined with antibodies against cell
marker proteins, such as S-opsin, parvalbumin, or calbindin.
Results: Syntaxin-4 was densely clustered beneath S-cones in all
primates investigated and it was colocalized with the dendritic tips
of HII horizontal cells at these sites. As we have shown previously
in macaque, the staining of syntaxin-4 beneath S-cones exceeded
staining intensities beneath long (L-) or middle (M-) wavelength
sensitive cones. Similarly, discrete and dense NKCC puncta were
found to be enriched beneath S-cones when compared to the diffuse
and sparse staining at L/M-cones. The expression pattern of KCC2 is
currently being investigated.
Conclusions: Syntaxin-4 and chloride transporters are proposed
to be components of a GABA-mediated feed-forward circuit from
horizontal cells to bipolar cells. Although the corresponding synaptic
elements are expressed in mouse and ground squirrel retinas,
enrichment at S-cones was not observed in these species. Therefore,
our data points to a primate-specific adjustment within the HII
horizontal cell circuitry. We hypothesize that GABA directly acts on
bipolar cells in support of enhanced processing of spectrally opponent
signals.
Commercial Relationships: Christian Puller, None; Michael B.
Manookin, None; Maureen Neitz, None; Jay Neitz, None
Support: NEI R01EY009303, P30EY001730, NIH P51 OD010425,
Research to Prevent Blindness
Program Number: 4531
Presentation Time: 12:15 PM–12:30 PM
Effects of Ethanol and Low Concentration of Picrotoxin Suggest
Involvement of Extra-synaptic GABAa Receptors in Direction
Selectivity
Stuart C. Mangel, Andrey Dmitriev, Thomas Hirschauer. Dept of
Neuroscience, Ohio State Univ Coll of Med, Columbus, OH.
Purpose: Although GABAa receptors (GABAaRs) consisting of
various subunits are expressed by many retinal neurons, evidence
suggests that GABAaRs that contain the δ-subunit are unique to
starburst amacrine cells (SACs) (Brandstätter et al., 1995). GABAaδRs are high-affinity receptors that are tonically active under ambient
GABA concentrations and not located within synapses (Belelli et
al., 2009). At blood alcohol concentrations typically associated with
social consumption (50 mM), the GABAergic effects of ethanol
appear to be limited to these GABAa-δRs (Wallner et al., 2003),
which ethanol potentiates. Because SACs are pre-synaptic to many
ganglion cell (GC) types, including direction selective (DS) GCs, we
studied whether ethanol alters the light responses of DS- and non-DS
GCs.
Methods: The spiking activity of rabbit GCs to stationary (spots,
annuli) and moving light stimuli was recorded using the loose patch
technique. In addition to studying the effects of ethanol, we also
studied the effects of a low concentration (0.5-2.0 mM) of picrotoxin
(PTX), which preferentially blocks tonic, extra-synaptic GABAaR
currents due to its greater affinity for GABA-bound GABAaRs than
for unbound ones. Concentrations of PTX 10 – 30 times higher are
needed to block synaptic GABAa and GABAcRs (Walker, Semyanov,
2007).
Results: Ethanol (20-30 mM) dramatically decreased and PTX
(0.5-2.0 mM) increased the responses of ON-OFF- and ON-DS
GCs to stimuli moving in all directions, thereby decreasing but not
eliminating direction selectivity. Neither drug at these concentrations
had an observable effect on the responses of non-DS GCs to moving
stimuli. ON-DS GCs exhibited OFF responses in the presence of 2
μM PTX. Additionally, ON-DS GCs treated with low PTX began to
respond to large (600 x 600 μm) moving rectangles that did not evoke
responses in control conditions.
Conclusions: Ethanol (20-30 mM) significantly decreased and
low PTX (0.5-2 μM) enhanced the responses of rabbit DS GCs to
stimuli moving in all directions, but neither drug had an effect on the
motion responses of non-DS GCs. These results are consistent with
the idea that the extra-synaptic GABAa-δRs of SACs mediate the
DS responses of ON-OFF- and ON-DS GCs. Ethanol may impair
our sense of balance in part by disrupting ON-DS GC input to the
accessory optic system, which mediates image stabilization (Pu,
Amthor, 1990).
Commercial Relationships: Stuart C. Mangel, None; Andrey
Dmitriev, None; Thomas Hirschauer, None
Support: NIH grants EY005102 and EY014235
462 Development, adaptation, modulation
Wednesday, May 07, 2014 3:45 PM–5:30 PM
S 210DE Paper Session
Program #/Board # Range: 5003–5009
Organizing Section: Visual Neuroscience
Program Number: 5003
Presentation Time: 3:45 PM–4:00 PM
Expression of extracellular matrix proteins in the developing
mouse retina
Mrinal K. Dewanjee, Matthew J. Brooks, Soo-Young Kim, JungWoong Kim, Robert N. Fariss, Anand Swaroop. NeurobiologyNeurodegeneration & Repair, National Eye Institute, Bethesda, MD.
Purpose: Extracellular matrix proteins (ECMPs) play a critical role
in the three-dimensional (3D) construction of distinct interconnected
cell layers in the developing retina. ECMPs participate in remodeling
and damage repair during aging and disease conditions. Cell
replacement therapies that include transplantation of photoreceptors
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
(PRs) in degenerating retina would also greatly benefit from the
better understanding of ECMPs that are associated with early
lamination in developing retina.
Methods: We carried out the high-throughput transcriptome profiling
of developing and mature mouse retina using RNA-seq, Affymetrix
exon arrays and quantitative reverse transcription polymerase chain
reaction (qRT-PCR) methods. Of approximately 16,000 expressed
genes from developmental RNA-seq data, we focused on the analysis
of about 50 genes that encode the ECMPs, such as Vitronectin: Vtn,
Collagen subtypes: Col, Fibronectin: Fn, Proteoglycans and Integrin
subtypes. We also examined the expression of ECMP genes in RNAseq data from purified rod photoreceptors. Immunohistochemistry
was performed for selected ECMPs using mouse, primate and human
retina sections.
Results: We analyzed a subset of mouse retinal transcriptome
(MRT) for cytoskeletal proteins, tubulins, actins and their crosslinking factors, microtubulin-actin crosslinking factor, MACF, cell
adhesion molecules and ECMPs that are all threaded in a spatially
self-organized 3D cell cluster and are very dynamic in orchestrating
the 3D structure from birth, development and degeneration till death
of an organism. Transcription dynamics was evaluated by plotting
the normalized expression value of transcription with the age of
developing mice. With some exceptions, the transcription levels
reached a plateau in 30 days, suggesting a possible homeostasis of
the corresponding proteins (ECM-proteostasis). The mean transcript
ratios of ECMPs to Integrins are about 9 and 6 for mice and human
respectively.
Conclusions: RNA expression data for ECMPs and photoreceptor
integrins are concordant with immunohistochemistry of retina. Our
studies for the first time, uncover the developmental expression
profile of ECMPs that are critical for retinal lamination and
architecture. Identification of rod photoreceptor ECMPs can provide
new avenues for exploring better strategies for integration of
transplanted cells in the degenerating retinal milieu.
Commercial Relationships: Mrinal K. Dewanjee, None; Matthew
J. Brooks, None; Soo-Young Kim, None; Jung-Woong Kim, None;
Robert N. Fariss, None; Anand Swaroop, None
Program Number: 5004
Presentation Time: 4:00 PM–4:15 PM
A Circadian Clock in the Retina is Required for Normal Retinal
Development and Visual Function
Zhijing Zhang1, Alexia Vidal1, Rachel Zimmerman2, Christophe
Ribelayga1. 1Ophthalmology & Visual Science, UT Health - Med
School, Houston, TX; 2Undergraduate Program, Rice University,
Houston, TX.
Purpose: Circadian clocks intrinsic to the retina are central regulators
of retinal function. Although a clear link has been established
between the presence of circadian clocks within the retina and retinal
processing, the importance of these clocks in retinal development and
visual function remain largely unknown. We studied the morphology
and distribution of specific retinal cell types as well as aspects of
visual behavior in a retina-specific circadian-clock-deficient mouse
model at 2, 8 and 40 weeks of age.
Methods: A retina-specific circadian-clock-deficient mouse line
was generated by conditionally silencing the essential circadian
clock component Bmal1 in retinal cells using the Cre-loxP system.
Specifically, we crossed Bmal1f/f mice with CHX10Cre mice.
Immunohistochemistry was used to label cell markers and assay the
expression of BMAL1 in retinal cells. Light entrainment and masking
of the voluntary locomotor activity rhythm was tested using activity
monitoring with wheeled cages. In addition, we measured spatial
frequency threshold (i.e. acuity) and contrast sensitivity of freely
moving mice by observing their optomotor responses to moving sinewave gratings using the Optomotry system.
Results: In the Bmal1f/f;CHX10Cre retina, BMAL1 was knockedout in >99% of the cells. Compared to wild-type retinas, most
cell types were present in the Bmal1f/f;CHX10Cre retinas but
showed altered density and/or morphology. In addition, the Bmal1f/
f;CHX10Cre retinas were thinner and their architecture showed gross
lamination defects, including a “waveform”-like structure of the
ONL. These defects were observed as early as 2 weeks of age, before
the retina becomes fully mature (P20), indicating that they likely
result from early developmental problems rather than maintenance/
aging-related processes. In addition, we found that silencing clock
activity in the retina impaired some aspects of visual function,
including contrast sensitivity, but not acuity, as well as the negative
masking of light on the locomotor activity rhythm.
Conclusions: Our data provide evidence that functional circadian
clocks in the retina are required for normal retinal development and/
or maintenance of normal visual function.
Commercial Relationships: Zhijing Zhang, None; Alexia Vidal,
None; Rachel Zimmerman, None; Christophe Ribelayga, None
Support: This work was supported by the National Institutes of
Health (EY018640, EY010608, OD010768), the Hermann Eye Fund
and a Challenge Grant to The University of Texas Medical School at
Houston from Research to Prevent Blindness.
Program Number: 5005
Presentation Time: 4:15 PM–4:30 PM
The Role of Circadian Rhythms in Regulating the Cone
Phototransduction Cascade Shutoff in Zebrafish Retina
Jingjing Zang, Jennifer Keim, Stephan C. Neuhauss. Institude of
Molecular Life Science, University of Zurich, Zurich, Switzerland.
Purpose: A variety of visual behaviours in different species are
regulated by the circadian clock. Zebrafish, as a model organism for
studying vertebrate circadian timing system, is used to investigate
the molecular mechanism underlying how the circadian rhythms
influence photoresponse shutoff which requires the phosphorylation
of visual pigment by opsin kinase and subsequent binding of Arrestin,
and hydrolysis of PDE-TαGTP complex by GTPase.
Methods: qRT-PCR has been used to quantitatively evaluate mRNA
expression level at 8 time points during the day. In situ hybridization
has been used to compare the difference in mRNA expression level
at different time points. Western Blot was preformed to show the
expression changes at the protein level. Electroretinography was
recorded for the functional analysis.
Results: qRT-PCR resultes show that in both 5 days post fertilization
larvae and adult eyes, the expression of several key genes which
are involved in regulating phototransduction deactivation fluctuates
in a 24-hour rhythm. These key genes encode Recoverin, cone
opsin kinase, cone specific Arrestin and the GTPase-accelerating
protein. Changes in the mRNA expression level throughout the day
are maintained when larvae are kept in continuous darkness for 5
days starting the first evening after fertilisation, indicating that this
oscillation is endogenous and circadian rhythm dependent. The qRTPCR results are confirmed by in situ hybridization staining for the
larvae or adult eyes fixed at different time points. Preliminary western
blot results show that protein levels also change at 24-hour cycle.
Electroretinography recording from 5 days post fertilization larvae
demonstrates the photoresponse recovery is delayed in the evening
and accelerated in the morning. This phenomenon has been observed
by spectrum electroretinography which stimulates only the UV cones
and white light electroretinography which is dominated by double
cone response, suggesting a common feature for cone photoresponse
recovery.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Conclusions: The expression level of several important regulators for
cone photoresponse recovery shows robust circadian rhythms, which
may be correlated with the photoresponse kinetic change throughout
the day.
Commercial Relationships: Jingjing Zang, None; Jennifer Keim,
None; Stephan C. Neuhauss, None
Program Number: 5006
Presentation Time: 4:30 PM–4:45 PM
Melatonin Modulates Rod Photoreceptor Electrical Coupling in
the Mouse Retina
Nange Jin, Christophe Ribelayga. Ophthalmology and Visual
Science, Univ of Texas Med School at Houston, Houston, TX.
Purpose: In the vertebrate retina, melatonin, whose levels are under
the control of a retinal clock and peak at night, plays a key role in the
regulation of circadian physiology. In the melatonin-proficient CBA/
Ca mouse, a circadian clock controls the state of electrical coupling
between rod photoreceptors, so that rod coupling is weak during the
day and strong at night (Jin and Ribelayga, 2013 ARVO abstract).
Here we tested 1) whether the state of rod coupling is sensitive to
melatonin, and 2) whether a circadian rhythm in rod coupling exists
in the retina of the melatonin-deficient C57/Bl6 mouse strain.
Methods: Perforated patch clamp recordings from single rod inner
segments were performed in intact mouse retinas maintained by
superfusion at 32°C. The kinetics and reliability of the rod light
responses were recorded using brief dim full-field stimuli, and the
receptive field was mapped using a computer-generated stimulus
projected onto the retina by a Lucivid camera. Recorded cells were
labeled by iontophoresis of the biotinylated tracer Neurobiotin.
Melatonin was dissolved and applied in the perfusion system.
Results: In CBA/Ca retinas, application of physiological doses (3300 pM) of melatonin during the subjective day, when endogenous
melatonin levels are low and rod coupling is weak, mimicked the
nighttime state. That is, the rod light responses became more reliable
compared to the daytime control, indicating an increase in rod
electrical coupling; the space constant, a measure of the receptive
field size of the recorded rod, increased by 2- to 3-fold; and tracer
coupling was observed in many neighboring rods while it was
consistently restricted to the recorded cell under control conditions.
In the C57/Bl6 mouse, the rod light response reliability and kinetics,
receptive field size and tracer coupling size were similar to the
daytime state observed in the CBA/Ca mouse, during both day and
night. Yet, physiological doses of melatonin increased rod electrical
and tracer coupling in C57/Bl6 retinas, regardless of time of day.
Conclusions: Our data provide electrophysiological and tracer
coupling evidence that melatonin increases rod electrical coupling in
the mouse retina. Together with the constitutively weak rod coupling
observed in the C57Bl/6J retina, our results suggest that melatonin is
a nighttime effector of the circadian clock that controls rod electrical
coupling.
Commercial Relationships: Nange Jin, None; Christophe
Ribelayga, None
Support: This work was supported by the National Institutes of
Health (EY018640, EY010608, OD010768), the Hermann Eye Fund
and a Challenge Grant to The University of Texas Medical School at
Houston from Research to Prevent Blindness.
Program Number: 5007
Presentation Time: 4:45 PM–5:00 PM
Spatial inhibitory input to the retinal OFF pathway narrows with
light adaptation
Reece Mazade3, Erika D. Eggers1, 2. 1Physiology, University of
Arizona, Tucson, AZ; 2Biomedical Engineering, University of
Arizona, Tucson, AZ; 3Physiological Sciences GIDP, University of
Arizona, Tucson, AZ.
Purpose: Retinal OFF cone bipolar cells (OFF BCs) bridge the rod
and cone pathways by receiving both excitatory input from cones
and inhibitory input via amacrine cells (ACs) from both rod and
cone pathways. While OFF BC inhibition is primarily glycinergic in
the dark, we previously found that there was a compensatory switch
to larger GABAergic input with light adaptation. However, it is
unknown how this switch will affect the spatial inhibitory input to
OFF BCs as it underlies a switch from morphologically narrow-field
glycinergic to wide-field GABAergic ACs.
Methods: Using whole-cell voltage clamp, light-evoked inhibitory
postsynaptic currents (L-IPSCs) were recorded from dark-adapted
mouse OFF BCs, identified via fluorescent labeling, while holding at
the reversal potential for nonspecific cation currents. The magnitude
of L-IPSCs was measured as charge transfer. A white OLED screen
was used to set the background light and to generate 25 μm bars
of light flashed for 500ms to map spatial inhibition. The spatial
distributions were averaged and compared between light conditions
where significance was p<0.05.
Results: Due to larger GABAergic input to OFF BCs in the light,
we predicted that OFF BC spatial inhibition would widen with light
adaptation as a result of the wide spatial extent of GABAergic ACs.
Surprisingly, we found that the spatial inhibition to OFF BCs became
narrower with light adaptation (n=9, p<0.05). When specific spatial
receptor inputs were isolated, we found that under both dark- and
light-adapted conditions, GABAergic spatial input (dark n=6, light
n=3) to OFF BCs was not different than glycinergic input (dark n=9,
light n=6, p>0.05). However, unexpectedly, both GABAergic and
glycinergic specific input to OFF BCs also became narrower in the
light (p<0.05).
Conclusions: Here we show that light adaptation narrows OFF BC
spatial inhibitory input. Though anatomical measurements imply
a change to wider inhibitory surrounds, our initial results suggest
opposite effects. Factors in addition to the spatial extent of ACs may
play a role in determining the spatial sensitivity of inhibition, such as
dopamine modulation of circuits and receptor properties and specific
BC-AC and AC-AC interactions. Adjusting the inhibitory surround of
BCs may be part of the mechanism for ganglion cell center-surround
changes seen with light adaptation.
Commercial Relationships: Reece Mazade, None; Erika D.
Eggers, None
Support: NIH grant EY018131 (EDE), University of Arizona
NIH Systems and Integrative Training Grant (REM), the ARCS
Foundation (REM), and the Science Foundation Arizona (REM).
Program Number: 5008
Presentation Time: 5:00 PM–5:15 PM
The output of the retina qualitatively changes at different light
levels
Thomas A. Munch, Alexandra Tikidji-Hamburyan. Center for
Integrative Neuroscience & Bernstein Center for Computational
Neuroscience, University Tubingen, Tubingen, Germany.
Purpose: Vision functions over a large range of light intensities. It
is not known how the ambient light level might influence response
properties of retinal ganglion cells to an otherwise identical stimulus.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
We characterized retinal output over scotopic, mesopic, and photopic
light levels.
Methods: We used multi-electrode arrays to record from ganglion
cells of whole-mount mouse retinas. We characterized their responses
to 2s full-field steps of positive and negative contrast. The polarity
of the cells (ON, OFF) was determined based on their linear filters,
obtained from responses to Gaussian white noise. We tested the
responses of ganglion cells at 8 different light levels separated by 1
log unit each, ranging from scotopic to high photopic level.
Results: Cells robustly increased spiking activity to full-field steps of
their preferred contrast (light decrements for OFF cells). Background
luminance affected only amplitude and duration of these responses.
Surprisingly, all OFF ganglion cells also had excitatory ON
responses (to light increments) at least at one light level. The overall
ON response pattern in OFF cells was very diverse: short-latency
suppression, as well as short- and long-latency excitatory responses
(peaking at ~170ms and ~680ms, respectively). The long-latency
ON response could be preceded by either suppression or the shortlatency ON response. Interestingly, the pattern was robust for each
cell at a given light level, but could qualitatively change at different
luminance. APB (an agonist of mGluR6 receptors that prevents
synaptic activation of ON bipolar cells) blocked some, but not all ON
responses in OFF cells, and even revealed some ON responses that
were absent without APB. Robust luminance-induced changes of the
response pattern were also observed for a naturalistic movie stimulus.
Conclusions: Our results suggest that retinal output strongly depends
on ambient luminance. Responses change not only quantitatively, but
qualitatively for many ganglion cells. The source of the ON response
in OFF cells appears to be diverse in different cell types, and at
different luminance levels within a cell type.
Commercial Relationships: Thomas A. Munch, None; Alexandra
Tikidji-Hamburyan, None
Support: DFG EXC307, BMBF FKZ 01GQ1002
Program Number: 5009
Presentation Time: 5:15 PM–5:30 PM
Age-related change in adaptation recovery of the full-field flash
ERG
Megan Tillman1, Athanasios Panorgias1, Erich E. Sutter2, John
S. Werner1, 3. 1Ophthalmology & Vision Science, University of
California, Davis, Sacramento, CA; 2Electro-Diagnostic Imaging
Inc., Redwood City, CA; 3Neurobiology, Physiology and Behavior,
University of California, Davis, Davis, CA.
Purpose: To quantify age-related change in adaptation recovery by
extracting responses to multiple flashes from an m-sequence using
full-field flash ERGs.
Methods: The flash ERG was recorded from and compared between
normal younger (n=6, 22 ± 2 years, mean ± SD, range: 20-25 years)
and older (n=5, 74 ± 10 years, range: 66-85 years) adults using an
m-sequence flash stimulus. The flash intensities ranged from 0.0001
to 0.01 cd s/m2 under scotopic conditions and from 0.7 to 10 cd s/
m2 under photopic conditions. The base intervals of the m-sequence
for the scotopic and photopic stimulation were 65ms and 10ms,
respectively. We obtained the complete binary kernel series from the
responses to an m-sequence cycle. From the kernel series we derived
the response to the adapting stimulus by itself and that of the adapting
flash followed by a single flash response at different inter-stimulus
intervals (ISIs). The adapting stimulus was a single flash under
scotopic conditions and a double flash for the photopic conditions.
Subtracting the response of the adapting flash from the responses
to the following flashes, we isolated the contribution of the adapted
flash and plotted its amplitude as a function of the ISI. We fitted a
straight line through the linear portion of the recovery curve and used
its slope as an estimate of the rate of recovery. We compared the rates
of recovery between the younger and older groups.
Results: The younger and older adults showed a linear response
recovery as a function of inter-stimulus interval followed by
saturation in both photopic and scotopic conditions. A statistically
significant difference between the recovery slopes of the younger
and older group was found with a two-way ANOVA for the scotopic
condition (p<0.001, α=0.05), but no significant difference was found
for the photopic condition (p=0.426, α=0.05).
Conclusions: The results of this study suggest significant reductions
in the rate of recovery of fast adaptation mechanisms to a full-field
flash under dark-adapted conditions but not under light-adapted
conditions, consistent with reports that show the rod-dominated
system to be more vulnerable in aging. M-sequence stimulation of
the full-field flash ERG provides a rapid assessment of the retinal
response dynamics. The use of rate of adaptation recovery as a
means to distinguish normal, healthy eyes from those with retinal
pathologies must still be explored.
Commercial Relationships: Megan Tillman, None; Athanasios
Panorgias, None; Erich E. Sutter, Electro-Diagnostic Imaging Inc.
(E), Electro-Diagnostic Imaging Inc. (I), Electro-Diagnostic Imaging
Inc. (P); John S. Werner, None
Support: NIH Grant AG04058
472 ERG and VEP techniques
Wednesday, May 07, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 5114–5130/A0152–A0168
Organizing Section: Visual Neuroscience
Program Number: 5114 Poster Board Number: A0152
Presentation Time: 3:45 PM–5:30 PM
A Glasses-Mounted Platform for Optimized Ocular
Electrophysiology
Amy E. Canham, Ramesh Raskar. MIT Media Lab, Cambridge, MA.
Purpose: The aim of this work is to develop a first prototype for
performing electroretinography in a unified interface which can be
mounted into a pair of glasses and conducted with minimal training
required, to better integrate electrophysiology into routine ophthalmic
care and improve outcomes in pediatric patients.
