GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol

Abstract

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to "tonic" inhibition that can control levels of excitability and network activity. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

Fig. 1.

GABA_A-R α4 protein is absent in KO mice. (A) Western blot analysis of hippocampal, thalamic, and cortical membranes from WT and KO mice. The ≈67-kDa immunoreactive α4 protein present in WT samples is completely absent from KO samples. Stripped blots probed for β-actin show equal loading of samples. (B) α4 Subunit immunoreactivity in sagittal sections from WT and KO mouse brain. In WT mice, α4 labeling is highest in the thalamus (T), moderate in the molecular layer of the dentate gyrus (DG) and striatum (S), and slightly lower in the outer layers of the cerebral cortex (Cx) and the external plexiform layer of the olfactory bulb (OB). α4 Labeling is essentially absent from the cerebellum (CB). No specific labeling is evident in KO mouse brain.

Fig. 2.

Decreased magnitude and potentiation of GABA_AR-mediated tonic current in KO mouse DGCs. (A) The GABA uptake inhibitor, NO-711, potentiates the tonic current (I_hold) in a DGC from a WT mouse. The kinetics of mIPSCs (Upper) averaged over the indicated 100-sec periods during continuous voltage clamp (V = 0 mV throughout) recordings (Lower) are only slightly affected by NO-711 (3 and 10 μM). Picrotoxin (50 μM) application reveals a GABA_AR-mediated tonic current component (I_tonic). In a DGC from a KO mouse (Lower), potentiation of I_hold by NO-711 is reduced and I_tonic is very small. (B) Summary graphs of I_hold and total charge transfer of averaged mIPSCs before and after NO-711 application in WT and KO mice. Each point represents a mean ± SEM value from 5–6 neurons (4–5 mice per group). ∗, (P < 0.05) between WT and KO groups; †, (P < 0.05) from pre-NO-711 value (two-way repeated measures ANOVA). (C) Summary graph of differences in the picrotoxin-sensitive tonic current between WT and KO groups. ∗, (P < 0.05, t test, n = 12–14, 3–4 mice per group). The rms of current noise was as follows: basal, 3.5 ± 0.8 pA (WT) and 3.2 ± 0.9 pA (KO); after picrotoxin, 2.6 ± 0.4 pA (WT) and 2.5 ± 0.5 pA (KO).

Fig. 3.

Summary graphs of differences in mIPSC kinetics between DGCs from WT and KO mice. mIPSC amplitude and frequency were significantly reduced in neurons from KO mice compared with WT controls. Also note the slower rise and early decay of mIPSCs in DGCs from KO mice. Each bar represents a mean ± SEM value from 10–11 neurons (5 mice per group). ∗, P < 0.05, t test.

Fig. 4.

Reduced tonic currents in KO VB thalamic neurons. (A Left) The current recorded from a thalamic VB neuron from a WT mouse before and after application of 20 μM SR95531. SR95531 abolished spontaneous IPSCs and also induced a positive shift in the holding current due to blockade of a tonic inward current. (A Right) The all-points histograms corresponding to the 30-sec traces. The black and gray histograms illustrate the holding current in the absence and presence of SR95531, respectively. The dashed lines represent best-fit curves to a single Gaussian distribution. SR95531 caused a rightward shift and reduced the width of the all-point distribution. (B Left) The current recorded from a KO mouse VB neuron before and after application of 20 μM SR95531. SR95531 abolished spontaneous IPSCs without causing a shift in the holding current. (B Right) The corresponding all-points histograms. (C) Summary data for the WT and KO mice show that thalamic VB neurons from KO mice have no tonic inhibition (n = 9 and 17 for WT and KO, respectively). (D) VB neurons from KO mice also were insensitive to currents elicited by 0.1 and 0.3 μM GBX (n = 7–12; ∗, P < 0.05). Averaged data are expressed as mean ± SE.

Fig. 5.

SR95531 and GBX modulate membrane properties of VB thalamic neurons. (A) A VB neuron from a WT mouse was hyperpolarized by nearly 2 mV after addition of 0.1 μM GBX. (B) GBX (0.1 μM) produced no significant membrane potential change in VB neuron from a KO mouse. (C) The summary data shows that VB neurons from WT mice are hyperpolarized by 0.1 and 0.3 μM GBX, whereas neurons from KO mice are insensitive to GBX. SR95531 (20 μM) also induces a depolarization in WT but not in KO neurons (n = 7–12 per genotype; ∗, P < 0.05). (D) Similar differences in modulation by GBX and SR95531 also are observed in measurements of membrane input resistance of VB thalamic neurons (n = 7–12 per genotype; ∗, P < 0.05).

Fig. 6.

KO mice are insensitive to the behavioral effects of GBX. The fixed speed rotarod measured GBX's ataxic effects at 10 mg/kg (n = 19 KO and 14 WT) (A) and 15 mg/kg (n = 18 KO and 12 WT) (B). Ataxic effects of GBX are dramatically reduced in KO mice (filled circles) compared with WT mice (open circles) at both 10 (P < 0.0001) and 15 mg/kg (P < 0.001). (C) The radiant tail-flick assay was used to measure the analgesic properties of 10 mg/kg GBX (n = 15 KO and 17 WT). Baseline latency to flick tail (BSL) or latency after GBX injection is displayed. GBX produced a marked analgesic effect in WT mice by increasing latency to flick tail (∗, P < 0.005) but only had a small but marginally significant (†, P = 0.05) effect in KO mice. (D) The open-field assay was used to measure the sedative effect of 10 mg/kg GBX (n = 7–10 mice per genotype per treatment). Total locomotor activity after injection with saline (SAL) or GBX is displayed. GBX depressed locomotor activity in WT mice (‡, P < 0.02) but had no effect in KO mice. No genotypic differences were observed between saline-treated groups.