Introduction

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The anxiolytic effect of EtOH is prominent in the reinforcing nature of EtOH consumption. However, the neurobiological substrates underlying this effect of EtOH consumption remain largely unknown, in part due to the multiplicity of EtOH effects reported. We therefore have chosen to study EtOH effects in the amygdala, a brain region associated with anxiogenesis and the action of anxiolytic agents (Aggleton, 1992), including EtOH (Rassnick et al., 1993; Koob et al., 1994). As a prerequisite to examining EtOH effects on the unique neural circuitry of the amygdala, we have begun to characterize the inhibitory action of EtOH on excitatory synaptic transmission in the basolateral nucleus.

Previous studies have shown that low-dose EtOH (corresponding to anxiolytic blood levels) produces relatively selective inhibition of the NMDA receptor-mediated currents in cultured hippocampal neurons (Lovinger et al., 1989), acutely isolated sensory neurons (White et al., 1990), and adult hippocampal slices (Weight et al., 1991; Morrisett and Swartzwelder, 1993). In these preparations EtOH had little effect on non-NMDA glutamate-mediated currents or GABA-mediated Cl currents, even at relatively high doses.

To characterize the action of EtOH on synaptic transmission in the amygdala, we examined effects of EtOH under voltage clamp conditions, using a range of Mg⁺⁺ concentrations. We have found that low-dose EtOH can significantly inhibit NMDA receptor-mediated synaptic currents in basolateral amygdala. In addition, this inhibition requires the presence of magnesium. EtOH produces comparable inhibition of NMDA-mediated currents elicited by exogenously applied glutamate, suggesting that this effect may be primarily postsynaptic. This synergistic interaction of EtOH and Mg⁺⁺ suggests a possible molecular site of the action of EtOH in the vicinity of Mg⁺⁺ binding sites on the NMDA receptor-channel complex.

	Methods

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Coronal brain slices were prepared from 14- to 21-day-old male Sprague-Dawley rats. Rats were decapitated under brief halothane anesthesia, the brains were rapidly removed, and coronal slices (300 µm) were prepared on a Vibratome tissue slicer. Slices containing the basolateral amygdala were identified and incubated in a holding chamber at room temperature for at least 60 min before use.

Whole cell patch electrodes (3-6 M) contained the following internal solution in mM: Cs-gluconate 85, CsFl 40, HEPES buffer 10, Cs₄BAPTA 10, QX314 2, MgATP 2; pH = 7.25. The extracellular solution (artificial cerebrospinal fluid; ACSF) contained in mM: NaCl 119.8, NaHCO₃ 22, dextrose 25, MgCl₂ 1, KCl 3.3, CaCl₂ 2. To isolate NMDA receptor-mediated events, the ACSF also contained BMI (20 µM) and DNQX (20 µM). In some experiments the ACSF also contained APV (50 µM). EtOH was applied by superfusion in concentrations of 11 to 44 mM. All drugs were obtained from Sigma Chemical Co. (St. Louis, MO).

For each experiment, slices were transferred to a submerged recording chamber with fluid temperature held at 30 ± 1°C. Using a Zeiss Axioscope fitted with differential interference contrast optics, the basolateral amygdala nucleus was identified under low power, and individual pyramidal cells were visualized with a 40X water immersion objective. Whole cell patch-clamp recordings were made of visualized cells using a Warner PC-501A amplifier. Series resistance was continuously monitored and experiments were discarded if this value varied by more than 20%. No adjustment was made for a 5 to 10 mV junction potential at the electrode tip. Afferent fibers were stimulated with a monopolar tungsten electrode placed within the basolateral nucleus adjacent to the external capsule. Data are expressed as means ± S.E.M.

	Results

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Recordings were taken from 74 neurons in the central region of the basolateral nucleus. Based on electrophysiological properties and morphological features (under high-power microscopy), the neurons we selected appeared to meet criteria for pyramidal neurons as previously described (Rainnie et al., 1991a, 1991b; Washburn and Moises, 1992). In dose ranges of 11 to 44 mM, superfused EtOH had no significant effect on resting membrane potential or input resistance (data not shown), consistent with previous reports in other brain areas (Siggins et al., 1987a, 1987b).

We recorded synaptically evoked potentials and currents at a stimulation rate of 1/60 sec. In our preliminary studies using normal ACSF, most neurons showed complex synaptic responses, consisting of both excitatory and inhibitory components (Rainnie et al., 1991a, 1991b). With the addition of QX-314 and Cs⁺ to the pipette solution and BMI and DNQX to the ACSF, only an APV-sensitive EPSP remained and was presumed to be mediated by the NMDA receptor (fig. 1A). In voltage clamp mode, the reversal potential of the NMDA-mediated EPSCs was approximately +10 mV. EPSCs were recorded from each neuron at holding potentials of 0, 20 and 40 mV.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Voltage-dependent inhibition of NMDA-mediated synaptic currents by EtOH. A, NMDA receptor-mediated synaptic currents in basolateral amygdala. Synaptic currents elicited by single pulses (7 µA, 0.3 msec) delivered from a stimulating electrode positioned in the lateral/basolateral juncture. Currents were elicited at three different holding potentials and were isolated by superfusion of 20 µM BMI and 20 µM DNQX (left column); the remaining responses were blocked by 50 µM APV (center column). Right column displays responses after 10 min. of APV washout. Scale = 200 pa, 100 msec. B, Effect of EtOH (44 mM) on individual synaptic responses at holding potentials of 0, 20 and 40 mV. Measurements of EtOH effect were taken after 10 min. of EtOH superfusion. Scale = 50 pA, 100 msec. C, Averaged percent attenuation of synaptic currents at three holding potentials, using three EtOH concentrations. Measurements were taken after 10 min of EtOH superfusion.

