The In Vitro Ethanol Sensitivity of Hippocampal Synaptic g-Aminobutyric AcidA Responses Differs in Lines of Mice and Rats Genetically Selected for Behavioral Sensitivity or Insensitivity to Ethanol

Previous work has demonstrated that in the hippocampal CA1 region of Sprague-Dawley rats, there are ethanol-sensitive and ethanol-insensitive populations of GABAergic synapses on pyramidal neurons. The present experiments characterized the ethanol sensitivity of these pathways in lines of rats and mice genetically selected for sensitivity or insensitivity to the behavioral effects of ethanol. In ethanol-sensitive inbred long sleep mice, GABAA IPSCs induced by stimulation of proximal (probably somatic) synapses were enhanced by 80 mM ethanol, whereas the distal (i.e., dendritic) pathway was unaffected. Thus, the relative sensitivity of these pathways (proximal . distal) is the same in both Sprague-Dawley rats and in inbred long sleep mice. However, in the ethanol-insensitive inbred short sleep mice, neither proximal nor distal IPSCs were affected by 80 mM ethanol. The ethanol sensitivity of the proximal pathway was also examined in replicate lines of rats selected for either high ethanol sensitivity or low ethanol sensitivity. GABAA IPSCs in the high ethanol sensitivity lines were significantly enhanced by 80 mM ethanol, whereas IPSCs in the low ethanol sensitivity lines were unaffected. Thus, IPSCs evoked via the proximal pathway were enhanced by ethanol in all the sensitive mouse and rat lines, and unaffected in all the insensitive lines. These experiments demonstrate that GABAA synapses in brain differ in their sensitivity to enhancement by ethanol, and the sensitivity to such enhancement is under the control of genes that can be selected for using classical genetic selective breeding based on a behavioral phenotype. 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, voltageas 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 GABAA receptor function, the GABAA 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 GABAA receptor-mediated responses by intoxicating concentrations of ethanol, as have electrophysiological studies of GABAA 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 Received for publication March 31, 2000. 1 Current address: Neurophysiologie, POB 101007, Heinrich-Heine-Universitat, D-40001 Dusseldorf, Germany. ABBREVIATIONS: GABA, g-aminobutyric acid; IPSC, inhibitory postsynaptic current; ILS, inbred long sleep mice; HAS1 and HAS2, high alcohol sensitivity rat lines; LAS1 and LAS2, low alcohol sensitivity rat lines; ISS, inbred short sleep mice; DNQX, 6,7-dinitroquinoxaline-2,3-dione; APV, DL-(2)-2-amino-5-phosphonovaleric acid; CGP 35348, 3-aminopropyl-(diethoxymethyl)phosphinic acid; LS, long sleep; PK, protein kinase; SS, short sleep. 0022-3565/00/2952-0741 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 295, No. 2 U.S. Government work not protected by U.S. copyright 2769/856034 JPET 295:741–746, 2000 Printed in U.S.A. 741 at A PE T Jornals on M ay 5, 2017 jpet.asjournals.org D ow nladed from 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 GABAA receptors can differ depending on which subset of GABAA 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 GABAA receptor subunits, which leads to differences in ethanol sensitivity. At least some of the differences in the ethanol sensitivity of GABAA responses appear to be under genetic control. Biochemical measures of GABAA receptor function, such as muscimol-stimulated Cl 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 GABAA 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 GABAA responses. However, the sensitivity of specific populations of GABAA 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 GABAA 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 GABAA 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 GABAA receptors. Materials and Methods Transverse hippocampal slices (400 mm) were prepared from 4to 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. GABAA IPSCs were recorded from CA1 neurons using the wholecell 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 MV 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 5 7.3 adjusted with KOH; 290 mOsm) and was kept on ice until immediately before use. Series resistances ranged from 10 to 41 MV (average 30 6 1.5 MV). 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 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 mM 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 50 mM DL-(2)-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 mm 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 265 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 (GABAA 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 100fold 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.