All procedures were performed under infrared (~1 mm) illumination with dual-unit Nitemare (BE Meyers, Redmond, WA) infrared scopes. The whole-cell patch-clamp recording (Pang et al., 2010a (link), 2012 (link)), preparation of living retinal slices (Werblin, 1978 (link); Wu, 1987 (link)), light simulation, immunofluorescence, and confocal microscopy (Pang et al., 2018 (link); Gao et al., 2019 (link)) essentially followed procedures described in previous publications.
Animals were dark-adapted for 1–2 h before the related experiment. The Ames medium in the recording chamber was oxygenated and maintained at 34°C with a temperature control unit (TC 324B, Warner Instruments, CT). The controller was wired with DigiData1322A to record and monitor the temperature. Axopatch 700A and 700B amplifiers were connected to DigiData 1322A interfaces and operated by the pClamp software v9.2 and v10.3 (Axon Instruments, Foster City, CA). Patch pipettes had 9–12 MΩ tip resistance when filled with an internal solution containing 112 mM Cs-methanesulfonate, 12 mM CsCl, 5 mM EGTA, 0.5 mM CaCl2, 4 mM ATP, 0.3 mM GTP, 10 mM Tris, and 0.5% Lucifer yellow, adjusted to pH 7.3 with CsOH. For current-clamp and some voltage-clamp recordings, the pipettes were filled with internal solutions containing: 112 mM K-gluconate, 10 mM KCl, 10 mM EGTA, 10 mM HEPES, 0.5 mM CaCl2, 1 mM MgCl2, 4 mM Na2-ATP, 0.3 mM Na3-GTP, and 0.5% Lucifer yellow, adjusted to pH 7.3 by KOH. The internal solution and external normal Ringer’s solution yield a chloride reversal potential (ECl) of −59 mV at room temperature. Recorded cells were visualized by Lucifer yellow fluorescence with a confocal microscope (LSM 510 and LSM 800, Carl Zeiss, Germany).
A photostimulator delivered light spots of a diameter of 600–1,200 μm and 500 nm wavelength (λmax = 500 nm, full width-half max 10 nm) at a series of intensities (−10 to −1 log I) to stimulate the retina via the epi-illuminator of the microscope (Maple and Wu, 1998 (link); Pang et al., 2002 (link), 2010b (link)). Since we delivered uncollimated light beams through an objective lens of a large numerical aperture (Zeiss 40x/0.75 water), the incident light could enter the retina in many directions and, thus, had a minor photoreceptor self-screening effect (Field and Rieke, 2002 (link)). The intensity of unattenuated (0 in log unit (log I)) 500 nm light from a halogen light source was 4.4 × 105 photons.μm−2.sec−1. The light intensity was transformed into the unit of photoisomerization per rod per second (Rh*rod−1 s −1) with a rod cross-section of 0.5 μm−2 (Howes et al., 2002 (link)) and a rod integration time of 0.4 s (Baylor, 1987 (link)).
Animals were dark-adapted for 1–2 h before the related experiment. The Ames medium in the recording chamber was oxygenated and maintained at 34°C with a temperature control unit (TC 324B, Warner Instruments, CT). The controller was wired with DigiData1322A to record and monitor the temperature. Axopatch 700A and 700B amplifiers were connected to DigiData 1322A interfaces and operated by the pClamp software v9.2 and v10.3 (Axon Instruments, Foster City, CA). Patch pipettes had 9–12 MΩ tip resistance when filled with an internal solution containing 112 mM Cs-methanesulfonate, 12 mM CsCl, 5 mM EGTA, 0.5 mM CaCl2, 4 mM ATP, 0.3 mM GTP, 10 mM Tris, and 0.5% Lucifer yellow, adjusted to pH 7.3 with CsOH. For current-clamp and some voltage-clamp recordings, the pipettes were filled with internal solutions containing: 112 mM K-gluconate, 10 mM KCl, 10 mM EGTA, 10 mM HEPES, 0.5 mM CaCl2, 1 mM MgCl2, 4 mM Na2-ATP, 0.3 mM Na3-GTP, and 0.5% Lucifer yellow, adjusted to pH 7.3 by KOH. The internal solution and external normal Ringer’s solution yield a chloride reversal potential (ECl) of −59 mV at room temperature. Recorded cells were visualized by Lucifer yellow fluorescence with a confocal microscope (LSM 510 and LSM 800, Carl Zeiss, Germany).
A photostimulator delivered light spots of a diameter of 600–1,200 μm and 500 nm wavelength (λmax = 500 nm, full width-half max 10 nm) at a series of intensities (−10 to −1 log I) to stimulate the retina via the epi-illuminator of the microscope (Maple and Wu, 1998 (link); Pang et al., 2002 (link), 2010b (link)). Since we delivered uncollimated light beams through an objective lens of a large numerical aperture (Zeiss 40x/0.75 water), the incident light could enter the retina in many directions and, thus, had a minor photoreceptor self-screening effect (Field and Rieke, 2002 (link)). The intensity of unattenuated (0 in log unit (log I)) 500 nm light from a halogen light source was 4.4 × 105 photons.μm−2.sec−1. The light intensity was transformed into the unit of photoisomerization per rod per second (Rh*rod−1 s −1) with a rod cross-section of 0.5 μm−2 (Howes et al., 2002 (link)) and a rod integration time of 0.4 s (Baylor, 1987 (link)).
