Synaptic Plasticity in the
MyosinVa Mutant Mouse
by
MASSACHUSETTS INSTITUTE'
OF TECHNOLOGY
Cansu Tunca
FEB 2 4 2009
B.S. Chemical Engineering,
Bogazici University (1999)
S.M. Chemical Engineering,
Massachusetts Institute of Technology (2001)
LIBRARIES
Submitted to the Department of Brain and Cognitive Sciences
in partial fulfillment of the requirements for the degree of
Master of Science in Brain and Cognitive Sciences
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2009
@ Massachusetts Institute of Technology 2009. All rights reserved.
Author
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Departmen
C ertified by ..............
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Brain and /Pnitive Sciences
/
Jnuary 5Q.-9
...........
/Martha Constdine-Paton
Professor "f BiolSoy and Brain an ognitive Sciences
Supervisor
The
Accepted by ........... ,.....
.
r' /
Earl Miller
Chairman, Department Committee on Graduate Students
ARCHIVES
Synaptic Plasticity in the
MyosinVa Mutant Mouse
by
Cansu Tunca
Submitted to the Department of Brain and Cognitive Sciences
on January 30, 2009, in partial fulfillrent of the
requirements for the degree of
Master of Science in Brain and Cognitive Sciences
Abstract
The trafficking of essential proteins into spines is an important aspect of synaptic plasticity. MyosinVa, an actin-based motor protein, has been implicated in the synaptic
delivery of AMPARs during LTP [1]. However an earlier study showed that LTP and
LTD were unaffected in the MyosinVa-null dilute-lethal mice [2]. To evaluate the role
of MyosinVa in synaptic plasticity, we studied different forms of LTP and LTD in
the CA1 region of the hippocanipus from MyosinVa dominant negative mutant flailer
mouse using field potential recordings. Flailer mice showed no impairment of LTP or
NMDAR-dependent LTD, consistent with the findings of the study on dilute-lethal.
In addition, MyosinVa has been implicated in the transport of an RNA-binding protein into the spines upon mGluR activation [3]. We explored protein synthesis and
mGluR,-dcpendent LTD in flailer. The preliminary data we obtained show a transient impairment in mGluR-LTD, suggesting a role for MyosinVa in protein synthesis
dependent plasticity.
Thesis Supervisor: Martha Constantine-Paton
Title: Professor of Biology and Brain and Cognitive Sciences
Acknowledgments
First of all, I would like to express my appreciation and gratitude to my advisor, Prof.
Dr. Martha Constantine-Paton for her encouragement, help and guidance.
My sincere thanks to all the group mnembers, Akira Yoshii, Jian-Ping Zhao, Yasunobu
Murata, Rory Kirchner, Andrew Bolton, Marnie Phillips, Paula Feinberg-Zadek and
Julie Goldberg for their generous assistance and valuable friendship.
Finally, I would like to express my profound gratitude to all my friends, especially
Ayca Darcan, Brigitte van Zundert, Oguz Gunes and Esra Atilla, and to my family
Onur Kuzucu, Canan, Burchan and Batu Bayazit, and my parents Gunel and Tamer
Tunca for their endless love.
Contents
1 Introduction
2 Methods
..................
2.1
Animals.
2.2
Slice Preparation
2.3
Field potential recordings
2.4
LTP/LTD protocols and presynaptic efficacy measurelments ......
2.5
Drugs
...............
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.....................
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3 Results
3.1
LTP is normal in flailer mice .......................
3.2
NMDAR-dependent LTD is normal in flailer .
3.3
Flailer mice show impaired PPLFS-LTD
.............
................
4 Discussion
22
A Spine Morphology Analysis in Flailer
23
A.1 DiI Labeling . . . . . ...............................
.
A.1.1
Methods
A.1.2
Preliminary results .
A.2 GFP-expressing CA neurons .
A.2.1 Methods ....
A.2.2 Preliminary results .
23
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...
24
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26
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26
...
.......................
27
B L-LTP studies with interface chamber
30
List of Figures
15
........
3-1
Flailer and WT mice have similar presynaptic efficacy.
3-2
Flailer mice show normal LTP and NMDAR-dependent LTD ....
3-3
Flailer mice show impairment in PPLFS-induced LTD .........
.
17
.
18
A-1 Sample images of Dil filled dendritic branches in flailer (A) and WT (B) 25
A-2 Spine densities in flailer and WT
.
...................
26
A-3 GFP-expressing CA1 neurons imaged in a GFP homozygous mice (A)
. .
and a GFP heterozygous mice (B) . ..............
A-4 Cortical neurons expressing GFP .......
.
28
.
.
...........
29
A-5 Sample images for dendritic branches of GFP expressing CA1 neurons
in W T mice ......
.....