Methods: The device incorporates both rigidly affixed electrodes and
low-profile stimulator into a modified glasses frame. The electrodes
are gold casted discs which are positioned and stabilized by the
frame. The stimulator is a DLP projector driven, low-profile dome
coupled with a fresnel lens. Analog signal processing and capture
via Arduino microprocessor is also mounted within the glasses
frame. The function of the 0.3Hz to 300Hz, 1000 V gain filter and
amplification circuitry was evaluated by delivering sample signals
generated computationally and rendered by a Realtek High Definition
Audio sound card. The sample signals consisted of continuous,
30mV peak-to-peak, sine waveforms at 3Hz, 15Hz, 30Hz, 100Hz
and 1kHz and decaying, oscillatory impulse signals with total voltage
offset from first peak to trough of 30mV. The luminous emittance
of the stimulus was measured to calibrate the device against ISCEV
standards. Preliminary evaluation of the system was conducted
by administering scotopic 3.0 ERG with both our system and a
Diagnosys ColorDome ERG system.
Results: The analog filter and amplification stage demonstrated 1000
fold amplification of signals within cutoff frequencies and effectively
attenuated the 1kHz waveform. The programmable stimulus system
demonstrated the ability to render both full-field and patterned stimuli
at multiple wavelengths, with adjustable intensity from 0.1 to 3.0
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
candela second per meters squared over the surface of the dome.
Preliminary evaluation demonstrated comparable results from both
our system and the Diagnosys ERG system.
Conclusions: The integration of the system into a glasses-mounted
interface simplifies electrode alignment while delivering comparable
results and the potential for administering a wide range of tests.
Ophthalmic electrophysiology has tremendous potential as an
asset in diagnosis and monitoring. Accessible technologies which
deliver objective evaluations of retinal function can impact routine
ophthalmic practice and large scale screening scenarios.
Commercial Relationships: Amy E. Canham, None; Ramesh
Raskar, None
Program Number: 5115 Poster Board Number: A0153
Presentation Time: 3:45 PM–5:30 PM
What monitor can replace cathode ray tube for visual stimulation
to elicit multifocal electroretinograms ?
Toshiko Arai, Celso S. Matsumoto, Kei Shinoda, Takaaki Kondo,
Makoto Kawashima, Atsushi Mizota. Ophthalmology, teikyo
Univercity school of medicine, Tokyo, Japan.
Purpose: To compare organic electro-luminescence (OEL) and liquid
crystal display (LCD) screens as visual stimulators to elicit mfERGs.
Methods: Multifocal ERGs (mfERGs) were recorded from 7
eyes of 7 healthy volunteers (21±2 years). The mfERGs elicited
by a conventional cathode ray tube (CRT) screen (S710,Compaq
Computer Co., USA) were compared with those elicited by a
studio grade master OEL monitor (BVM-E170, Sony, Japan), and
conventional LCD (S1721, Flexscan, Eizo Nanao Corp, Japan).
The luminance changes of each monitor were measured with a
photodiode. The CRT, LCD, and OEL screens with frame frequency
of 60 Hz were studied. A hexagonal stimulus array with 61 stimulus
elements was created on each monitor.
Results: The serial white stimuli of the OEL screen at 60 Hz did not
overlap, while that of the LCD screens overlapped. The amplitude
of P1 and P2 of the first-order kernels of the mfERGs were not
significantly different between the CRT and OEL screens, while the
P1 amplitude of the first-order kernel elicited by the LCD stimuli was
significantly smaller than that elicited by CRT in all the groups of the
averaged hexagonal elements. The implicit time was approximately
10 msec longer in almost all components elicited by LCD compared
to those elicited by CRT.
Conclusions: The mfERGs elicited by monitors other than CRT
should be carefully interpreted especially those elicited by LCD
screens. The OEL had good performance and we conclude that it can
replace CRT as a stimulator for mfERGs, however normative data
collection is recommended.
Commercial Relationships: Toshiko Arai, None; Celso S.
Matsumoto, None; Kei Shinoda, None; Takaaki Kondo, None;
Makoto Kawashima, None; Atsushi Mizota, None
Program Number: 5116 Poster Board Number: A0154
Presentation Time: 3:45 PM–5:30 PM
Detecting Early Signs of Hydroxychloroquine and Chloroquine
Retinopathy: A Meta-analysis Evaluating Multifocal
Electroretinogram as a Screening Test under the 2011 Revised
American Academy of Ophthalmology Guidelines
Sina Ahmadi Pirshahid, Chloe C. Gottlieb, Stuart G. Coupland,
Adrian Tsang, Brian C. Leonard, Bernard Hurley. Ophthalmology,
University of Ottawa, Ottawa, ON, Canada.
Purpose: To determine a measure of sensitivity and specificity for
multifocal electroretinography (mfERG) as a screening tool for
detecting Chloroquine and Hydroxychloroquine toxicity. To evaluate
and compare mfERG to automated visual fields (AVF), fundus
auto fluorescence (FAF) and spectral domain optical coherence
tomography (sd-OCT).
Methods: Following PRISMA statement guidelines a literature
search was conducted in EMBASE and EMBASE Classic (1947 to
July 2013), Ovid MEDLINE (1946- July 2013) and PubMed (July
2013) with the assistance of a research librarian.
After amalgamating the individual data for each eye, the sensitivity
and specificity of mfERG was estimated using AVF and a
combination of two of three of AVF, FAF and sdOCT as the reference
test. The percent agreement between tests was calculated. An
ANOVA was performed to determine if there was a difference in the
rate of positive test results between the testing modalities at different
cumulative dose ranges.
Results: Using AVF as the reference test, the sensitivity and
specificity of mfERG was estimated to be 84.97% and 63.38%
respectively .When agreement between at least two of AVF, sdOCT
or FAF was used as the reference test the sensitivity and specificity
of mfERG was estimated at 96.55% and 91.30% respectively.
mfERG had the greatest test agreement with sdOCT (94.34%) and
the combined reference test (92.45%) followed by FAF (88%) and
AVF(74.58%).mfERG had the highest rate of positive test results.
The rate of positive test results differed significantly between the four
screening modalities in both the group of subject eyes exposed to a
cumulative dose of less than 1000g of HCQ (F (3, 327) = 2.901, p =
0.035 ) and the group with a cumulative dose of greater than 1000g of
HCQ ( F (3, 570) = 5.683, p = .0008)
Conclusions: mfERG has an important role in screening for CQ/
HCQ toxicity before a cumulative dose of 1000g and annually
after this critical cumulative dose. Further prospective studies are
warranted to determine a safe cumulative dose and to guide CQ/HCQ
therapy
Commercial Relationships: Sina Ahmadi Pirshahid, None; Chloe
C. Gottlieb, None; Stuart G. Coupland, None; Adrian Tsang,
None; Brian C. Leonard, None; Bernard Hurley, None
Program Number: 5117 Poster Board Number: A0155
Presentation Time: 3:45 PM–5:30 PM
Impact of the spectral output of “white” LEDs on the murine
flash ERG
Mathias W. Seeliger1, Kristina Narfstrom2, Naoyuki Tanimoto1. 1Div
of Ocular Neurodegeneration, Ctr Ophthal Inst Ophthalmic Rsrch,
Tuebingen, Germany; 2Dept. of Vet & Med Surgery, University of
Missouri, Columbia, MO.
Purpose: Electroretinography (ERG) is a technique that uses
standardized equipment and examination protocols to assess retinal
function. Until recently, Xenon bulbs have been the best available
technology to generate stimulus flashes, but now these are more and
more replaced by LED stimulators. Here, we focus on the spectral
differences between these stimulators, and discuss the impact on the
outcome of scotopic and photopic recordings.
Methods: Functionally normal wild-type mice (C57Bl/6), mice with
rod function only (Cnga3-/-) and mice with cone function only (rho-/-),
were examined in vivo with Xenon and LED flash ERG systems. The
results were related to the spectral properties of each stimulus type.
Results: The spectral data of “white” LEDs reveal that their output
peaks in the blue and yellow range, with a deep trough in the green
range where rod sensitivity has its maximum (data from 5 different
LED manufacturers). Due to this output spectrum being far from flat,
it was tested whether a pure “white” LED stimulator does stimulate
cones more efficiently than rods. This was indeed confirmed in in
vivo recordings. In comparison to the cone system responses, the rod
system responses came out relatively smaller with a LED system than
with a Xenon system.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Conclusions: Comparing a LED stimulator (“white” LEDs) with
a Xenon flash system, we found a definite impact on the outcome
of scotopic but not photopic recordings. This does have a number
of potential consequences for clinical and experimental ERGs. In
diseases where rod sensitivity is lowered, a less efficient stimulation
of the rod system may reduce the diagnostic performance. Further,
to obtain a certain flash effect or a sufficiently rod-desensitizing
background with such types of LEDs, the cone system would be
stimulated more than necessary, with possibly altered behavior. The
“unbalanced” stimulation of rods and cones due to an “unnatural”
spectrum of the stimulus may further alter the interaction of rod and
cone systems at different lighting conditions, and lead to distorted
results in case of diseases that have a different impact on those
systems (either intrinsically or as part for the degenerative process).
Commercial Relationships: Mathias W. Seeliger, None; Kristina
Narfstrom, None; Naoyuki Tanimoto, None
Support: DFG Se837/6-2
Program Number: 5118 Poster Board Number: A0156
Presentation Time: 3:45 PM–5:30 PM
New handheld, non-mydriatic ERG device to screen for diabetic
retinopathy and other eye diseases
Byongdo Kim1, Evelyn Ross2, Kimberly Pham1, Jenny Nguyen3,
Vinna Nam1, Vidhya Gunasekaran4, Scott E. Brodie5, Gloria Wu6.
1
University of California, Berkeley, Berkeley, CA; 2University
of Iowa College of Public Health, Iowa City, CA; 3University of
Southern California, Los Angeles, CA; 4Ophthalmology, Aravind Eye
Hospital, Madurai, India; 5Ophthalmology, Mount Sinai School of
Medicine, New York City, NY; 6Ophthalmology, Stanford Hospital &
Clinics, Stanford, CA.
Purpose: To evaluate the use of the 30 Hz RETeval(TM) handheld
ERG device in diabetic and glaucoma patients in the office setting.
Methods: The RETeval(TM) (LKC Gaithersburg, MD) is a small
handheld ERG device using adhesive skin electrodes in lieu of
contact lens electrodes to assess cone function in patients without
mydriasis. The RETeval(TM) is currently in Phase 2 and 3 clinical
trials (US FDA, and EC, respectively). RETeval(TM) (REv) was used
in patients with diabetes mellitus and glaucoma patients in a retina
practice in San Jose, CA. Inclusion criteria: Diabetic pts HbA1c >
6.0 mg/dL or FBS > 100 mg/dL; Glaucoma patients were verified
by visual field findings. Visual acuity was 20/15-20/40. The Stata
statistical software program was used. For each patient, ERG data
from only one eye was used, based on randomization by coin toss.
Informed consent was obtained.
Results: A total of 50 patients and controls were enrolled over 3
months: Control (C): n=22: age range 23-75 yrs, avg=47.9, sd=16.3;
Diab (DM): n=12: age range 23-77 yrs, avg=55.8,sd=14.6; Glaucoma
(G) n=17: age range=37-76 yrs, avg=59.2, sd=11.95. ERG photopic
implicit times were prolonged in both diabetic and glaucoma patients:
2 tailed t-test: Control mean 33.2 msec vs DM mean 34.6 msec,
implicit time p=0.045; Control mean 33.2 msec vs G mean 35.4
msec, implicit time p=0.0009. No significant differences were noted
between implicit times in the diabetic and glaucomatous patients or
for difference in response in amplitude: C vs DM: p=0.26.
Conclusions: This small study suggests that prolongation of flicker
implicit times in diabetes and glaucoma can be discerned with the
RETeval(TM) in a clinical setting. The RETeval(TM) may thus be
of value as a screening tool in nursing homes or facilities where
ophthalmic exams are not available.
Commercial Relationships: Byongdo Kim, None; Evelyn Ross,
None; Kimberly Pham, None; Jenny Nguyen, None; Vinna Nam,
None; Vidhya Gunasekaran, None; Scott E. Brodie, None; Gloria
Wu, None
Program Number: 5119 Poster Board Number: A0157
Presentation Time: 3:45 PM–5:30 PM
Effect of Pupil Size on ERGs Recorded by New Mydriasis-Free
Full-Field Flicker ERG System (Reteval™)
Mineo Kondo, Kumiko Kato, Ryunosuke Nagashima, Yoshitsugu
Matsui, Masahiko Sugimoto, Hisashi Matsubara. Ophthalmology,
Mie Univ Graduate School of Med, Tsu, Japan.
Purpose: To study the effect of pupil size on the amplitude and
implicit time of 28.3-Hz flicker electroretinograms (ERGs) recorded
with a new mydriasis-free full-field flicker ERG system (RETeval™,
LKC Technologies, Gaithersburg, MD). This system was designed to
deliver constant retinal illumination by adjusting the light intensity to
compensate for the pupil size.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Methods: Ten healthy volunteers (age, 25 to 46 years) were studied.
After 10 minutes of light adaptation, 28.3-Hz flicker ERGs were
repeatedly recorded every three minutes after starting the pupil
dilation by drops of 0.5% tropicamide and 0.5% phenylephrine
over a period of 21 minutes. The pupil size was measured by an
automated pupil area measuring device. The amplitudes and implicit
times of 28.3-Hz flicker ERG were plotted against the time after the
installation of the mydriatics.
Results: The average pupil area was 14.39 ± 4.78 mm^2 at the
beseline, and it gradually increased to 46.50 ± 12.20 mm^2 at 21
minutes after starting the mydriatic drops. There was no statistically
significant change in the amplitude of flicker ERG during the 21
minutes. However, the average implicit time of the flicker ERGs
was 32.80 ± 1.03 ms at the baseline, and it significantly increased
with time, then reached 34.20 ± 1.12 ms at 21 min (p<0.05, two-way
layout ANOVA and Bonferroni type multiple comparisons). There
was a significant positive correlation between the pupil area and
implicit time (Pearson product-moment correlation: p<0.001, r =
0.62). A simple regression equation showed that a 1 mm^2 increase
of pupil area caused approximately 0.05 msec implicit time delay.
Conclusions: These results suggest that the implicit time of 28.3Hz flicker ERGs recorded with mydriasis-free flicker ERG System
(Reteval™) can be influenced by the pupil size.
Commercial Relationships: Mineo Kondo, None; Kumiko Kato,
None; Ryunosuke Nagashima, None; Yoshitsugu Matsui, None;
Masahiko Sugimoto, None; Hisashi Matsubara, None
Support: Ministry of Education, Culture, Science and Technology
(No. 18591913), Japan
Program Number: 5120 Poster Board Number: A0158
Presentation Time: 3:45 PM–5:30 PM
Corneal ERG Topography: Visualizing Multi-Electrode
Electroretinogram (meERG) Data Using a Three-Dimensional
Surface Spline
Brian Kunzer, Zahra Derafshi, Hadi Tajalli, John R. Hetling.
Bioengineering, University of Illinois at Chicago, Chicago, IL.
Purpose: The impact of meERG recording lies in the ability to
observe and interpret spatial differences in corneal potentials
(ERG topography) following a light stimulus. An initial step is to
interpolate between the meERG measurement locations to form a
smooth topographic map, analogous to high-density EEG mapping. A
MATLAB-based tool was developed that performs the interpolation
at each time step in the ERG response. The visualization tool is used
to differentiate healthy-eye responses from those obtained from eyes
with retinal lesions. Robustness of the spline-interpolated maps was
evaluated by calculating error introduced by missing values (due to
noisy or inoperable electrode channels) in the meERG data set.
Methods: meERG data sets were obtained from Long Evans rat eyes
using a contact lens electrode array. Photocoagulation lesions were
created adjacent to the optic disk but restricted to one hemisphere.
A three-dimensional spline interpolation approach was adapted to
the meERG data structure (25 measurement locations) to create a
smooth corneal potential map. Potential maps were converted to a
dimensionless quantity, standard deviations from the spatial mean,
in order to facilitate pooling and comparison of responses from
different experiments. Robustness of the interpolation was evaluated
by calculating an average percent difference between measured
values and interpolated values when individual channels, or groups of
channels, were not included in interpolation calculations.
Results: The interpolation approach provides the first ERG
topographic maps derived from meERG data sets. Topographic
maps of eyes with retinal lesions are visually and quantitatively
distinguishable from healthy eye responses. Interpolation based on
incomplete data sets (< 25 channels) results in mean error levels of
less than 10% until ten or more channel values are removed, though
maximum error rate is sensitive to the position of missing channels.
Conclusions: The spline interpolation approach was successfully
adapted to the meERG data structure, and proved robust in the case
of moderate numbers of missing channels. The resulting corneal ERG
topographic color maps can present absolute amplitudes or relative
spatial differences, and can be used to visually and quantitatively
compare results from healthy and unhealthy eyes.
Commercial Relationships: Brian Kunzer, None; Zahra Derafshi,
None; Hadi Tajalli, None; John R. Hetling, RetMap, Inc. (P)
Program Number: 5121 Poster Board Number: A0159
Presentation Time: 3:45 PM–5:30 PM
Effect of varying conductive fibre electrode position between
fornix and lid margin on electroretinogram amplitudes and
implicit times
Ambreen Tariq1, Ibrahim Sheriff1, Taha Bhatti1, Ahmed Sankoh1,
Hong Gao1, Christopher J. Hammond1, Omar A. Mahroo1, 2.
1
Ophthalmology, King, London, United Kingdom; 2Physiology,
Cambridge University, Cambridge, United Kingdom.
Purpose: Techniques for recording the electroretinogram (ERG)
include using contact lens, gold foil, and conductive fibre electrodes;
different methods yield different response amplitudes. We explored
the effect of varying the position of the conductive fibre electrode
on responses recorded from the same subject, elicited by standard
stimuli.
Methods: Full-field ERG responses were recorded from both eyes
in 6 healthy subjects: in one session, electrodes were placed in the
lower conjunctival fornix in both eyes; in a different session, the
electrode was placed at the lid margin in one eye and left at the fornix
in the other eye (which served as a control). Stimuli were delivered
according to ISCEV standards, using the full set of scotopic and
photopic stimuli in 3 subjects and only the photopic stimuli in 3
subjects. Pupils were pharmacologically dilated and pupil diameters
were monitored throughout.
Results: When moving from the fornix to the lid margin the mean
increase in 30 Hz flicker amplitudes was 44.9% (range 5.5 to 82.7%).
Mean increases in photopic a-wave and b-wave amplitudes were
respectively 47.3% (range 29.4 to 68.0%) and 44.3% (range 17.2 to
86.7%). Mean increases in implicit times were between 0.4 and 2.5%
for all photopic stimuli. For scotopic stimuli, the mean increase in
dim-flash b-wave amplitude was 64.8% (range 18.2 to 98.9%), and
the mean increase in a-wave and b-wave amplitudes elicited by the
3 cd m-2s flash were 47.3% (range 4.0 to 82.1%) and 44.0% (range
14.6 to 61.9%). For the 10 cd m-2s flash, mean increases were 49.2%
(range 11.5 to 83.6%) and 44.6% (range 14.7 to 65.1%) respectively.
Mean increases in implicit time ranged from 1.4% to 6.9% for
scotopic stimuli. In the control eye, mean amplitudes did not vary by
more than 21%, and mean implicit times did not vary by more than
5% between the two sessions.
Conclusions: Compared to the fornix, ERG response amplitudes
were over 40% higher when the fibre electrode was placed at the
lid margin. Implicit times did not appear to change more than in the
control eye. This would be consistent with changes in position having
a scaling effect on response amplitudes, with little effect on latencies,
and shows the importance of consistent electrode positioning in
recordings.
Commercial Relationships: Ambreen Tariq, None; Ibrahim
Sheriff, None; Taha Bhatti, None; Ahmed Sankoh, None; Hong
Gao, None; Christopher J. Hammond, None; Omar A. Mahroo,
None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 5122 Poster Board Number: A0160
Presentation Time: 3:45 PM–5:30 PM
The Use of Photoresponse Kinetics as a Molecular Thermometer
Marja Pitkänen, Ari O. Koskelainen. Department of Biomedical
Engineering and Computational Science, Aalto University School of
Science, Espoo, Finland.
Purpose: Heating of the retinal pigment epithelium (RPE) has been
considered as a potential treatment for degenerative retinal diseases.
During the treatment, the temperature of the RPE must be monitored
continuously in order to reach the therapeutic effect while avoiding
damaging the cells. The photoreceptors are located at the proximal
side of the RPE so that the outer segments (OSs) are partially
embedded in the RPE layer leading to the temperatures of OSs and
RPE being nearly equal.
The photoreceptor light response kinetics are set by the
phototransduction machinery located in the OS. The kinetics
depend on temperature and, at least in principle, could be used as a
temperature indicator during the heat treatment. The objective of this
work is to find out whether the temperature of the photoreceptors
can be determined accurately enough from the leading edge of
ERG responses in the temperature-range 37 – 42 °C and to define a
parameter that best represents the temperature. This method could be
used for the RPE temperature determination in animal models.
Methods: We recorded ERG flash responses from isolated mouse
retinas (C57BL/6J) at three temperatures: 37, 39.5, and 42 °C. The
retinas were perfused with Ringer’s solution containing DL-AP4 and
BaCl2 to isolate the photoreceptor response. Three fixed stimulus
intensities were used to give three different responses: a linear
range, a half-saturated and a saturated response. The temperaturedependence was determined for two parameters: the time and the
slope at the steepest point of the leading edge (Fig. 1).
Results: The results from three retinas are given in Table 1. Relative
changes of the parameter values were quite similar between the
different response types. The relative shortening of the inflection
point time was on average 20% per 2.5 °C. The relative increase of
the slope at the inflection point was greater, on average 39% per 2.5
°C, but the variance between the results was also higher.
Conclusions: Temperature elevations of 2.5 and 5 °C have a
considerable effect on the parameter values which implies that the
temperature could be determined accurately enough with this method.
The next phase of this study is to test the method on synaptically
active retina and later in vivo.
Table 1. The relative change of the parameter value (compared to 37
°C), mean ± stdev % (n).
Commercial Relationships: Marja Pitkänen, None; Ari O.
Koskelainen, None
Support: Academy of Finland Grant #269747, Finnish Cultural
Foundation
Program Number: 5123 Poster Board Number: A0161
Presentation Time: 3:45 PM–5:30 PM
Visual Evoked Potentials as an Extension of Electroretinography
in Non-Clinical Ocular Toxicity Studies
Margaret E. Collins. Toxicology, Charles River, Reno, NV.
Purpose: Electroretinography (ERG) has been used to assess retinal
function and identify changes that may be associated with ocular
toxicity during the conduct of nonclinical studies. Routine ERGs
consist of measuring retinal responses to a series of light stimuli, with
the ISCEV standards for ERG generally being used. However, ERGs
only assess electrical impulse generation and transmission to the
level of the retinal ganglion cell (RGC) and are not affected by loss
of RGC function. Visual evoked potential (VEP) assesses impulse
generation at the level of the visual cortex. The combination of ERGs
and VEPs can give a full picture of visual impairment if there are test
article-related effects on vision.
Methods: Cynomolgus monkeys were assessed via ERG and
VEP measurements. All measurements were conducted using the
LKS UTAS E-3000 Visual Electrodiagnostic System. ERGs were
measured in accordance with ISCEV standards. To mimic the
effect of reduced rod/cone function, a second round of ERGs were
collected immediately following the first set of flicker measurements.
