2.4 ∣. Ex vivo rod recordings from isolated mouse retinas

Mice were dark-adapted overnight and sacrificed by CO₂ asphyxiation. The whole retina was removed from each mouse eyecup under infrared illumination and stored in oxygenated aqueous L15 (13.6 mg/mL, pH 7.4) solution (Sigma-Aldrich) containing 0.1% BSA, at room temperature. The retina was mounted on filter paper with the photoreceptor side up and placed in a perfusion chamber³⁰ between two electrodes connected to a differential amplifier. The tissue was perfused with bicarbonate-buffered Ames medium (Sigma-Aldrich) supplemented with 40 μM DL-2-amino-4-phosphonobutyric acid to block postsynaptic components of the photoresponse,³¹ and with 100 μM BaCl₂ to suppress the slow glial PIII component.³² The perfusion solution was continuously bubbled with a 95% O₂/5% CO₂ mixture and heated to 36-37°C.

The photoreceptors in the retina were stimulated with 20-ms test flashes of calibrated 505 nm LED light. The light intensity was controlled by a computer in 0.5 log unit steps. Intensity-response relationships were fitted with the following Naka-Rushton hyperbolic functions:

where R is the transient-peak amplitude of the rod or cone response, R_max is the maximal response amplitude, I is the flash intensity, n is the Hill coefficient (exponent), and I_1/2 is the half-saturating light intensity. Photoresponses were amplified by a differential amplifier (DP-311, Warner Instruments), low-pass filtered at 300 Hz (8-pole Bessel), and digitized at 1 kHz. Data were analyzed with Clampfit 10.4 and Origin 8.5 software.