Introduction

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Despite the fact that ethanol is one of the most frequently abused drugs, the molecular targets through which it exerts its actions on the nervous system are uncertain. Although previous research focused on ethanol interactions with membrane lipids, voltage- as well as ligand-gated ion channels have now been shown to be important candidates for ethanol action (Lovinger, 1997; Mihic, 1999). So many effects of ethanol on ion channels have been described that an important issue confronting this field is identifying which of these many actions underlie the disturbances of higher order nervous function induced by ethanol (Harris, 1999).

Because the behavioral effects of ethanol resemble those initiated by other central anesthetic compounds known to be specific modulators of GABA_A receptor function, the GABA_A receptor complex has been hypothesized to be an important target of ethanol action (Mihic et al., 1997; Harris, 1999). This receptor is the primary mediator of fast inhibitory neurotransmission in the central nervous system, and enhancement of the effects of GABA on these receptors would have significant inhibitory effects on neuronal activity. However, there is a great deal of variability in the reported effects of ethanol on this receptor complex. Biochemical studies in brain synaptosome and microsac preparations (Allan and Harris, 1987) and in cultured neurons (Mehta and Ticku, 1994) have reported enhancement of GABA_A receptor-mediated responses by intoxicating concentrations of ethanol, as have electrophysiological studies of GABA_A receptor-mediated currents (Aguayo, 1990; Reynolds et al., 1992; Weiner et al., 1997a; Soldo et al., 1998). However, even in these studies, ethanol-sensitive and ethanol-insensitive responses have been reported, and there have also been numerous studies in which potentiation by ethanol was not observed (Morelli et al., 1988; Osmanovic and Shefner, 1990; White et al., 1990).

To account for such variability in ethanol enhancement, a number of hypotheses have been proposed. One possibility is that differences in ethanol sensitivity may be attributable to differences in receptor subunit composition. Weiner et al. (1997a) showed that even on single hippocampal CA1 pyramidal neurons, ethanol sensitivity of GABA_A receptors can differ depending on which subset of GABA_A synapses is activated. Electrical stimulation of GABAergic afferents in the pyramidal cell layer (proximal responses) evoked inhibitory postsynaptic currents (IPSCs) that were potentiated by intoxicating concentrations of ethanol, whereas IPSCs evoked by stimulation within the stratum radiatum (distal responses) were less sensitive to all concentrations of ethanol tested. One possible explanation for these results is that different synapses have postsynaptic receptors that incorporate different GABA_A receptor subunits, which leads to differences in ethanol sensitivity.

At least some of the differences in the ethanol sensitivity of GABA_A responses appear to be under genetic control. Biochemical measures of GABA_A receptor function, such as muscimol-stimulated ³⁶Cl flux, are differentially sensitive to modulation by ethanol in lines of animals that differ in their behavioral sensitivity to ethanol (Allan and Harris, 1986). If the GABA_A receptor is a specific target of ethanol action that subserves at least part of the behavioral response to ethanol, and if there are forms of this receptor that differ in their ethanol sensitivity, then genetic selection experiments could result in animal lines that also differ in the effects of ethanol on synaptically mediated GABA_A responses. However, the sensitivity of specific populations of GABA_A synapses to ethanol has never been characterized in selected lines of animals. More specifically, the ethanol sensitivities of the proximal (ethanol-sensitive) and distal (ethanol-insensitive) subpopulations of GABA_A receptors, which were initially characterized by Weiner et al. (1997a) in Sprague-Dawley rats, have not been examined in selected lines of animals.

To address this issue, the present experiments examined the effect of ethanol on IPSCs in hippocampal slices from rodents selectively bred based on their behavioral sensitivity to ethanol. We have examined proximal and distal GABA_A IPSCs in six lines of animals, which include the inbred long sleep (ILS) mice and the replicate high alcohol sensitivity (HAS₁ and HAS₂) lines of rats (all bred for ethanol sensitivity), and the low alcohol sensitivity (LAS₁ and LAS₂) rats and inbred short sleep (ISS) mice (all ethanol insensitive), to determine whether the sensitivity of GABAergic synapses to ethanol is altered in these selected lines of animals. If such differences could be observed, this would suggest that there are genetically controlled factors that can regulate ethanol sensitivity of GABA_A receptors.