Following ERG assessment, pattern and flash VEPs were measured
by placing reference, ground and recording electrodes on the scalp.
All data were analyzed by measuring amplitude and latency of
waveforms.
Results: Ophthalmic findings that were clearly limited to the retina
upon ophthalmoscopy produced alterations in ERG responses, but not
in VEPs. When a second round of ERGs were collected immediately
after the flicker assessment (i.e. in the absence of dark adaptation),
reduced amplitude was noted for most parameters. Animals noted
as having reduced vision based on behavior without the presence of
findings during ophthalmoscopy variably showed either alterations
in ERG and/or VEP responses, or no alterations in response. Due to
the nature of pattern VEPs, which require directed focusing by the
subject, flash VEPs were less variable than pattern VEPs.
Conclusions: While ERGs remain the primary assessment of retinal
function in nonclinical ocular toxicity studies, VEPs can provide an
additional assessment. In some cases, VEPs may be more appropriate.
The nature of the test article effect should be taken into account when
determining the most appropriate method for elucidating the cause of
vision changes in non-human primates
Commercial Relationships: Margaret E. Collins, Charles River
Laboratories (F)
Figure 1. The time and the slope at the steepest point (inflection
point) of the leading edge of a photoresponse.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 5124 Poster Board Number: A0162
Presentation Time: 3:45 PM–5:30 PM
Objective Assessment of Visual Acuity using Visual Evoked
Potentials – the Visus VEP revised
Torsten Strasser, Fadi Nasser, Gudrun Haerer, Ditta Zobor, Anne
Kurtenbach, Eberhart Zrenner. Institute for Ophthalmic Research,
University of Tuebingen, Tuebingen, Germany.
Purpose: Visual acuity (VA) is an important parameter to test visual
function. It is commonly assessed with eye (e. g. Snellen-) charts,
which use optotypes of different sizes. This technique relies on the
subjects’ cooperation, and hence, its results are always subjective.
VA can also be estimated using Visual Evoked Potentials (VEP):
amplitude size of VEPs is correlated to the spatial frequency of a
stimulus pattern. This provides an objective method for assessing VA,
especially in subjects with low compliance. Two different paradigms
are commonly used: the Visus VEP and the Sweep VEP. Here we
present a best-of-breed approach, which combines advantages of both
techniques. It can be used with current electrophysiological recording
systems.
Methods: A custom software was developed to present repetitive
sequences of 11 checkerboards with increasing spatial frequencies
(.6/.9/1.4/2.1/3.3/4.9/7.3/10.4/18.2/24.3/36.5cpd) using a 21”
CRT monitor (92% contrast, on/off: 40/300 ms, isoluminant).
Sweep VEPs were recorded using an Espion e2 system (Diagnosys
LLC). Averaged results (n=50), were evaluated for trough-topeak amplitudes of the spindle-shaped signal. The limiting spatial
frequency and the resulting VA, were estimated by fitting modified
Gaussian functions to the data. Goodness of fit was evaluated using
root-mean-square error (RMSE). VA estimated by Gaussian fit, 2nd
order polynomial fit and subjectively measured VA were compared.
Results: Nine healthy, non-myopic volunteers (4f/5m, 30±9.3y)
were tested. Best corrected visual acuity (BCVA) and simulated
deterioration of VA using lenses (1, 2, 3dpt) was measured using an
eye chart. Sweep VEP was recorded and objective VA was estimated.
Goodness of fit showed better results for modified Gaussian
compared to 2nd order polynomial (RMSE 4.14, SD3.61 vs. 5.74,
SD2.93). Comparison between subjective and objective VA was
performed by Bland-Altman plot (r=0.73).
Conclusions: The Sweep VEP allows for an objective assessment
of VA almost independent of subject compliance. Using custom
developed software we were able to extend the method and to
implement it in our electrophysiologyl recording system. By
combining advantages of two well-established techniques we
improved the objective estimation of the VA. Next steps include
automated marker placement, improvement of curve fitting and
definition of a mapping between spacial frequency and VA in a larger
parameter space.
Commercial Relationships: Torsten Strasser, None; Fadi
Nasser, None; Gudrun Haerer, None; Ditta Zobor, None; Anne
Kurtenbach, None; Eberhart Zrenner, None
Program Number: 5125 Poster Board Number: A0163
Presentation Time: 3:45 PM–5:30 PM
Binocular interaction of visually evoked cortical potentials
(VEPs) elicited by dichoptic binocular stimulation
Makoto Kawashima1, Celso S. Matsumoto1, Ryota Nakagomi2,
Kei Shinoda1, Takaaki Kondo1, Toshiko Arai1, Atsushi Mizota1.
1
Ophthalmology, Teikyo University, School of Medicine, Tokyo,
Japan; 2Orthoptics, aculty of Medical Technology Teikyo University,
Tokyo, Japan.
Purpose: To determine the interaction of cortical potentials elicited
by dichoptic stimulation of the dominant and fellow eyes at different
frequencies.
Methods: A pair of programmed power supply units were used to
drive a light emitting diode (LED) mounted in the right and left
eyes of light-proof goggles to elicit the VECPs. The right eye was
stimulated at 11.5 Hz and the left eye at 11.0 Hz. The stimulus
duration was 5 ms. The duration of collection was 200 ms and about
200 responses were averaged. The visually evoked cortical potential
(VEP) of each eye was extracted separately. The components of the
VEPs following monocular or binocular stimuli of the two eyes were
compared.
Results: Individual VEPs could be recorded separately after
simultaneous dichoptic stimulation of each eye. The amplitudes
of the VEPs were not significantly different after stimulating the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
dominant eye and the fellow eye separately. The implicit times of
N-2 and P-2 were shorter after stimulation of the dominant eye
than after stimulation of the fellow eye but the difference was not
significant. However, the implicit time of N-2 elicited by stimulating
the dominant eye was significantly shorter when the stimulation rate
was 11.5 Hz.
Conclusions: The VEPs elicited by stimulating the two eyes can
be separately recorded with simultaneous dichoptic stimulation.
Dichoptic simultaneous stimulation required a shorter time and
may be a more sensitive method of analyzing binocular interactions
compared to the classic VEPs using monocular stimulation.
Commercial Relationships: Makoto Kawashima, None; Celso S.
Matsumoto, None; Ryota Nakagomi, None; Kei Shinoda, None;
Takaaki Kondo, None; Toshiko Arai, None; Atsushi Mizota, None
Program Number: 5126 Poster Board Number: A0164
Presentation Time: 3:45 PM–5:30 PM
Using Multifocal Steady-State Visual Evoked Potentials for
Objective Assessment of Visual Field Loss: A Pilot Study
Yuan-Pin Lin1, Yijun Wang1, Tzyy-Ping Jung1, Felipe A. Medeiros2.
1
Institute for Neural Computation, University of California, San
Diego, La Jolla, CA; 2Department of Ophthalmology, University of
California, San Diego, La Jolla, CA.
Purpose: To develop an objective electroencephalogram (EEG)based brain sensing technique for visual-field examination by using
high-density EEG to associate the dynamics of multifocal steadystate visual-evoked potentials (mfSSVEPs) with visual field defects.
Methods: The working hypothesis was that presenting multiple
frequency-tagged flickering (alternating black/white) sectors in the
monocular visual field, a sector(s) corresponding to a visual field
deficit(s) would be less perceivable or unperceivable and thereby
would have weaker SSVEP amplitude, compared to other normal
visual spots. To test the hypothesis, we designed a layout of visual
stimuli consisting of 20 sectors in three concentric rings (subtending
6°, 15°, and 25° in the visual field). All sectors flickered concurrently
at different frequencies ranging from 8 Hz to 11.8 Hz with a
frequency resolution of 0.2 Hz. The visual field DEFICIT condition
was mimicked by replacing the 9 Hz sector (the 0-45° patch in
the middle ring) with a black patch in contrast to the CONTROL
condition in which all 20 sectors flickered concurrently. The EEG
data from five normal participants were recorded using a 128-channel
BioSemi ActiveTwo EEG system during visual stimulation (5
seconds per trial, 100 trials for each condition).
Results: The empirical results showed that four of five participants
consistently exhibited a significant deterioration of the 9 Hz SSVEP
amplitude in the DEFICIT condition compared to the CONTROL
condition (Figure). The inconsistency from one participant was
likely attributed to the absence of gaze-attentive fixation to the visual
stimulus according to his self-report after the experiment.
Conclusions: These preliminary results demonstrated that visual field
deficit mimicked by disabling the 9 Hz sector did result in significant
SSVEP attenuation at the corresponding frequency. Furthermore,
this pilot study suggests that the dynamics of mfSSVEP amplitude is
capable of serving as an objective biomarker to assess potential visual
field deficits in conditions such as glaucoma.
Commercial Relationships: Yuan-Pin Lin, None; Yijun Wang,
None; Tzyy-Ping Jung, None; Felipe A. Medeiros, None
Support: RO1-EY021818-03
Program Number: 5127 Poster Board Number: A0165
Presentation Time: 3:45 PM–5:30 PM
Method to detect visual field loss using multifocal visual evoked
potential
Givago S. Souza1, 2, Iraquitan Cordeiro Filho3, 6, Lorena Botelho
Vergara4, Alexandre Rosa4, Hideraldo Cabeça5, Schubert Carvalho3.
1
Insituto de Ciencias Biologicas, Universidade Federal do Para,
Belem, Brazil; 2Nucleo do Doenças Tropicais, Universidade Federal
do Para, Belém, Brazil; 3Instituto Tecnologico VALE, Belem, Brazil;
4
Instituto de Ciências da Saúde, Universidade Federal do Para,
Belem, Brazil; 5Hospital do Estado Ophir Loyola, Belem, Brazil;
6
Instituto de Ciencias Exatas e Naturais, Universidade Federal do
Para, Belem, Brazil.
Purpose: To develop a supervised learning model with decision trees
to act as a decision support system in the task to identify visual field
losses by using multifocal visual evoked cortical potential (mfVECP).
Methods: We studied mfVECP data from 22 eyes of healthy
subjects, 23 eyes of subjects with neuromyelitis optica (NMO), and
16 eyes from subjects with multiple sclerosis (MS). Dartboards
with 60 checkboard sectors were used as stimuli. The SNR from the
waveforms of the 60 sectors was used as input into a decision tree.
A combination of two-sample t-test feature selection and forward
sequential feature selection was used to obtain the best representative
features or sectors that sorted out the three subject classes. With
these selected features, decision tree models were fitted and crossvalidated using leave-one-out technique. For evaluation criteria, we
used the classification of accuracy and Cohen’s Kappa coefficient.
The following comparisons were analyzed: healthy subjects versus
NMO subjects; healthy subjects versus MS subjects; NMO subjects
versus MS subjects; healthy subjects versus subjects suffering for
both illnesses.
Results: We found a number of visual field sectors that better
differentiate mfVEP data for each comparison: 12 sectors (healthy
subjects vs NMO subjects); 24 sectors (healthy subjects vs MS
subjects); 21 sectors (healthy subjects vs both illnesses); none sector
(NMO subjects vs MS subjects). With the selected features, decision
trees were fitted following the same combinations of the feature
selection process. For each combination, two decision trees were
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
fitted, one without the feature selection process and another with
the selected features. In the healthy vs NMO dataset the results after
cross-validation shows that the accuracy of the decision tree and
the Kappa value were 80% and 0.6002, respectively; for this same
dataset after the feature selection the results were 84% and 0.6884,
respectively. For healthy vs MS dataset, the accuracy and Kappa
value were 71.05% and 0.4011, respectively (no feature selection),
and after feature selection the results were 76% and 0.5100,
respectively. For healthy vs both illnesses as one class, the accuracy
and Kappa value were 68.85% and 0.3039, respectively, after feature
selection the results were 68.85% and 0.3178, respectively.
Conclusions: In general, the use of feature selection improved
classification accuracy.
Commercial Relationships: Givago S. Souza, None; Iraquitan
Cordeiro Filho, None; Lorena Botelho Vergara, None; Alexandre
Rosa, None; Hideraldo Cabeça, None; Schubert Carvalho, None
Support: CAPES, CNPq, UFPA, FINEP
Program Number: 5128 Poster Board Number: A0166
Presentation Time: 3:45 PM–5:30 PM
Comparison of multifocal photopic negative response (mfPhNR)
with structural and functional measures in experimental
glaucoma
Lakshmi Rajagopalan1, Nimesh B. Patel1, Suresh Viswanathan2,
Ronald S. Harwerth1, Laura Frishman1. 1College of Optometry,
University of Houston, Houston, TX; 2College of Optometry, State
University of New York, New York, NY.
Purpose: To utilize the multifocal electroretinogram (mfERG)
technique to assess local loss of function, as reflected by the
mfPhNR, in a nonhuman primate model of experimental glaucoma
and to compare the results with standard clinical structural and
functional measures.
Methods: mfERGs, were recorded longitudinally (5-10 visits) in 3
macaques with unilateral elevated intraocular pressure (IOP) induced
by laser photocoagulation of the trabecular meshwork. Optical
coherence tomography (OCT) and standard automated perimetry
tests were done concurrently. The mfERG (VERIS 4.1) stimulus
display, 35° x 34°, was an array of 19 unstretched hexagons, each 7°
across. For each hexagon the stimulus consisted of 5 bright frames,
each occurring on 50% of the frame changes (75 Hz), followed
by 25 dark frames, repeating every 400 ms for 7 min. Global and
regional mfPhNR amplitudes were compared in experimental (Exp)
and fellow control (Con) eyes using a repeated measure ANOVA
corrected with a post hoc Tukey test. Relationships between local
mfPhNR amplitude and corresponding sectoral retinal nerve fiber
layer thickness (RNFLt), retinal ganglion cell/inner plexiform
thickness (RGC/IPLt) and local subjective visual sensitivity (VS)
were analyzed using linear regression for individual subject data.
Results: Significant reductions in mfPhNR amplitudes occurred early
after lasering in all Exp eyes, compared to fellow Con eyes (P<0.005,
ANOVA). Local mfPhNR amplitudes were strongly correlated with
corresponding sectoral RNFLt. The Pearson correlation coefficient
r was >0.78 (P<0.0001) and 0.86 (P<0.0001) for Exp and Con eyes
respectively. Similarly good correlations were seen between mfPhNR
and RGC/IPLt; r >0.9 (Exp eye; P<0.0001) and 0.77 (Con eye;
P<0.0001). Local mfPhNR amplitudes were correlated moderately
with local VS; r >0.41 (Exp; P<0.001) and 0.42 (Con; P<0.001).
Reductions in mfPhNR amplitude in Exp eyes that exceeded the
test-retest variability in Con eyes, preceded parallel changes in
corresponding sectoral RNFLt in two subjects (superior optic nerve
head in one, and temporal in the other) and the reduction in macular
RGC/IPLt in one subject.
Conclusions: The current findings indicate that the mfPhNR can be
used as an additional tool to detect and monitor functional changes in
primate eyes with elevated IOP.
Commercial Relationships: Lakshmi Rajagopalan, None; Nimesh
B. Patel, None; Suresh Viswanathan, None; Ronald S. Harwerth,
None; Laura Frishman, None
Support: NIH grants EY EY01139, EY07551 and Fight for Sight
summer student fellowship 2013.
Program Number: 5129 Poster Board Number: A0167
Presentation Time: 3:45 PM–5:30 PM
Macular cone photoreceptor density distribution in fellow eyes of
young adults
Tianjiao Zhang1, 2, Pooja Godara2, Russell Griffin3, Ernesto Blanco1, 2,
Xiaolin Wang2, Christine A. Curcio2, Yuhua Zhang1, 2. 1Department of
Biomedical Engineering, The University of Alabama at Birmingham,
Birmingham, AL; 2Department of Ophthalmology, The University
of Alabama at Birmingham, Birmingham, AL; 3Department
of Epidemiology, The University of Alabama at Birmingham,
Birmingham, AL.
Purpose: Quantitative estimation of cone packing density
distribution in fellow eyes of young subjects with good retinal health
may provide a baseline for understanding age- or disease-related cone
loss. We examine macular cone photoreceptor density differences
in fellow eyes using adaptive optics scanning laser ophthalmoscopy
(AOSLO).
Methods: Twenty subjects aged 19-29 years were enrolled. All
subjects underwent assessment of best-corrected visual acuity
and measurement of refractive error to ensure no participant had
refractive errors worse than -3 D or fellow-eye refractive error
difference greater than 0.25 D. The retinal magnification factor
for each subject was calculated using the Liou and Brennan eye
model with the corneal curvature, anterior chamber depth, and axial
length of the eyes measured using an ocular biometer. AOSLO was
performed to image the maculae. Cone density was assessed over the
central 2400 mm x 2400 mm macula, and evaluated statistically with
a mixed model approach. Each eye was divided into four quadrants:
superior/nasal (SN), superior/temporal (ST), inferior/nasal (IN),
and inferior/temporal (IT). Within each quadrant, the association
between cone density and eccentricity was compared between
fellow eyes. A three-way interaction term was included in models to
examine whether the association between eyes and cone density by
eccentricity varied by quadrant.
Results: The isodensity lines of macular cone density of fellow
eyes have the same shapes (Fig.1). There was no difference in the
association between eccentricity and cone density in fellow eyes for
the quadrants SN (p=0.8503), ST (p=0.1551), IN (p=0.8609), and IT
(p=0.6662). Associations did not vary between quadrants (p=0.6772).
In all subjects, the maximum cone density difference [assessed by
(DOS-DOD)/DOD] at a single sample point was 23.6%, and the rootmean-square difference was 6.78%.
Conclusions: We have characterized the macular cone density
distribution in fellow eyes including the fovea in 2 dimensions,
and estimated the maximum and average cone density differences.
Overall, macular cone distributions in fellow eyes are radially and
bilaterally symmetric, but considerable difference may exist at single
points. The range of differences is consistent with data previously
reported by Lombardo et al. for the parafoveal horizontal meridian.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Figure 1. Macular cone density distribution in subject 84. Plots are
centered on the fovea.
Commercial Relationships: Tianjiao Zhang, None; Pooja Godara,
None; Russell Griffin, None; Ernesto Blanco, None; Xiaolin Wang,
None; Christine A. Curcio, None; Yuhua Zhang, None
Support: This research is funded by EyeSight Foundation of
Alabama (YZ), International Retina Research Foundation (YZ), and
institutional support from Research to Prevent Blindness, EyeSight
Foundation of Alabama, Buck Trust of Alabama, and NIH P30
EY003039.
Program Number: 5130 Poster Board Number: A0168
Presentation Time: 3:45 PM–5:30 PM
Gender Differences in Anesthetized Primate ERG and Full-field
Flash VEP
James N. Ver Hoeve1, Brittany Glatting1, Kimberly B. McIntyre1,
Brian J. Christian2, T Michael Nork1, Charlene B Y. Kim1.
1
Ophthalmology & Visual Science, Univ of Wisconsin-Madison,
Madison, WI; 2Covance Laboratories, Inc, Madison, WI.
Purpose: To describe gender-related differences in the flash-evoked
cortical potential and the electroretinogram in the anesthetized
primate.
Methods: As part of baseline screening, a total of 300 male and 268
female cynomolgus macaque monkeys (Macaca fascicularis) was
evaluated using an ERG protocol based on the ISCEV standard with
the addition of cortical evoked response to flash stimuli. Animals
were sedated with ketamine and medetomidine . Rod dark-adapted
0.01 cd-s m-2 ERG (DA 0.01), mixed rod/cone dark-adapted (DA
2.7), and light-adapted cone (LA 2.7, LA 30 Hz) ERGs were recorded
from each animal. ERG signals were recorded from dilated eyes via
monopolar contact lens electrodes. Flash visual evoked potentials
(FVEPs) were elicited by LA 2.7 flashes delivered at 4.1 Hz. FVEPs
were recorded from two channels over the occipital scalp referenced
to vertex. The distributions of response parameters were evaluated
and the means were compared using parametric statistics.
Results: The distribution of ERG B-wave amplitudes in both females
and males was normalized with a log transform. Mean female
ERG B-wave amplitudes exceeded those of males by 7-9% for DA
0.01, DA 2.7 and LA 2.7 conditions (p’s < 0.001). Latency-to-peak
(implicit time, IT) of the A- and B-waves did not differ by gender
with the exception of shorter ITs in females for the DA 2.7 flash
A-wave. The FVEP waveform consisted of a small voltage negative
(~1 mcV) wave with an IT of 25 ms (N25), a positive voltage wave
peaking at 50 ms (P50), a large voltage N61 wave, and a prominent
P94 wave. Female P94 amplitudes exceeded males’ on average
by 33 percent. Female FVEP RMS exceeded that of males by
56% (p<0.001). No significant correlations existed between FVEP
measures and any ERG measure.
Conclusions: This study provides normative data on full-field ERG
and the FVEP from a large sample of male and female monkeys. The
FVEP is a technically relatively simple test that can play an important
role in the functional assessment of visual pathways in pre-clinical
pharmaceutical development. These data provide a reference for
evaluating the FVEP in this setting. As found similarly in studies
of awake humans, female monkeys have a slightly larger amplitude
ERG B-wave compared with males. The basis of the difference
between males and females in ERG and VEP amplitude remains to be
elucidated.
Commercial Relationships: James N. Ver Hoeve, OSOD, LLC
(C); Brittany Glatting, None; Kimberly B. McIntyre, None; Brian
J. Christian, Covance, Inc (E); T Michael Nork, OSOD, LLC (C);
Charlene B Y. Kim, OSOD, LLC (C)
Support: NEI Grant P30EY016665, Research to Prevent Blindness
514 ipRGCs
Thursday, May 08, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 5761–5768/B0069–B0076
Organizing Section: Visual Neuroscience
Program Number: 5761 Poster Board Number: B0069
Presentation Time: 8:30 AM–10:15 AM
Effect of age on the electrical response from intrinsically
photosensitive retinal ganglion cells
Manami Kuze1, 2, Hisashi Matsubara1, Masahiko Ayaki3, Kazuo
Tsubota3, Mineo Kondo1, Takeshi Morita4. 1Department of
Opthalmology, Mie University School of Medicine, Tsu, Japan;
2
Dept of Ophthalmology, Mie Central Medical Center, Tsu, Japan;
3
Department of Ophthalmology, Keio University School of Medicine,
Tokyo, Japan; 4Department of Living Enviromental Science, Fukuoka
Women’s University, Fukuoka, Japan.
Purpose: We have previously succeeded in recording intrinsically
photosensitive ganglion cells (ipRGC) response to light stimuli from
human eyes using four-primary illumination system, which modulates
stimulus levels to the ipRGC and other cones independently (Fukuda
et al. 2010; 2012). The purpose of this study was to investigate
the effect of age on the electroretinogram (ERG) from ipRGCs in
humans.
Methods: We used the four-primary illumination system (stimulus
duration, 250 ms) to stimulate ipRGCs independently of other
photoreceptors using a silent-substitution technique (Fukuda et al. J
Physiol Anthropol. 2012). Three elder subjects (age, 54.6±5.7 years)
and five younger subjects (23.0±1.7 years) are recruited. The implicit
times were measured from stimulus onset and offset to the positive
peaks and the amplitudes were measured from the baseline to the
positive peaks.
Results: Two distinct positive peaks were recorded after the onset
(on-response) and offset (off-response) for ipRGCs responses in both
groups. The implicit times of on- and off-responses were significantly
longer in elder group (on-response,97.3±2.2 ms; off-response,
282.3±5.2 ms) than those in younger group (on-response,79.0±6.5
ms; off-response, 279.0±13.4ms; P<0.05). In addition, the amplitudes
of on- and off-responses in elder group were significantly lower (onresponse,1.5±0.2μV; off-response, 1.5±0.1μV) than those in younger
group (on-response, 2.5±1.6μV; off-response,2.9±1.8μV;P<0.05).
