Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina methods
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Intravitreal AAV injections in mice
Rd1 mice, 4-5 weeks old, were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg). Intravitreal injection was performed bilaterally: Pupils were dilated, and an ultrafine needle was passed through the sclera, at the equator and next to the limbus, into the vitreous cavity in order to inject 2 µl AAV2-hSyn:ReaChR-mCitrine AAV (2.53 × 10 13 vector genomes ml -1 ).
Human retina and lentivirus infection
Postmortem human ocular globes from donors were acquired from the School of Surgery (Ecole de Chirurgie, Assistance Publique Hôpitaux de Paris) via a protocol approved by the institutional review boards of the School of Surgery and the Quinze-Vingts National Ophthalmology Hospital (CPP Ile-de-France V). All experiments on postmortem human retinal explants were performed according to local regulations as well as the guidelines of the Declaration of Helsinki. Eyes were collected from six anonymous donors (ranging from 63 to 95 years of age) at postmortem delays ranging from 9 to 38 h. The eyes were transported in CO 2 -independent medium and upon arrival were dissected under room light and processed for tissue culture exactly according to the procedure used for the non-human primate retinae. From each donor retina, peripheral pieces were test...
Fluorescence and confocal microscopy
Eyecups from ReaChR-treated rd1 mice were harvested at 2-4 months post-injection for immunofluorescence. The eyecups were fixed in 4% paraformaldehyde for 30 minutes at room temperature followed by three washes in PBS at room temperature and stored overnight in PBS containing 30% (w/v) sucrose (Sigma-Aldrich, St. Louis, MO). The eyecups were then dissected in PBS, and the anterior segments of the eye were removed. The neural retina together with the retinal pigment epithelium were embedded in 4% agarose and 30% sucrose (w/v in PBS) and frozen in -80°C until sectioning. The frozen blocks were then sectioned in a cryostat, and 12-µm retinal sections were mounted on slides for confocal imaging. Prior to imaging, the retinal sections were fixed in 4% PFA for 30 min, rinsed 5 × with PBS, incubated with DAPI (1:5,000, Invitrog...
2-photon imaging and patch-clamp recordings
A custom-made 2-photon microscope equipped with a 25 × water immersion objective (XLPlanN-25x-W-MP/NA1.05, Olympus) and a pulsed femto-second laser (InSight ™ DeepSee ™, Newport Corporation, Irvine, CA) was used for imaging and patch-clamp recording of mCitrine or GFP-positive ganglion cells. Mice were sacrificed by CO 2 inhalation followed by quick cervical dislocation, and eyeballs were removed. ReaChR-treated retinae from rd1 mice were isolated in oxygenated (95% O 2 /5% CO 2 ) Ames medium (Sigma-Aldrich) and placed in the recording chamber of the microscope at 36°C for the duration of the experiment. For live 2-photon imaging, a whole-mount retina with ganglion-cell-side up was placed in the recording chamber of the microscope. Image acquisition was made using the excitation l...
Multi-electrode array recordings and data analysis
All multi-electrode array recordings were performed on a 252 channel multi-electrode array system (USB-MEA256-System, Multi Channel Systems, Reutlingen, Germany), and data were acquired using the MC_Rack software (MC_Rack v4.5, Multi Channel Systems). Ex vivo isolated flat-mounted retinae of ReaChR-treated rd1 mice aged 2 months or older as well as ReaChR-treated primate ex vivo retinal explants in tissue culture were tested. Mice were euthanized as described above and post-dissection, the retinae were placed on a cellulose membrane soaked overnight in poly-L-lysine (Sigma-Aldrich) and superfused in oxygenated Ames medium (A1420, Sigma-Aldrich) containing sodium bicarbonate (Sigma-Aldrich) at 34°C, with the RGC side gently pressed against a 60-µm electrode spacing multi-electr...
