Electroretinograms were recorded from PKC-KO and WT mouse littermates. Mice were dark-adapted overnight (>12 hours) and prepared for recording under dim red light. Initial anesthesia was achieved via intraperitoneal injection of ketamine:xylazine (100:10 mg/kg), and maintained with supplemental 30:3 mg/kg anesthesia injections approximately every 35 minutes. Body temperature was maintained between 35.5°C to 36.5°C by placing the mouse on a water-circulated heating pad and monitoring its temperature with a rectal thermometer. Before ERG recording, the pupil was dilated with phenylephrine (2.5%) and tropicamide (1%) and the cornea was anesthetized with topical proparacaine (1.0%). A custom-made cone placed over the snout allowed delivery of 95% O2 and 5% CO2, which helped minimize breathing artifacts during recording. A platinum needle electrode bent at 90° was placed in contact with the center of the cornea with a small amount of 2.5% methylcellulose gel, and covered with a contact lens to serve as the active electrode. Similar platinum reference and ground electrodes were placed in the forehead and tail, respectively. Following setup, the mouse and heating plate were advanced into the Ganzfeld in complete darkness and ERG recording began following an additional 12 minutes of dark adaptation. Light stimuli were provided by custom-made light-emitting diode (LED) photoflash units. The stimulus strength could be controlled by altering the flash duration under computer control. A 3.0 log unit glass neutral density filter was used to further extend the stimulus strength range. Flash intensities were measured using a photometer (Model IL1700; International Light, Newburyport, MA, USA) fitted with a scotopic filter in integrating mode that gave results as scotopic (sc) candela second per square meter. Scotopic and photopic ERGs were amplified at a gain of 5000, and were band-pass filtered (0.1–1 kHz). Data were acquired with a National Instruments data acquisition board (sampling rate: 10 kHz; National Instruments, Austin, TX, USA). Traces were recorded with customized software (ERGTool 1.0a20; Richard Weleber, Casey Eye Institute, Portland, OR, USA).
Full-field, scotopic ERGs were recorded to a series of flashes ranging between −4.5 and 2.27 log(cd-s/m2). Electroretinograms were the averages of 8 to 25 responses for dim intensities, up to −1 log(cd-s/m2). For intensities above −1 log(cd-s/m2), three trials were averaged. For brighter stimuli above 1 log(cd-s/m2), each ERG was recorded to two flashes. The interflash intervals were 10 seconds for the −4.5 to −1 log(cd-s/m2) flashes, 20 seconds for the −1 to 0 log(cd-s/m2) flashes, 30 seconds after the 0 log(cd-s/m2) flash, 60 seconds after the 1 log(cd-s/m2) flash, and 120 seconds after 2 log(cd-s/m2) flash.