	Materials and Methods

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Transverse hippocampal slices (400 µm) were prepared from 4- to 6-week-old HAS and LAS rats, and ILS and ISS mice using a Sorvall (Newtown, CT) tissue chopper. Submerged slices were incubated in a submersion chamber consisting of a grid of small, square compartments with plastic netting attached to the bottom, suspended in a 250-ml beaker and covered with a loose-fitting plastic lid. This chamber was maintained at a constant temperature of 31-33°C in aerated (95% O₂, 5% CO₂) artificial cerebrospinal fluid containing 126 mM NaCl, 3 mM KCl, 1.5 mM MgCl₂, 2.4 mM CaCl₂, 1.2 mM NaH₂PO₄, 11 mM glucose, and 26 mM NaHCO₃. Slices were left in this chamber for at least 90 min after the dissection. For recordings, slices were transferred to a submersion recording chamber maintained at a constant temperature of 31-33°C and superfused with aerated artificial cerebrospinal fluid at 2 ml/min. Slices were allowed to equilibrate in the recording chamber for a few minutes before electrophysiological recordings were begun.

GABA_A IPSCs were recorded from CA1 neurons using the whole-cell patch-clamp technique. Recording electrodes were constructed from borosilicate glass (1.5 mm o.d., 0.86 i.d.; Sutter Instrument Co., Novato, CA) and had resistances of 6 to 9 M when filled with the patch pipette solution. The patch pipette solution contained 125 mM potassium-gluconate (Fluka, Buchs, Switzerland), 5 mM KCl, 10 mM HEPES (Fluka), 0.1 mM CaCl₂, 1 mM potassium-EGTA (Fluka), 2 mM MgCl₂, 2 mM magnesium-ATP, and 0.2 mM Tris-GTP (pH = 7.3 adjusted with KOH; 290 mOsm) and was kept on ice until immediately before use. Series resistances ranged from 10 to 41 M (average 30 ± 1.5 M). The average change in the series resistance for all cells from the beginning of the control to the end of the washout period was 15.6 ± 1.2%, and all cells in which the change was >25% were excluded from subsequent analysis.

All recordings were made in the presence of 20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 50 µM DL-()-2-amino-5-phosphonovaleric acid (APV) to block excitatory postsynaptic currents. Synaptic stimulation was delivered using a bipolar twisted nichrome wire electrode (0.2-ms pulses of 7-30 V) positioned within 250 µm of the recording pipette and placed directly over stratum pyramidale, with an interstimulus interval of 30 to 60 s, as previously described (proximal stimulation; Weiner et al., 1997a). All cells were clamped to 65 mV (after correction for the liquid junction potential) and recorded in the voltage-clamp mode. After superfusion with DNQX and APV to block the glutamatergic components of the synaptic current, the strength of the stimulation pulse was adjusted so that the peak amplitude of the residual GABAergic component (GABA_A IPSC) was about 50 to 100 pA.

All drugs were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Drugs applied to slices were made up as 100-fold concentrates and added to the superfusion buffer via calibrated syringe pumps (Razel Scientific Instruments, Stamford, CT). A 4 M solution of ethanol (Aaper, Shelbyville, KY; diluted in deionized water) was prepared immediately before each experiment from a 95% stock solution kept in a glass storage bottle.

Drug effects were quantified as the percentage of change in amplitude or area under the curve of IPSCs relative to the mean of control and washout values. Statistical analyses of drug effects were carried out using two-tailed student's paired and unpaired t tests, or two-way ANOVAs as indicated, with a level of significance of P < .05.