........................
.
29
List of Tables
A.1 The spine densities (spine nulmber per 10tin) for flailer and WT . . .
25
Chapter 1
Introduction
Myosins are a motor protein family that transport proteins along actin filaments. In
particular, MyosinVa transports proteins from dendritic shafts into spines and trans-
ports vesicles within presynaptic axon terminals. Mutation of the MyosinVa gene
in humans causes Griscelli and Elejalde syndromes, neurological disorders characterized by severe neurologic deficits including hypotonia, a marked delay in motor
cevelopment, ataxia, seizures and mental retardation [4, 5, 6, 7]. Spontaneous Myo5a
mutations in mice and rats, such as dilute-lethal, dilute-opisthoto'nus (dop) and flailer,
provide animal models for MyosinVa dysfunction. Both the mouse line dilute-lethal
[8. 9] and the rat line dop [10, 11] are MyosinVa null mutants. These mutants show
severe neurological abnormalities and die within 2-3 weeks after birth. The MyosinVa
mutant flailer expresses a novel gene that combines the promoter and the first two
exons of guanine nucleotide binding protein beta, 5 (Gnb5) with the C-terminal exons
of the Myo5a gene, derived by exon shuffling. The hybrid flailer protein is composed
of MyosinVa lacking the actin-binding head region, thus acting as a dominant negative. Flailer mice have ataxia and seizures similar to dilute-lethal but recover from
seizures and show mild ataxia after 4 weeks of age [12].
In dop rats, textitdilute-lethal and flailer mice, smooth endoplasmic reticulum
(sER) and IP3 receptors are lacking in the dendritic spines of cerebellar Purkinje cells,
suggesting a role for MyosinVa in transport of sER, into spines [13, 14, 12, 15, 16].
Due to the deficiency of IP3 receptor-mediated calcium release in cerebellar spines,
cerebellar LTD is absent in dilute-lethal mice and dop rats [15].
A recent study have suggested that MyosinVa plays a role in AMIPA receptor
trafficking in LTP. They have shown that MyosinVa associates with AMPAR,s and is
responsible for the activity-dependent transport of AMPAR,s into spines. The same
study also showed that infecting organotypic cultures of hippocampal slices with
either a MyosinVa dominant-negative construct or with a siRNA targeting MyosinVa abolished LTP, which was induced by pairing presynaptic stimulation (3Hz, 1.5
min) with postsynaptic depolatization (OmV). These findings together indicate that
MyosinVa-dependent AMPAR, transport into spines is necessary for hippocampal LTP
[1]. However, this result conflicts with an earlier study of plasticity in dilute-lethal
mice, which showed that LTP and LTD were unaffected in acute hippocampal slices
measu-red by field potential recordings [2].
In addition to a possible role in AMPAR transport into spines, MyosinVa is involved in transportation of an RNA-binding protein, TLS (translocation in liposarcoma), to spines from the dendritic shaft upon mGluR stimulation. The transport
of TLS to the spine is not affected by AMPAR or NMDAR blockers and is absent
in dilute-lethal neurons, showing that MyosinVa is the motor protein to transport
TLS to the spine upon mGluR activation [3]. Local protein synthesis in dendritic
spines is an important aspect of synaptic plasticity [17]. Given the role of MyosinVa
in trafficking mRNA complexes into spines upon mGluR activation [3], we explored
the possibility that MyosinVa may play a role in local protein synthesis dependent
mnGluR-LTD.
mGluR-dependent LTD
NMDAR-dependent and mGluR-dependent LTD are two distinct forms of LTD.
mGluR-LTD depends on activation of group I mGluRs, which consist of two sub-
types: mGluR1 and mGluR5. To abolish mGluR-LTD, a simultaneous blockade of
mGluR1 and an mGluR5 is required, meaning that both subtypes are required for
mGhluR-LTD [18]. mGluR-LTD is expressed presynaptically in young ( postnatal day
(P)8-P15) animals and has a postsynaptic mechanism of expression in older animals
(P21-P35), corresponding to ages used in this study [19].
Group I mGluRs are linked to G proteins of the Gq and G11 types. Gq but not
on G11 is required for mGluR,-LTD [20]. Group I mGluRs activate phospholipase
C (PLC) and thereby produce diacylglycerol (DAG) and inositol triphosphate (IP3).
DAG and IP3 then trigger calcium release from the intracellular stores and activa2+ from
tion of PKC. However, activation of PKA or PKC [21, 22] or release of Ca
intracellular stores is not required for DHPG-induced mGluR-LTD in CA1 [21]. A
recent study, however, presented a role for calcium release from intracellular stores in
mGluR-LTD based on their findings in perirhinal cortical slice cultures [23].
