Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina methods

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    "name": "Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina methods",
    "description": "Evidence-backed execution summary for Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina methods from Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Intravitreal AAV injections in mice",
        "text": "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 )."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Human retina and lentivirus infection",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Fluorescence and confocal microscopy",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "2-photon imaging and patch-clamp recordings",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Multi-electrode array recordings and data analysis",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Multi-electrode array recordings and data analysis",
        "text": "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..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Visual cortex extracellular recordings",
        "text": "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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    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "Human retina and lentivirus infection"
      },
      {
        "@type": "HowToTool",
        "name": "Fluorescence and confocal microscopy"
      },
      {
        "@type": "HowToTool",
        "name": "2-photon imaging and patch-clamp recordings"
      },
      {
        "@type": "HowToTool",
        "name": "Multi-electrode array recordings and data analysis"
      },
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        "@type": "HowToTool",
        "name": "Multi-electrode array recordings and data analysis"
      },
      {
        "@type": "HowToTool",
        "name": "Visual cortex extracellular recordings"
      },
      {
        "@type": "HowToTool",
        "name": "Light-induced behavior"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Plasmids, AAV and lentivirus production"
      },
      {
        "@type": "HowToSupply",
        "name": "Primate retinal explant culture and AAV infection"
      },
      {
        "@type": "HowToSupply",
        "name": "Human retina and lentivirus infection"
      },
      {
        "@type": "HowToSupply",
        "name": "Human retina and AAV infection"
      },
      {
        "@type": "HowToSupply",
        "name": "Fluorescence and confocal microscopy"
      },
      {
        "@type": "HowToSupply",
        "name": "2-photon imaging and patch-clamp recordings"
      },
      {
        "@type": "HowToSupply",
        "name": "Multi-electrode array recordings and data analysis"
      },
      {
        "@type": "HowToSupply",
        "name": "Multi-electrode array recordings and data analysis"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina",
      "datePublished": "2016",
      "author": [
        {
          "@type": "Person",
          "name": "Abhishek Sengupta"
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        {
          "@type": "Person",
          "name": "Antoine Chaffiol"
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        {
          "@type": "Person",
          "name": "Emilie Macé"
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        {
          "@type": "Person",
          "name": "Romain Caplette"
        },
        {
          "@type": "Person",
          "name": "Mélissa Desrosiers"
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        {
          "@type": "Person",
          "name": "Maruša Lampič"
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        {
          "@type": "Person",
          "name": "Valérie Forster"
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        {
          "@type": "Person",
          "name": "Olivier Marre"
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        {
          "@type": "Person",
          "name": "John Y Lin"
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        {
          "@type": "Person",
          "name": "José-Alain Sahel"
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        {
          "@type": "Person",
          "name": "Serge Picaud"
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        {
          "@type": "Person",
          "name": "Deniz Dalkara"
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        {
          "@type": "Person",
          "name": "Jens Duebel"
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      "identifier": "10.15252/emmm.201505699"
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