	Results

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Recordings were made from CA1 pyramidal neurons in hippocampal slices from ILS and ISS mice, and from the replicate lines of HAS₁ and HAS₂ and LAS₁ and LAS₂ rats. In most cases, the responses evoked in the presence of DNQX + APV were mediated primarily via GABA_A receptors, but in some instances there was a small, late component on the falling phase of the IPSC (Fig. 1A) that could be blocked by superfusion with the GABA_B receptor antagonist CGP 35348 (data not shown). However, this secondary component did not overlap significantly with the peak of the GABA_A response, so it was not blocked pharmacologically in the instances where it was observed. To examine the ethanol sensitivity of GABA_A responses, slices were superfused with 80 mM ethanol, and changes in the amplitude and area of the IPSC response were determined. This concentration of ethanol was used because this is the mean blood ethanol concentration at regain of righting reflex in the heterogeneous stock of animals from which the HAS and LAS rat lines were originally selected (Draski et al., 1992). At generation 12 of selection, the LAS and HAS animals regained their righting reflexes with mean blood ethanol concentrations of 87 and 75 mM, respectively (Draski et al., 1992).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effects of bath superfusion with 80 mM ethanol on GABAA IPSCs evoked from selected lines of rats and mice. IPSCs were evoked by proximal stimulation in the presence of DNQX + APV in each of the six lines of animals tested. Each set of traces was obtained from a single pyramidal neuron from the indicated line of animals. Averaged responses were recorded under control conditions (C), in the presence of 80 mM ethanol (E), and after ethanol washout (W), and the control and washout responses were generally indistinguishable. Responses from HAS1, HAS2, and ILS mice all showed enhancement comparable with what we have previously observed in the Sprague-Dawley rats (Weiner et al., 1997a), whereas the ethanol-insensitive lines (LAS1, LAS2, and ISS) were all unaffected. A, illustrates one example of a response where there was a small GABAB component to the evoked response, seen as a slowly decaying tail on the GABAA response; this component could be blocked by the GABAB antagonist CGP 35348 (data not shown). There was also considerable variability in the time course of the GABAA component (note the different time scales in B and C versus the other records), but there did not appear to be any relationship between the time course and the ethanol sensitivity of the response.

Under these experimental conditions, bath superfusion with ethanol did not significantly change either the holding current or the input resistance of CA1 pyramidal neurons in any of the lines of animals tested (Table 1). However, 80 mM ethanol significantly enhanced the GABA_A IPSC in both of the lines of rats selected for behavioral sensitivity to ethanol (HAS₁, HAS₂; Fig. 1, A and C) compared with the baseline response amplitude (average of pre-ethanol and washout periods). This was observed both as an increase in the amplitude of the response, as well as an increase in the area under the curve (Table 1). The falling phases of the IPSCs with no GABA_B component were fitted to single exponential functions, and the time constants for the decay were compared for control and ethanol responses. Ethanol had no significant effect on the time constants in any of the individual lines tested, but across all the animals there was a significant 22 ± 7.9% (n = 23; P < .02) increase in the average time constant, suggesting that ethanol did prolong the IPSCs to a limited extent.

The time course of the effect of bath superfusion with ethanol on GABA_A responses in HAS animals was similar to what we have previously reported in Sprague-Dawley rats, i.e., it usually took 5 to 10 min of superfusion with ethanol to achieve the maximal enhancement of the IPSC (Fig. 2A). There did not appear to be any short-term tolerance occurring during the superfusion with ethanol, and recovery to baseline was observed within 5 to 10 min of ethanol washout (Fig. 2). The enhancement of GABA_A IPSCs induced by ethanol was also repeatable, and in some slices, potentiation was observed with as many as three successive applications of ethanol with no apparent decrement in the ethanol enhancement (Fig. 2B). Unlike the HAS rats, GABA_A IPSCs elicited in hippocampal slices from the two independent strains of rats with low behavioral sensitivity to ethanol (LAS₁, LAS₂) showed no significant change in response to ethanol superfusion (Fig. 1, B and D). In addition to there being no effect on the peak amplitude of the IPSC, this concentration of ethanol had no effect on the area under the curve for the IPSC as well (Table 1). When the changes in the peak IPSC induced by ethanol were compared between the pooled HAS and LAS animals, there was a highly significant difference between the ethanol-sensitive and ethanol-insensitive groups of rats (LAS: 107 ± 4.7%, n = 29; HAS: 141 ± 9.6%, n = 30; P < .01)

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Time course and repeatability of ethanol responses. A, illustrates the time course of ethanol action on GABAA IPSCs evoked from cells from a HAS2 and a LAS2 rat. The rate of onset and washout of the effect in the slice from the HAS2 animal was comparable with what we have previously observed in Sprague-Dawley rats (Weiner et al., 1997a; Fig. 3). As shown in these examples, there was no significant effect of ethanol on the LAS2 IPSC, but a significant enhancement of the HAS2 IPSC. B, shows the repeatability of the ethanol response in a single cell from a HAS1 animal that was superfused repeatedly with the same concentration of ethanol. Although there was some decline in the absolute magnitude of the GABAA response over time, the relative enhancement of the IPSC by ethanol was maintained throughout the experiment.