Group I mGluRs activate the two signaling pathways, ERK and PI3K-Akt-mTOR
pathways, which are required for mGluR-LTD [24, 25, 26, 27, 28, 29, 30]. Signaling
mechanisms that mediate the induction of mGluR-LTD involve activation of protein
tyrosine phosphatases [31, 32, 33, 34], which leads to the tyrosine dephosphorylation
of AMPARs and their subsequent internalization [35, 36, 37].
mGluR-LTD has been shown to be dependent on local protein synthesis [38, 39]
and recent studies have elucidated some of the mechanisms associated with protein
synthesis involvement in LTD induction through AMPAR, internalization. mGluR
activation increases dendritic synthesis of MAP1B and Arc/Arg3.1 proteins in an
eEF2K manner [40, 41]. Arc/Arg3.1 protein mediates mGluR-LTD through increasing AMPAR endocytosis rate [41, 42]. In addition, mGluR activation increases the
translation of STEP, a protein tyrosine phosphatase which also mediates AMPAR
endocytosis [34]. Moreover, a recent study indicated that mGluR induction involves
miRNA-mediated suppression of mRNA translation [43].
In this study we wanted to assess any changes in hippocampal synaptic plasticity
in the MyosinVa dominant negative mutant flailer mouse using field potential recordings. Flailer mice have advantages over both the dilute-lethal mice and infection based
knockdown methods for the study of the role of MyosinVa in plasticity. While studies
on dilute-lethal mice are limited to young mice of ages 12-19 days due to early death,
flailer mice have a normal life span, allowing later ages to be studied. In addition,
flailer mice do not require infection-based knockdown methods, which have the risk
of off-target effects [44]. Given that MyosinVa transports an RNA-binding protein
into spines upon mGluR activation, we also explored protein synthesis and mGluRdependent LTD in flailer mice. We obtained preliminary data suggesting transient
impairment in mGluR-dependent LTD suggesting a role of MyosinVa in protein synthesis dependent plasticity. In addition, we assayed several different forms of LTP and
NMDAR-dependent LTD in flailer mice using field potential recordings and found no
differences in LTP or LTD compared to wild type (WT) mice.
Chapter 2
Methods
2.1
Animals
WT and flailer mice were bred and maintained in Massachusetts Institute of Technology facilities. Experimental protocols were approved by Massachusetts Institute
of Technology Animal Care and Use Committee.
2.2
Slice Preparation
Transverse hippocamnpal slices (400 pm) were prepared from 2-4 week old flailer and
WT male mice in ice-cold dissection solution saturated with 95% 0, and 5% CO 2.
The slices were transferred into a humidified interface chamber filled with ACSF. The
slices were allowed to recover for 30 minutes at 320 C and for at least 1 hour at room
temperature. They were transferred to a submerged recording chamber and perfused
with ACSF. Experimental conditions used in different protocols are listed below.
* LTP stimulations and NMDAR-LTD in age group P13-P15:
Dissection solution contained (in inM): 240 sucrose, 3 KC1, 22 NaHCO 3 , 1.25
NaH 2PO 4 , 10 glucose, 0.5 CaC12 and 7 MgCl 2.
ACSF contained (in mM): 124 NaC1, 3 KC1, 22 NaHCO 3 , 1.25 NaH 2 PO 4 , 10
glucose, 2 CaC12 and 2 MgCl 2.
Recording solution also included 0. 1 mM picrotoxin. The CA3 region was removed from the slice to reduce epileptiform activity. Recording temperature
was 300 C.
* NMDAR.-LTD in age group P18-P26:
Dissection solution contained (in rmM): 240 sucrose, 3 KC1, 22 NaHCO 3, 1.25
NaH 2 PO 4 , 10 glucose, 0.5 CaC12 and 7 MgSO 4.
ACSF contained (in mM): 124 NaC1, 3 KC1, 22 NaHCO 3 , 1.25 NaH 2 PO 4 , 10
glucose, 2 CaCl 2 and 2 MgSO 1 .
No drugs were added. The CA3 region was not removed. Recording temperature
was 28°C.
* PPLFS:
Dissection solution contained (in mM): 240 sucrose, 2.5 KC1, 26 NaHCO 3 , 1
NaH 2 PO 4 , 11 glucose, 1 CaC12 and 5 MgSO 4.
ACSF contained (in rmM): 119 NaC1, 2.5 KC1, 26 NaHCO 3 , 1 NaH 2 PO 4 , 11
glucose, 2.5 Ca,C12 and 1.3 MgSO 4.
Recording solution included 50 p2M APV. The CA3 region was not removed.
Recording temperature was room temperature.
