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. 2008 Aug;326(2):475-82.
doi: 10.1124/jpet.108.139303. Epub 2008 May 13.

Ethanol modulates synaptic and extrasynaptic GABAA receptors in the thalamus

Fan Jia  1 , Dev Chandra, Gregg E Homanics, Neil L Harrison
Affiliations

Ethanol modulates synaptic and extrasynaptic GABAA receptors in the thalamus

Fan Jia et al. J Pharmacol Exp Ther. 2008 Aug.

Abstract

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. The thalamus is a brain structure that plays a pivotal role in sleep regulation and rhythmicity. A number of studies have implicated GABA(A) receptors (GABA(A)-Rs) in the anxiolytic, amnestic, sedative, and anesthetic effects of ethanol. In the present study, we examined the effects of ethanol on both synaptic and extrasynaptic GABA(A)-Rs of relay neurons in the thalamus. We found that ethanol (> or =50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABA(A)-R alpha(4) subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. On the other hand, ethanol had no effect on spontaneous or evoked inhibitory postsynaptic currents (IPSCs) at 50 mM but did prolong IPSCs at 100 mM. Ethanol had no effect on the paired-pulse depression ratio, suggesting that the release of GABA from presynaptic terminals is insensitive to ethanol. We conclude that ethanol, at moderate (50 mM) but not low (10 mM) concentrations, can inhibit thalamocortical relay neurons and that this occurs mainly via the actions of ethanol at extrasynaptic GABA(A)-Rs containing GABA(A)-R alpha(4) subunits.

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Figures

Fig. 1
Fig. 1
Ethanol (50 mM) decreases the excitability of VB neurons via GABAA-Rs. A, representative current clamp traces demonstrate AP firing evoked by current steps (40–160 pA, duration 500 ms) in a VB neuron. After ethanol (50 mM) perfusion, AP firing decreased, and the input-output curve shifted rightwards. B, representative current clamp traces demonstrating AP firing evoked by current steps in the presence of gabazine (20 μM) in another VB neuron. Ethanol (50 mM) perfusion failed to change the input-output curves when GABAA-Rs were blocked. C, exemplar current trace demonstrating that 20 mM ethanol induced no change on firing rate evoked by depolarized current steps. On average, ethanol (20 mM) also makes no change on the numbers of APs (9.6 ± 1.5 versus 9.4 ± 1.6, p > 0.05, n = 5). N.S., not significant. D, pooled data show that 50 mM ethanol significantly reduced the number of evoked action potentials from 10.9 ± 1.4 to 8.5 ± 1.4 (**, p < 0.01, n = 6). E, when GABAA-Rs are blocked by gabazine, 50 mM ethanol fails to inhibit tonic action potential firing (12.5 ± 1.7 to 12.4 ± 1.9, p > 0.05, n = 5).
Fig. 2
Fig. 2
Ethanol (≥50 mM) enhances tonic currents mediated by extrasynaptic GABAA-Rs. A, typical voltage-clamp recordings of four VB neurons in response to the applications of different concentrations (10–100 mM) of ethanol. Ethanol (≥50 mM) induced substantial current-shifts. B, the averaged current-shifts elicited by ethanol are dose-dependent (10 mM: 0.8 ± 0.3 pA, n = 12; 20 mM: 1.8 ± 0.7 pA, n = 18; 30 mM: 2.4 ± 1.2 pA, n = 10; 50 mM: 9.5 ± 1.5 pA, n = 19; 100 mM: 19.0 ± 3.6 pA, n = 12). C, gabazine (20 μM) occluded the enhancement of tonic currents by 50 mM ethanol and revealed the background tonic current. The dotted trace and Gaussian fittings were made from the raw trace as described under Materials and Methods. Tonic currents before and after ethanol application in this case are 43.4 and 62.2 pA, respectively. D, each point corresponds to the tonic currents before (x-axis) and after (y-axis) ethanol application from individual experiment similar to the one shown in C. The points analyzed from twelve experiments were fitted by a straight line pretty well (r = 0.98). The slope of the fitted line is 1.50, well above the unitary line (y = x, the gray dashed line).
Fig. 3
Fig. 3
Ethanol-induced current-shift is absent in VB neurons from mice lacking the GABAA-R α4 subunit. A, ethanol (50 mM) evoked a holding current shift (∼10 pA) in a VB neuron from a wild-type mouse. In contrast, ethanol produced no current shift in a VB neuron from a Gabra4−/− mouse. WT, wild-type; KO, knockout. B, bar graph demonstrates that ethanol (50 mM) induced current shifts in wild-type, but not α4 knockout, VB neurons (knockout: 1.7 ± 0.9 pA, n = 10; wild type: 8.6 ± 2.6 pA, n = 6; **, p < 0.01).
Fig. 4
Fig. 4
The tonic currents evoked by acamprosate at high concentrations. A, a typical recording of a VB neuron in response to 1 μM acamprosate. B, pooled data show that 200 and 500 μM, but not 1 to 100 μM, acamprosate elicits significant current-shift in VB neurons.
Fig. 5
Fig. 5
The effects of ethanol on spontaneous IPSCs. A, a typical recording of spontaneous IPSCs in a VB neuron in the absence and presence of 50 mM ethanol. Averaged spontaneous IPSC traces before (black) and after (gray) ethanol application are superimposed to illustrate the similarity in amplitude and decay time. B, a representative experiment shows that the frequency, amplitude, and decay time of spontaneous IPSCs did not change during the ethanol application for more than 20 min. C, pooled data demonstrate that spontaneous IPSCs are largely insensitive to ethanol. Only 100 mM ethanol significantly increases the decay time of spontaneous IPSCs (**, p < 0.01).
Fig. 6
Fig. 6
The effects of ethanol on evoked IPSCs and paired-pulse depression. A, exemplar evoked IPSC traces demonstrating that 100 mM ethanol increase the decay time, but not the amplitude of evoked IPSCs. B, average data show that evoked IPSCs are insensitive to ethanol less than 100 mM. Only 100 mM ethanol increased the decay time of evoked IPSCs significantly (***, p < 0.001). C, sample traces showing paired-pulse responses before (in dark) and after 100 mM ethanol (in gray) application. The superimposed traces clearly show the similar degree of paired-pulse depression. D, bar graph demonstrates that paired-pulse ratio is insensitive to ethanol (20–100 mM), which indicates that the presynaptic GABA release probability is not modified by ethanol.
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