Abstract
Purpose :
Light-evoked inhibition to Rod Bipolar Cells (RBC) is reduced by light adaptation and activation of dopamine receptor 1 (D1R). However, the circuit location of this modulation is not clear. To test this, amacrine cell (AC) inputs to BCs were isolated with direct optogenetic activation of inhibitory ACs. Using this we determined 1) how D1Rs modulate AC inputs to RBCs 2) the role that GABAR and GlycineR serve in the optogenetic evoked inhibitory currents. 3) the role of photoreceptor inputs between RBCs and ON cone (ON)BCs.
Methods :
Retinas from 6 - 12 week old B6.Cg-Tg(Slc32al-COP4*H134R/EYFP) male and female mice, which contain ChR2 under the VGAT promoter in the inhibitory ACs and horizontal cells, were cut into 250µm thick slices. Whole-cell voltage clamp recordings were made from RBCs and ONBCs. ChR2-expressing cells were stimulated with a 1s full-field light stimulus projected through a 60x objective (λ = 470 nm). Photoreceptor inputs were pharmacologically blocked with CNQX (25 µM), APV (50 µM), ACET (1 μM), and L-AP4 (50 µM). D1Rs were activated with SKF-38393 (20 µM). GlycineRs were blocked with Strychnine (1 µM). All experiments were performed under room light. The Peak Amplitude (PA) and charge transfer (Q) were analyzed for all evoked responses and paired t tests were performed.
Results :
Robust inhibitory currents were recorded from RBCs after ChR2 stimulation that were not significantly affected by the photoblocking cocktail (PA p = 0.34, Q p = 0.08, n=3) or by the photoblocking cockail + strychnine (PA p = 0.12, Q p = 0.12, n=4). Subsequent application of SKF did not significantly alter total (PA p = 0.79, Q p = 0.17, n = 3) or GABAergic inhibition (PA p = 0.22, Q p = 0.34, n = 4). In contrast, the photoblock cocktail significantly reduced ChR2 evoked inhibitory currents on ONBCs (PA 58%, p = 0.025; Q 18%, p = 0.03, n=3). Application of SKF had no additional effect (PA p = 0.57, Q p = 0.36).
Conclusions :
In light adapted conditions ONBCs and RBCs had significant optogenetically evoked inhibitory currents, but only ONBCs received significant input from photoreceptors that was blocked by the photoblocking cocktail. This agrees with previous data showing limited inhibition to RBCs after light adaptation. Neither cell type showed significant modulation by a D1R agonist. These results suggest direct optogenetic activation of ACs is a powerful tool to investigate retinal inhibition.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.