Conclusions: These results suggested that the electrical function of
ipRGC is significantly influenced by the age in humans.
Commercial Relationships: Manami Kuze, None; Hisashi
Matsubara, None; Masahiko Ayaki, None; Kazuo Tsubota, None;
Mineo Kondo, None; Takeshi Morita, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 5762 Poster Board Number: B0070
Presentation Time: 8:30 AM–10:15 AM
Role for intrinsically photosensitive retinal ganglion cells in
modulating dopaminergic amacrine cell development
Simon Pan1, Samer Hattar1, 2, Tiffany M. Schmidt1. 1Department of
Biology, Johns Hopkins University, Baltimore, MD; 2Department of
Neuroscience, Johns Hopkins University, Baltimore, MD.
Purpose: A subset of retinal ganglion cells (RGCs) express the
photopigment melanopsin and are intrinsically photosensitive
(ipRGCs). A subtype of ipRGC, the M1 ipRGC, has been shown to
costratify their dendrites with DAC processes in the OFF sublamina
of the interplexiform layer. Additionally, ipRGCs have been
demonstrated to signal to DACs in a retrograde manner. However,
the effects of ipRGC inputs on DAC development and survival is
unknown.
Methods: We performed whole mount immunohistochemistry in the
mouse retina for tyrosine hydroxylase to identify the DAC population
throughout development on several animal models where mice lack
either ipRGCs or the melanopsin photopigment. Animals raised in
either constant light or constant darkness were also examined.
Results: We report a significant deficit in the total number of tyrosine
hydroxylase (TH) positive DACs in mice whose ipRGCs have been
genetically ablated by a diphtheria toxin (DTA) early in development.
This deficit can be detected as early as P14 and persists into
adulthood. Surprisingly, adult mice where ipRGCs are not ablated
until adult stages via expression of an attenuated diphtheria toxin
(aDTA) show a similar deficit to adult DTA animals, demonstrating
that loss of ipRGCs at any developmental stage causes a loss of TH
positive amacrine cells. Examination of the processes of DACs in
DTA animals reveal that the morphology of the remaining cells was
unaffected. Interestingly, rearing of WT animals in constant darkness
did not affect the number of TH positive cells.
Conclusions: Our findings implicate ipRGCs in a novel role in
regulating the total number of DACs in the retina. DAC loss by
ipRGC ablation can be detected in early postnatal development
but can also be induced in adult animals, suggesting possible
involvement in both the development and maintenance of the DAC
population.
Commercial Relationships: Simon Pan, None; Samer Hattar,
None; Tiffany M. Schmidt, None
Program Number: 5763 Poster Board Number: B0071
Presentation Time: 8:30 AM–10:15 AM
Ocular surface damage and migraine preclinical mouse models of
photophobia
Anna Matynia, Sachin Parikh, Samer Habib, Paul Kim, Steven
Nusinowitz, Michael Gorin. Jules Stein Eye Institute, UCLA, Los
Angeles, CA.
Purpose: Light sensitivity negatively impacts productivity and
quality of life, and is a clinical problem of increasing concern and
interest. As many as 50% of mild traumatic brain injury patients,
80% of migraine patients and many patients with ocular inflammation
or trauma experience photoallodynia, a painful response to normal
light. We aim to establish mouse models of ocular (corneal surface
injury) and central (migraine) etiologies using light aversion as
an endophenotype of photoallodynia, and ultimately use them to
investigate molecular and neural mechanisms.
Methods: A customized light aversion behavioral test was used
to assess photosensitivity in wild type and ipRGC-ablated mice
with corneal application of benzalkonium chloride (BAC, ocular
damage) or injection of nitroglycerin (NTG, migraine). Full-field
electroretinography (ERG) was performed on NTG-injected animals.
Corneal sensitivity was tested using von Frey Fibers.
Results: Corneal damage from BAC causes increased ipRGCdependent light aversion (2% BAC, 1 day acute treatment, n=7).
Lower levels of BAC (0.25%, n=5 and 0.50%, n=4) show a trend
towards increased ipRGC-dependent light aversion after 1 day
but not 7 days of treatment. There is a trend for decreased corneal
sensitivity with 2% BAC (n=6 eyes) after 1 day but not with 0.25%
(n=8 eyes) or 0.5% (n=10 eyes) after 7 days. Additional animals
will be tested for light aversion and corneal sensitivity after BAC
treatment. Mice with NTG-induced migraine (n=9) exhibit increased
ipRGC-independent light aversion that is resistant to treatment with
sumatriptan (n=8), a migraine medication. No differences in ERGs
were observed using the same dose of NTG. Chronic treatment
with NTG (n=6) compared to vehicle (n=6) does not sensitize light
aversion.
Conclusions: These studies establish two clinically relevant mouse
models of photoallodynia. In BAC-treated mice, light aversion
may be an early symptom of damage. In migraine, the mechanisms
that underlie light aversion may be different from other migraine
symptoms such as headache. The results indicate that ipRGCs
mediate some but not all forms of light aversion, indicating that
more than one retinal-brain circuit can elicit light aversion. Their
role in specific etiologies of photoallodynia may help elucidate these
differential retinal-brain circuits and provide insights to potential
clinical classification and management of photoallodynia.
Commercial Relationships: Anna Matynia, None; Sachin Parikh,
None; Samer Habib, None; Paul Kim, None; Steven Nusinowitz,
None; Michael Gorin, None
Support: Knights Templar Eye Foundation, EY00331-43
Program Number: 5764 Poster Board Number: B0072
Presentation Time: 8:30 AM–10:15 AM
Influence of Stimulus Size and Luminance on Rod-, Cone-,
Melanopsin-mediated Pupillary Light Reflexes
Jason C. Park, J Jason McAnany. Ophthalmology, University of
Illinois at Chicago, Chicago, IL.
Purpose: The human steady-state pupil size is thought to be jointly
dependent on adapting field luminance and area (i.e. corneal flux
density [CFD]; luminance x area). The purpose of this study was to
determine if the pupillary light reflex (PLR) driven by brief stimulus
presentations can also be accounted for by CFD under conditions
biased toward the rod, cone, and melanopsin pathways.
Methods: Pupil size was recorded using an infrared camera from
5 visually-normal subjects. Stimuli were presented in the central
visual field and consisted of short-wavelength flashes of 1-s duration
presented in the dark (rod and mealnospin condition; recorded after
10-min of dark adaptation; luminance range of -4 to 2.6 log cd/m2)
and against a rod-suppressing blue background (cone condition;
recorded after 2-min of light adaptation; luminance range of -1 to
2.6 log cd/m2). The stimuli subtended four sizes (4°, 16°, 32° and
full-field). PLR was defined as the ratio of the steady-state pupil
size (baseline) to post-stimulus pupil size. Rod- and cone-mediated
PLRs were measured at the time of maximum constriction following
stimulus presentation, whereas the melanopsin-mediated PLR was
measured at 6-8 s (median value) after stimulus offset.
Results: The rod- and melanopsin-mediated PLRs were well
accounted for by CFD, such that a lower intensity stimulus of a
larger area produced the same PLR as a higher intensity stimulus
of a smaller area when CFD was kept constant. The rod-mediated
PLR increased as CFD increased. Melanopsin-mediated PLRs were
elicited only in the higher luminance range (> 0 log cd/m2) and
for larger stimulus size (> 16°), but when present, the melanopsinmediated PLR was well accounted for by CFD. However, CFD
could not account for the cone-mediated PLR, due to an approximate
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
independence of the PLR on stimulus size, but a strong dependence
of the PLR on stimulus luminance.
Conclusions: The rod- and melanopsin-mediated PLRs had a similar
dependence on CFD, both acting as a flux integrator. This was not the
case for the cone-mediated PLR, which was strongly dependent on
stimulus luminance but not size. The finding that the cone-mediated
PLR is not dependent on CFD, but the steady-state pupil size under
photopic conditions is dependent on CFD suggests that the spatial
summation characteristics differ for these two pupil responses.
Commercial Relationships: Jason C. Park, None; J Jason
McAnany, None
Support: NIH research grand R00EY019510 (JM), NIH core
grant P30EY001792, and an unrestricted departmental grand from
Research to Prevent Blindness.
Program Number: 5765 Poster Board Number: B0073
Presentation Time: 8:30 AM–10:15 AM
Melanopsin-driven responses in the human brain
Manuel Spitschan1, Long Luu1, Ritobrato Datta2, David H. Brainard1,
Geoffrey K. Aguirre2. 1Department of Psychology, University
of Pennsylvania, Philadelphia, PA; 2Department of Neurology,
University of Pennsylvania, Philadelphia, PA.
Purpose: Photopic vision arises from L, M and S cones as well
as from the recently discovered intrinsically photosensitive retinal
ganglion cells (ipRGCs) containing the photopigment melanopsin.
Much is known about the brain pathways of signals originating
from cones. Considerably less is known about the projections of the
ipRGCs, in particular in the human brain. Here we used BOLD fMRI
to measure neural responses in humans to spectral modulations that
selectively target melanopsin while minimizing the responses of the
cones, and compare these to responses generated using cone-targeted
spectral modulations.
Methods: Four observers viewed large-field (27.5° diameter, central
5° obscured) sinusoidal flicker (1-16 Hz, log spaced) at 470 cd/m2
during BOLD fMRI. Using the method of silent substitution and
a digital light synthesizer, spectral modulations were directed at
melanopsin, L+M, L-M, or S cones. An isochromatic modulation
(L+M+S+melanopsin) was also studied. Estimates of photopigment
spectral sensitivities used to produce modulations accounted for
observer age and stimulus extent. Anatomical regions of interest were
defined for the lateral geniculate nucleus (LGN; using volumetric
templates), for primary visual cortex (V1) and for extrastriate areas
(V2, V3 and hV4) (Benson VSS2013). Cortical voxels were restricted
to >5° eccentricity as an extra precaution against contamination from
changes in cone spectral sensitivity at the fovea. Temporal transfer
functions (TTFs), describing the BOLD response as a function of
temporal frequency for each modulation direction, were averaged for
the four observers.
Results: We found robust melanopsin-driven responses in both
LGN and V1-hV4. In LGN, the melanopsin response was bandpass,
maximal at 8 Hz with no measurable response below 2 Hz. The
melanopsin response in V1 and V2 through hV4 was also bandpass.
Across areas the responses to cone-directed stimuli differed with the
direction of the spectral modulation, and differed as well from the
responses to melanopsin stimulation. S-cone responses were distinct
from melanopsin responses in both LGN and cortex.
Conclusions: Signals originating in the melanopsin-containing
ipRGCs produce robust responses in the LGN and visual cortex
of the human brain. The TTFs for melanopsin-driven responses
are bandpass in both LGN and visual cortex. The responses
tomelanopsin-driven stimulation have a temporal signature distinct
from that of cone-driven responses.
Commercial Relationships: Manuel Spitschan, U.S. Provisional
Patent Application No. 61/876,756 (P); Long Luu, None;
Ritobrato Datta, None; David H. Brainard, U.S. Provisional
Patent Application No. 61/876,756 (P); Geoffrey K. Aguirre, U.S.
Provisional Patent Application No. 61/876,756 (P)
Support: R01 EY10016, R01 EY020516, P30 EY001583
Program Number: 5766 Poster Board Number: B0074
Presentation Time: 8:30 AM–10:15 AM
Melanopsin-Derived Signals in the dLGN of the Light-Adapted
Mouse
Katherine E. Davis, Annette E. Allen, Robert J. Lucas. Faculty of
Life Sciences, The University of Manchester, Manchester, United
Kingdom.
Purpose: A subset of melanopsin-expressing intrinsically
photosensitive retinal ganglion cells (ipRGCs) project directly to the
dorsal lateral geniculate nucleus (dLGN). As ipRGCs are generally
considered to be global irradiance detectors, the significance for
primary vision remains unclear. To determine the extent to which
they contribute to tracking changes in luminance over time and space,
we set out to define the sensory characteristics of melanopsin-derived
responses under light-adapted conditions.
Methods: To examine melanopsin-derived signals in vivo, a
multi electrode array was lowered into the dLGN of the urethaneanaesthatised mouse. Extracellular spiking activity was recorded in
response to stimulation of the contralateral eye to full field stimuli.
To fully isolate the contribution of melanopsin, we used transgenic
mice lacking all cone activity (Cnga3 -/-). LEDS generated blue and
yellow stimuli that were isoluminant for rods. By switching from
yellow to blue for 30s we were able to present a 5-6 fold increase in
effective light intensity for melanopsin (75% Mickelson contrast)
that was silent for rods. This stimulus was presented over a 5 log unit
range of background light intensity.
Results: Approximately 15 % of light responsive dLGN neurons
responded to the melanopsin-isolating stimulus with increased firing
at irradiances >1.2 x 10^13 melanopic photons/cm2/s (equivalent
to mid-photopic illuminance). Latency to peak was long compared
to rod/cone derived responses; however, at <1 sec it was faster than
those traditionally associated with ipRGCs and/or those recorded in
rodless+coneless mice. A second set (17%) of dLGN neurons showed
irradiance dependent increases in background firing (a feature
associated with ipRGCs), however these did not show a detectable
response to the melanopsin step.
Conclusions: We provide the first description of melanopsin-derived
responses in the dLGN under light-adapted conditions and in the
presence of outer-retinal photoreception. We find cells that encode
irradiance but do not track transient changes in melanopsin excitation
while a separate population use melanopsin to encode such higher
frequency events. The relatively fast kinetics of the latter group
allows the possibility that melanopsin contributes information to
spatial structure, identifying an alternative/complimentary route to
the known rod and cone infrastructure in signalling structure in the
primary visual system.
Commercial Relationships: Katherine E. Davis, None; Annette E.
Allen, None; Robert J. Lucas, None
Support: ERC-MeloVision
Program Number: 5767 Poster Board Number: B0075
Presentation Time: 8:30 AM–10:15 AM
Influences of melanopsin on cone visual pathways
Annette E. Allen, Riccardo Storchi, Timothy Brown, Robert J. Lucas.
University of Manchester, Manchester, United Kingdom.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Purpose: Intrinsically photosensitive retinal ganglion cells
(ipRGCs), which express the photopigment melanopsin, are known
to efficiently encode ambient light levels in order to drive many
neurophysiological responses to light, such as modulating pupil size
and synchronising circadian clocks to the light-dark cycle. In addition
to their central projections, there is evidence that ipRGCs signal to
neighbouring cells within the retina via dendritic glutamate release
and gap junctions. ipRGCs thus have both the sensory capabilities
and intra-retinal connections to provide an independent mechanism
for adjusting retinal circuits according to ambient illumination.
Methods: To determine whether ipRGCs do indeed perform such
a function, we presented cone-activating stimuli to the mouse eye
against backgrounds that were equiluminant for cones but differed
in their relative activation of melanopsin. Responses were recorded
concurrently at two physiological levels; in the retina, via the
electroretinogram (ERG), and centrally, by measuring the lightevoked change in firing rate within the dorsal Lateral Geniculate
Nucleus (dLGN).
Results: A direct comparison of the ERG response under different
backgrounds revealed that the amplitude of the cone–driven ERG was
modulated according to the level of melanopsin activation. Changes
were also detected downstream in the dLGN, where the amplitude
and timing of cone-driven responses were also significantly affected
by melanopsin activity. These differences were absent in mice lacking
melanopsin.
Conclusions: These data indicate that the irradiance-coding functions
of ipRGCs are employed to adjust the retinal circuitry according
to ambient light levels and that this impacts the quality of visual
information available to the brain.
Commercial Relationships: Annette E. Allen, None; Riccardo
Storchi, None; Timothy Brown, None; Robert J. Lucas, None
Support: ERC Grant, BBSRC Grant
Program Number: 5768 Poster Board Number: B0076
Presentation Time: 8:30 AM–10:15 AM
Human opsin–G-protein fusion proteins as potential light
sensitizers
Doron Hickey1, Steven Hughes1, Wayne L. Davies2, 1, Robert E.
MacLaren1, Mark Hankins1. 1Nuffield Lab of Ophthalmology,
University of Oxford, Oxford, United Kingdom; 2School of Animal
Biology and Oceans Institute, University of Western Australia, Perth,
WA, Australia.
Purpose: Opsins are light-sensitive G-protein coupled receptor
proteins, essential for vision, circadian rhythmicity and eye
development. Opsins function by activating G-protein second
messenger systems. Rod and cone opsins activate Gαt, while
melanopsin activates Gαq/11. If inner retinal neurons, such as bipolar
cells, were engineered to become light sensitive, these cells could
act as substitute photoreceptors in patients who have lost their
photoreceptors. We have fused human melanopsin to different Gαproteins to test whether such a fusion modifies the coupling of an
opsin to a G-protein second messenger system. An opsin–Gα-protein
fusion could provide an optogentic tool for restoring sight in humans.
Methods: Two melanopsin–Gα-protein fusion constructs were
cloned into pMT4 by removing the stop codon from melanopsin and
inserting the in-frame coding sequence of either GNAQ (encoding
Gαq) or GNA11 (Gα11). Calcium kinetics were observed using
Rhod-2 fluorescent dye. Small interfering RNAs (siRNA) targeting
endogenous Gα-protein transcripts (including GNAQ and GNA11)
were applied to HEK293T cells expressing wild type melanopsin and
melanopsin–Gα-protein fusions to determine the relative importance
of the fused Gα-protein to activating the intracellular signalling
cascade.
Results: Melanopsin–Gαq/Gα11 fusion proteins exhibited a similar
response rate (41% and 40%, respectively) and time course of
calcium kinetics compared to non-fused melanopsin (48%; no
statistically significant differences between groups on ANOVA with
post hoc Tukey HSD). Using siRNA to knock down endogenous
levels of Gα-proteins in HEK293T cells showed melanopsin–Gαprotein fusion transfected cells to have a higher response rate than
wild type melanopsin transfected cells (melanopsin–Gα11 response
rate 36%, wild type melanopsin 13%, p>0.05).
Conclusions: Fusing melanopsin to either of its native Gα subunits,
Gαq or Gα11, shows that melanopsin can maintain coupling to the
Gαq/11 second messenger system in the presence of a fused Gαq or
Gα11 subunit. Furthermore, transient expression of melanopsin–Gα
protein fusions in HEK293T cells with siRNA-induced knock
down of endogenous Gα subunits suggests that fusing a Gα subunit
to melanopsin enables greater efficiency of coupling to the second
messenger pathway. Melanopsin–Gα protein constructs may therefore
offer advantages over wild type melanopsin as a potential optogenetic
gene therapy for photoreceptor loss.
Commercial Relationships: Doron Hickey, None; Steven Hughes,
None; Wayne L. Davies, None; Robert E. MacLaren, None; Mark
Hankins, None
Support: NIHR Biomedical Research Centre, Wellcome Trust,
Medical Research Council, UK Department of Health, Special
Trustees of Moorfields Eye Hospital, the Royal College of Surgeons
of Edinburgh, Woolf Fisher Trust
525 Photoreceptors
Thursday, May 08, 2014 12:00 PM–1:45 PM
S 210DE Paper Session
Program #/Board # Range: 5955–5961
Organizing Section: Visual Neuroscience
Program Number: 5955
Presentation Time: 12:00 PM–12:15 PM
Light regulates the outer segment protein transport and disc
renewal of mammalian photoreceptors
Ching-Hwa Sung2, 1, Ya-Chu Hsu2, Jen-Zen Chuang2. 1Cell and
Developmental Biology, Weill Med College of Cornell University,
New York, NY; 2Ophthalmology, Weill Med College of Cornell
University, New York, NY.
Purpose: The vertebrate photoreceptor outer segment (OS)
is a modified cilium containing ~1,000 membranous discs to
accommodate rhodopsin for light detection. Mammalian rod OS
undergo constant and rapid renewal (every ~10 days). Nascent
discs are formed at the base of the OS via the incorporation of
proteins synthesized at and transported from the inner se through the
connecting cilium, while distal discs are shed and phagocytosed by
neighboring RPE cells. To date, the mechanism and environmental
cues that regulate the disc renewal and OS protein transport of
remains elusive
Methods: We used both constitutive and Cre-lox inducible
expression system in transfected rods to trace the path and fate of two
essential OS proteins, rhodopsin and peripherin-2/rds, in rats reared
under different conditions. Both confocal and electron microscopies
were employed to investigate the distribution of the reporter proteins.
Results: Our results show that newly synthesized rhodopsin appears
on Rab11-positive recycling endosome prior to reaching the OS.
Rhodopsin primarily enters the OS in the dark. Photoexcitation of
post-Golgi rhodopsins retains them in the inner segment. Dailysynthesized rhodopsins are packed into distinct “segments”; each OS
has ~10 segments. The OS entry of disc-rim protein peripherin-2/
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
rds follows a rhythm complementary from that of rhodopsin’s.
Thus, cyclic light serves as a mechanism to alternate rhodopsin- and
peripherin-2/rds- rich disc and “divide” the OS into ~10 segments.
Conclusions: Our data showed that the post-Golgi rhodopsin
transits through intermediate compartments before entering the OS.
Furthermore, the trafficking of rhodopsin from the inner segment
to the OS is inhibited by light. Light differentially regulates the OS
transport of rhodopsin and peripherin-2/rds in a complementary
fashion. As a result, discs characterized by distinguishable protein
composition are alternately stacked. We propose a model explaining
how this specialized cytostructure of the OS participates the balance
the quanta of discs added and removed daily, and, hence, maintaining
the OS homeostasis.
Commercial Relationships: Ching-Hwa Sung, None; Ya-Chu Hsu,
None; Jen-Zen Chuang, None
Support: NIH Grant EY11307 & EY16805
Program Number: 5956
Presentation Time: 12:15 PM–12:30 PM
Photoreceptor cells with profound structural deficits can support
useful vision in mice
Stewart Thompson1, 2, Frederick R. Blodi2, 3, Pratibha Singh1, 2,
XiuYing Liu1, 2, Robert Mullins1, 2, Budd A. Tucker1, 2, Steven F.
Stasheff2, 3, Edwin M. Stone1, 2. 1Ophthalmology & Visual Sciences,
University of Iowa, Iowa City, IA; 2The Stephen A. Wynn Institute
for Vision Research, University of Iowa, Iowa City, IA; 3Pediatrics,
University of Iowa, Iowa City, IA.
Purpose: In animal models of degenerative photoreceptor
diseases, there has been some success restoring photoreception by
transplanting new stem-cell-derived photoreceptor cells into the
subretinal space. However, only a small proportion of transplanted
cells develop extended outer-segments, considered critical for
photoreceptor cell function. Photoreceptor cells of mice homozygous
for the Rds mutation in Peripherin-2 never develop a fully formed
outer-segment. The objective of this study was to determine whether
photoreceptor cells that lack a fully formed outer-segment could
usefully contribute to vision.
Methods: Retinal function in wild-type and Rds mice at 90 days of
age (RdsP90) was measured by electroretinogram and multielectrode
array recording. Visual capabilities were assessed by three distinct
visual behavior tests: the optokinetic tracking response; the
discrimination based visual water task; and a wheel running based
test of vision augmented mobility.
Results: RdsP90 mice had reduced but measurable electroretinogram
responses to light, and exhibited light-evoked responses in multiple
types of retinal ganglion cells, the output neurons of the retina. In the
optokinetic response and visual water task, acuity was measurable
but reduced, most notably when contrast was decreased. The wheel
running based test showed that RdsP90 mice needed 3-log units
brighter luminance than wild-type for vision augmented mobility
(10cd/m2).