2.3
Field potential recordings
The Schaffer collateral pathway of hippocampal slices was stimulated at 0.02 Hz with
a bipolar tungsten stimulating electrode. Field potentials (FP) were recorded with
glass micropipettes (3-5 MQ) filled with 4M NaC1l. Both stimulating and recording
electrodes were placed in CA1 stratum radiatum, approximately same distance from
the cell body layer. The stimulation intensity was set to produce 40-60% of the
maximal spike-free response. The initial slope of the FP was used to quantify the
magnitude of the response. After obtaining a stable baseline for at least 15 min,
conditioning stimulation was applied. Mean FP slopes were calculated for the 15min baseline and the last 15 minutes of the post-conditioning period. Any changes
in synaptic strength were calculated as the ratio of these mean FP slopes.
Data
are presented as the percent change in FP slope ± SEM. Statistical significance was
evaluated using a two-tailed, Student's t-test. P < 0.05 was considered statistically
significant.
2.4
LTP/LTD protocols and presynaptic efficacy
measurements
Conventional high frequency stimulation consisted of two 100Hz trains of is with an
interval of 20 s. NMDAR-LTP was induced by 900 pulse-stimulation of 25 Hz. Saturating levels of LTP was induced by 100 Hz stimulus for 1 s, repeated six times with
5 min intervals. 1 Hz 15 min stimulation was used to induce LTD. Paired pulse low
frequency stimulation consisted of 1 Hz for 15 min paired pulses with 50 ms interval.
Presynaptic efficacy was measured as the ratio of the field potential slope to the amplitude of the presynaptic afferent fiber volley. Only recordings with picrotoxin were
used to measure presynaptic efficacy.
2.5
Drugs
D-APV was purchased from Sigma and picrotoxin was purchased from Tocris.
Chapter 3
Results
First, we tested the excitatory synaptic efficacy in the flailer mouse. The ratio of the
field potential slope to the amplitude of the prcsynaptic afferent fiber volley associated
with Schaffer collateral stimulation was not different in flailer, suggesting that there
are no presynaptic changes. (WT: 3.2 ± 0.6, n = 6 slices, 5 animals; Flailer: 3.6 +
0.6, n = 7 slices, 7 animals; Fig. 3-1).
I
WT
Flailer
Figure 3-1: Flailer and WT mice have similar prcsynaptic efficacy.
3.1
LTP is normal in flailer mice
We next tested different forms of hippocampal LTP in the mutant. We started with
conventional high frequency stimulation (two 100 Hz trains of I s with an interval
of 20 s). Both flailer and WT showed similar magnitudes of potentiation (WT: 168
+ 14%, n = 6 slices, 5 animals; Flailer: 181 + 17%, n = 6 slices, 6 animals; ages:
P17-P24; Fig.3-2A). Cavus and Teyler have reported that LTP induced by 100Hz
stimulation is a compound potentiation mediated by both NMDAR and voltage dependent calcium channels (VDCC). They also showed that NMDAR-only LTP can be
induced by a weaker stimulation of 25 Hz [45]. In order to test whether NMDAR.-only
LTP was intact in flailer, we used 25 Hz stimulation protocol. There was no difference
in the magnitudes of LTP between the two groups, suggesting that NMDAR-only LTP
is intact in flailer (WT: 139 ± 12%, n = 8 slices, 7 animals; Flailer: 128 ± 17%, n =
10 slices, 8 animals; ages: P16-P24; Fig.3-2B).
Previous work in our lab has shown that in flailer, the scaffolding protein PSD95 is reduced in the spines of cultured pyramidal neurons from the visual cortex
[46]. While PSD-95 typically becomes enriched in synaptosomes upon eye-opening
paradigm in WT animals, this does not occur in flailer [47]. PSD-95 is critical in
regulating surface expression of AMPA receptors [48, 49], and mutant mice lacking
PSD-95 express enhanced LTP saturating at a higher level [50]. We wanted to assess
LTP saturation levels in hippocampal slices from flailer mice. Saturating levels of
LTP were induced by 100 Hz stimulus for 1 s, repcated six times with 5 min intervals.
Potentiation was saturated at similar levels in both flailer and the WT (WT: 201
± 13%, n = 3 slices, 3 animals; Flailer: 200 ± 20%, n = 6 slices, 6 animals; ages:
P17-P33; Fig.3-2C).
3.2
NMDAR-dependent LTD is normal in flailer
In addition to LTP, we examined NMDAR-dependent LTD. LTD is age dependent,
decreasing as animals get older [51]. We used two different age groups: P13-P15
and P18-P26. LTD was induced by delivering low frequency stimulation at 1 Hz for
15 min. In both age groups, LTD in flailer was similar to WT. (P13-P15, WT: 66 ±
12%, n = 3 slices, 3 animals; Flailer: 65 ± 8%, n = 4 slices, 3 animals; Fig.3-2D),
(P18-P26, WT: 50 ± 5%, n = 6 slices, 4 animals; Flailer: 55 ± 9%, n = 5 slices,
5 animals; Fig.3-2E). Taken together, these results reveal that flailer mice display
similar synaptic plasticity characteristics to WT animals.
