Extrasynaptic GABA(A) receptors and tonic inhibition in rat auditory thalamus

Abstract

Background: Neural inhibition plays an important role in auditory processing and attentional gating. Extrasynaptic GABA(A) receptors (GABA(A)R), containing α(4)and δ GABA(A)R subunits, are thought to be activated by GABA spillover outside of the synapse following release resulting in a tonic inhibitory Cl(-) current which could account for up to 90% of total inhibition in visual and somatosensory thalamus. However, the presence of this unique type of inhibition has not been identified in auditory thalamus.

Methodology/principal findings: The present study used gaboxadol, a partially selective potent agonist for δ-subunit containing GABA(A) receptor constructs to elucidate the presence of extrasynaptic GABA(A)Rs using both a quantitative receptor binding assay and patch-clamp electrophysiology in thalamic brain slices. Intense [(3)H]gaboxadol binding was found to be localized to the MGB while whole cell recordings from MGB neurons in the presence of gaboxadol demonstrated the expression of δ-subunit containing GABA(A)Rs capable of mediating a tonic inhibitory Cl(-) current.

Conclusions/significance: Potent tonic inhibitory GABA(A)R responses mediated by extrasynaptic receptors may be important in understanding how acoustic information is processed by auditory thalamic neurons as it ascends to auditory cortex. In addition to affecting cellular behavior and possibly neurotransmission, functional extrasynaptic δ-subunit containing GABA(A)Rs may represent a novel pharmacological target for the treatment of auditory pathologies including temporal processing disorders or tinnitus.

Figure 1. Receptor Binding Assay Indicating High Levels of δ-containing GABA_ARs on MGB Neurons:

Representative autoradiographs of [³H] gaboxadol binding in young adult rats. Warm colors (red) indicate higher levels of binding while cooler colors (blue) represent lower levels (referenced to Relative Optical Density spectrum at left). At all three concentrations shown here (75 nM, 125 nM and 250 nM), [³H]gaboxadol binds selectively to GABA_ARs in MGB with little binding in brain regions shown in this coronal section, except for hippocampus and upper layers of neocortex. The MGB and hippocampus are indicated by arrows labeled “MGB” and “HP”, respectively with primary auditory cortex labeled as “A1”.

Figure 2. GABA_AR Mediated Tonic Inhibition in MGB Neurons:

A) Representative traces of gaboxadol-induced tonic Cl⁻ currents (outward) revealed by gabazine block, resulting in an inward shift in baseline current for MGB neurons held at −10 mV. The solid black line above the first trace represents the continuous focal application of (50 µM) gabazine for all traces. B) Bar graph of tonic current amplitude changes revealed by focal application of gabazine in the presence of increasing concentrations of GABA_AR agonist, gaboxadol (GBX), applied to the ACSF. Current amplitudes are represented on the y-axis with the concentration of gaboxadol on the x-axis. (*p<0.005 when compared to Control using Dunnett's post-hoc analysis, data underwent first-order Winsorization; n = control: 8; 0.1 µM: 4; 0.3 µM: 6; 1 µM: 6; 2 µM: 6; 5 µM: 6).

Figure 3. Summary Illustration of GABA_ARs on MGB Neurons:

Note receptor location relative to the presynaptic GABAergic terminal (IC or TRN) with classic α₁γ-subunit containing GABA_ARs located within the synapse and α₄δ-subunit containing GABA_ARs outside of the synapse. The concentration of GABA to which each receptor type is typically exposed and the nature of the current mediated by each subtype of receptor is also depicted. For sample traces, the internal solution used here is CsCl-based (140 mM) and the membrane potential is clamped at −60 mV. As a result, GABA_AR currents are inward and blocked by gabazine. The phasic response (left trace) is expanded from within the trace of the tonic response (right trace). The inward shift in baseline current is induced by upregulating extracellular GABA through inhibition of GABA uptake via the application of neuronal and glial GABA transporters with NNC-711 and SNAP 5114, respectively (solid line, right trace). These sample recordings were obtained from an MGB neuron of a 7-month-old FBN rat.