Conclusions: Photoreceptor cells that lack fully formed outersegments can support useful vision. This challenges the idea that
normal cellular structure needs to be completely reproduced for
transplanted cells to contribute to useful vision.
Commercial Relationships: Stewart Thompson, None; Frederick
R. Blodi, None; Pratibha Singh, None; XiuYing Liu, None; Robert
Mullins, None; Budd A. Tucker, None; Steven F. Stasheff, None;
Edwin M. Stone, None
Support: Howard Hughes Medical Institute (EMS), Stephen A.
Wynn Institute for Vision Research (ST/BAT/SFS/EMS), National
Institutes of Health (1DP2OD007483, BAT), Foundation Fighting
Blindness (EMS/BAT), and Grousbeck Family Foundation (ST/BAT/
SFS/EMS).
Program Number: 5957
Presentation Time: 12:30 PM–12:45 PM
The Role of Na+/Ca2+, K+ exchanger 1 in Mammalian Vision
Frans Vinberg1, Tian Wang2, Robert S. Molday3, Jeannie Chen2,
Vladimir J. Kefalov1. 1Ophthalmology and Visual Sciences,
Washington University School of Medicine, St Louis, MO; 2Cell and
Neurobiology, University of Southern California, Los Angeles, CA;
3
Biochemistry/Molecular Biology, University of British Columbia,
Vancouver, BC, Canada.
Purpose: Ca2+ homeostasis in rod and cone outer segments is
important for visual adaptation and the health of photoreceptors.
Rods and cones are thought to use different isoforms of Na+/
Ca2+, K+ exchangers (NCKX) to extrude Ca2+ from their outer
segments. We sought to determine the physiological importance
of Nckx1 in mammalian rods by generating Nckx1 knockout mice
and characterizing the functional, morphological, and molecular
properties of its rods.
Methods: To remove Nckx1, most of the first exon of Slc24a1
gene was deleted and replaced by neomycin cassette. Standard
methods were used to produce Nckx1+/- mice which were crossed to
produce Nckx1-/- and WT control mice. We used transretinal ERG
to study photoreceptor function from intact isolated retinas perfused
with Ringers’ solution supplemented with L-15 (0.72 g/L), 40 μM
DL-AP4 to block the b-wave, and 100 μM BaCl2 to remove glial
component. Contrast sensitivity as a function of mean luminance was
determined with the OptoMotry system.
Results: The light responses from Nckx1-/- rods were 100-fold
smaller and with slower shut-off kinetics compared to those of
WT controls. However, fractional sensitivity of Nckx1-/- rods was
over 2-fold larger than in the control rods. Weber-like background
adaptation of Nckx1-/- rods was preserved. In behavior experiments,
optimal contrast sensitivity of Nkcx1-/- mice required more light than
in WT mice but still less than in Gnat1-/- controls. Cone response
amplitudes were comparable to those of Gnat1-/- mice and photopic
visual acuity and contrast sensitivity were normal in Nckx1-/mice. Retinal structure and rod morphology in Nckx1-/- mice were
slightly degenerated and expression analysis revealed a significant
downregulation of CNG channels in the Nckx1 KO mouse retinas.
Conclusions: Nckx1 is required for normal rod function but not for
Weber adaptation. The removal of Nckx1 had a subtle effect on rod
morphology. We find a novel mechanism of regulation by Nckx1
of CNG channels expression in mouse rods that likely preserves a
normal steady state Ca2+ and prevents large scale rod degeneration
in Nckx1-/- mice. Channel regulation by Nckx1 expression levels
could be a therapeutic target for preventing degeneration in rod
channelopathies.
Commercial Relationships: Frans Vinberg, None; Tian Wang,
None; Robert S. Molday, None; Jeannie Chen, None; Vladimir J.
Kefalov, None
Support: EY019312, EY021126, EY002687 to the Department
of Ophthalmology and Visual Sciences at Washington University,
Research to Prevent Blindness, EY12155 and EY02422
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 5958
Presentation Time: 12:45 PM–1:00 PM
Time course of dark adaptation under conditions of persistent
rhodopsin phosphorylation in mouse rods
Justin Berry1, Rikard Frederiksen1, Yun Yao2, Jeannie Chen2, M
C. Cornwall1. 1Department of Physiology and Biophysics, Boston
University School of Medicine, Boston, MA; 2Department of Cell
and Neurobiology and Department of Ophthalmology, University of
Southern California, Keck School of Medicine, Los Angeles, CA.
Purpose: Exposure of retinal rods to light leads to
photoisomerization of the rhodopsin chromophore, resulting in
activation of the visual transduction cascade. This activation is
terminated by the phosphorylation of a cluster of serine and threonine
residues located at the carboxyl terminus of rhodopsin and by
subsequent binding of arrestin. Conventionally, it is thought that
recovery of sensitivity following bleaching requires, among other
factors, dephosphorylation of rhodopsin and unbinding of arrestin.
The purpose of our study was to test this view by determining the
extent to which rhodopsin dephosphorylation affects rhodopsin
regeneration and recovery of dark-adapted sensitivity.
Methods: Flash sensitivity, rhodopsin concentration, and rhodopsin
phosphorylation were compared before and following exposure
to bright light that bleached a large fraction of rhodopsin, and
during rhodopsin regeneration fueled by exposure to exogenous
11-cis retinal. All measurements were made in rods of transgenic
mice lacking cone transducin (Gnat2-/-) to isolate rod responses.
Sensitivity was determined via transretinal ERG. Rhodopsin
concentration was determined by microspectrophotometry on intact
rods in isolated retina. The extent of rhodopsin phosphorylation at its
6 phosphorylation sites was determined by isoelectric focusing.
Results: Bleaching 50% of the rhodopsin in retinal rods isolated from
the retinal pigment epithelium resulted in persistent desensitization
as well as persistent phosphorylation at all 6 phosphorylation sites
on rhodopsin. Little dephosphorylation of rhodopsin was observed
in darkness during three hours subsequent to bleaching or following
total pigment regeneration. Surprisingly, near complete recovery of
sensitivity was observed in spite of persistent phosphorylation in a
large fraction of the regenerated visual pigment, The kinetics of dim
flash responses recovered to previous dark-adapted levels.
Conclusions: Our results demonstrate that, despite the presence of
substantial amounts of regenerated phosphorylated rhodopsin, the
flash sensitivity recovers, almost completely. Furthermore, dim flash
kinetics were fully restored, indicating that the basal PDE and cyclase
activity were at dark-adapted rates.
Commercial Relationships: Justin Berry, None; Rikard
Frederiksen, None; Yun Yao, None; Jeannie Chen, None; M C.
Cornwall, None
Support: EY01157
Program Number: 5959
Presentation Time: 1:00 PM–1:15 PM
Meta III Limits Opsin Availability During Pigment Regeneration
in Bleached Mouse Rods
Rikard Frederiksen1, Soile Nymark1, 2, Justin Berry1, Alexander V.
Kolesnikov3, Vladimir J. Kefalov3, M C. Cornwall1. 1Department of
Physiology and Biophysics, Boston University School of Medicine,
Boston, MA; 2Department of Electronics and Communications
Engineering, University of Technology and BioMediTech, Tampere,
Finland; 3Department of Ophthalmology and Visual Sciences,
Washington University School of Medicine, St. Louis, MO.
Purpose: The purpose was to determine if Meta III, a product of
rhodopsin bleaching, limits the availability of opsin for rhodopsin
regeneration in mouse rods. We did this by varying the decay rate of
Meta III in intact rod outer segments, and then determined differences
in pigment regeneration rate.
Methods: Experiments were performed in isolated intact retinae
of WT, Arr1-/-, and Grk1-/- mice. We used microspectrophotometry
to measure the rates of Meta III production and decay following
bleaching. Subsequently, we measured the amount of rhodopsin
regenerated after exogenous treatment with 11-cis retinal at different
stages of Meta III decay.
Results: In WT retinae exposed to light that bleached >90% of
rhodopsin, Meta III concentration rose to a peak in ~6 min and
decayed with a time constant of ~18 min. This time course could be
slowed by post-bleach exposure to 390 nm light, producing a stable
Meta III like photoproduct. Similarly, Meta III decay was remarkably
slowed in bleached Arr1-/- rod outer segments, with a correspondent
decay time constant of ~44 min. In contrast, Meta III decay in
bleached Grk1-/- rod outer segments was accelerated (time constant:
~12 min). When treated exogenously with 11-cis retinal at different
stages of Meta III decay, the amount of pigment regenerated in
these three models was consistent with their rates of Meta III decay.
Rhodopsin regeneration was most rapid in Grk1-/- rod outer segments
compared to WT, but much slower in Arr1-/- rod outer segments.
However, once Meta III had decayed fully, treatment with exogenous
11-cis retinal resulted in complete pigment regeneration in all models.
Conclusions: Our data demonstrate that post-bleach exposure to
bright UV light or the deletion of Arr1 or Grk1 can dramatically alter
the time course of Meta III production and decay. The presence of
Meta III prevents the regeneration of rhodopsin whose rate could be
regulated by these two proteins in vivo.
Commercial Relationships: Rikard Frederiksen, None; Soile
Nymark, None; Justin Berry, None; Alexander V. Kolesnikov,
None; Vladimir J. Kefalov, None; M C. Cornwall, None
Support: EY01157
Program Number: 5960
Presentation Time: 1:15 PM–1:30 PM
Foveolar cones of monkeys and humans have a unique, still
unknown morphology with impact for understanding the StilesCrawford effect
Ulrich Schraermeyer1, Sebastian Schmelzle2, Sigrid Schultheiss1,
Sylvie Julien1. 1Experimental Vitreoretinal Surgery, Centre for
Ophthalmology, Tubingen, Germany; 2Institute of Evolution and
Ecology, Eberhard Karls University Tübingen, Tübingen, Germany.
Purpose: The Stiles–Crawford (SC) effect is a property of the
cone photoreceptors of the human eye and was first described 8
decades ago. It was found that foveal cones have a less pronounced
directional sensitivity than parafoveal cones. It was speculated that a
change in the shape or the orientation of foveal cones was responsible
for the SC-effect. Until now, no morphologic evidence for this
assumption has been found.
Methods: The eyes from 52 cynomolgus monkeys (Macaca
fascicularis Raffles) and 2 human eye donors were fixed and
embedded for electron microscopy. Semithin sections were cut
from 22 foveae. Serial sections were made from individual foveae.
The image stack was aligned and segmented using Amira® 4.0.1
(Visualization Sciences Group, SAS ) resulting in a three-dimensional
model. Focused ion beam electron microscopy was also performed.
Results: Three-dimensional reconstruction of serial sections from
humans and monkeys clearly showed that in the foveola (200 – 300
mm in diameter) the inner segments of the cones were curved whereas
in the fovea and parafovea they were oriented along the optical axis.
Inner and outer segments together were S shaped in the foveola. The
orientation of inner and outer segments from cones in the foveola in
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
well-fixed specimens was extremely regular and nearly crystalline
and not orientated parallel to the optical axis.
Conclusions: The shape and orientation of the foveolar cones is
different to parafoveal cones in both human and monkey eyes. This
orientation contributes to the directional sensitivity of the StilesCrawford effect.
Commercial Relationships: Ulrich Schraermeyer, None;
Sebastian Schmelzle, None; Sigrid Schultheiss, None; Sylvie
Julien, None
Program Number: 5961
Presentation Time: 1:30 PM–1:45 PM
Rods may actively drive ganglion cells at surprisingly high light
levels in mouse retina
Alexandra Tikidji-Hamburyan1, Thomas Muench2. 1Neurosurgery,
Stanford University, Stanford, CA; 2Center for intergrative
neuroscience, Tuebingen, Germany.
Purpose: Recent studies have suggested the possibility that rod
photoreceptors may participate in vision at higher light levels than
previously suspected. We used wild type and transgenic mice to
investigate the limits of rod-mediated vision.
Methods: Multi-electrode-array recordings were made from ganglion
cells of flat-mounted mouse retinas in response to full-field light
stimuli (Gaussian white noise). Every ~30 minutes, the luminance
was increased by 1 log unit, while the contrast of the stimulus was
kept constant. 8 log units of light intensity were tested, ranging from
scotopic to high photopic levels. A linear-nonlinear model framework
was used to characterize the responses.
Results: In wild type mice, every brightness increase by 1 log unit
led to a step-wise decrease of time-to-peak of the linear filters (LFs).
However, at a certain light level (10^5 R*/rod/s), where rods are
thought to be saturated, the time-to-peak of the LFs slowly (within
15min) increased again, returning to the dark-adapted values. LFs
in mice lacking functional cones (Cnga3-/-) had high amplitude at
scotopic light levels, medium in mesopic, and were flat at the lowest
photopic light level (rods saturated). During 15 min after switch to
10^5 R*/rod/s light level, their amplitude gradually increased from 0
to the maximum. At the same light level, linear filters in mice lacking
functional rods (Rho-/-) slowly decreased in amplitude by 50%. The
results from all 3 mouse models suggest that the light level of 10^5
R*/rod/s slowly shifts ganglion cells responses from being conedriven to being (predominantly) rod-driven.
A computational model shows that if the active photopigment
concentration drops (when bleaching is much stronger than the
regeneration rate), isomerization rate may drop so low that it
will mimic dark-adapted conditions. This will lead to only weak
modulation of the cone output and strong modulation of the rod
output.
Conclusions: Taken together, our results suggest that rods may
indeed drive the responses of ganglion cells at very high light levels,
above levels at which rods are thought to be saturated. This effect
has most drastic consequences for in-vitro measurements of isolated
retina where pigment regeneration rate is very low. However, there
is a potential that similar effects at bright light may also take place
in-vivo (Abstract #736.01, SfN meeting 2013).
Commercial Relationships: Alexandra Tikidji-Hamburyan,
None; Thomas Muench, None
Support: BMBF FKZ 01GQ1002, DFG EXC307
542 ERG and VEP animal models
Thursday, May 08, 2014 12:00 PM–1:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 6172–6199/B0077–B0104
Organizing Section: Visual Neuroscience
Program Number: 6172 Poster Board Number: B0077
Presentation Time: 12:00 PM–1:45 PM
Analysis of SIRT3 function in the mouse retina
Norimitsu Ban1, 2, Takaaki Inaba2, Seiji Miyake1, 2, Kazuo Tsubota2,
Yoko Ozawa1, 2. 1Laboratory of Retinal Cell Biology, Keio University,
Shinjyuku, Japan; 2Ophthalmology, Keio University, Shinjyuku,
Japan.
Purpose: Sirtuins are a family of NAD-dependent deacetylase that
are involved in a variety of cellular functions, including metabolism,
DNA repair, apoptosis, neuronal survival, and inflammation.
SIRT3 is one of the seven mammalian sirtuins, localized mainly
in mitochondria and plays an important role in regulating cell
metabolism. However, functions of SIRT3 in the retina are almost
unknown. In this study, we analyzed the retinal phenotype of SIRT3
knockout (KO) mice.
Methods: 10-week-old male SIRT3 KO and the litter mate wild type
(WT) mice backcrossed to C57BL/6 were used. The mRNA levels of
mitochondria-related genes and antioxidant genes in the retina were
analyzed by real-time PCR. In the retinal sections, retinal thickness
was measured, and the expressions of Rhodopsin, Glial fibrillary
acidic protein (GFAP), Synaptophysin and Tom20 were analyzed
immunohistochemically. We also analyzed visual function by ERG.
Results: No significant differences in mRNA levels of mitochondriarelated and antioxidant genes, retinal thickness, and immunostaining
pattern of each molecule were observed in the retina of SIRT3 KO
mice compared with WT mice. ERG also showed no significant
differences both in the a-wave and b-wave.
Conclusions: The retina of SIRT3 KO mice showed no significant
phenotype at the stage of 10-week-old.
Commercial Relationships: Norimitsu Ban, None; Takaaki Inaba,
None; Seiji Miyake, None; Kazuo Tsubota, None; Yoko Ozawa,
None
Program Number: 6173 Poster Board Number: B0078
Presentation Time: 12:00 PM–1:45 PM
Electrophysiological and Histological Characterizations of
Retinal Changes in a Mouse Model of Sanfilippo Syndrome
Dennis Y. Tse1, Parisa Lotfi2, David L. Simons1, Marco Sardiello2,
Samuel M. Wu1. 1Dept of Ophthalmology, Baylor College of
Medicine, Houston, TX; 2Department of Human and Molecular
Genetics, Neurological Research Institute, Baylor College of
Medicine, Houston, TX.
Purpose: Sanfilippo syndrome or Mucopolysaccharidosis III (MPSIII) is a neurodegenerative autosomal recessive lysosomal storage
disorder in which patients suffer progressive vision loss. Here we
sought to study the underlying functional and morphological changes.
Methods: B6.129S6-Naglutm1Efn/J, the mouse model of the
MPSIIIB, and age-matched wildtype (WT) mice were purchased
from the Jackson lab. Scotopic flash ERG and paired flash ERG
were recorded bilaterally from 8 knockout (KO) and 7 WT mice
when they were 28 and 46-week-old. Rod a-wave leading edges
were modeled as described by Cideciyan and Jacobson (1996). Rod
b-wave was modeled using the Naka-Rushton equation. Mice (4/
group) were sacrificed at the 46th week for immunohistochemistry,
in which staining was performed using double-labeling procedures
on vertical vibratome retinal sections with antibodies for PKCα (rod
bipolar cell), GNAT2 (cones) and the fluorescent nuclear dye TO-
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
PRO3 (photoreceptor somas). Segments of the section taken from
designated central and peripheral regions temporal to the optic disc
were imaged under confocal microscope for cell counting.
Results: At the 28th week, rod a- and b-wave were found
significantly diminished in the KO compared to the WT (a-wave
Rmax± SE: -125±6 vs -316±8μV; b-wave Rmax: 276±13 vs
357±27μV; unpaired t-test, p<0.05). The cone a- and b-waves of the
KO were not significantly different from those of the control at the
28th week, but were significant diminished at the 46th week (Mean
a-wave amplitude± S.E: -27.0±2.7 vs -42.1±3.9μV. Mean b-wave±
S.E: 204.8±17.2 vs 252.9±11.8μV).
KO mice have a reduced mean ONL thickness (number of cells)
in central and peripheral retina (5.75±0.36 vs 10.5±0.001 cells;
and 5±0.31 vs 8.75±0.30 cells; p<0.05). They also have a reduced
number of rod bipolar cells (per 200μm horizontally) in both regions
(5.5±0.36 vs 16.75±0.0001; and 7±0.31 vs 15.5±0.31; p<0.05). There
was no difference in the number of cones in either region.
Conclusions: In an early stage (28th week) of the MPSIIIB model,
function of rods was first affected and was suppressed by about 60%.
Cones became dysfunctional only in a later stage (46th week) and
was suppressed by 36% compared to the control. Deaths of rods and
rod bipolar cell, but not cones, were evident at the 46th week. The
relationship between the rods and the cones pathways during the
degeneration is under investigation.
Commercial Relationships: Dennis Y. Tse, None; Parisa Lotfi,
None; David L. Simons, None; Marco Sardiello, None; Samuel M.
Wu, None
Support: NIH EY004446 & EY019908, NIH Vision Core EY02520,
the Retina Research Foundation (Houston), Research to Prevent
Blindness Inc, Team Sanfilippo Foundation, Swiss Sanfilippo
Foundation, and the International Retinal Research Foundation Loris
and David Rich Postdoctoral Scholar Award.
Program Number: 6174 Poster Board Number: B0079
Presentation Time: 12:00 PM–1:45 PM
Morphology of the Retina in Early Diabetes.
J. Beckman, J. M. Moore-Dotson, M. J. Romero-Aleshire, H. L.
Brooks and E.D. Eggers. Physiology and Biomedical Engineering,
University of Arizona, Tucson, AZ
Jamie Beckman1, Johnnie Moore-Dotson1, Erika D. Eggers1, 2,
Heddwen Brooks1, Melissa Romero-Aleshire1. 1Physiology, University
of Arizona, Tucson, AZ; 2Biomedical Engineering, University of
Arizona, Tucson, AZ.
Purpose: Recent studies have shown early changes in diabetic
retinal activity in vivo. These deficits suggest changes in the activity
or survival of retinal bipolar cells or amacrine cells. The purpose of
this study is to determine if there is an early loss of retinal neurons in
diabetic mice, and if a particular type of bipolar cell is targeted.
Methods: 5 week old C57BL/6J and transgenic Mito-CFP mice were
injected i.p. with streptozotocin (STZ, 3 injections of 75 mg/kg, n=
7 mice) or control vehicle citrate buffer (n= 7 mice). In STZ mice,
diabetes was confirmed by blood glucose levels >200 mg/dL. Six
weeks post injections, eyes were enucleated, retinas removed and
fixed with 3% paraformaldehyde. TOPRO-3 was used in all retinas to
stain nucleic acid in order to count cells in the retinal layers. Retinas
from Mito-CFP mice express CFP in one type of OFF cone BC and
were stained with an anti-GFP antibody. For each retina, four sections
(143 μm2) 500 μm from the optic nerve head in each direction were
imaged with a confocal microscope. The cell numbers from each
section were averaged for each retina. Stained cells were counted in
ImageJ and statistics were done using the Student’s T-test.
Results: The average cells/area in the ganglion cell layer were not
different between control (10563 + 411 cells/mm2, n=7) and STZ
(10571 + 840 cells/mm2, n=7, p= .99) mice. There was no significant
difference in cell numbers of in the inner nuclear layer for control
(38169 + 1642 cells/mm2, n=7) versus the STZ (34592 + 891 cells/
mm2, n=7, p= .08). There was also no significant difference in cell
numbers in the outer nuclear layer of control (56695 + 784 cells/
mm2, n=7) versus STZ (56524 + 747 cells/mm2, n=7, p= .87). OFF
bipolar cell staining from Mito-CFP retinas show no significant
differences cell number in control (3657 + 218 cells/mm2, n=3)
versus STZ (3848 + 469 cells/mm2, n=3, p= .73).
Conclusions: These results suggest that there is no significant loss of
retinal cells after a short duration of diabetes. At this same early stage
of diabetes significant changes in in vivo (ex. Aung et al, 2013) and in
vitro (ARVO abstract Moore-Dotson et al, 2013) retinal activity have
been reported. This indicates that there are changes in neural circuits
before there is any significant cell loss.
Commercial Relationships: Jamie Beckman, None; Johnnie
Moore-Dotson, None; Erika D. Eggers, None; Heddwen Brooks,
None; Melissa Romero-Aleshire, None
Support: Juvenile Diabetes Research Foundation Innovative Award
Program Number: 6175 Poster Board Number: B0080
Presentation Time: 12:00 PM–1:45 PM
Retinal Physiology Is Altered In Glucose-Treated Zebrafish
Retinas
Victoria P. Connaughton1, Zaid Tanvir1, Ralph F. Nelson2. 1Biology,
American University, Washington, DC; 2Neural Circuitry Unit,
NINDS/NIH, Bethesda, MD.
Purpose: To determine changes in retinal physiology (ERG a- and
b-waves) in zebrafish following 1 month exposure to alternating
glucose/hyperglycemic conditions
Methods: Adult, wildtype zebrafish were exposed to alternating 2%
glucose/0% glucose solution for 24hr. Control fish were alternately
exposed to either 0% glucose/0% glucose every 24hr or 2%
mannitol/0% glucose every 24hr (osmotic control). After ~4 weeks
of exposure, physiological responses in superfused retinal eye cups
were examined using ERG. Eye cups were perfused with oxygenated
MEM containing 50mm CNQX to allow isolation of a- and b-waves.
