Pharmacological Analysis of the Activation and Receptor Properties of the Tonic GABACR Current in Retinal Bipolar Cell Terminals

Stefanie M Jones,Mary J Palmer,Steven Barnes

doi:10.1371/journal.pone.0024892

Abstract

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABAA receptors (GABAARs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABACRs in their axon terminal, in addition to synaptic GABAAR and GABACR currents, which strongly regulate BC output. The tonic GABACR current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABACRs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABACR current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABACRs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABACR ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABACRs are thought to be ρ1-ρ2 heteromers. To investigate whether GABACRs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABAARs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABACR currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.

Highlights

GABA, the major inhibitory neurotransmitter in the central nervous system (CNS), evokes transient postsynaptic currents (IPSCs) via ionotropic GABAA and GABAC receptors, as well as slower synaptic responses via metabotropic GABAB receptors (GABARs)
The role of anion channels: Effects of nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and flufenamic acid In order to investigate the role of Bestrophin 1 (Best1) and other anion channels in non-vesicular GABA release in the retina, we tested the effect of anion channel inhibitors on GABACR-mediated currents in Bipolar cell terminals (BCTs)
The potentiated current in NPPB and/or flufenamic acid (FFA) was subsequently inhibited by the addition of the GABAR antagonist picrotoxin (200 medium comprising (mM); 2.5 mM Ca2+ NPPB n = 1, FFA n = 1; Ca2+-free NPPB n = 1, NPPB+FFA n = 2; fig. 1A), confirming that it was mediated by GABACRs

Summary

Introduction

GABA, the major inhibitory neurotransmitter in the CNS, evokes transient postsynaptic currents (IPSCs) via ionotropic GABAA and GABAC receptors, as well as slower synaptic responses via metabotropic GABAB receptors (GABARs). There is increasing evidence that GABA evokes a tonic increase in membrane conductance by activating extrasynaptic GABA receptors, either as a result of spill-over from synapses or via a non-synaptic mechanism [1]. A GABACR-mediated tonic current occurs in the synaptic terminals of bipolar cells (BCs), which regulates membrane excitability [4,5]. Bipolar cell terminals (BCTs) exhibit rapid synaptic GABAAR and GABACR currents that mediate feedback inhibition and limit BC glutamate release, thereby modulating the light responses of ganglion cells, the output cells of the retina [6]. Volume-regulated anion channels (VRACs) have been implicated in the non-vesicular release of neurotransmitters [15].

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