On-line Supplementary Methods
Animals
Mice were maintained in the animal facility at University College London. All experiments
have been conducted in accordance with the Policies on the Use of Animals and
Humans in Neuroscience Research, revised and approved by the Society for
Neuroscience in January 1995. Animal strains used included: Cba.gfp+/+, Ck6.cfp
(Jackson Laboratories), Nrl.gfp+/+, rd, rds, rho-/- (kind gift of P. Humphries). These have
been described elsewhere6,7,13,24,24,26,27. Mice defined as “adult” were at least 6, but not
more than 12, weeks old.
Dissociation of retinal cells and transplantation
Dissociated retinal cells were prepared from transgenic mice that are hemizygous for a
ubiquitously expressed gfp transgene7 or from Nrl.gfp+/+ transgenic mice6. Mice were
sacrificed by cervical dislocation and neural retinas dissected free from surrounding
tissues. Cells were dissociated using a papain-based kit (Worthington Biochemical,
Lorne Laboratories UK) and diluted to a final concentration of ~4 x 105 cells / l. Where
appropriate, retinas from P1 Nrl.gfp+/+ mice were dissociated, as described above, before
being sorted into Nrl.gfp-positive and Nrl.gfp-negative populations, using FACS. The
final concentration of sorted Nrl.gfp-positive cells was ~2 x 105 cells / l. Surgery was
performed under direct ophthalmoscopy control through an operating microscope.
Recipient mice were anaesthetised with a single intra-peritoneal injection of 0.15 ml of a
mixture of Dormitor (1 mg/ml medetomidine hydrochloride, Pfizer Pharmaceuticals, Kent
UK), ketamine (100 mg/ml, Fort Dodge Animal Health, Southampton, UK) and sterile
water for injections in the ratio of 1:0.6:84 for P1 pups and 5:3:42 for adult mice. The tip
of a 1.5 cm, 34-gauge hypodermic needle (Hamilton, Switzerland) was inserted through
the sclera into the intravitreal space to reduce intraocular pressure. The needle was then
withdrawn and loaded with cells before re-inserting tangentially through the sclera into
the sub-retinal space, causing a self-sealing wound tunnel. Cell suspensions were
injected (0.5 l per eye for P1 recipients, 2 l per eye for adults) slowly to produce a
retinal detachment in the superior and/or inferior hemisphere around the injection sites.
Mice were sacrificed at least 21 days after transplantation and eyes were fixed in 4 %
paraformaldehyde in phosphate-buffered saline (PBS). Retinal sections were prepared
by cryoprotecting fixed eyes in 20 % sucrose, before embedding in OCT (TissueTek)
and frozen in isopentane cooled in liquid nitrogen. Cryosections (18 µm thick) were cut
and affixed to poly-L-lysine coated slides. All sections were collected for analysis.
Histology and Immunohistochemistry
Retinal sections were pre-blocked in Tris-buffered saline (TBS) containing normal goat
serum, bovine serum albumin and 0.1 % Triton-X 100 for 1h before being incubated with
primary antibody overnight at 4°C. After rinsing 3 x 10 mins with TBS, sections were
incubated with secondary antibody for 2 hrs at room temperature (RT), rinsed and
counter-stained with Hoechst 33342. Negative controls omitted the primary antibody.
The following antibodies were used: rabbit anti-peripherin-2 (kind gift of G. Travis),
mouse anti-rhodopsin (Rho4D2; kind gift of R. Molday), sheep anti-phosducin (kind gift
of V. Arshavsky), rabbit anti-bassoon (Stressgen) and rabbit anti-PKC (AbCam), with
appropriate Cy3- (Jackson ImmunoResearch) or Alexa- (Molecular Probes, Invitrogen)
tagged secondary antibodies.
BrdU labelling
Labelling dividing cells post-transplantation
P1 cells were prepared as described above. Recipient adult mice received intraperitoneal injections of bromodeoxyuridine, BrdU (100 ng/g body weight) immediately
following transplant and every other day for the next 8 days.