Stimulating wavelengths, 570, 490, 410, and 370nm, corresponded to
the wavelengths closest to maximal stimulation for each of the cone
types in zebrafish. Each wavelength was tested at 7 light intensities.
The stimulation protocol was run four times with each eye cup,
2x with a blue (418nm) background and 2x with a red (627nm)
background to prevent dark adaptation and to better isolate individual
cone mechanisms. Traces were analyzed using pCLAMP and Origin
software.
Results: ERG a-wave and b-wave components were clearly evident
in retinas from all treatment groups. No delay in the onset of either
ERG component was evident at any of the stimulating wavelengths.
b-wave amplitude was consistently reduced in glucose-treated retinas
compared to water-treated controls while b-wave amplitude in
mannitol-treated retinas was increased. Similarly, a-wave amplitude
in glucose-treated retinas was decreased in response to most
stimulating wavelengths (vs. water-treated controls), while a-wave
amplitude in mannitol-treated retinas was increased. The percent
change in mean b-wave amplitude observed in glucose-treated tissue
(30-40%) was consistently larger than the percent change in mean
a-wave amplitude (< 20% change).
Conclusions: Glucose-treated zebrafish retinas show a reduction in
photoreceptor and on-bipolar cell responses after one month. These
results correspond to previous studies showing a thinning of inner
retina (INL, IPL) (Gleeson et al., 2006) and a loss of cones (Alvarez
et al., 2010) after one month of hyperglycemic conditions. These
results suggest that one month of glucose treatment alters physiology
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
in zebrafish retina. These findings are consistent with clinical studies
showing physiological changes in retinas of diabetic patients.
Commercial Relationships: Victoria P. Connaughton, None; Zaid
Tanvir, None; Ralph F. Nelson, None
Support: AU Helmlinge Graduate Award (ZT)
Program Number: 6176 Poster Board Number: B0081
Presentation Time: 12:00 PM–1:45 PM
Visual impairment in mice expressing glaucoma-associated E50K
mutant optineurin
Henry Tseng1, Thorfin Riday2, Howard Bomze1, Ian Barak1, Carrie
Marean-Reardon1, Ben Philpot2, Michael Ehlers3. 1Ophthalmology,
Duke Eye Center, Durham, NC; 2Cell and Molecular Physiology,
University of North Carolina, Chapel Hill, NC; 3Neuroscience
Research Unit, Pfizer, Inc, Cambridge, MA.
Purpose: Glaucoma is a leading cause of irreversible blindness that
results from degeneration of retinal ganglion cells (RGCs). Primary
open angle glaucoma (POAG) is the predominant form of glaucoma
in which the intraocular pressure can be high or normal. A major
hurdle in studying RGC neurodegeneration in normal pressure POAG
is the lack of robust animal disease models. Here, we report a novel
mouse model based on the disease-associated E50K mutation in the
human OPTN gene. This transgenic mouse recapitulates key clinical
characteristics of normal pressure POAG.
Methods: Mice with high over-expression of the E50K mutant
optineurin have been reported, but resulted in diffuse loss of
photoreceptors which is not observed in clinical glaucoma. In
contrast, we generated bacterial artificial chromosome (BAC)
transgenic mice with low overexpression of the E50K human
optineurin (hOPTN) that is closer to normal physiological level.
These mice were aged for at least 1.5 years. Gross ocular anatomy,
intraocular pressure, retinal histology, and normal central targeting
of RGC axons in the brain were assessed. Finally, visual function
was assessed by measuring visually-evoked potentials (VEPs) in the
primary visual cortex.
Results: These E50K optineurin BAC transgenic mice exhibit
normal ocular anatomy, intraocular pressure, and normal RGC axonal
projections in the brain as seen in patients. Despite ~30% loss of
RGCs, the remainder of the retina appears normal histologically.
Functionally, via VEP electrophysiological assessment, these mice
exhibit a significant reduction in contrast detection, but not visual
acuity or motion detection.
Conclusions: As in human disease, mild overexpression of E50K
hOPTN in our BAC transgenic mice resulted in selective loss of
RGCs and functional visual impairment. These transgenic mice
provide a novel disease model for normal-pressure POAG that may
provide mechanistic insights into the RGC degeneration associated
with this poorly understood disease, as well as providing a platform
to test novel therapeutics.
Commercial Relationships: Henry Tseng, None; Thorfin Riday,
None; Howard Bomze, None; Ian Barak, None; Carrie MareanReardon, None; Ben Philpot, None; Michael Ehlers, Pfizer (E)
Support: K12-EY016333 (HT), K08-EY021520 (HT), R01EY018323 (BP), Howard Hughes Medical Institute (ME)
Program Number: 6177 Poster Board Number: B0082
Presentation Time: 12:00 PM–1:45 PM
Visual phenotyping of Wfs1 mutant mice, models of Wolfram
syndrome neuronal and diabetic symptoms
Cecile Delettre1, Christian P. Hamel1, Sulev Koks2, Marie Seveno1,
Guy Lenaers1, Delphine M. Bonnet Wersinger1. 1INSERM U1051,
Montpellier, France; 2University of Tartu, Tartu, Estonia.
Purpose: Wolfram syndrome is an early onset genetic disease
(1/160,000) featuring diabetes mellitus and optic neuropathy,
associated to mutation in the WFS1 gene. Mouse model with deleted
exon 8 of Wolframin shows pancreatic beta cell atrophy, but its visual
performance has not been investigated, prompting us to study its
visual function and the histopathology of the retina and optic nerve.
Methods: Electroretinogram (ERG, retinal function) and visual
evoked potentials (VEPs, visual pathway) were performed in
Wfs1-/- and Wfs1+/+ mice at 3, 6 and 9 months of age. Fundi were
pictured with Micron III apparatus. Retinal ganglion cell (RGC)
proportion was determined from Brn3a immuno-labeling of retinal
sections. RGC axonal loss was quantified by electron microscopy in
transversal optic nerve sections.
Results: ERG showed a sex-dependent alteration in Wfs1 mutant
mice at 3 months. Photoreceptor response amplitude (a-wave) was
increased by 25.5% by Wfs1 mutation in females, while reduced by
28.2% in males. In contrast, positive scotopic threshold responses
(pSTR) at the same age were found increased in mutant group by
20.5%. A preliminary study of 7 months male samples showed a
severe loss of RGC somas (-50%) and axons in retina and optic nerve
respectively. Finally, 7-8 months knocked-in mice presented a severe
ocular hypertension.
Conclusions: Electrophysiological phenotyping of Wfs1 deleted
mouse exon 8 visual function indicate a significant loss of RGC in
mutant mouse at 7 month. Structural analysis of retinal ganglion cell
somas and axons are conducted to characterize optic neuropathy in
these animals.
Commercial Relationships: Cecile Delettre, None; Christian
P. Hamel, None; Sulev Koks, None; Marie Seveno, None; Guy
Lenaers, None; Delphine M. Bonnet Wersinger, None
Support: INSERM, FRM, Retina France, Association Syndrome de
Wolfram
Program Number: 6178 Poster Board Number: B0083
Presentation Time: 12:00 PM–1:45 PM
Collagen 4a1 deficiency induces a vascular leakage in the retina
Alix Trouillet1, Emmanuelle Plaisier2, Brahim El Mathari1, Henri
Lorach1, Ivana Ivkovic1, Julie Degardin1, Michel Paques1, Pierre
Ronco2, Jose A. Sahel1, Serge A. Picaud1. 1Institut de la Vision, Paris,
France; 2APHP- Hopital Tenon, Paris, France.
Purpose: HANAC syndrome is an autosomal dominant hereditary
angiopathy with nephropathy, aneurysms, and muscle cramps. People
with HANAC syndrome can also experience occasional visual
problems due to arterial retinal tortuosity, cataract or Axenfeld-Rieger
abnormalities. It has been shown that a mutation in the gene COL4A1
is responsible for these symptoms. COL4A1 gene codes for a subtype
of collagen protein mainly located in the basement membrane
surrounding blood vessels. We recently found rod and cone
dysfunction in a COL4A1 mutant mouse at 9 months. In the present
paper, we have analyzed vascular changes in this animal model to
better understand the disease physiopathology and investigated retinal
cell dysfunction at earlier stages.
Methods: ERG was performed to evaluate retinal function on Col4a1
deficient mice. Retinal structure was first examined in vivo using
OCT, SLO and Micron III and then on histological sections. Blood
vessels were stained on the flat-mounted retina and measured by an
automated analysis. Retinal vascular permeability was quantified with
the Evans blue dye method. Animals were examined at 3 months and
9 months old.
Results: ERG measurements showed greater cell dysfunction with
the animal aging. Mutation in col4a1 was also associated with retinal
vessel tortuosity, which was seen only in aging animals confirming
thereby age-related changes and vascular reorganization. When
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
the integrity of retinal vessels were examined at 9 months with
fluorescence angiography, local area of vascular leakage appeared
in different mutant animals but not in control mice. To further
investigate this question, Evans blue was perfused in the animal
vascular system. This demonstrated the increased albumin vascular
leakage in mutant mice with respect to control animals. Finally, we
examined retinal vessels at the ultrastructural level and found local
thinning of the basement membrane in mutant animals.
Conclusions: This study indicates that the col4a1 deficiency can
induce progressive phenotypic retinal features such as retinal cell
dysfunction, blood vessel tortuosity, vascular leakage and thinning
of the blood basement membrane, all symptoms also observed in
diabetic retinopathy. The mouse models may therefore be used to
asses further retinal symptoms reported in patients affected by the
HANAC syndrome but it could also become an interesting model for
vascular retinal pathologies especially diabetic retinopathy.
Commercial Relationships: Alix Trouillet, None; Emmanuelle
Plaisier, None; Brahim El Mathari, None; Henri Lorach, None;
Ivana Ivkovic, None; Julie Degardin, None; Michel Paques, None;
Pierre Ronco, None; Jose A. Sahel, None; Serge A. Picaud, None
Program Number: 6179 Poster Board Number: B0084
Presentation Time: 12:00 PM–1:45 PM
Characterization of a refined mouse model of retinal vein
occlusion
Andreas Ebneter, Cavit Agca, Sebastian Wolf, Volker Enzmann,
Martin S. Zinkernagel. Department of Ophthalmology, University of
Bern, Bern, Switzerland.
Purpose: Retinal vein occlusion represents a chronic disorder of the
inner retina with hypoxia, vascular leakage and neuronal degeneration
causing significant morbidity. A reliable retinal vein occlusion model
in mice would be a valuable tool for the investigation of specific
questions regarding this disease using genetically modified animals.
The aim of the current work was to refine and further characterize a
model of retinal vein occlusion in mice.
Methods: Retinal vein occlusion was induced in BALB/c mice
by indirect laser photocoagulation (532 nm) of 1-2 veins one disc
diameter from the optic nerve after intravenous tail vein injection
of Rose Bengal (25mg/kg), a photo-activator dye to enhance
thrombus formation. Color fundus photographs (Optomap), retinal
OCT imaging and fluorescein angiography using a 55° optic were
performed at baseline, days 3, 7 and 14 after induction of the venous
blockage. Mice were killed at various time-points and eyes processed
for histology.
Results: Experimental retinal vein occlusion caused significant
vascular change in the affected retina with remodeling of capillaries
and shunt-vessel formation (Fig. 1). However, significant retinal
thickening was not seen during the observational period using OCT
imaging in this model with relatively mild laser. Nonetheless, retinal
thinning (Fig. 2) was observed in the affected quadrant from day 7
onwards on OCT (p<0.001; one-way ANOVA for repeated measures).
Imaging data corresponded well with findings on hematoxylin/eosin
sections (Fig. 2).
Conclusions: Similar to the changes in humans suffering from retinal
vein occlusion, experimental venous blockage induced shunt-vessel
formation. Different intensities of laser application may mimic
different severities of disease. However, some facets of human
macular disease such as leakage and edema may not be appropriately
represented due to structural differences. Further studies are needed
to closer characterize this model, but the paradigm seems suitable
to gain valuable insight into patho-mechanisms occurring during
vascular remodeling after vein occlusion and other aspects of
ischemic retinal disease.
Figure 1: Fluorescein angiography showing vascular remodeling 7
days after experimental retinal vein occlusion
Figure 2: Retinal thickness (95% CI) in the affected quadrant at
different time-points after experimental retinal vein occlusion. H&E
image shows degeneration of inner retinal layers 6 weeks after the
insult
Commercial Relationships: Andreas Ebneter, Novartis (I),
Novartis (R); Cavit Agca, None; Sebastian Wolf, Allergan (C),
Allergan (F), Allergan (R), Bayer (C), Bayer (F), Bayer (R), Euretina
(S), Heidelberg Engineering (C), Heidelberg Engineering (F),
Novartis (C), Novartis (F), Novartis (R), Optos (C), Optos (F), Optos
(R); Volker Enzmann, None; Martin S. Zinkernagel, Allergan (F),
Bayer (F), Heidelberg (C), Heidelberg (F), Novartis (C), Novartis (F),
Novartis (I)
Support: OPOS Foundation, Switzerland
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 6180 Poster Board Number: B0085
Presentation Time: 12:00 PM–1:45 PM
Corneal ERG Topography in Healthy Rat Eyes and Eyes with
Focal Retinal Lesions
Zahra Derafshi1, Hadi Tajalli1, Sanitta Thongpang2, Justin Williams2,
John R. Hetling1. 1Bioengineering, University of Illinois at Chicago,
Chicago, IL; 2Biomedical Engineering, University of WisconsinMadison, Madison, WI.
Purpose: Spatial differences in ERG potentials recorded from
different locations on the cornea (ERG topography) reflect spatial
differences in retinal activity, and may therefore have diagnostic
value. Multi-electrode electroretinograms (meERG), consisting of 25
simultaneously recorded ERG waveforms, have been recorded from
rats using a Contact Lens Electrode Array (CLEAr Lens) to evaluate
sensitivity of ERG topography to local retinal lesions.
Methods: meERG responses were recorded from seven Long Evans
rats following full-field flash stimuli; animals were prepared as for
conventional ERG (dark adapted, general anesthesia, pupil dilation,
corneal anesthetic). Four out of the seven rats then received local
damage (adjacent to optic disk but restricted to one hemisphere)
using laser photocoagulation, and a second set of meERG responses
was recorded from all rats, resulting in nine healthy eye data sets and
4 lesion eye data sets.
Results: To evaluate spatial symmetry in ERG potentials, the a-wave
amplitudes measured on each of the 12 peripheral electrodes were
normalized to the average amplitudes on the central five electrodes,
which resulted in 12 ratios for each eye. These ratios were averaged
(by electrode position) for all nine healthy-eye meERG responses
to form a normative data set (containing 12 average ratios). In laser
damaged eyes, ratios were reduced compared to healthy eyes in
areas of the cornea closest to the area of laser damage at the retina.
A cluster analysis was performed, using the 12 ratios as coordinates
in a 12-dimensional space, and calculating the Euclidean distance
between each response and the normative mean response. Average (±
one SD) distances for healthy eyes (using a leave-one-out technique)
was 6 ± 2 and for laser damaged eyes was 12 ± 5. Using these
distances as the sole metric to distinguish healthy from laser-damaged
eyes resulted in the area under an ROC curve of ~90%.
Conclusions: Spatial differences in a-wave amplitudes across the
cornea are altered for rat eyes having a local lesion in the retina.
Lesions hear the posterior pole of the retina resulted in measureable
changes in corneal ERG topography (evaluated by the meERGderived ratios), yielding good sensitivity and specificity. Corneal
ERG topography is a novel source of information, independent of
absolute ERG amplitudes, and obtained using relatively simple fullfield stimuli.
Commercial Relationships: Zahra Derafshi, None; Hadi Tajalli,
None; Sanitta Thongpang, None; Justin Williams, None; John R.
Hetling, Retmap Inc. (P)
Support: 1R21EY018200-01A2
Program Number: 6181 Poster Board Number: B0086
Presentation Time: 12:00 PM–1:45 PM
Electroretinogram Findings in Animal Model of Concurrent
Diabetes and Hypothyroidism
Bruno D. Gomes1, Glenda F. Guimarães1, Natielle F. Rabelo1,
Moisés Hamoy1, Luiz Carlos L. Silveira1, 2, Anderson M. Herculano1,
Fernando Allan F. Rocha1. 1Instituto de Ciências Biológicas,
Universidade Federal do Pará, Belém, Brazil; 2Núcleo de Medicina
Tropical, Universidade Federal do Pará, Belém, Brazil.
Purpose: Investigate the retinal functional impairment due to
concurrent diabetes and hypothyroidism using electroretinogram
(ERG).
Methods: 16 Wistar rats (Rattus norvegicus) with two-monthsold, were divided in four groups: control (glucose: 80.7±15.2;
weight: 165.1±9.2); group with hypothyroidism induced by bilateral
thyroidectomy (glucose: 84.4±12.1mg/dl; weight: 157.2±9.7g);
group with diabetes induced by injection of 2% alloxan (200 mg/kg)
intraperitoneally (glucose: 389.6±87.3mg/dl; weight: 189.7±12.8g);
group with both, diabetes and hypothyroidism (glucose:
412.1±70.04mg/dl; weight: 187.53±14g). After 30 days of treatment
electroretinograms were obtained using flash stimuli to evaluate
physiological changes in scotopic and photopic light adaptation. Rod
driven ERGs were obtained with 10 cd/m2.s flash after two log units
attenuation. Mixed rod-cone driven ERGs were elicited with 10 cd/
m2.s stimulation after 12 hours scotopic adaptation. Cone driven
ERGs were obtained with 10 cd/m2.s stimulation after 10 minutes
photopic adaptation.
Results: There was a decrease in the average amplitude of the aand b-wave in animals with diabetes and both metabolic diseases in
all light adaptations. When comparing the group with diabetes and
the group with hypothyroidism, it was clear that diabetes provoked
greater decrease in ERG amplitude (one-way ANOVA, p= 0.05).
Moreover, the animals with both diseases showed a synergistic
action of diabetes and hypothyroidism as verified by ERG amplitude
decrease. In combined response (rods and cones) the highest
statistical differences among groups were found. The mean a- and
b-wave amplitude values were a-79.71 mV (±15.18) and b-99.95 mV
(±30.60) for the group with both diseases; a-100.4 mV (± 13.31) and
b-165.7mV (± 20.85) for the group with diabetes; a-160 mV (± 19.72)
and b-280.3 mV (± 54.47) for the group with hypothyroidism; a-179.3
mV (± 22.79) and b-332.3 mV (± 57.53) for control group.
Conclusions: The results support the hypotheses that both,
photoreceptors and inner layers of the retina were affected by
both diseases, but remarkably by diabetes. All ERG responses
were significantly impaired, mainly those recorded after scotopic
adaptation
Commercial Relationships: Bruno D. Gomes, None; Glenda F.
Guimarães, None; Natielle F. Rabelo, None; Moisés Hamoy, None;
Luiz Carlos L. Silveira, None; Anderson M. Herculano, None;
Fernando Allan F. Rocha, None
Support: CNPq#479500, CAPES, FINEP, FAPESPA, UFPA. LCLS
is a CNPq research fellow.
Program Number: 6182 Poster Board Number: B0087
Presentation Time: 12:00 PM–1:45 PM
A familial abnormal negative photopic ERG in Papillon dogs
Simon M. Petersen-Jones, Kristen J. Gervais, Paige A. Winkler, Freya
M. Mowat, Laurence M. Occelli, Joshua T. Bartoe. Small Animal
Clinical Sciences, Michigan State University, East Lansing, MI.
Purpose: To report a familial unusual negative photopic
electroretinographic waveform in the Papillon breed of dog. We
hypothesized that this was an inherited trait in the breed.
Methods: Electroretinographic studies were performed on Papillon
dogs from within an extended pedigree. They consisted of a darkadapted intensity response series recorded from below threshold up
to a bright flash. Then following light adaptation (10 mins; 30 cd/
m2) a light-adapted intensity response series was recorded. Vision
testing was also performed using a previously described four-choice
exit device. Tunnel choice and time to exit the device were recorded.
Ability to exit the device was tested under a series of lighting
conditions ranging from bright to very dim.
Results: The dark-adapted ERGs appeared of normal shape.
However, the amplitude of the STR of the dogs with the abnormal
photopic ERG was significantly greater than that of unaffected breed
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
matched control dogs. The dark-adapted b- to a-wave ratio was
significantly lower in affected dogs compared to controls.
The light-adapted waveforms of affected dogs had a negative
component that led to a large post b-wave negativity. In the more
severely affected dogs the b-wave was reduced to a small elevation
on a negative slope. The ratio between the amplitudes of post b-wave
negativity and the b-wave was significantly greater in affected dogs
compared to controls and increased with age.
Vision testing did not suggest a major effect on vision. However,
affected dogs took longer to exit the device at the brightest lighting
intensity compared to the second brightest light intensity. Whereas
the normal control dogs took the same time to exit the device at the
brightest and second brightest light intensities.
Crossbreeding an affected Papillon to a normal beagle produced a
litter with 2 of 8 puppies developing the negative photopic ERG.
Conclusions: We report an unusual familial negative photopic ERG
in Papillon dogs with evidence of increased negative components
in the scotopic ERG. Similar ERG changes have been reported to
develop in RCS rats as a result of aberrant amacrine cell signaling
(Machida et al IOVS 2008). Although the affected Papillons appeared
to have a slight reduction in visual performance in bright light there
was no evidence of a retinal degenerative process. Further studies are
required to show the origin of this negative component of the ERG
waveform.
Commercial Relationships: Simon M. Petersen-Jones, None;
Kristen J. Gervais, None; Paige A. Winkler, None; Freya M.
Mowat, None; Laurence M. Occelli, None; Joshua T. Bartoe, None
Support: Myers-Dunlap Endowment for Canine Health
Program Number: 6183 Poster Board Number: B0088
Presentation Time: 12:00 PM–1:45 PM
Dietary ω3 fatty acids & metabolic syndrome in the rat :
consequences on sensitivity and retinal functionality
Magalie Thierry1, Bruno Pasquis2, Valérie Febvret1, Stéphane
Grégoire1, Laurent Leclère1, Niyazi Acar1, Catherine P. Garcher1,
3
, Alain M. Bron1, 3, Lionel Bretillon1. 1Eye, Nutrition & Signalling
Research, CSGA, UMR 1324 INRA, 6265 CNRS, University of
Burgundy, Dijon, France; 2Animalerie expérimentale, CSGA, UMR
1324 INRA, 6265 CNRS, University of Burgundy, Dijon, France;
3
Department of Ophtalmology, University Hospital, Dijon, France.
Purpose: High fructose diets have been widely used to trigger
metabolic syndrome (MetS) in rodents. Insulin resistance is a major
risk factor for the development of type 2 diabetes leading in 30
to 40% of the cases to the development of diabetic retinopathy in
humans. In the other hand, ω3 fatty acids have been associated with
the prevention of MetS. Few studies have been interested in the
early changes occurring in the retina after induction of MetS and to
potential of ω3 fatty acids to reverse those effects.
