Labeling of individual RGCs was performed using a previously described protocol (Beier et al., 2013 (link); Dhande et al., 2013 (link): Cruz-Martin et al., 2014 (link); El-Danaf and Huberman, 2015 (link)). Mice were anesthetized with inhalant isoflurane and the eyes were enucleated. The dorsal pole of the left and right eyes were marked before removing them from the animal, using waterproof color markers thus ensuring that knowledge about retinal location was preserved (Wei et al., 2010 (link)). Retinas were cut in half along the dorsal-ventral (D-V) or nasal-temporal (NT) axes using vascular landmarks (Wei et al., 2010 (link)), and kept in oxygenated (95% O2/5% CO2) NaHCO3 (23 mM) containing Ame’s medium (Sigma-Aldrich, catalog #A1420). The fluorescent RGC somas were localized using differential interference contrast (DIC) and epifuorescence microscopy. RGCs were targeted with borosilicate glass electrodes (Sutter Instruments; 15–20 MΩ) containing Alexa Fluor 555 hydrizide dye (10 mM in 200 mM KCl; Invitrogen, catalog #A20501MP), and were completely filled by applying hyperpolarizing current pulses ranging between 0.1– 0.9 nA for ≤1 minute.
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Labeling and Imaging of Individual Retinal Ganglion Cells
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Alexa fluor 555
Animal
Axes
Eyes
Inhalant
Isoflurane
Mice
Microscopy
Nahco3
Nasal
Pulses
Retinas
Somas
Vascular
Corresponding Organization : Neurosciences Institute
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Variable analysis
independent variables
- Labeling of individual RGCs
- Localization of fluorescent RGC somas using differential interference contrast (DIC) and epifuorescence microscopy
- Filling of RGCs with Alexa Fluor 555 hydrizide dye
- Anesthesia with inhalant isoflurane
- Removal of eyes from animal with dorsal pole marked for retinal location preservation
- Cutting of retinas along dorsal-ventral (D-V) or nasal-temporal (NT) axes using vascular landmarks
- Keeping retinas in oxygenated (95% O2/5% CO2) NaHCO3 (23 mM) containing Ame's medium
- Targeting of RGCs with borosilicate glass electrodes (15–20 MΩ) containing Alexa Fluor 555 hydrizide dye (10 mM in 200 mM KCl)
- Application of hyperpolarizing current pulses (0.1–0.9 nA for ≤1 minute) to completely fill RGCs
- No positive or negative controls were explicitly mentioned in the protocol.
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