Regulation of mouse horizontal cell ρ-subunit containing GABA receptors by light and dopamine via intracellular calcium

Abstract

Purpose : Light adaptation and dopamine (DA) acting at D1Rs in horizontal cells (HC) increase the strength of inhibitory feedback to photoreceptors. Here, actions of light and DA that regulate ρ-subunit containing GABA receptors in HCs, that have a key role in feedback, are investigated.

Methods : Immunolabelling of GABAR ρ2 subunits at HC synapses was confirmed using super-resolution confocal imaging under light adapted conditions, but was weak in dark adapted retinas. Patch clamp recording in mouse tangential retinal slices showed that TPMPA-sensitive GABAρR mediated currents in HCs are larger when light adapted than dark adapted .

Results : Light and DA induced GABAR subunit expression at HC synapses was tested in dark-adapted mice exposed for 5 or 30 min to light levels of 10, 10³ and 10⁴ lux, and in eyecups incubated DA alone or with SCH23390, a D1R antagonist. Brighter light, both exposure times and D1R activation increased ρ2 immunoreactivity on HC endings. Testing with brief GABA puffs, patch clamp recordings showed that when light-adapted, SR96631 (non-GABA ρR blocker) blocked 66% of GABA-activated currents while TPMPA blocked 33%. In dark-adapted HCs, TPMPA produced no reduction. HCs respond to their own tonic release of GABA. We assessed these currents using TPMPA, finding no effect on tonic GABA currents in dark-adapted HCs while there was reduction in light-adapted HCs. In dark-adapted mice DA levels are low, and TPMPA-sensitive currents in HCs were greatly increased by the D1R agonist, SKF38393, with TPMPA blocking 33% of the GABA-activated current, as in light-adapted retinas. CNQX, which hyperpolarizes HCs, increased current blocked by TPMPA by 25%. High intracellular [Ca²⁺] reducing GABAR activity was tested with cyclosporin A, which blocks Ca²⁺activated calcineurin. In dark-adapted retinas with cyclosporin, TPMPA reduced GABAR currents by ~30%, similar to light-adapted conditions.

Conclusions : Feedback inhibition is reduced under dark-adaption due to elevated [Ca²⁺] caused by HC depolarization via photoreceptor glutamate. Depolarization produces higher [Ca²⁺] via Ca_V channel activation, but is down-modulated by D1Rs, contributing to lower [Ca²⁺] levels. The effects of these actions, together with the autaptic activation by GABA of HC GABARs, known to modulate photoreceptor Ca channels, mediate changes in feedback signaling during light- and dark-adaptation.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.