Espion visual electrophysiology system

Scotopic electroretinography (ERG) was conducted in 12-week old WT C57BL/6 J mice before and after STF31 treatment using Espion Visual Electrophysiology system (Diagnosys LLC, Cambridge, UK) as described previously [23 (link)]. Briefly, mice were anesthetized and pupils dilated as above. The mouse was placed on the heat-pad (38 °C). ERG was recorded using mouse corneal ERG electrodes in response to single white light flash, delivered by a standard Ganzfeld Stimulator (Diagnosys LLC). ERG signals were bandpass filtered between 0.3–500 Hz (without notch filtering), amplified 500-fold and digitized at 2 kHz using the CMGS-1 electrophysiology system (Diagnosys LLC).

Full-field electroretinography (ERG) recordings were conducted as described previously (Xu et al., 2012 (link)). Briefly, after overnight dark adaptation, mice were anesthetized by intraperitoneal injection of 85 mg/kg ketamine and 14 mg/kg xylazine. ERGs were recorded using the Espion Visual Electrophysiology System (Diagnosys) with the ColorDome Advanced Performance Ganzfeld Dome system (Diagnosys). Potentials were recorded using a gold-wire electrode to contact the corneal surface through a layer of 2.5% hypromellose (Gonak, Akorn Pharmaceuticals). For the assessment of scotopic responses, a stimulus intensity of 1.89 log cd · s m⁻² was presented to dark-adapted dilated mouse eyes. To evaluate photopic responses, mice were adapted to a 1.48 log cd · s m⁻² light for 7 min, and then a light intensity of 1.89 log cd · s m⁻² was given. Responses were differentially amplified, averaged, and analyzed using Espion 100 software (Diagnosys).

ERG recordings were performed with the Espion visual electrophysiology system (Diagnosys LLC, Lowell, MA, USA) that includes a ColorDome Ganzfeld. ERG was performed one week after BLD. After overnight dark adaptation, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). Eye drops were used to dilate the pupils (0.5 % tropicamide+ 5% phenylephrine hydrochloride) and anesthetize the cornea (0.4 % oxybuprocaine chlorhydrate). Body temperature was maintained at 37°C using a circulating hot-water heating pad. Corneal electrodes (Ocuscience, a subsidiary of Xenolec Inc., USA) were placed on the corneal surface of each eye. Lubrithal eye gel was used to maintain good contact and corneal moisture. Needle electrodes placed subcutaneously in cheeks served as reference and a needle electrode placed in the back served as earth. The ERG was recorded from both eyes simultaneously after placing the animal into the Ganzfeld bowl. Five responses to light stimulus at increasing intensities (0.01, 0.1, 1, 10 and 30 cd.s.m^-2) were averaged for scotopic response. After 5 min of light adaptation, the photopic response was recorded at the highest stimulus (average of 5 measurements at 30 cd.s.m^-2).

Ganzfeld ERG was performed using an Espion visual electrophysiology system (Diagnosys LLC, Littleton, MA, USA), according to manufacturer’s instructions and as previously described by our group [31 (link),32 (link)]. Briefly, four responses were averaged at each light intensity (0.8, 2.5, 8, and 25 cdxs/m²). A-wave and B-wave amplitudes were measured using the Espion analysis software (Diagnosys Technologies, Littleton, MA, USA).

Electroretinography (ERG) analysis was performed on AAV5-hNR2E3 treated and untreated mouse eyes with the Espion Visual Electrophysiology System (Diagnosys LLC, Lowell, MA, USA). Mice were dark-adapted overnight in a dark chamber and anesthetized with ketamine/xylazine by IP injection using a 0.5” 25G needle. Pupils were dilated with a drop of 1% Tropicamide and kept moist using a topical application of Genteal. Mice were positioned on to the platform and the ground electrode was placed at the base of the tail followed by the reference electrode placed just underneath the skin of the scalp. Two gold loop electrodes (Diagnosys LLC) were aligned and placed flush on the eyes of the mouse. Dark- and light-adapted ERGs were performed and recorded using the previously described protocol [19 (link)]. Peak scotopic A- and B-wave amplitudes were taken from each animal and averaged for each dose and timepoint compared to the untreated and wild-type amplitudes.