Labelling donor cells prior to transplantation
P1 received x 3 intra-peritoneal injections of BrdU (100 ng/g body weight) 4 hrs apart, in
order to label the DNA of the nucleus of a cohort of donor cells. Cells were dissociated,
as described above, and transplanted into adult wildtype recipients.
Immunohistochemistry for BrdU labelling
Retinal sections were washed in dH20 before incubating in 2M HCl for 2 hrs at 37°C,
0.1M Na-Borate for 20 mins at RT and 3 x 10 mins wash in TBS. Sections were then
blocked in TBS containing normal goat serum, bovine serum albumin and 0.1% Triton-X
100 for 1h at RT, prior to incubation with anti-BrdU (rat) primary antibody overnight at
RT. Following 3 x 10 mins wash with TBS, sections were incubated with secondary
antibody (goat anti-rat Cy3; Jackson ImmunoResearch) for 2h at RT, washed in TBS
and counter-stained with Hoechst 33342. Negative controls omitted the primary
antibody.
Confocal microscopy
Histology/immunohistochemistry
Retinal sections were viewed on a confocal microscope (Leica SP2 or Zeiss LSM510).
GFP-positive cells were located using epifluorescence illumination before taking a series
of XY optical sections, approximately 0.2 - 0.4 µm apart, throughout the depth of the
section. Individual XY scans were built into a stack to give an XY projection image. The
fluorescence of Hoechst, GFP and Cy3/Alexa-546 were sequentially excited using the
350 nm line of a UV laser, the 488 nm line of an argon laser and the 543 nm line of a
HeNe laser, respectively. In each case, projections of the XYZ stacks were generated,
as described above. Unless otherwise stated, images show (i) show merged Nomarski
and confocal fluorescence projection images of GFP (green) and the nuclear counter
stain, Hoechst 33342 (blue) (or propidium iodide, in some instances), and
immunolabelling where appropriate and (ii) the same region showing GFP signal only.
For co-localisation assessments, single confocal sections were taken at the level of GFP
signal from the integrated cell, in addition to the standard projection images. For
simplicity, only the ONL is shown, unless otherwise stated.
To visualise GFP cells transplanted into CFP recipients, the fluorescence of GFP and
CFP were excited sequentially. GFP fluorescence was excited, as described above, and
the emission collected at 505-550 nm, whilst that of CFP was excited using the 405 nm
line of a blue diode laser and the emission collected at 450-485 nm. Separation of the
fluorescence signals of the two proteins is complete when acquired at these
wavelengths.
Calcium Imaging
Retinas transplanted with Nrl.gfp+/+ P1 cells were dissected free of all surrounding tissue.
Nrl.gfp+/+ donors were used to ensure responses were recorded from rod
photoreceptors. Whole-mount neural retinas were loaded with Fura Red-AM (15 M,
Molecular Probes) and the dispersant Cremophor-EL (0.03%, Sigma) for 1.5 hrs at 36°C
and then de-esterified in fresh DMEM-F12 (without phenol red) for 30 mins at 36°C.
Retinas were transferred to the stage of an inverted microscope (SP2, Leica, UK) and
held flat under a nylon-strung platinum wire ‘harp’. XY images were taken through the
cell bodies in the inner region of the ONL, nearest the outer plexiform layer, where rod
(as opposed to cone, which lie at the outer edge of the ONL) photoreceptor nuclei
reside.
Images were acquired at 3 sec intervals and analysed off-line. Cells were
selected at random and the mean fluorescence of individual cells was calculated and
normalised against the fluorescence at time 0s. Drugs were applied by micro-pipette
injection into the bathing solution. NB, when collected at 660 50 nm, the emission of
Fura-Red undergoes an increase in fluorescence as [Ca2+]i decreases.
Drugs
DCPG ((S)-3,4-dicarboxyphenylglycine) (20 M; final concentration in bath), CPPG
((RS)-alpha-cyclopropyl-4-phosphonophenylglycine) (100 M) and NMDA (N-methyl-Daspartate) (200 M) were supplied by Tocris (UK).