Methods: 96 male Brown Norway rats (6 weeks of age) were fed
for 3 and 8 days with either of the four following diets (n=8 in each
group): a regular chow diet (S), a 60% fructose enriched diet (F),
a regular chow diet enriched with ω3 fatty acids (Sω3) or a 60%
fructose + ω3 fatty acids-enriched diet (Fω3). Flicker (8Hz) and
scotopic single flash electroretinograms were recorded from both
eyes to study respectively the sensitivity of the photoreceptors and
the functionality of the retina. At the time of euthanasia, blood was
collected to measure glycaemia and plasma circulating insulin and
leptin levels. The fatty acid profile of the retina was analyzed by gas
chromatography.
Results: We reported a significant loss of cone sensitivity (Δ=1.5 log)
after 8 days of fructose feeding which was not counterbalanced by ω3
supplementation. However, ω3 supplementations reduced the latency
time of the a- and b-waves at high light intensities after 8 days of
feeding (b-wave: S/Sω3 -7ms F/Fω3 -9ms ; a-wave : S/Sω3 -6ms F/
Fω3 -3ms ). The amplitude of the a-and b-waves was not affected by
the different diets. Plasma analyses showed a significant increase of
insulin and leptin levels after 8 days of fructose feeding (respectively
+64%, +173%). Fω3 diet significantly increased plasma insulin but
restored leptin levels. ω3 fatty acids were expectedly incorporated in
the retina and in the brain of rats supplemented with ω3 FA (+0.65%
DHA ). The levels were surprisingly increased to higher levels in Fω3
fed rats.
Conclusions: Our findings in rats fed with fructose suggested that
the early steps of MetS were characterized by hyperinsulinemia and
hyperleptinemia, and were associated to the loss of cone sensitivity.
ω3 FA, that have been associated with the prevention of MetS,
showed beneficial although marginal effects on photoreceptors and
inner retinal cells.
Commercial Relationships: Magalie Thierry, None; Bruno
Pasquis, None; Valérie Febvret, None; Stéphane Grégoire, None;
Laurent Leclère, None; Niyazi Acar, None; Catherine P. Garcher,
None; Alain M. Bron, None; Lionel Bretillon, None
Program Number: 6184 Poster Board Number: B0089
Presentation Time: 12:00 PM–1:45 PM
Photoreceptor and post-photoreceptoral contribution to
reduction of photopic b-wave ERG in light-adapted Pikachurin
null-mutant mice
Masatoshi Nagaya1, Shinji Ueno1, Mineo Kondo2, Takahisa
Furukawa3, Hiroko Terasaki1. 1Ophthalmology, Nagoya University
Graduate School of Medicine, Nagoya, Japan; 2Ophthalmology, Mie
University Graduate School of Medicine, Tsu, Japan; 3Institute for
Protein Research, Osaka University, Osaka, Japan.
Purpose: The amplitude of the b-wave of the ERG increases during
light-adaptation, but the mechanism for this increase has not been
determined. The Pikachurin null-mutant mice (Pika -/-) have a
misalignment of the bipolar cell dendritic tips to the photoreceptor
ribbon synapses which alters the post-receptoral responses. We
have shown that the photopic b-wave did not increase but decreased
during light-adaptation in Pika -/- mice (ARVO 2013). The purpose
of this study was to determine the cellular components that cause the
decrease in the b-waves during light-adaptation in Pika -/- mice .
Methods: Pika -/- and control C57 BL6J mice (WT) of 8 to 12-weeks
of-age were studied. After at least 6 hours of dark-adaptation, animals
were anesthetized and placed in a Ganzfeld bowl. ERGs were
elicited by stroboscopic stimuli, and 20 to 30 ERGs were averaged
with a repetition rate of 1 sec. The photopic stimulus intensity was
1.0 log cd-s/m2 under 30 cd/m2 of background light. ERGs were
recorded immediately after the beginning of light-adaptation and
recorded periodically for 10 minutes during the light-adaptation.
Eleven Pika -/- and 11 WT mice were injected toDL-2-amino-4phosphonobutyric acid (APB) and cis-2, 3-piperidine-dicarboxylic
acid (PDA) intravitreally to block the post-receptoral ON and OFF
bipolar cell components. The post-receptoral contributions were
calculated by subtracting the ERGs after APB+PDA injection from
ERGs after PBS injection.
Results: The amplitude of photopic b-waves of WT mice increased
by 50% of the pre-light-adaptation amplitude after 10 minutes of
light-adaptation. On the other hand, the amplitude of the b-wave of
Pika -/-mice decreased by 20%. After the APB+PDA intravitreal
injection, which blocked the post receptor potentials, only the a-wave
remained in both Pika -/- and WT mice. In both types of animals, the
a-waves increased by 30% during the 10 minutes of light-adaptation.
The post-receptoral contribution, i.e., the difference between the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
PBS-treated eyes and APB+PDA-treated eyes, increased by 70% in
WT mice and decreased by 30% in Pika -/-mice.
Conclusions: Our results suggest that the reduction of the b-wave
amplitudes in Pika -/- mice during light-adaptation was caused
mainly by a reduction of the post-receptoral potentials.
Commercial Relationships: Masatoshi Nagaya, None; Shinji
Ueno, None; Mineo Kondo, None; Takahisa Furukawa, None;
Hiroko Terasaki, None
Program Number: 6185 Poster Board Number: B0090
Presentation Time: 12:00 PM–1:45 PM
Effects of Quensyl on the ERG a-wave amplitude from the
isolated superfused vertebrate retina
Siarhei Siapich, Andrea Goebel, Peter Walter. RWTH UKAachen,
Aachen, Germany.
Purpose: Long-term therapy with quensyl is known to cause
neurodegenerative changes in the retina. In our present research
we study acute toxic effects of quensyl on the a-wave response of
electroretinogram (ERG) of isolated superfused bovine retinae
Methods: Isolated bovine retinae were mounted in a temperaturecontrolled recording chamber. After light stimulation electric field
potentials were recorded as a transretinal potential using Ag/AgClelectrodes. Isolated bovine retinas were perfused with phosphate
buffered saline (PBS) containing 1mM L-aspartate to block further
synaptic transmission in order to record the effects of quensyl
on photoreceptors. We tested the low and high light intensities
of 100 mlux and 10 lux. After reaching a stable ERG amplitude,
quensyl (190 mM, 570 mM or 1,9 mM) was added to the perfusing
solution. After 90 min quensyl was washed out for 90 min with PBS
containing 1mM L-aspartate. Changes in a-wave amplitude were
calculated and plotted
Results: 190 mM quensyl showed a 1,3 fold stimulation of the
a-wave amplitude at 100 mlux, the effect at 10 lux was not
significant. 570 mM quensyl reduced the a-wave amplitude by 3-folds
independent of light-intensity. The inhibition was good reversible
by washing with PBS containing 1mM L-aspartate only at low light
intensity (2 folds), there was almost no recovery at 10 lux. 1,9 mM
quensyl showed a massive depression of a-wave amplitude (6 to 7
folds) and no wash out effect over 90 minutes at both light intensities
Conclusions: Quensyl has a toxic effect on photoreceptors, even
with slight increase of concentration showing a huge progression of
inhibition and reduction of recovery. An exact dosage of Quensyl is
of great importance to avoid an irreversible neuronal damage
Commercial Relationships: Siarhei Siapich, None; Andrea
Goebel, None; Peter Walter, None
Program Number: 6186 Poster Board Number: B0091
Presentation Time: 12:00 PM–1:45 PM
Differentiating between Ischemic and Non-Ischemic Origins of
the “Negative” Electroretinogram
Naoyuki Tanimoto1, James D. Akula2, Anne Fulton2, Bernhard
H. Weber3, Mathias W. Seeliger1. 1Division of Ocular
Neurodegeneration, Ctr Ophthalmol Inst Ophthalmic Res, Tuebingen,
Germany; 2Department of Ophthalmology, Boston Children’s
Hospital and Harvard Medical School, Boston, MA; 3Institute of
Human Genetics, University of Regensburg, Regensburg, Germany.
Purpose: Strong attenuation of the electroretinographic (ERG)
b-wave in the presence of a normal or less reduced a-wave (‘negative
ERG’) is typically caused either by defects in synaptic transmission
between photoreceptors and ON-bipolar cells or by ischemia of the
inner retina. The purpose of this study was to explore whether ERG
flicker responses permit discrimination of synaptic and ischemic
cases.
Methods: Three murine models of ophthalmic disease demonstrating
negative ERG, the nob1 mouse model of congenital stationary night
blindness characterized by deficits in the ON-pathway, the oxygeninduced retinopathy rat model of retinopathy of prematurity (‘ROP
rat’) characterized by inner retinal ischemia, and the Rs1hy/- mouse
model of X-linked juvenile retinoschisis, were studied. After a
dark-adapted single-flash ERG intensity series (-4 to 1.5 log cd s/
m2), a flicker ERG frequency series (12 steps from 0.5 to 30 Hz) at
the International Society for Clinical Electrophysiology of Vision
standard flash (ISCEV SF) intensity (0.5 log cd s/m2) was performed.
This series was considered in three frequency ranges that are
dominated by activity in the A) rod system (<5 Hz), B) cone ONpathway (5-15 Hz), and C) cone OFF-pathway (>15 Hz).
Results: In ROP rats, photoreceptors are supplied by the choroid but
both ON- and OFF-bipolar cells are ischemic due to loss of retinal
vasculature; correspondingly, ROP rats had nearly normal a-waves
but reduced flicker responses in all ranges (A-C). nob1 mice likewise
have normal photoreceptor function and, thus, a-waves were not
reduced. As only the ON-pathway is defective in nob1 mice, flicker
signals were reduced in ranges A and B but not C. In Rs1hy/- mice,
there are patchy alterations in both the photoreceptor and bipolar
cell layers, but not in the same vertical column. Consequently, the
fraction of functional photoreceptors is larger than that of functional
bipolar cells receiving photoreceptor signals. Therefore, there was
a noticeably reduced a-wave, an even more reduced b-wave, and
attenuated flicker responses in all ranges (A-C).
Conclusions: The response to flickering ISCEV SF light at specified
frequencies can be compared to the size of the a-wave to discriminate
the pathophysiology of the negative ERG, including synaptic and
ischemic conditions.
Commercial Relationships: Naoyuki Tanimoto, None; James D.
Akula, None; Anne Fulton, None; Bernhard H. Weber, None;
Mathias W. Seeliger, None
Support: DFG Se837/5-2 (KFO134), Se837/6-2, Massachusetts
Lions Eye Research Fund
Program Number: 6187 Poster Board Number: B0092
Presentation Time: 12:00 PM–1:45 PM
The neuroprotection of Angiotensin-(1-7) in Tg(RHO P347S) and
rd10 mice
Ping Zhu, Pollob K. Shil, Wen-tao Deng, Jie Li, Amrisha Verma,
Tuhina Prasad, Qiuhong Li. Ophthalmology, University of Florida,
Gainesville, FL.
Purpose: Ang-(1-7) is an endogenous peptide that counterregulates the effect of Angiotensin II and produces vasodilation,
anti-inflammation and cardioprotection. In addition, Ang-(1-7) also
confers cerebroprotection against ischemia and improves learning and
memory. These effects are mediated by Mas receptor. We recently
show that Mas receptor is expressed in both adult and developing
mouse retina, more abundant in retinal neurons than endothelial and
muller glial cells. We hypothesize that increased Ang-(1-7) would
confer retinal neuroprotection and slow photoreceptor degeneration.
We tested this hypothesis in two different animal models of
photoreceptor degeneration: Tg(RHO P347S) and rd10 mice, by
intraocular delivery of AAV vector expressing Ang-(1-7).
Methods: The Tg(RHO P347S) pups were injected with 1 μl (1×
1012 vg/ml) of AAV2 vector expressing secreted form of Ang-(1-7)
intravitreally in right eyes on postnatal day 10-15 (P10-15) and rd10
pups on P9. The left contralateral eyes served as untreated controls.
Full-field rod- and cone-mediated ERG were recorded at P60 for
Tg(RHO P347S) and 6 wk for rd10 mice. A series of increasing
flash intensities (−3.7,-1.7,−0.7, 0.3, 2.3, and 2.8 log cds/m2) were
recorded after overnight dark adaptation. Photopic recordings were
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
taken after mice were adapted to a white background light of 2.8
log cd/m2 for 5 min. Rod and cone photoreceptor degeneration was
evaluated by immunofluorescence staining, in situ apoptosis detection
and histology.
Results: There was no significant difference between treated and
untreated eyes in rod-driven scotopic ERG responses in both models.
However cone-driven photopic ERG was significantly improved in
Ang-(1-7)-AAV treated eyes. The average cone b-wave amplitude
of Tg(RHO P347S) mice was 140.38 ± 32.76 μV in injected eyes
versus 119.65 ± 24.78 μV in untreated eyes at 2.8 log cds/m2(n=6).
The average cone b-wave amplitude of rd10 was 206.37 ± 24.87
μV in injected eyes versus 173.67 ± 24.42 μV in untreated eyes at
2.8 log cds/m2 (n=3). Ang-(1-7)-AAV vector treated eyes also show
significantly reduced cone photoreceptor loss detected by cone opsin
staining compared to untreated eyes.
Conclusions: Increased ocular expression of Ang-(1-7) slowed
the progression cone photoreceptor degeneration in both Tg(RHO
P347S) and rd10 mice, supporting the protective role of Ang-(1-7)
in the retina and this approach may provide a general strategy for
neuroprotection.
Commercial Relationships: Ping Zhu, None; Pollob K. Shil, None;
Wen-tao Deng, None; Jie Li, None; Amrisha Verma, None; Tuhina
Prasad, None; Qiuhong Li, None
Support: American Diabetes Association, Research to prevent
blindness, NIH grants EY021752 and EY021721
Program Number: 6188 Poster Board Number: B0093
Presentation Time: 12:00 PM–1:45 PM
Non-metric Multidimensional Scaling of Electroretinogram
Oscillatory Potential Data achieves Early Diagnosis of Retinal
Degeneration in a Mouse Model of Mitochondrial Dysfunction
Eric Dolinar1, Thomas MacPherson1, Alex Laliberté1, Jolien Van
Gaalen1, Cindy M. Hutnik2, Kathleen Hill1. 1Biology, University of
Western Ontario, London, ON, Canada; 2Ivey Eye Institute, London
Health Sciences Center, London, ON, Canada.
Purpose: Early mechanisms of retinal degeneration (RD) in humans
are poorly understood. As a proxy, we study the harlequin (hq)
mouse which carries an X-linked Apoptosis-inducing factor mutation
leading to mitochondrial dysfunction and subsequent RD. The hqY
genotype exhibits severe disease with early onset whereas female
carriers (hqX) exhibit delayed onset and moderate disease. We
reported parainflammation in hqY retinas with functional deficits at
two months and structural deficits by four months of age. We seek
a non-invasive, in vivo diagnostic for assessing retinal integrity at
high resolution. Components of an electroretinogram (ERG) for four
flash intensities (0.63, 4, 10, 25 cd*s/m2) are used to assess retinal
layer function. Oscillatory potentials (OPs) are associated with inner
plexiform (IPL) and ganglion cell layer (GCL) function. We used
OPs to track retinal integrity with hq disease onset and progression.
Methods: Two mutant genotypes (hqY and hqX) were compared
to gender-matched WT mice at multiple ages relevant to disease
progression. A 5th order Butterworth filter (65-300Hz) was applied to
smooth OPs and filter out the a and b-wave contribution. Latencies
and amplitudes of OPs1-4 (OP parameters) were measured as they
model neural activity and response. A Fast Fourier Transform (FFT)
converted OP waveforms from time-domain to frequency-domain.
The major frequency and total power were recorded. Total energy
of the neural-retinal response was calculated by integration of the
frequency-domain OP waveform. Due to the longitudinal attritive
nature of the study, a non-metric multidimensional scaling analysis
(NMDS) was applied.
Results: In NMDS, early hqY disease (2 months) cluster due to
energy, power and frequency changes. By three months, the OP
parameters influencing distinct hqY NMDS clustering are reduced
amplitude and increased latency. With age, hqX retinas show specific
NMDS clustering reflective of reduced FFT power and energy. hqX
NMDS clustering for OP parameters reflects reduced amplitude and
increased latency with disease progression.
Conclusions: We established a sensitive and comprehensive
experimental framework using 44 parameters associated with ERG
OPs, that with NMDS detects IPL and GCL deficits arising early in
hq mitochondrial dysfunction that lead to later photoreceptor losses.
Commercial Relationships: Eric Dolinar, None; Thomas
MacPherson, None; Alex Laliberté, None; Jolien Van Gaalen,
None; Cindy M. Hutnik, None; Kathleen Hill, None
Support: Plunkett Foundation, CIHR-Canadian Institutes of Health
Research
Program Number: 6189 Poster Board Number: B0094
Presentation Time: 12:00 PM–1:45 PM
Daily optokinetic testing improves contrast sensitivity through
BDNF-mediated pathways
Amanda Mui1, Brian C. Prall2, Moe H. Aung1, 2, Tracy Obertone1,
Hanna Park1, Jeffrey H. Boatright1, Machelle T. Pardue1, 3.
1
Ophthalmology, Emory University, Atlanta, GA; 2Neuroscience,
Emory University, Atlanta, GA; 3Veterans Affairs Medical Center,
Atlanta, GA.
Purpose: Daily exposure of rodents to optokinetic tracking (OKT)
assessment in early development leads to hypernormal visual
acuity thresholds, an effect associated with upregulation of brain
derived neurotrophic factor (BDNF) in the retina. In this study, we
investigated (1) whether visual benefit after daily OKT was localized
in the retina and generalizable to other visual tasks and (2) whether
these visual improvements were mediated by BDNF pathway
activation.
Methods: C57BL/6J mice (n=29) received daily intraperitoneal
injections of TrkB receptor antagonist, ANA-12, or vehicle (1%
DMSO and 16.5% Cremphor EL) from P16 to P23. At 1.5-2 hours
after the injection, mice were placed in an OptoMotry© OKT
chamber (CerebralMechanics, Lethbridge, Alberta, Canada) to be
stimulated with full OKT testing (stimulated group) or exposed to
gray background screens (non-stimulated, control group). Dark and
light-adapted electroretinograms (ERGs) were performed on P21
to compare retinal function of these mice (n=13). At P23, contrast
sensitivity thresholds (at optimal gradient 0.103 c/d) along with
visual acuity of these animals were obtained to assess the potential
benefit of OKT exposure to other visual tasks. Immediately following
the final testing, mice were sacrificed and eyes enucleated and fixed.
Radial sections were labeled with a BDNF antibody and imaged with
fluorescent microscopy.
Results: Daily OKT testing resulted in 60% greater visual acuity
thresholds (p<0.001) and 300% greater contrast sensitivity (p<0.01)
in vehicle-injected, stimulated mice. In ANA-12 injected mice,
improvements were diminished with only 20% greater visual acuity
thresholds (p<0.01) and 5% greater contrast sensitivity (p<0.05)
compared to control vehicle mice. There were no statistical difference
between the ANA-12 and the vehicle-injected, non-stimulated control
mice. Mean ERG amplitudes were not significantly different among
the different treatment types.
Conclusions: Using daily stimulation with OKT to elicit a visual
acuity threshold produced enhanced visual acuity levels and also
improved contrast sensitivity compared to control mice. These effects
were mediated, at least in part, by activation of BDNF pathway in
the retina. Since retinal function did not change in stimulated mice,
the observed visual benefits may be result of plasticity occurring in
higher order visual processing.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Commercial Relationships: Amanda Mui, None; Brian C. Prall,
None; Moe H. Aung, None; Tracy Obertone, None; Hanna Park,
None; Jeffrey H. Boatright, None; Machelle T. Pardue, None
Support: NIH EY016435 (MTP), NIH P30 EY006360, Research
to Prevent Blindness, and the Department of Veterans Affairs, The
Abraham and Phyllis Katz Foundation (JHB) NIH R01EY14026
(JHB)
Program Number: 6190 Poster Board Number: B0095
Presentation Time: 12:00 PM–1:45 PM
Spatiotemporal mapping of retinal phototropic response evoked
by oblique light stimulation
Xincheng Yao, Rongwen Lu, Benquan Wang, Qiuxiang Zhang.
Biomedical Engineering, Univ of Alabama at Birmingham,
Birmingham, AL.
Purpose: This study was designed to map transient phototropic
response in the retina evoked by oblique visible light stimuli, and
to compare the orientation dependent dynamics in rod and cone
photoreceptors.
Methods: Both frog (Rana pipiens) and mouse (Mus musculus)
retinas were used to demonstrate the transient phototropic response
in the retina. Animal handling was approved by the Institutional
Animal Care and Use Committee of the University of Alabama at
Birmingham. The imaging system consisted of two light sources: a
near infrared (800-1000 nm) light for retinal imaging and a visible
(450-650 nm) light for retinal stimulation. Visible light flashes with
oblique incident angle were used to test the effect of the stimulus
direction. The duration of each visible flash was 5 ms. Near infrared
images of the retina were acquired at 200 frames/s. Localized retinal
movements were calculated at cellular resolution. Rod and cone
photoreceptor dynamics were quantitatively compared.
Results: High-spatial (micrometer) and high-temporal (millisecond)
resolution NIR imaging revealed that retinal rods could rapidly
(onset: ~10 ms for frog and 5 ms for mouse; time-to-peak: ~200 ms
for frog and 30 ms for mouse) shift toward the direction of the visible
light. In contrast, such directional movement was negligible in retinal
cones.
Conclusions: Rod-dominant transient phototropic response in frog
and mouse retinas was observed. Such transient phototropic response
might compensate for the loss of illumination efficiency under
oblique stimulation in the rod system, which can explain the absence
of the Stiles-Crawford effect in rod system. Moreover, the observed
transient rod movement promises a characteristic biomarker to enable
selective mapping of retinal rod dysfunction, which is valuable for
early detection of age-related macular degeneration and other eye
diseases.
Commercial Relationships: Xincheng Yao, University of
Alabama at Birmingham (P); Rongwen Lu, University of Alabama
at Birmingham (P); Benquan Wang, University of Alabama at
Birmingham (P); Qiuxiang Zhang, University of Alabama at
Birmingham (P)
Support: NSF CBET-1055889, NIH R21EB012264, and UASOM I3
Pilot Award
Program Number: 6191 Poster Board Number: B0096
Presentation Time: 12:00 PM–1:45 PM
Anesthesia usage confounds in vivo pharmacological studies
using electrophysiology
Jason Charng1, Zheng He1, Algis J. Vingrys1, Bang V. Bui1, Rebecca
L. Fish2, Rachel Gurrell2, Christine T. Nguyen1. 1Optometry and
Vision Sciences, The University of Melburne, Melbourne, VIC,
Australia; 2Pfizer Neusentis, Cambridge, United Kingdom.
Purpose: Anesthesia related confounds during in vivo
pharmacological testing are poorly understood. This project employs
conscious recordings to quantify the influence of ketamine:xylazine
anesthesia on isoguvacine (GABAa agonist) induced changes to the
electroretinogram (ERG) and visual evoked responses (VEP).
Methods: Long-Evans rats (n=8, male, 3-month old) were implanted
with telemetry transmitters to record electrophysiology under
conscious conditions. ERG and VEP electrodes on the sclera and
above the visual cortex were referenced to a forehead electrode.
Isoguvacine was administered via intramuscular (IM 0, 10, 30mg/
kg), intracerebroventricular (ICV 6ml, 45mM) or intravitreal (IV 3ml,
180mM) routes and electrophysiology recorded with 5 day washout
period in between. Conventional anaesthetized ERG and VEP
recordings with chlorided silver electrodes were also recorded in age
matched cohort (n=5) following same isoguvacine injections as the
conscious group. Responses were measured at 1.52 log cd.s.m-2 and
analyzed to return peak amplitudes and timing (mean±SEM).