After a 5- to 10-min period of baseline recording, we superfused ACSF containing EtOH (11-44 mM) for a period of 10 min, followed by continued recording during the washout period. Responses were averaged as percent of corresponding responses of a control group of non-EtOH treated neurons. At 10 min of superfusion, 44 mM EtOH reduced the mean amplitude of the NMDA-mediated EPSCs current by 31 ± 5% when the cell membrane was clamped at 40 mV (fig. 1, B and C). However, the inhibitory effect of EtOH was significantly lessened when the cell membrane was depolarized to 0 mV (16 ± 5%, fig. 1C), although this difference was less for the first 5 min of EtOH superfusion. The EtOH effect was usually reversible upon washout, though in some neurons (n = 5/14 at 44 mM, 3/8 at 22 mM, 1/4 at 11 mM) the washout was accompanied by an apparent hyperexcitability characterized by an increased amplitude over baseline. No significant voltage dependency was noted with 22 mM EtOH, with little observable effect of 11 mM EtOH at any membrane potential (fig. 1C).

For comparison to baseline responses we used the final synaptic responses during EtOH application (just before washout), restricting total EtOH application to 10 min to avoid acute tolerance (Grover et al., 1994). However, in some cells (9/14 at 44 mM, 4/8 at 22 mM) the peak EtOH inhibitory effect occurred before the final measurements, suggesting development of rapid tolerance. This acute tolerance to 44 mM EtOH was most apparent when holding the neurons at depolarized potentials and may account for a significant portion of the voltage dependency of the EtOH effect seen at later time points.

One explanation for the above findings is that the action of EtOH in the amygdala is primarily presynaptic, reducing release of an excitatory amino acid neurotransmitter. To test this possibility, we examined the effect of EtOH superfusion on responses to pressure-applied NMDA although cells were held at a resting potential of 40 mV. EtOH (44 mM; n = 8) consistently reduced the inward current resulting from exogenous NMDA (100 µM, 500-1000 msec pulse duration) applied to the dendritic region by 21 ± 6% (fig. 2). As the magnitude of this inhibition is comparable to the effect of EtOH on synaptically mediated responses, we interpret the inhibitory effect of EtOH in this preparation as predominantly postsynaptic.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   EtOH inhibits NMDA-mediated currents elicited by exogenous glutamate. A, Raw traces of EtOH (44 mM) inhibition of NMDA-mediated currents elicited by pressure ejection of 100 µM NMDA (500-msec pulse duration) delivered from a glass micropipette positioned over the dendrites. Neuron was held at 40 mV. Scale = 200 pa, 2 sec. B, Time course of averaged responses to NMDA delivered by pressure ejection.

Based on previous studies showing a synergistic action of EtOH and Mg⁺⁺ in inhibiting of field potentials in hippocampus (Schummers et al., 1997; Martin et al., 1991; Morrisett et al., 1991), we hypothesized that the voltage-dependent effect of EtOH may require the presence of Mg⁺⁺. We therefore switched to ACSF containing 0 mM or 0.3 mM Mg⁺⁺ without replacement. Under conditions of 0 mM Mg⁺⁺, 44 mM EtOH caused a potentiation of NMDA-mediated EPSCs. Synaptic currents recorded in ACSF containing 0.3 mM Mg⁺⁺ were weakly inhibited by 44 mM EtOH, significantly less so than in ACSF containing 1.0 mM Mg⁺⁺ (fig. 3).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of Mg++ concentration on inhibition of NMDA-mediated synaptic currents by EtOH. A, Raw traces of EtOH (44 mM) potentiation of NMDA-receptor mediated synaptic currents in ACSF containing 0 mM Mg++. Scale = 150 pa, 100 msec. B, Averaged percent attenuation of synaptic currents at three holding potentials, using three Mg++ concentrations. Measurements were taken after 10 minutes of 44 mM EtOH superfusion. C, Time course of EtOH effect at different concentrations of external Mg++. Graphs depict EtOH effect at holding potentials of 0 mV (open squares), 20 mV (open circles) and 40 mV (closed circles).

