Whole-Mount Retina Electrophysiology

Mice were maintained in darkness overnight before being sacrificed. After the cornea and lens were removed, a piece of retina was flattened on a membrane filter (Merck Millipore, HAWP01300). Then the whole-mounted retina was incubated in Ames’ medium and maintained at 31°C to 33°C.³³ (link) TdTomato-expressing cells were identified with a brief snapshot of fluorescence excitation light.³⁴ (link) An intracellular electrophysiological recording was then performed at tdTomato-positive cells. Light spots at different size gradients with a 2-second ON and 8-second OFF repeated three times and generated by a customized projector were casted over the retina through the 40× objective lens. The responses to spots were recorded under the current clamp. The intracellular solution contained: 121 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 4 mM MgATP, 0.3 mM Na₃GTP, 5 mM sodium phosphocreatine, and 13.4 mM biocytin (pH adjusted to 7.25 with KOH). The light intensity was calibrated to 6.2 log photons/µm²/s.²⁴ (link)^,26 (link) The latency time to peak response is defined by the time from light onset or offset to peak response.