Developmental window cell counts
To assess the integration potential of donor cells from a range of developmental stages,
adult animals received a single 1l injection of 4 x 105 cell / l in each eye. Three weeks
post-transplantation, animals were sacrificed and the eyes prepared for analysis as
described above. Cells were considered integrated if the whole cell body was visible
together with at least one of the following; spherule synapse and/or inner/outer
segments. The average number of integrated cells per section was determined by
counting all the integrated GFP-positive cells in every 1 in 4 serial sections through the
site of injection in each eye. This was multiplied by the total number of sections that
encompassed the injection site to give an estimate of the mean number of integrated
cells per eye.
Assessment of light sensitivity
Pupillometry
Following dark-adaptation for at least 1h during the light phase of their light/dark cycle,
un-anaesthetized were manually held with the eye to be recorded perpendicular to an
infrared sensitive camera fitted with a macro lens. Background illumination was provided
by infrared LEDs throughout the experiment. Animals were subjected to a series of 10
second white light exposures of ascending irradiance controlled by neutral density filters
provided by a fiber optic from a 100W halogen lamp (Zeiss). At least 2 mins elapsed
between exposures, during which time the animal was unrestrained.
A complete
intensity series was performed for one eye before retesting the other eye at identical
intensities, with at least an hour of darkness between exposures of the 2 eyes.
Subsequently, pupil area was determined from individual video frames captured 5s after
light exposure, at which time constriction was maximal. The effective intensity of each
exposure was calculated by measuring the spectral irradiance (photons/s/m2/nm)
incident on the cornea, at 1nm intervals between 300-870nm with a Ocean Optics
USB2000 spectrometer fitted with a P-600-5-UV/Vis fiber optic and CC-3-UV irradiance
collector (previously calibrated with reference to an Ocean Optics DH-2000-CAL
calibration light) and weighting these data by the spectral sensitivity of the wildtype
murine pupil response29. To facilitate comparisons between individuals, pupil areas (ai)
were expressed relative to the dilated area immediately prior to each exposure (a0). A 4
term sigmoid was fitted to the pupil area vs irradiance data for each eye and the
irradiance required to give 50% of the dilated pupil area taken as a measure of that eye’s
sensitivity. Following pupil assessment, animals were sacrificed and eyes prepared for
analysis as described above. The total number of integrated cells per eye was
determined by counting all the GFP-positive cells in the ONL of every section. Slide
identity was masked by an independent observer prior to assessment.
Extracellular Field Potential Recordings
Three weeks after transplantation, mice were dark-adapted for 1h prior to sacrifice in the
dark. Eyes were removed under infra-red light and the lens and vitreous were dissected
away, but the RPE was left intact. Four small cuts were made to allow the retinal
wholemount to lie flat. Preparations were mounted GCL side uppermost in a blacked-out
interface recording chamber where they were continually perfused with oxygenated
Krebs’ solution (containing, in mM: NaCl, 124; KCl, 3; KH2PO4, 1.25; MgSO4, 1; CaCl2, 2;
NaHCO3, 26 and glucose, 10), maintained at 34°C. Extracellular recordings were made
approximately 30mins after the retina was positioned in the chamber, from the GCL
using glass microelectrodes (1-3 M-Ohm) filled with the same Krebs’ medium as that
used to maintain the slices in the recording chamber. Recordings were made in at least
8 independent regions around the optic nerve head. Light-evoked potentials were
stimulated by flashes (100ms duration, 0.5s interval) of increasing intensity emitted by a
green LED (562nm peak wavelength) positioned 8mm above the retina. Voltage
responses, evoked by 10-20 flashes at each intensity, were recorded via an Axoprobe
1A amplifier (Axon Instruments), digitized via a CED1401 interface (Cambridge
Electronic Design), and stored on a computer system running Spike2 software
(Cambridge Electronic Design). Average responses (10-20 responses) were computed
and average light intensity plots were drawn for each eye by determining the average
voltage change from all regions of interest (ROIs) at each stimulus intensity. The
stimulus threshold for a light-evoked response was determined as being the stimulus
intensity that evoked a response magnitude that was 10% of the potential evoked by the
maximum stimulus. Quantitative results are expressed as mean ± SEM.
N numbers
N=number of eyes, n=number of cells examined, where appropriate.