Results: Under anesthesia, IM isoguvacine produced a significant
decrease in a-wave (10mg/kg -10±1%, 30mg/kg -16±2%, p<0.05)
and b-wave (10mg/kg -7±1%, 30mg/kg -14±1%, p<0.05) amplitudes
compared to vehicle injection. Timing parameters were similar except
a significantly slowed a-wave with 30 mg/kg isoguvacine (24.4±0.4
vs 25.5±0.7ms). IM isoguvacine in conscious animals yielded no %
difference in a- and b-wave amplitudes compared to vehicle (a-wave:
10mg/kg 33±19%, 30mg/kg 21±14%; b-wave: 10mg/kg 21±9%,
30mg/kg 13±7%) and timings (a-wave: 10mg/kg 6±14%, 30mg/kg
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
-14±7%; b-wave: 10mg/kg 3±4%, 30mg/kg 5±5%). IV isoguvacine in
anesthetized animals decreased a-wave (-30±8%, p<0.05), increased
b-wave amplitudes (15±5%, p<0.05) and produced faster a-wave
(-50±3%, p<0.05) and b-waves (-22±3%, p<0.05). In contrast, IV
isoguvacine in conscious animals did not affect ERG amplitude
(a-wave 21±20%, b-wave -13±11%) or timing (a-wave -25±11%,
b-wave -11±5%). ICV isoguvacine in anesthetized rats reduced P2N1 amplitude (-52±4%, p<0.05). In conscious rats ICV isoguvacine
slowed P2 (26±10%) and N1 (14±4%) peak time but did not affect
amplitudes.
Conclusions: We show that ketamine:xylazine anesthesia confounds
in vivo electrophysiology. The ability to conduct pharmacology
studies without anaesthesia may provide superior clinical translation
than conventional techniques.
Commercial Relationships: Jason Charng, None; Zheng He,
None; Algis J. Vingrys, None; Bang V. Bui, None; Rebecca L.
Fish, Pfizer Neusentis (E); Rachel Gurrell, Pfizer Neusentis (E);
Christine T. Nguyen, None
Support: Australian Postgraduate Award
Program Number: 6192 Poster Board Number: B0097
Presentation Time: 12:00 PM–1:45 PM
Long-term bilateral microglial responses in rat retina after
unilateral optic nerve transection
Ling Ping Cen, Miaomiao Hang, Mingzhi Zhang. Joint Shantou
International Eye Center, Shantou, China.
Purpose: To characterize long-term bilateral retinal microglia
responses after unilateral optic nerve (ON) transection.
Methods: Unilateral optic nerve transections were performed
intraorbitally on the right side 0.5mm behind the eyeballs of adult
Fischer rats. After allowed to survive for various periods of time
from 3 days to 12 weeks, retinas from both eyes of the animals were
obtained for immunohistochemistry to label microglial cells. Density
and morphology of microglial cell were evaluated in retinal flat
mounts under fluorescent microscope.
Results: Quantitative analysis of microglia in flat mount retinas
showed that, 3 days after ON transection, the average number of
microglial cells in the injured side (1463±137/mm2) was similar
to that of the control group (1407±134/mm2). The number was
dramatically increased on day 7 (2016±122/mm2), and kept stable
for at least 3 weeks before dropped to the control level by 6 weeks
(1494±80/mm2). In retinas of the contralateral side, however, no
obvious change of the average number of microglial cells was seen.
For morphology analysis, average numbers of branch points of
microglial processes in bilateral sides were shown decreased by 5
folds on day 7, and kept stable for at least 3 weeks. The numbers of
branch points were seen gradually increased in 6 weeks and reached
the control level by 12 weeks.
Conclusions: Proliferation of microglial cells was seen only in the
injury side after unilateral ON transection. However, morphology
changes of microglial cells were shown in retinas from both eyes.
Commercial Relationships: Ling Ping Cen, None; Miaomiao
Hang, None; Mingzhi Zhang, None
Program Number: 6193 Poster Board Number: B0098
Presentation Time: 12:00 PM–1:45 PM
Characterization of light toxicity on retinal ganglion cells in vivo
and in vitro
Emilie Arnault1, 2, Coralie Barrau3, Pauline Gondouin1, 2, Celine
Nanteau1, 2, Karine Bigot1, 2, Thierry Villette3, Denis A. CohenTannoudji3, Jose-Alain Sahel1, 4, Serge A. Picaud1, 2. 1U968, INSERM,
Institut de la Vision, Paris, France; 2UMR_S968, UPMC Université
Paris 06, Institut de la Vision, Paris, France; 3Essilor International,
Charenton-le-Pont, France; 4Centre Hospitalier National
d’Ophtalmologie des Quinze-Vingts, Paris, France.
Purpose: Light has not yet been identified as a risk factor in retinal
diseases involving retinal ganglion cell degeneration such as
glaucoma or diabetic retinopathy. Recently, we showed that retinal
ganglion cells (RGCs) degenerate in taurine-depleted animals,
in which a light-dependent degeneration of photoreceptors had
previously been shown in the 70-80s.
Here, we tested if RGCs are preserved in taurine-depleted mice
maintained in darkness. In vitro, we characterized the most
phototoxic wavelengths for purified retinal ganglion cells in culture
using our specific cell illumination device within the blue-green
range, which has already enabled us to define the spectral band from
415 to 455 nm as the most toxic for A2E-loaded RPE cells.
Methods: For in vivo experiments, mice were treated for 2 months
with GES, a taurine transporter blocker and maintained or not in
darkness. Retinal cells density was then quantified on sections
following immunolabeling.
To identify the most toxic wavelengths, purified rat RGCs were
exposed to 10 nm illumination bands centered from 390 to 520 nm
in 10 nm increments for 15 hours. Light irradiances were normalized
with respect to the natural sunlight reaching the retina after ocular
media filtering. Control cells were maintained in darkness during the
experiment. Cell viability was assessed using CellTiter-Glo Assay
(Promega).
Results: GES-treated mice showed a significant reduction in the
densities of both cone photoreceptors and RGCs. The RGC cell
loss did not appear as a secondary process to the cone degeneration
because these degenerative processes were occurring at the same rate.
Degeneration of both RGC and cone was not detected in GES-treated
animals maintained in darkness. Following this in vivo demonstration
of toxicity of light on RGCs, we defined the phototoxic action
spectrum on purified RGCs. After light exposure, morphological
changes were associated with a loss of cell viability. Quantification
of viable cells revealed that the RGC phototoxicity was significantly
higher in a 60 nm spectral band centered at 460 nm.
Conclusions: These results demonstrated for the first time light
effect on RGC degeneration in vivo. Furthermore, the in vitro study
suggests that blue light is more toxic to RGCs. Interestingly, this
RGC phototoxicity action spectrum is at higher blue wavelengths
compared to the RPE action spectrum that we have previously
reported on A2E-loaded RPE cells.
Commercial Relationships: Emilie Arnault, Essilor International
(P); Coralie Barrau, Essilor International (E), Essilor International
(P); Pauline Gondouin, None; Celine Nanteau, None; Karine
Bigot, None; Thierry Villette, Essilor International (E), Essilor
International (P); Denis A. Cohen-Tannoudji, Essilor International
(E), Essilor International (P); Jose-Alain Sahel, Essilor International
(P); Serge A. Picaud, Essilor International (P)
Support: OSEO Grant for Descartes research consortium
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 6194 Poster Board Number: B0099
Presentation Time: 12:00 PM–1:45 PM
Determination of Injury Thresholds for Torsional Indirect
Traumatic Optic Neuropathy in a Rat Model
Brooke I. Asemota1, Randolph D. Glickman2, William E. Sponsel1,
Matthew A. Reilly1. 1Biomedical Engineering, University of Texas at
San Antonio, San Antonio, TX; 2University of Texas Health Science
Center at San Antonio, San Antonio, TX.
Purpose: Traumatic optic neuropathy (TON) occurs in up to 5% of
all head traumas resulting in severe visual deficit or blindness. In
this study we imposed torsional indirect TON in a physiologically
relevant rat model, which may be used for development of novel
therapeutics.
Methods: Flash visual evoked potentials (fVEPs) were used before
and after injury stimulus to characterize the visual performance of the
visual track. A torsional indirect TON insult was applied using a robot
described previously (Reilly et al., ARVO 2013 E-abstract 54:5757).
The amplitude and velocity of this insult was varied to modulate the
degree of irreversible TON. Histopathology was also used to examine
optic nerve sections.
Results: Application of super-saccade rotation induced TON (Fig. 1).
The torsion parameters correlated with fVEP amplitude and latency
(Fig. 2).
Conclusions: The difference between pre- and post-traumatic event
fVEPs directly corresponds to optic nerve damage because the signal
relay producing the fVEP is dependent on the conductivity of the
optic nerve with regards to amplitude, period, and phase number.
Because the optic nerve is a part of the Central Nervous System
(CNS), the neuroprotectives that are effective in preventing blindness
in our TON rat model may be applicable to other neurodegenerative
diseases and disorders.
Figure 1: Difference in fVEP between Normal (Pre-TITON) and
Injured (Post-TITON) Eye
Commercial Relationships: Brooke I. Asemota, None; Randolph
D. Glickman, None; William E. Sponsel, None; Matthew A. Reilly,
None
Program Number: 6195 Poster Board Number: B0100
Presentation Time: 12:00 PM–1:45 PM
Heat shock protein 25 kDa gene therapy alleviates retinal
ganglion cell dysfunction after optic nerve crush in mice
Henri O. Leinonen1, Symantas Ragauskas2, 3, Jooseppi Puranen2,
Adrian Smedowski2, 4, Kari Airenne5, Seppo Ylä-Herttuala5, 6, Heikki
Tanila1, Giedrius Kalesnykas2, 7. 1Department of Neurobiology, A.I.
Virtanen institute, University of Eastern Finland, Kuopio, Finland;
2
Department of Ophthalmology, Institute of Clinical Medicine,
School of Medicine, University of Eastern Finland, Kuopio, Finland;
3
Institute of Innovative Medicine, Vilnius, Lithuania; 4Department
of Physiology, Medical University of Silesia, Katowice, Poland;
5
Department of Biotechnology and Molecular Medicine, A.I. Virtanen
Institute, University of Eastern Finland, Kuopio, Finland; 6Research
Unit and Gene Therapy Unit, Kuopio University Hospital, Kuopio,
Finland; 7Experimentica Ltd., Kuopio, Finland.
Purpose: Heat shock proteins (HSPs) are molecular helper proteins,
chaperones, which are known to be induced after various forms of
chemical and environmental insults. HSP expression is upregulated
in retinal neurons and glial cells in experimental glaucoma, cerebral
ischemia and hypoperfusion models. Thus, we asked the question
whether HSP25 gene therapy could prevent retinal ganglion cell
(RGC) dysfunction after optic nerve crush (ONC) which is known to
cause retrograde damage to RGCs.
Methods: Serotype 2 recombinant adeno-associated viral vectors
encoding HSP25 were intravitreously, unilaterally, injected three
months before the optic nerve crush into C57Bl/6J mice. The control
group of mice received saline injections. ONC was performed to the
same eye as injections. Electroretinography (ERG) was performed
30 days after ONC in response to patterned (pERG) and flash
stimuli (fERG). After ERG recordings, the animals were deeply
anesthetized and perfused. The eyes and optic nerves were collected
for morphological analysis.
Results: As expected, ONC strongly decreased pERG responses
without affecting fERG. However, virus-injected eyes preserved
approximately 60% better function compared to saline-injected
controls as measured with pERG amplitude. No other statistically
significant differences in other analyzed amplitude parameters were
found. Currently, we are performing morphological analysis with the
collected retinas and optic nerves.
Conclusions: HSP25 gene therapy alleviates retinal function
deficit after ONC. Our preliminary results suggest that specific
HSP induction might serve as a novel treatment strategy for optic
neuropathies.
Commercial Relationships: Henri O. Leinonen, None; Symantas
Ragauskas, None; Jooseppi Puranen, None; Adrian Smedowski,
None; Kari Airenne, None; Seppo Ylä-Herttuala, None; Heikki
Tanila, None; Giedrius Kalesnykas, Experimentica Ltd. (E)
Figure 2: Correlation of Torsion Parameters with fVEP Amplitude
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Program Number: 6196 Poster Board Number: B0101
Presentation Time: 12:00 PM–1:45 PM
Morphological and functional evaluation of retinal ganglion cells
in R6/2 mice
Symantas Ragauskas1, 2, Henri O. Leinonen3, Jooseppi Puranen1,
Soile Nymark4, Arto Lipponen3, Kestutis Gurevicius3, Outi
Kontkanen5, Jukka Puoliväli5, Heikki Tanila3, Giedrius Kalesnykas1,
6 1
. Department of Ophthalmology, University of Eastern Finland,
Kuopio, Finland; 2State Reaserch Institute for Innovative Medicine,
Vilnius, Lithuania; 3Department of Neurobiology, A.I. Virtanen
Institute, Kuopio, Finland; 4Department of Electronics and
Communications Engineering, BioMediTech, Tampere University of
Technology, Tampere, Finland; 5Charles River DRS Finland, Kuopio,
Finland; 6Department of Ophthalmology, Kuopio University Hospital,
Kuopio, Finland.
Purpose: To study mutated Huntigtin (mHtt) protein deposition and
its effect on retinal ganglion cell (RGC) survival and function in R6/2
mice.
Methods: R6/2 mice and their wild-type littermates (WT) were
used at the age of 4 to 19 weeks. Immunohistochemistry and
stereology were employed to quantify the total number of RGC layer
(RGCL) cells, the total number of retinal astrocytes, and RGCL
cells that contained soluble and aggregates forms of mHtt. The
deposition of mHtt was studied in retinas and optic nerves using
immunohistochemistry and confocal microscopy. Optic nerve axons
from R6/2 and WT mice were quantified. Electroretinography (ERG)
recordings were used to assess the functional status of retinal cells.
Results: The total number of RGCL cells, the total number of GFAPpositive retinal astrocytes, and the total number of RGC axons in the
optic nerve did not differ between 18-week old R6/2 mice and their
littermate controls. Mutant Htt deposits were localized in nuclear
layers of retina. At the age of 4 weeks R6/2 mice had predominantly
soluble mHtt in the RGCL cells, whereas the aggregated form of
mHtt was found in the majority of cells from the 12-week old R6/2
mice onwards. Retinal astrocytes did not contain mHtt deposits.
However, mHtt deposits were found to localize in the GFAP-ir
astrocytes of the optic nerve. The ERG recordings showed a deficit in
the cone-related function already at the 4-week of age in R6/2 mice
with a complete loss of pattern ERG signal at the 8 week of age as
compared to the WT controls. The rod-related measurements showed
significant deficit at the age of 8 week.
Conclusions: Deposition of mHtt does not cause RGC degeneration
or retinal astrocyte loss in R6/2 mice even at the late stage of
HD-related pathology. However, the R6/2 mice experience visual
functional deficits already at the age of 8 weeks.
Commercial Relationships: Symantas Ragauskas, None; Henri
O. Leinonen, None; Jooseppi Puranen, None; Soile Nymark,
None; Arto Lipponen, None; Kestutis Gurevicius, None; Outi
Kontkanen, None; Jukka Puoliväli, None; Heikki Tanila, None;
Giedrius Kalesnykas, None
Program Number: 6197 Poster Board Number: B0102
Presentation Time: 12:00 PM–1:45 PM
Targeted modulation of the glial inflammatory response in
Retinitis Pigmentosa attenuates photoreceptor cell death.
Enrique J. de la Rosa1, Catalina Hernández-Sánchez1, Alberto M.
Hernández-Pinto1, María Platón1, Miguel Marchena1, Noemí ÁlvarezLindo1, Ana I. Arroba1, Sean Jmaeff2, Pablo F. Barcelona2, H Uri
Saragovi2. 1Cell & Molecular Medicine, Centro de Investigaciones
Biologicas, Madrid, Spain; 2Lady Davis Institute-Jewish General
Hospital Montreal, McGill University, Montreal, QC, Canada.
Purpose: Retinitis Pigmentosa (RP) is a heterogeneous group of
genetic retinal dystrophies. In most forms of RP, photoreceptor
cell death is associated with disease progression. Reactive Müller
cell gliosis and microglial activation are also found, often prior
to photoreceptor death. Here, we studied inflammatory pathways,
arising from Müller cell and microglia, that regulate neuronal cell
death.
Methods: We analyzed the expression of pro-inflammatory
cytokines and of markers of reactive Müller cell gliosis and
microglial activation by qRT-PCR and immunohistochemistry in
retinas from wild type and RP mouse models. Growth factors and
pharmacological agents were tested in retinal explants ex vivo and by
intravitreal injection in vivo. The agents were selected to modulate
the glial inflammatory response. The treatments included clodronateliposomes (to cause depletion of microglia), IGF-I (to polarize
microglia response), and antagonists of the P75(NTR) receptor
(a receptor present in Müller and glial cells and whose activity
stimulates TNFα production).
Results: A marked increase in GFAP and TNFα transcription
preceded photoreceptor cell death in RP retinas. Reduced
photoreceptor cell death, better preservation of the outer nuclear
layer, and decreased gliosis were observed upon treatment.
Conclusions: Our results suggest the possible existence in the RP
retinas of a pro-inflammatory loop mediated by the activation of
P75(NTR) in Müller glial cells, which causes production of TNFα.
Modulation of the P75(NTR) inflammatory response is a possible
target to attenuate RP progression.
Commercial Relationships: Enrique J. de la Rosa, None; Catalina
Hernández-Sánchez, None; Alberto M. Hernández-Pinto, None;
María Platón, None; Miguel Marchena, None; Noemí ÁlvarezLindo, None; Ana I. Arroba, None; Sean Jmaeff, None; Pablo F.
Barcelona, None; H Uri Saragovi, McGill University (P)
Support: SAF2010-21879 (Spain)
Program Number: 6198 Poster Board Number: B0103
Presentation Time: 12:00 PM–1:45 PM
Changes of Receptive Field Properties of Ganglion Cells in a Rat
Model of Retinitis Pigmentosa
Wan-Qing Yu1, Eun-Jin Lee2, Greg Field3, 4, Norberto Grzywacz1, 2.
1
Neuroscience Graduate Progam, University of Southern California,
Los Angeles, CA; 2Department of Biomedical Engineering,
University of Southern California, Los Angeles, CA; 3Zilkha
Neurogenetic Institute, University of Southern California, Los
Angeles, CA; 4Department of Cell and Neurobiology, University of
Southern California, Los Angeles, CA.
Purpose: In some models of Retinitis Pigmentosa (RP), cone mosaics
undergo rearrangement following the death of rods. In S334ter-line3
rats, cones migrate out of a semi-regular lattice to form ring-like
patterns. However, light responses of retinal ganglion cells (RGCs)
persist after cone migration. Our study identified the consequences
of cone reorganization on the receptive fields (RFs) of RGCs. In
particular, RGCs located in the center of cone rings may have a
minimal light response if the cones are disconnected. Alternatively,
these RGCs may exhibit a displaced RF with a Gaussian shape if the
RGC reconnects (via bipolar cells) to cones in the rings. Finally, RGC
RFs may exhibit arch-like shapes if RGCs maintain their original
connections as the cones migrate. To resolve these possibilities, we
measured RFs at high resolution across large populations (~300) of
RGCs in normal and RP retinas.
Methods: Extracellular recordings were made from P60 Long Evans
and heterozygous S334ter-line3 rat RGCs using a 512-electrode
array. Spatiotemporal RFs were estimated as spike-triggered averages
using binary white noise stimuli. Cones were labeled with PNA and
confocal images were taken after the recordings.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Visual Neuroscience
Results: Recordings from normal and RP retinas exhibited similar
numbers of RGCs, suggesting that they were connected to the cone
mosaic despite its reorganization. Most RGCs in RP rats showed nonGaussian RFs, with arch- or star-like shapes. The abnormal RFs were
observed across all types of RGCs. Gaps in light sensitivity of RGC
RFs matched the ring-like organization of the cones.
Conclusions: RGCs continue to function after the cone
rearrangement in RP retinas. However, not surprisingly, RFs are
abnormal. This suggests that RGCs do not disconnect from the
cone mosaics as the cones migrate. Further, it suggests that bipolar
dendrites or cone axons extend to maintain their contacts while the
inner retinal circuitry is largely maintained.
Commercial Relationships: Wan-Qing Yu, None; Eun-Jin Lee,
None; Greg Field, None; Norberto Grzywacz, None
Support: VSoE Research Innovation Fund (E.-J.L.); National Eye
Institute, EY016093 and EY11170 (N.M.G.).
Commercial Relationships: Masayoshi Yukita, None; Shigeki
Machida, None; Kazuko Omodaka, None; Kazuichi Maruyama,
None; Toru Nakazawa, None
Program Number: 6199 Poster Board Number: B0104
Presentation Time: 12:00 PM–1:45 PM
Brimonidine Enhances Electrophysiological Activity of Retinal
Ganglion Cells through Trk-PI3K Pathway
Masayoshi Yukita1, Shigeki Machida2, Kazuko Omodaka1, Kazuichi
Maruyama1, Toru Nakazawa1. 1Ophthalmology, Tohoku University
Graduate School of Medicine, Miyagiken, Japan; 2Ophthalmology,
Iwate Medical University School of Medicine, Iwateken, Japan.
Purpose: It has been reported that an intravitreal injection of
brimonidine elevates BDNF level in the retinal ganglion cell
(RGC) layer and protects RGCs from cell death in rats. However,
electrophysiological change of RGCs induced by the intravitreal
brimonidine remains unknown. In this study, we investigate the
changes of RGC activity by measuring the positive scotopic threshold
response (pSTR) of the electroretinogram (ERG) in eyes injected
with brimonidine.
Methods: We used 40 adult Sprague-Dawley rats. The right eyes
were injected with DPBS (1μl) as control, and the contralateral left
eyes were injected with brimonidine (0.10nmol). Scotopic ERGs
were recorded simultaneously from both eyes at 1, 2, 3, 7 and 10
days after the intravitreal injection. Amplitudes of the a- and b-waves
and the pSTR were measured. In other experiments to elucidate the
mechanism of the ERG changes induced by intravitreal brimonidine,
we injected K252a (an inhibitor of tyrosine kinase phosphorylation of
Trk receptor: 0.06 pmol), U0126 (MAPK/ERK kinase inhibitor: 0.15
nmol) or LY294002 (phosphoinositide 3-kinases(PI3Ks): 0.60 nmol)
with brimonidine into the left eyes and recorded ERGs with a same
protocol. One-way repeated measures ANOVA was used to determine
statistical significance.
Results: In the brimonidine-injected eyes, the pSTR amplitudes
were significantly increased to 133.5±27.4%, 147.1±21.2% and
130.2±19.4% (5 animals at each time point, P<0.01) as compared
to those of the control eyes at the day 1, 2 and 3 after the injection,
respectively. However, the enhanced amplitudes of the pSTR
returned to the normal level (109.0±21.7%) at the day 7. The
intravitreal injections of K252a or LY294002 significantly reduced
the enhancement of the pSTR induced by the intravitreal brimonidine
(P<0.01). In contrast, the U0126 injection did not affect the
enhancement of the pSTR.
Conclusions: The intravitreal brimonidine enhanced
electrophysiological activity of RGCs in rats. Activation via Trk
receptor and PI3K signals were involved in the mechanism of the
electrophysiological change. It would be interesting to investigate
an association between this neuroactivation and neuroprotection of
brimonidine in the future.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.