The Use of Optogenetics to Study Inhibition in the Ex Vivo Mouse Electroretinogram

Abstract

Purpose : The mouse Electroretinogram is a well characterized tool used to measure hyperpolarization of photoreceptors followed by excitatory activation of bipolar cells and ganglion cells across the entire retina. While the interaction of inner retinal neurons can be measured, we are not able to measure inhibition in isolation across the entire retina. Previous data has shown that inhibition likely plays an important role in light adaptation. Finding a way to measure inhibition across the entire retina could be an important tool to understanding the mechanisms of light adaptation and the role inhibition plays.

Methods : Retinas were isolated from 7-10 week old B6.Cg-Tg(Slc32al-COP4*H134R/EYFP) male and female mice and placed in a custom ERG chamber. Retinas were perfused with Ringers solution and bubbled with 95% O₂-5% CO₂. These mice contain ChR2 in the inhibitory amacrine and horizontal cells under the VGAT promoter and were optogenetically activated with 5ms, (20 hz frequency) light stimulations (λ 470 nm) at 3.19x10⁹ photons/µm². ERGs were recorded under ambient room light. Photoreceptor inputs were pharmacologically blocked with CNQX (25 µM), APV (50 µM), ACET (1 μM), and L-AP4 (50 µM). Inhibitory responses were blocked with TPMPA (50 µM), Gabazine (20 µM), and Strychnine (5 µM). D1 receptors were agonized with SKF-38393 (20 µM). Normalized peak amplitudes of the 1^st (fast) and 2^nd (slow) responses were calculated for each response before and after antagonizing the GABA_C, GABA_A and Glycine receptors.

Results : Treatment with GABA_C, GABA_A and Glycine receptor antagonists (n=5) did not cause significant changes to the fast response. This suggests the response is a measurement of the depolarization of amacrine and horizontal cells. However, the inhibitory block did cause a significant decrease in the normalized peak amplitude of the slow response. This suggest the slow response is likely a measure of inhibition generated by the activation of amacrine cells in the INL. Furthermore, the application of a D1 agonist (n=4) did not cause a change in the normalized peak amplitude of the slow response.

Conclusions : Optogenetic activation of ChR2 in the inhibitory amacrine and horizontal cells may be a novel way to measure inhibition across the entire retina. It is also possible to isolate the amacrine cell mediated inhibitory circuits in the mouse retina by blocking upstream photoreceptors and recording from bipolar cells.

This is a 2021 ARVO Annual Meeting abstract.