Ethanol potentiation of GABAergic synaptic transmission may be self-limiting: role of presynaptic GABA(B) receptors

Abstract

Ethanol enhances GABAergic synaptic inhibition, and this interaction contributes to many of the behavioral and cognitive effects of this drug. Most studies suggest that ethanol enhances GABAergic neurotransmission via an allosteric potentiation of the postsynaptic GABA(A) receptors that mediate fast synaptic inhibition in the mammalian CNS. Despite widespread acceptance of this hypothesis, direct support for such a mechanism has been difficult to obtain. Ethanol does not enhance GABA(A) receptor function in all brain regions or under all experimental conditions, and factors responsible for this variability remain mostly unknown. Notably, blockade of GABA(B) receptors dramatically enhances ethanol potentiation of hippocampal GABA(A) IPSPs and IPSCs, suggesting that some unknown GABA(B) receptor mechanism limits the overall potentiating effect of ethanol on GABAergic synapses. In this study, we demonstrate that, at perisomatic synapses in the rat hippocampus, ethanol enhances presynaptic GABA(B) autoreceptor function and that this interaction reduces the overall potentiating effect of ethanol at these synapses. We further show that ethanol significantly elevates basal presynaptic GABA(B) receptor tone, possibly via an increase in spontaneous GABA release, and that pretreatment with a subthreshold concentration of the GABA(B) receptor agonist baclofen blocks ethanol but not flunitrazepam or pentobarbital potentiation of GABA(A) IPSCs. These data suggest that an interaction between ethanol and presynaptic GABA(B) autoreceptor activity regulates the ethanol sensitivity of GABAergic synapses. Given that the in vitro ethanol sensitivity of these synapses correlates with in vivo ethanol responsiveness in a number of rodent lines, our data further suggest that presynaptic GABA(B) receptor activity may play a role in regulating behavioral sensitivity to ethanol.

Figure 1.

Pretreatment with a GABA_B receptor antagonist enhances ethanol potentiation of proximal GABA_A IPSCs. A, Representative time course of the effect of 80 mm ethanol (EtOH) on the area of proximal GABA_A IPSCs recorded in the absence and presence of the GABA_B receptor antagonist SCH 50911. Responses were pharmacologically isolated using the competitive NMDA and AMPA/kainate receptor antagonists APV (50 μm) and DNQX (20 μm). Traces above the graph are averages of five to seven IPSCs evoked at the times indicated by the letters. B, Bar graph summarizing the mean effect of 80 mm ethanol on the area of proximal GABA_A IPSCs recorded in the absence and presence of 20 μm SCH 50911. STD, Standard. ^*p < 0.05, unpaired Student's t test. Numbers in parentheses represent the number of cells tested under each experimental condition.

Figure 2.

Ethanol increases presynaptic GABA_B receptor function. A, Representative time course illustrating the effect of 1.25 μm baclofen (BAC) in the absence and presence of 80 mm ethanol (EtOH) on the area of proximal GABA_A IPSCs. Responses were pharmacologically isolated as in Figure 1. Traces above the graph are averages of six to eight IPSCs evoked at the times indicated by the corresponding letters. B, Bar graph summarizing the effect of 1.25 μm BAC on proximal GABA_A IPSCs when applied alone or in the presence of 20, 40, or 80 mm EtOH or 1 μm FLU. ^*p < 0.05, relative to BAC alone, post hoc Neuman-Keuls test. Numbers in parentheses represent the number of cells tested under each experimental condition.

Figure 3.

Ethanol does not potentiate postsynaptic GABA_B receptor function in rat CA1 pyramidal neurons. A, Representative traces illustrating the effect of 80 mm ethanol (EtOH) and 20 μm SCH 50911 on the area of GABA_B IPSCs evoked by stimulation of the stratum lacunosummoleculare in the presence of APV, DNQX, and 20 μm bicuculline methiodide. Traces are averages of five GABA_B IPSCs recorded under the conditions indicated. The bar graph summarizes the effect of EtOH and SCH 50911 on the area of GABA_B IPSCs. CTL, Control; WASH, washout. B, Representative time course of the outward current generated by bath application of 20 μm baclofen (BAC) in the absence and presence of 80 mm EtOH. The bar graph summarizes the maximal amplitude of BAC-evoked currents recorded in the absence and presence of EtOH. Numbers in parentheses represent the number of cells tested under each experimental condition.

Figure 4.

Ethanol increases presynaptic GABA_B receptor tone. Traces from representative experiments illustrate that 20 μm SCH 50911 potentiates the area of proximal GABA_A IPSCs in the presence (B) but not the absence (A) of ethanol (EtOH). Note that this concentration of SCH 50911 is sufficient to completely block the inhibitory effect of 5 μm BAC on GABA_A IPSCs. CTL, Control; WASH, washout. C, Summary of the effect of 20 μm SCH 50911 on the area of GABA_A IPSCs recorded in the absence and presence of EtOH. ^*p < 0.05, relative to control. Numbers in parentheses represent the number of cells tested under each experimental condition.

Figure 5.

Pretreatment with a GABA_B receptor antagonist selectively enhances ethanol potentiation of sIPSC frequency. A, sIPSCs recorded from a representative CA1 neuron voltage clamped at -70 mV before (CTL), during, and after bath application of 80 mm ethanol (EtOH, WASH). B, sIPSCs recorded from another representative neuron under control conditions, during bath application of 20 μm SCH 50911 alone and with 80 mm EtOH and after ethanol washout (WASH). C, Summary of the effect of ethanol, SCH 50911, and SCH 50911 plus EtOH on sIPSC frequency (FREQ), amplitude (AMP), and area. ^*p < 0.05, paired Student's t test; ^# significant difference between two conditions, one-way ANOVA followed by post hoc Neuman-Keuls test; NS, no significant difference. Numbers in parentheses represent the number of cells tested under each experimental condition. Note that SCH 50911 pretreatment significantly increases ethanol potentiation of sIPSC frequency but not their amplitude or area.

Figure 6.

Time course from a representative cell illustrating the effect of 80 mm ethanol (EtOH), in the presence and absence of a subthreshold concentration of baclofen (BAC), on the area of proximal GABA_A IPSCs. Note that EtOH inhibits GABA_A IPSCs in the presence of 500 nm BAC but potentiates GABA_A IPSCs under standard recording conditions. Traces are averages of five to eight IPSCs recorded at the times indicated by the letters above the graph.

Figure 7.

A subthreshold concentration of baclofen selectively blocks ethanol potentiation of proximal GABA_A IPSCs. A summary of the effect of 500 nm baclofen (BAC) pretreatment on the effect of 80 mm ethanol (EtOH), 1 μm FLU, and 50 μm pentobarbital (PENTO) on the area of proximal GABA_A IPSCs is shown. ^*Significant difference relative to control, paired t tests; ^# significant difference between two conditions, unpaired t tests; NS, no significant difference. Traces are averages of 6-10 GABA_A IPSCs recorded from representative cells under the conditions indicated. Note that 500 nm BAC alone has no effect on proximal GABA_A IPSCs and does not interact with FLU or PENTO potentiation of these responses. In contrast, 500 nm BAC pretreatment reverses the effect of ethanol on proximal IPSCs from potentiation to inhibition.