Because it is not known whether there are proximal and distal GABA_A responses in mice such as we have previously described in rats (Weiner et al., 1997a), all of the experiments in ILS and ISS mice were conducted with two independent stimulating electrodes positioned to activate each of these pathways selectively. IPSC responses evoked by stimulation in stratum radiatum (distal stimulation) were unaffected by 80 mM ethanol in both lines of mice. The amplitude of the IPSCs in ethanol were 109 ± 13% (n = 17) of control in ISS mice, and 95 ± 6.8% (n = 17) of control in ILS mice. As far as the proximal pathway was concerned, the ethanol-insensitive mouse strain (ISS) did not show any change either in the amplitude or the area of the hippocampal GABA_A IPSC during ethanol superfusion (Fig. 1F; Table 1), but IPSCs in the ethanol-sensitive ILS line were significantly enhanced relative to their baseline and washout values (Fig. 1E). When results from proximal stimulation experiments in the two lines of animals were compared with each other, there was a highly significant difference in the effects of ethanol on IPSC amplitude in the two groups (Fig. 3; P < .003).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of ethanol on peak IPSC amplitude in selected lines of mice and rats. The average effect of 80 mM ethanol is illustrated on the peak amplitude of the IPSC evoked by proximal stimulation for each of the six lines of animals tested. Although the magnitude of the enhancement in the ILS and HAS1 animals was somewhat less than in the HAS2 rats, the effect of ethanol was statistically significant in each of the ethanol sensitive lines, whereas it was not in any of the lines selected for ethanol insensitivity (*, indicates P < .05 compared with control/washout responses in the same line of animals, paired t test).

	Discussion

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Ethanol Effects in Selected Lines of Rodents. Ethanol activates or inhibits the activity of a number of different ligand-gated ion channels, and a key issue in understanding the cellular mechanisms of ethanol action is to determine which of these effects underlie specific pharmacological actions of ethanol. One approach to making such determinations is to use animals developed using classical selective breeding approaches to segregate genes that are associated with specific behavioral phenotypes, such as ethanol sensitivity. Comparisons of ethanol responses in animals developed by this approach can then be used to identify the specific molecular targets of ethanol that are linked to the behavioral phenotype.

Ethanol Effects on Synaptic GABA_A Responses. Studies of ethanol effects on GABA_A responses in nonselected lines of rodents have found considerable variability in ethanol sensitivity, even in studies of what would seem to be the same receptors in the same population of cells (Proctor et al., 1992a,b; Peoples and Weight, 1994; Wan et al., 1996; Weiner et al., 1997a; Peoples and Weight, 1999). This suggests that ethanol sensitivity must depend on a rather specific combination of factors. One such variable is the subpopulation of GABA_A receptors activated by synaptic stimulation. Previous work by Pearce and colleagues has demonstrated that there are populations of GABA_A synapses on CA1 pyramidal neurons that differ in a variety of respects, including their kinetic properties, and sensitivity to pharmacological agents such as furosemide (Pearce, 1993). Our studies in Sprague-Dawley rats have shown that distal GABA_A-mediated IPSCs are less sensitive to ethanol action than are proximal IPSCs (Weiner et al., 1997a). This study demonstrated such differential ethanol sensitivity of GABA_A receptors even in single hippocampal pyramidal neurons, confirming that the differences in sensitivity were not merely due to unknown variables in the experimental approach. The present studies found that proximal GABA_A IPSCs are sensitive to ethanol in the HAS₁ and HAS₂ lines of rats, and in the ILS mice. Further support for the conclusion that somatically located GABA_A receptors are particularly sensitive to ethanol comes from local application experiments in which GABA was applied either directly to the somata of CA1 pyramidal cells, or to their dendrites in stratum radiatum. Ethanol enhanced GABA_A responses mediated by the somatic receptors, while having no effect on dendritic currents (W. R. Proctor, unpublished data). Similarly, Soldo et al. (1998) concluded based upon local application experiments that ethanol had a selective effect on somatic GABA_A receptors in cortical neurons, whereas dendritic receptors showed little or no sensitivity. These experiments support the conclusion that at least a part of the action of ethanol is postsynaptic, either on the receptor itself, or on other cellular constituents that can modulate GABA_A receptor sensitivity.