LTP induced by lOOHzx2
2
,
P
2 40-
LTD (P13-P15)
153
1sWT
s.
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LL23
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63
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LTP induced by 25Hz
23
23
35
5 iO
0 8 10 IS 20 25 30 35
05 5' E 60
4.
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14
LTD (P1 8-P26)
2.
4r
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LTP induced by 100Hzx6
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Figure 3-2: Flailer mice show normal LTP and NMDAR-dependent LTD
3.3
Flailer mice show impaired PPLFS-LTD
Given the role of MyosinVa in trafficking mRBNA complexes into spines upon mGluR,
activation [3], we explored the possibility that MyosinVa may play a role in local
protein synthesis dependent mGluR-LTD. mGluR-LTD can be induced by bath application of group I mGluR agonist DHGP [52, 53, 38] as well as by delivery of paired
pulse low frequency stimulation (PPLFS) in the presence of NM DAR blocker D-APV
[54, 38].
We tested if flailer could exhibit normal levels of PPLFS-LTD. PPLFS (1Hz for 15
min paired pulses with 50ms interval) was delivered in the presence of 50 pM APV.
Flailer showed reduced LTD in the first 15 min after PPLFS stimulation. However
after 45 min, the difference between flailer and WT was not significantly different
(15-30 min: WT: 49 ± 5%, n = 6 slices, 4 animals; Flailer: 70 ± 4%, n = 10 slices,
7 animals; p = 0.01), (45-60 min: WT: 78 ± 7%, n = 6 slices, 4 animals; Flailer: 95
± 7%, n = 10 slices, 7 animals; p = 0.12; ages: P20-P31), (Fig.3-3).
120
110
-90
60.
_
30
*
-
-o
Failer
-5
5
1
5
20
20
so
o
o
s
time(rin)
Figure 3-3: Flailer mice show impairment in PPLFS-induced LTD
e
Chapter 4
Discussion
We showed that the flailer mouse shows no impairment of LTP or NMDAR-LTD in
field potential recordings of hipp)ocampal acute slices, consistent with the findings
of a. previous study on the cdlute-lethal mouse [2].
A different study argued that
MyosinVa, is the motor protein required for synaptic delivery of AMPAR in LTP
Conflicting results may be due to the differences in experimental conditions.
[1].
Their experiments involved infection-based knockdown methods and organotypic slice
preparation. Infection-based knockdown methods are known to have the risk of offtarget effects [44]. Moreover, in organotypic slice preparation, hippocampus develops
in the total absence of patterned input from other brain areas. If MyosinVa is the
motor protein involved in the activity dependent AMPAR, trafficking to the spine,
then we would have expected to find an impairment of LTP in the flailer mouse.
Since we find no impairment in LTP, our finding does not support the hypothesis
that MyosinVa is the motor protein involved in the synaptic delivery of AMPAR in
LTP.
We also studied protein synthesis dependent mGluR,-LTD induced by PPLFS in
the flailer mouse. We found a significantly reduced LTD in the flailer mouse in the
first 15min after stimulation. However, after 45 min, there remained no significant
difference between flailer and WT. Previous studies have showed that MyosinVa trafficks proteins important in synaptic plasticity such as PSD-95 [46, 47] as well as a
protein involved in activity dependent protein synthesis (TLS) [55, 3]. The imnpair-
ment of mGluR-LTD induced by PPLFS in flailer may be due to decreased transport
of synaptic plasticity proteins or decreased protein synthesis in spines. In addition,
dynamin3 is reduced in flailer mouse (A. Yoshii, personal communication, 2008).
Dynamin3 interacts with Arc, which is a rapidly translated protein that stimulates
AMPAR endocytosis upon mGluR activation [42].
Decreased dynamin3 levels in
flailer may mnediate the impairment of Arc mediated AMPAR endocytosis and may
explain the decreased mGluR-LTD in flailer. Another reason underlying the impairment of mGluR-LTD in flailer may be the decreased transport of sER into spines,
resulting in decreased calcium release from intracellular stores in CA1 neurons of the
hippocampus. It will be important to elucidate whether flailer mice have less sER in
the dendritic spines of CA1 neurons as in Purkinje cells and whether mnGluR-LTD
requires calcium release from intracellular stores in CA1 region of hippocampus as in
perirhinal cortex.