	Discussion

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This study indicates that low concentrations of EtOH have a potent inhibitory effect on NMDA-receptor mediated excitatory synaptic transmission in the basolateral amygdala. We therefore have confirmed similar findings from other brain areas (White et al., 1990; Weight et al., 1991; Morrisett and Swartzwelder, 1993) and extended the demonstration of this phenomenon to the amygdala, a brain region associated with action of anxiolytic agents. Consistent with reports in other brain areas, we found relatively little effect on resting membrane potential or input resistance (Siggins et al., 1987a, 1987b).

Our data indicate that the inhibitory potency of EtOH in the amygdala is increased under conditions of physiological Mg⁺⁺ concentrations. This synergism of EtOH and Mg⁺⁺ suggests a potential molecular site of action of EtOH within the NMDA receptor complex (Michaelis and Michaelis, 1994). Only one other study has reported direct effects of acute EtOH in the amygdala. In that study, EtOH inhibited NMDA receptor-mediated burst activity (at 25-100 mM EtOH) and reduced synaptic potentials (at 100 mM EtOH) in Mg⁺⁺-free medium (Gean, 1992). We observed an inhibitory effect on NMDA receptor-mediated synaptic currents in normal ACSF with lower, anxiolytic concentrations of EtOH (22-44 mM); thus EtOH appeared more potent in our preparation. Because we averaged measures of the inhibition of synaptic currents after 10 min of EtOH superfusion, as opposed to measures of peak inhibition, we may have actually underestimated the potency of the acute EtOH application. In addition, in our study, removing Mg⁺⁺ from the medium abolished the inhibitory effect of low-dose EtOH on NMDA receptor-mediated synaptic currents. We observed no bursting activity in Mg⁺⁺-free medium, presumably because we included DNQX in the bath medium and all measures were taken within 30 min of switching to Mg⁺⁺-free medium. Possible explanations for these contrasting effects of EtOH in Mg⁺⁺-free medium include differences in EtOH concentrations and differences in the age of the animals.

Our study contrasts with the reports showing lack of Mg⁺⁺ dependency for EtOH inhibition of NMDA receptor-mediated currents in cultured mouse hippocampal pyramidal cells (Peoples et al., 1997) or Xenopus oocytes (Chu et al., 1995). These conflicting results may possibly be accounted for by differences in species or preparation. For example, these studies measured steady-state NMDA receptor-activated currents over relatively prolonged (5-60 sec) exposure to agonist, whereas we measured peak currents associated with the briefer synaptic responses.

Under nonvoltage clamped conditions the net inhibitory effect of EtOH on synaptic responses may be greater than reported in our study. Activation of voltage-dependent Ca⁺⁺ channels during current clamp recordings may constitute a substantial portion of NMDA-mediated synaptic potentials (Miyakawa et al., 1992), although these channels would be largely inactivated during our voltage-clamp protocol. Preliminary current- and voltage-clamp data from our lab indicates that EtOH has an additional inhibitory effect on these channels (Calton et al., 1996); thus, the aggregate synaptic potential in Mg⁺⁺-free medium may still be decreased by EtOH.

Our data do not preclude the possibility of additional mechanisms relating to EtOH inhibition of excitatory synaptic transmission, including presynaptic inhibition of transmitter release by EtOH (Nie et al., 1994) or inhibition of voltage dependent channels (Calton et al., 1996; Carlen et al., 1982; Moore et al., 1990). However, to the extent that the effect of EtOH we observed varies with postsynaptic membrane potential, we interpret this effect on NMDA-mediated responses as primarily postsynaptic. Also, in our preparation, EtOH inhibited NMDA-receptor mediated currents produced by application of exogenous NMDA by pressure ejection. As the magnitude of this inhibition was comparable to the inhibition of synaptic currents, this also suggests a postsynaptic site of EtOH action.

This interaction between EtOH and Mg⁺⁺ may have important implications for understanding the behavioral effects of EtOH consumption. Acute EtOH exposure can cause significant, transient decreases in brain intracellular free Mg⁺⁺ (Altura et al., 1991). In our study we have shown that EtOH may potentiate excitatory synaptic transmission under conditions of zero Mg⁺⁺. As yet, little attention has been directed toward more subtle interactions between EtOH and Mg⁺⁺ that may underlie the behavioral effects acute EtOH consumption at moderate doses. We speculate that there may be a dynamic interaction, such that as EtOH consumption reduces free brain Mg⁺⁺, excitatory synaptic transmission may become less sensitive to the inhibitory effect of EtOH, thereby reducing the acute reinforcing effect of low-dose EtOH and, in susceptible individuals, causing increased EtOH consumption to overcome this loss of sensitivity.

NMDA receptor function is implicated in long-term synaptic potentiation in the basolateral amygdala (Gean et al., 1993a, 1993b) and this NMDA-mediated synaptic plasticity may underlie various forms of fear conditioning that have served as models for anxiety states (Fanselow and Kim, 1994). By acute inhibition of excitatory synaptic transmission and plasticity in the amygdala, EtOH may alter the underlying neurobiological associations of external and internal cues that produce anxiety (LeDoux, 1993), thereby exerting an anxiolytic effect.