Abstract
Purpose :
Retinal amacrine cells provide one of two sources of inhibition (GABA or glycine) to ganglion cells (GCs). Various roles for GABAergic inhibition have been discovered. For example, in ON-OFF Direction Selective GCs (ON-OFF-DSGCs), asymmetric GABAergic input restricts sensitivity to objects moving in only one direction. While ON-OFF DSGCs also receive glycinergic input, its function has not been examined. Here we examined GlyRα mediated synaptic inputs and their light evoked currents in WT and GlyRα knockout ON-OFF-DSGCs.
Methods :
We pharmacologically isolated glycinergic inputs by eliminating GABAA/GABAC inputs (PTX and TPMPA) and verified the remaining currents were glycinergic using strychnine. Whole-cell patch clamp recording assessed spontaneous glycinergic currents (sIPSCs) from GFP labeled ON-OFF DSGCs in TRHR mice. GlyRα subunit composition was predicted from known sIPSC decay τ's and tested in mice lacking specific GlyRα subunits (KO mice). Glycinergic light-evoked inhibitory postsynaptic currents (L-IPSCs) were compared between WT and GlyRα KO TRHR cells to evaluate the contribution of GlyRα 2, 3 or 4 inputs at light onset and offset.
Results :
Isolating glycinergic inputs reduced the L-IPSC amplitude by ~45% in TRHR ON-OFF DSGCs, indicating that glycine mediates a significant inhibitory input. In WT TRHR GCs, the median sIPSC decay τ was 11.3 ms and this shifted to a slower decay τ of 13.9ms in TRHR/GlyRα3-KO mice. This indicates that the remaining currents should be mediated by GlyRα2 or GlyRα4. The median decay τ was similar in TRHR/GlyRα2-KO and TRHR/GlyRα4-KO GCs, which is inconsistent with their predicted decay τ's (~20 vs 40 msec, respectively). Ongoing studies of glycinergic sIPSCs and L-IPSCs in TRHR double KOs: GlyRα2/GlyRα3 and GlyRα3/GlyRα4 will determine if one or both provide synaptic input.
Conclusions :
Our results clearly show that glycinergic currents represent a significant source of inhibition to TRHR ON-OFF-DSGCs. These GCs receive predominant synaptic input via GlyRα3 and an additional input from GlyRα's with slower decay τ's. The different decay kinetics between GlyRα3 (fast τ decays) and GlyRα2 and GlyRα4 (slow τ decays) could lead to subunit specific inhibition in ON-OFF DSGCs on different time scales. Ongoing analyses of light evoked responses will evaluate the functional contributions GlyRα mediated input in ON-OFF DSGCs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.