In this study, we used PPLFS to induce mGluR-LTD. Although PPLFS-LTD has
been shown to depend on mGluRs [38], protein synthesis [38, 56] and to occlude
DHPG-LTD [57], conflicting results have been reported. Volk et al. reported that
PPLFS-LTD is not dependent on mGluRs [18] since PPLFS induced LTD was normal
in mGluR1 KO mice and in mGluR5 KO mice. They confirmed that group 1 mGluR
antagonist 100 pM LY341495 abolish PPLFS-induced LTD as previously reported
[38], suggesting that LY341495 may have other unidentified targets [18].
However
Moult et al. showed that another mGluR5 antagonist, MPEP was able to prevent
PPLFS induced LTD [29], although they used grease gap recordings in adult (10-15
weeks of age) rats in contrast to field potential recordings in young adults. The same
study had two other conflicting findings with the previous studies. First, they showed
that new protein synthesis is not required in PPLFS-LTD as was previously reported
[38, 56]. Second, PPLFS-induced ULTD was not dependent on ERK, unlike DHPG
induced LTD [29], although a common mechanism between PPLFS-LTD and DHPGLTD was suggested through occlusion studies [57]. Moreover, R,onesi and Huber have
reported that nmGluR-Homer interactions are not necessary for PPLFS-LTD as they
are in DIIPG-LTD [30], indicating that different LTD induction pathways may be
involved in these two protocols. Therefore, it is important to investigate mGluR-LTD
in flailer mice with DHPG application as well as the protein synthesis dependence of
PPLFS-LTD using protein synthesis blockers.
Finally, a recent study showed that mGluR,-dependent LTD in the CA1 region is
reduced in hippocampus in rats after status epilepticus induction [58]. Humans with
Griscelli syndrome as well as dilute-lethal and flailer mnice suffer from severe epilepsy.
Therefore elucidation of the role MyosinVa plays in mnGluR-LTD may provide new
targets for the treatment of epilepsy.
Appendix A
Spine Morphology Analysis in
Flailer
We wanted to analyze the spine morphology of hippocampal pyramidal neurons in
the CA
region of flailer mice and compare against the WT mice. Previous studies
on TLS, an RNA-binding protein which is transported to spines from dendrites by
MyosinVa, showed that the TLS-deficient mice have significantly reduced density
of spines and increased number of filopodia in hippocampal pyramidal neurons [55].
Consistent with this study, previous work in our lab has revealed an increased density
of filopodia and fewer spines in flailer cortical neurons [47].
To analyze spine morphology, two approaches were used.
First, CA1 neurons
were labeled with Dil through ballistic delivery of Dil-coated particles. Preliminary
results showed no change in synaptic morphology between flailer and WT. However,
fine structure of synaptic morphology was difficult to obtain from the images of DiIlabeled cells. The second approach involved crossing flailer mice with GFP-expressing
mice. Images of dendritic spines in GFP-expressing CA1 neurons show that this is a
promising approach for dendritic spine analysis.
For spine morphology analysis, secondary branches of the apical dendrites in the
middle third of stratum radiatum, where Schaffer collateral afferents were stimulated,
were imaged. In field potential recordings, both the stimulation electrode (-70 jim
tip distance) and the recording electrode were placed in the middle part of stratumn
radiatum, which has a total length of 200 p~mn from the cell body layer in the 3-4
week old mice used in this study. To reduce variability, selection of dendritic branches
was restricted to lateral branches of the apical dendrite before branching of the apical
dendrite CA1 pyramidal neurons [59]. In addition, it was reported that the distance
of CA1 cell bodies from the border of stratum radiatum affects dendrite and spine
number [60]. Therefore, it is important to select dendritic branches of CA1 neurons
whose cell bodies are in approximately similar regions.
Dendritic protrusions were classified into four types (mushroom, thin, stubby
spines and filopodia) [61, 62]. Following criteria were used to classify these types:
1. A mushroom spine has a large distinguishable head and total spine length is
less than 1.3 /anm.
2. A thin spine has a distinguishable head and an elongated neck. Its total spine
length is more than 1.3 plm.
3. A stubby spine lacks a distinguishable neck.
4. A filopodium is a long protrusion lacking a distinguishable head.
A.1
A.1.1
DiI Labeling
Methods
Slice preparatzon
22-day-old male WT and flailer mice were anesthetized with isoflurane and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains
were dissected and post-fixed with 4% PFA for 20-30 min on ice. After postfixation,
the brains were coronally sectioned (200 ipm) using a vibratome. Brain sections only
from the dorsal part of hippocampus were collected and stored in PBS at 4C prior
to ballistic delivery of Dil-coated particles.
