Abstract
Purpose :
Most retinal amacrine cells provide inhibitory input to ganglion cells (GCs) via glycinergic or GABAergic synapses. In ON-OFF Direction Selective GCs (ON-OFF-DSGCs), asymmetric GABAergic input restricts sensitivity to objects moving in one direction. However, ON-OFF DSGCs also receive substantial glycinergic input whose function is not understood. Here, we examined which glycine receptor subunits (GlyRα) mediate input to ON-OFF-DSGCs.
Methods :
We performed whole cell patch clamp recordings from ON-OFF-DSGCs in TRHR mice. Glycinergic spontaneous or light-evoked inhibitory postsynaptic currents (sIPSCs or L-IPSCs) were isolated by pharmacologically eliminating GABAA/GABAC input (picrotoxin and TPMPA). In some experiments, Glycine (100µM) was puffed directly onto synaptically isolated ON-OFF-DSGCs (50µM DNQX, 50µM AP5, 4mM CoCl2). In all experiments, currents were confirmed to be GlyR-mediated by blocking with strychnine. To knockdown GlyRα’s within the RGCs themselves, AAV-GlyRα-shRNA was injected into superior colliculus and retrogradely transported to GCs. GlyRα subunit inputs were predicted from known sIPSC decay taus and tested in GlyRα subunit specific knockout mice.
Results :
Glycinergic sIPSCs in ON-OFF-DSGCs had a median decay tau of 21.2ms, predicting the presence of GlyRα2. In contrast to this hypothesis, no differences in glycinergic sIPSC frequency, decay, or L-IPSC amplitudes were observed in GlyRα2-KO retinas compared to WT. However, we observed reductions in glycine sIPSC frequency, decay, and L-IPSC amplitude in GlyRα2/α4-dKO retinas, suggesting both α2 and α4 may be present. Consistent with this, glycine puff-evoked IPSCs had significantly faster decay tau’s in GlyRα4-KO and GlyRα2/α4-dKO retinas compared to WT. Residual glycinergic sIPSCs in GlyRα2/α4-dKO had a median decay tau of 5.8ms.
Conclusions :
Altogether, data from sIPSCs, L-IPSCs, and glycine puff-evoked IPSCs suggest that GlyRα2 and GlyRα4 all mediate synaptic input to ON-OFF-DSGCs. The varying decay kinetics between GlyRα2 (medium decay) and GlyRα4 (slow τ decay) could lead to subunit specific inhibition of ON-OFF DSGCs on different time scales. Ongoing analyses of light-evoked responses will evaluate the functional contributions of GlyR-mediated input to ON-OFF DSGCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.