Multi-electrode array recordings and data analysis
For the primate experiments (macaque and human), retinal explants infected with AAV or lentivirus in tissue culture for 3-12 days were utilized by which time the innate light responses of the retina are undetectable in the multi-electrode array. For the primate experiments, immediately prior to the experiment, a macaque or human retinal explant piece that had been infected with the CAG:ReaChR AAV or hSyn:ReaChR lentivirus was dismounted from the Transwell membrane (Corning, Tewksbury, MA) and mounted on a cellulose membrane soaked overnight in poly-l-lysine (Sigma-Aldrich) and gently lowered on a 100 µm multi-electrode array (Multichannel systems). Tissues were superfused in oxygenated Ames medium (containing sodium bicarbonate) at 34 °C. Data are presented from recordings obtained after L-AP4 was added to the medium to blo...
Visual cortex extracellular recordings
As previously described in detail (Mace et al, ), mice (3-9 months old) were sedated with ketamine-xylazine injection and deeply anesthetized with urethane. Blocked by a stereotaxic holder, the mice were maintained at 37°C (heating pad controlled by a rectal probe). Both eyes (with pupils dilated) were covered with small coverslips to avoid dehydration. A 1-mm 2 craniotomy was performed above V1 in the left hemisphere (3 mm lateral and 0.5 mm rostral from the lambda point). The dura was removed, and the exposed area was maintained, cleaned, and hydrated. Extracellular recordings were made with a silicon multisite (16) electrode (A1 × 16-3 mm-50-703, NeuroNexus Technologies, Ann Arbor, MI), in order to record simultaneously multiple layers of cortex (spikes and LFP). The multi-electrode was gently i...
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Rd1 mice, 4-5 weeks old, were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg). Intravitreal injection was performed bilaterally: Pupils were dilated, and an ultrafine needle was passed through the sclera, at the equator and next to the limbus, into the vitreous cavity in order to inject 2 µl AAV2-hSyn:ReaChR-mCitrine AAV (2.53 × 10 13 vector genomes ml -1 ).
Postmortem human ocular globes from donors were acquired from the School of Surgery (Ecole de Chirurgie, Assistance Publique Hôpitaux de Paris) via a protocol approved by the institutional review boards of the School of Surgery and the Quinze-Vingts National Ophthalmology Hospital (CPP Ile-de-France V). All experiments on postmortem human retinal explants were performed according to local regulations as well as the guidelines of the Declaration of Helsinki. Eyes were collected from six anonymous donors (ranging from 63 to 95 years of age) at postmortem delays ranging from 9 to 38 h. The eyes were transported in CO 2 -independent medium and upon arrival were dissected under room light and processed for tissue culture exactly according to the procedure used for the non-human primate retinae. From each donor retina, peripheral pieces were tested on the multi-electrode array to identify viable retinae exhibiting spontaneous RGC activity. The macula from one retina (received at a postmortem delay of 10 h) exhibiting RGC spontaneous spiking was dissected through the foveal pit (see Fig A and B), and this piece was infected...
Eyecups from ReaChR-treated rd1 mice were harvested at 2-4 months post-injection for immunofluorescence. The eyecups were fixed in 4% paraformaldehyde for 30 minutes at room temperature followed by three washes in PBS at room temperature and stored overnight in PBS containing 30% (w/v) sucrose (Sigma-Aldrich, St. Louis, MO). The eyecups were then dissected in PBS, and the anterior segments of the eye were removed. The neural retina together with the retinal pigment epithelium were embedded in 4% agarose and 30% sucrose (w/v in PBS) and frozen in -80°C until sectioning. The frozen blocks were then sectioned in a cryostat, and 12-µm retinal sections were mounted on slides for confocal imaging. Prior to imaging, the retinal sections were fixed in 4% PFA for 30 min, rinsed 5 × with PBS, incubated with DAPI (1:5,000, Invitrogen, Carlsbad, CA) and washed in PBS three times, and cover-slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Primate retinal explants were also processed as above except that the retinal explant pieces were fixed prior to embedding in the sectioning medium. The hu...