Ballistic delivery of Dil-coated particles
A membrane filter was placed on the slice to prevent large clumps of particles from
landing on tissue. Using Helios Gene Gun System with 60-160 psi of helium pressure,
Dil-coated tungsten particles were delivered to the fixed hippocampal slices. After
delivery of the particles, sections were transferred to a plate containing 4% PFA and
fixed overnight at room temperatuire in the dark. Following day, slices were mounted
onto slides in Fluoromount-G for confocal imaging.
Confocal imaging
Images were obtained by using a confocal laser-scanning microscope (Nikon PCM2000
(MVI)) with SimplcPCI acquisition software (Cormpix). A 60x oil-immersion objective was used and images were collected at an additional electronic zoom factor of 2.
Multiple optical sections with z steps of 0.5 pm were collected.
Data analysis
The stack of images were exported to the image analysis software, softWoRx
(Applied Precision), and deconvolved. The software had the capability to include
the z direction for analyzing stacked images. Observing images in consecutive optical
sections facilitated classifying protrusions. Lengths and head diameters of individual
spines along the dendritic branch were measured to be used for classification. Length
of the dendritic branch was also measured.
Image analysis was performed blind,
without knowing the identity of the samples during the analysis.
A.1.2
Preliminary results
Secondary apical dendrites in DiI-filled CA1 neurons in dorsal hippocampus of 22-dayold male flailer and WT mice were imaged (Fig.A-1). In total, 6 dendritic segments
in 4 neurons from 2 flailer nmice an(t 5 dendritic segments in 2 neurons from 2 WT
mice were analyzed. Preliminary analysis of spine density revealed that the mean
densities of dendritic spines in WT and flailer were similar (WT: 11.6 ± 1.2; Flailer:
9.4 ± 1.1).
The measured densities of stubby, thin and mushroom spines revealed comparable quantities for flailer and WT. The spine densities (spine number per 10pim) are
Figure A-1: Sample images of DiI filled dendritic branches in flailer (A) and WT (B)
Table A.1: The spine densities (spine number per 10pm) for flailer and WT
Stubby
Thin
Mushroom
flailer
WT
1.0 ± 1.7 1.0 ± 1.0
3.2 ± 2.0 2.5 ± 1.6
4.9 ± 3.5 4.9 ± 2.7
tabulated in Table A.1 and plotted in Fig.A-2.
In this study, filopodia spine densities could not be reliably measured due to poor
resolution of DiI images, hence were not included in the analysis. While a large number of samples are required for a conclusive assessment of spine densities, a modest
number of samples show no obvious difference between WT and flailer densities. In
general, in order to characterize the spine morphology, a consistent labeling efficiency
and high-resolution images of the spines are needed. A good labeling efficiency could
not be obtained consistently in this study due to several practical issues. These include gas pressure, filter pore density and bullet dye density. For instance, the gas
pressure was adjusted such that it was high enough to avoid low labeling densities
and low enough to prevent tissue injury. Furthermore, the labeling efficiency was adversely affected by the inaccuracy of the ballistic method in targeting the small-sized
region of interest. Furthermore, the variability of the bullet dye density prevented
obtaining consistent high-resolution images. Image resolution was also limited by the
photobleaching problem of DiI, which prevented further resolution enhancements and
consequently reduced the signal-to-noise ratio.
7
-_______
--------
_
__
__
_
__-
6E WT
EFlailer
5
43
2
stubby spine
thin spine
mushroom spine
Figure A-2: Spine densities in flailer and WT
The aforementioned practical limitations render the Di approach inconvenient for
complete characterization of the spine morphology. Consistent labeling efficiencies
and higher resolution images could be obtained by the GFP method. Therefore, we
did not further proceed with the DiI approach and continued the spine morphology
analysis with the GFP method, which is explained in the following section.
A.2
GFP-expressing CA1 neurons
A.2.1
Methods
Mice
In order to generate flailer mice with CA1 pyramidal neurons labeled with GFP,
we crossed flailer mice [12] with thyl-eGFP-S mice [63], which express eGFP in CA1
pyramidal neurons under the control of thyl promoter. The background of flailer mice
is 75% C57BL/6J and 25% CBA, and C57BL/6 is the background for thyl-eGFP-S
mice.
Homozygous flailer mice were identified with the expression of flailer phenotype,
such as ataxia and seizures since these neurological abnormalities are observed only
in homozygous flailer mice. To identify wild type mice for flailer in these crosses,
genotyping was required.
The flailer gene was amplified using primers G1F and
M5R,; however, these primers were not ideal. New primers for genotyping have been
suggested by Meisler through personal communication. The genotype of the crossed
mice for GFP was identified by epifluorescent detection of GFP fluorescence in tail
tissues from these mice.
Slice preparation
Mice were anesthetized with isoflurane and transcardially perfused with PBS
followed by 4% PFA in PBS. Brains were dissected and post-fixed with 4% PFA
overnight. The following day, the brains were sectioned (200 pm) using a vibratome.