A custom-made 2-photon microscope equipped with a 25 × water immersion objective (XLPlanN-25x-W-MP/NA1.05, Olympus) and a pulsed femto-second laser (InSight ™ DeepSee ™, Newport Corporation, Irvine, CA) was used for imaging and patch-clamp recording of mCitrine or GFP-positive ganglion cells. Mice were sacrificed by CO 2 inhalation followed by quick cervical dislocation, and eyeballs were removed. ReaChR-treated retinae from rd1 mice were isolated in oxygenated (95% O 2 /5% CO 2 ) Ames medium (Sigma-Aldrich) and placed in the recording chamber of the microscope at 36°C for the duration of the experiment. For live 2-photon imaging, a whole-mount retina with ganglion-cell-side up was placed in the recording chamber of the microscope. Image acquisition was made using the excitation laser at a wavelength of 930 nm (Fig B). For patch-clamp recordings, a CCD camera (Hamamatsu Corp., Bridgewater, NJ) was used to visualize the retina under infrared light, and AAV-transduced fluorescent ganglion cells were targeted with a patch electrode under visual guidance u...
All multi-electrode array recordings were performed on a 252 channel multi-electrode array system (USB-MEA256-System, Multi Channel Systems, Reutlingen, Germany), and data were acquired using the MC_Rack software (MC_Rack v4.5, Multi Channel Systems). Ex vivo isolated flat-mounted retinae of ReaChR-treated rd1 mice aged 2 months or older as well as ReaChR-treated primate ex vivo retinal explants in tissue culture were tested. Mice were euthanized as described above and post-dissection, the retinae were placed on a cellulose membrane soaked overnight in poly-L-lysine (Sigma-Aldrich) and superfused in oxygenated Ames medium (A1420, Sigma-Aldrich) containing sodium bicarbonate (Sigma-Aldrich) at 34°C, with the RGC side gently pressed against a 60-µm electrode spacing multi-electrode array chip (256MEA60/10iR, Multi Channel Systems). Full-field light stimuli were presented using a Polychrome V monochromator (TILL Photonics, Munich, Germany) and driven by a STG2008 stimulus generator (Multichannel Systems), using custom-written stimuli in MC_Stimulus II (MC_Stimul...
For the primate experiments (macaque and human), retinal explants infected with AAV or lentivirus in tissue culture for 3-12 days were utilized by which time the innate light responses of the retina are undetectable in the multi-electrode array. For the primate experiments, immediately prior to the experiment, a macaque or human retinal explant piece that had been infected with the CAG:ReaChR AAV or hSyn:ReaChR lentivirus was dismounted from the Transwell membrane (Corning, Tewksbury, MA) and mounted on a cellulose membrane soaked overnight in poly-l-lysine (Sigma-Aldrich) and gently lowered on a 100 µm multi-electrode array (Multichannel systems). Tissues were superfused in oxygenated Ames medium (containing sodium bicarbonate) at 34 °C. Data are presented from recordings obtained after L-AP4 was added to the medium to block any remaining input from the photoreceptors to ON bipolar cells. L-AP4 was freshly diluted to a final concentration of 50 µM and bath-applied through the perfusion system for at least 15 min prior to recordings. The action spectrum of ReaChR was constructed in these ret...
As previously described in detail (Mace et al, ), mice (3-9 months old) were sedated with ketamine-xylazine injection and deeply anesthetized with urethane. Blocked by a stereotaxic holder, the mice were maintained at 37°C (heating pad controlled by a rectal probe). Both eyes (with pupils dilated) were covered with small coverslips to avoid dehydration. A 1-mm 2 craniotomy was performed above V1 in the left hemisphere (3 mm lateral and 0.5 mm rostral from the lambda point). The dura was removed, and the exposed area was maintained, cleaned, and hydrated. Extracellular recordings were made with a silicon multisite (16) electrode (A1 × 16-3 mm-50-703, NeuroNexus Technologies, Ann Arbor, MI), in order to record simultaneously multiple layers of cortex (spikes and LFP). The multi-electrode was gently inserted into the cortex perpendicularly to the surface of the brain and 600 µm deep. Then, the tissue surface was covered with agarose (1.2% in cortex buffer). Signals were amplified using a 16-channel amplifier from MultiChannel Systems (model ME16-FAI-µPA-Sys...
Machine-readable layer
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