Brains were oriented to obtain transverse sections. Brain sections only from the dorsal part of hippocainpus were collected and stored in PBS at 4oC. They were then
mounted on slides in Fluoroinount-G for confocal imaging.
Confocal Laser Microscopy
Images were obtained by using a confocal laser-scanning microscope (Nikon PCM2000
(MVI)) with SimplePCI acquisition software (Compix). A 60x oil immersion objective was used and images were collected at an additional electronic zoom factor of
4. To enhance the signal-to-noise ratio, four images of the same optical section were
averaged. Multiple optical sections with z steps of 0.3 pmi were collected. Intensity
of the argon laser was attenuated to 10% to reduce photobleaching.
Data analysis
The stack of images were exported to the image analysis software, softWoRx
(Applied Precision), and deconvolved.
Lengths and head diamneters of individual
spines along the dendritic branch were measured to be used for classification. Length
of the dendritic branch was also measured.
A.2.2
Preliminary results
Secondary branches of the apical dendrites in the middle third of the stratum radiatum were imaged in GFP-expressing CA1 neurons in slices from GFP heterozygous
and homozygous mice. Images taken from GFP homozygous mice show high density
of GFP-expressing CA1 neurons as seen in Fig.A-3A. Due to the overlap of dendritic
branches, analysis is extremely difficult. In GFP heterozygous mice, however, indi-
vidual dendrites can be imaged and analyzed (Fig.A-3B). In addition to CA1 region
of hippocampus, many other regions of the brain express GFP, including layer 5 but
not layers 2/3 of cortex (Fig.A-4).
The preliminary images from WT yielded promising results concerning the labeling efficiencies and resolution (Fig.A-5). The GFP method overcomes the practical
difficulties of the DiI method, thereby providing a promising way of spine morphology
characterization.
A
B
Figure A-3: GFP-expressing CA1 neurons imaged in a GFP homozygous mice (A)
and a GFP heterozygous mice (B)
Figure A-4: Cortical neurons expressing GFP
Figure A-5: Sample images for dendritic branches of GFP expressing CA1 neurons
in WT mice
Appendix B
L-LTP studies with interface
chamber
Activity dependent potentiation consists of three mechanistically different temporal
stages: short term potentiation phase lasting minutes, early phase of LTP that last
a few hours and late phase of LTP (L-LTP) that lasts from several hours to weeks.
Both translation [64] and transcription [65] are involved in maintenance of L-LTP.
Some protein translation occurs locally in dendritic spines [66]: L-LTP is impaired
by local application of protein synthesis inhibitors [67]. In addition, L-LTP involves
a shift of polyribosomes from the dendritic shaft into spines [68].
MyosinVa is implicated in transport of mRNA complexes from dendritic shafts
into spines. In order to investigate the role of MyosinVa in L-LTP, it is necessary
to maintain healthy slices for imany hours, providing reliable and stable electrophys-
iological recordings.
Slices can be maintained over long durations using interface
chambers where they are kept at the fluid-gas interface. In this type of chamber, oxy-
gen is supplied to the slice by heated and humidified carbogen gas administered to
the slice whereas in conventional submerged chambers, slices are not well oxygenated
due to the limited water solubility of oxygen. In fact, previous electrophysiological
and morphological studies have demonstrated that slices maintained in submerged
chambers deteriorate at a faster rate compared to those kept in interface chambers
[69, 70, 71].
To study L-LTP in flailer mice, we used MS3 Interface Chamber (Scientific Systems Design Inc.). In this chamber layout, slices rest on a piece of lens tissue while
perfusion solution flows through the tissue by capillary action. The perfusion solution
exits the chamber through the tissue that is tapered into the exit well and then removed by a peristaltic pump. A slow perfusion flow rate (0.5-1.5 ml/min) was used.
The adjustment of the solution height is extremely critical: It should be kept low
enough to maintain interface mode, whereas high enough to avoid slices becoming
dry. Small variations on lens paper tapering and the suction rate make it challenging
to maintain a consistent solution height.
The carbogen gas flows on to the slices after being deflected by a profiled acrylic
lid on the chaimber. In order to maintain a constant temperature and a high humidity
level, the carbogen gas is passed through an external humidification vessel in a heated
water bath. A high humidity was necessary to prevent the slices from drying. Excess
gas flow rates were to be avoided to prevent the surface of the slides becoming dry.
Obtaining stable recordings for a long period of time with an interface chamber
requires a fine balance of perfusion rate, gas flow rates, and humidification level. In
the recording time span of 23 days, an ideal parameter set for the interface chamber
has not been found. Even the most viable experimental settings yielded only a few
hours of stable recording, whereas longer time spans for stable baselines were required
for L-LTP experiments.
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