Loren H. Parsons, Marisa Roberto, and George R. Siggins
GABAergic transmission is sensitive to ETOH in distinct brain regions and is clearly involved in the acute actions of ethanol and the development of ethanol tolerance and dependence. Ethanol allosterically modulates the GABAA
receptor complex and is believed to potentiate the effects of GABA in some preparations, though inconsistent findings have contributed to a continuing controversy over this hypothesis. For example, in several brain regions the acute effects of ethanol on GABAA
synaptic responses have been reported to be negligible (Nie et al., 2000
; Proctor et al., 1992
), contingent on additional manipulations such as GABAB
receptor blockade (Nie et al., 2000
; Wan et al., 1996
) or stimulation in discrete segments of neuronal afferents (Weiner et al., 1997a
). In addition, the differential effects of acute and chronic ethanol exposure on GABAergic function (Davis and Wu, 2001
; Mihic, 1999
) have further confounded consensus on the effects of ethanol on GABAergic neurotransmission in the CNS.
In an effort to gain more insight into these issues we have conducted a series of experiments that examined the effects of both acute and chronic ethanol on GABAA
-mediated neurotransmission in the central nucleus of the amygdala (CeA). The CeA, together with its connections to the bed nucleus of the stria terminalis and nucleus accumbens is considered a major component of the “extended amygdala,” a limbic forebrain complex hypothesized to be a common neural substrate for the motivational effects of abused drugs and ethanol in particular. The GABAergic system in the CeA has been implicated in the expression of emotionality including behavioral states of fear and anxiety, as well as states associated with consummatory responses. Moreover, the CeA is considered to be integrally involved in mediating the behavioral effects of acute and chronic ethanol (Hyytia and Koob, 1995
; Roberts et al., 1996
). Many of the findings discussed below have recently been published (Roberto et al., 2003
) and reviewed in a broader context (Siggins et al., 2005
Using an intracellular recording procedure in CeA neurons of brain slices prepared from ethanol-naive male Sprague–Dawley rats we found that acute superfusion with a medium containing 44 mM ethanol clearly enhanced the amplitude of evoked GABA-mediated inhibitory postsynaptic currents (IPSCs). The magnitude of this effect was responsive to ethanol concentration (11–66 mM) with an apparent EC50 of 20 mM and was rapidly reversible by removal of ethanol from the super-fusion medium. Ethanol was also found to decrease the paired-pulse facilitation ratio of GABAA inhibitory IPSCs relative to the control condition. Changes in paired-pulse facilitation ratio are inversely related to transmitter release such that a reduction in paired-pulse facilitation is associated with an increased probability of transmitter release. Thus, these data suggest that acute ethanol exposure increases GABA release in the CeA of ethanol-naive rats. Using whole-cell patch-clamp measurements, ethanol was found to increase the frequency of spontaneously occurring mIPSCs in a bicuculline-sensitive manner, suggestive of an ethanol-induced enhancement of presynaptic GABA release. In addition, ethanol increased the amplitude of mIPSCs in approximately half the neurons evaluated, suggesting an influence of ethanol on postsynaptic mechanisms of some, but not all neurons.
In subsequent experiments, we sought to evaluate the effect of ethanol on GABA release in the CeA using an in vivo preparation. In these experiments ethanol was locally delivered to the CeA of ethanol-naïve rats by retrodialysis, and concurrent changes in dialysate GABA levels were determined using capillary electrophoresis coupled with laser-induced fluorescence. Based on previous work by others (Robinson et al., 2000
), a 10% retrodialysis efficiency was estimated in this experiment, and thus the ethanol concentrations delivered to the tissue immediately surrounding the dialysis probe were approximately 10 to 100 mM, with the concentration declining sharply as a function of distance from the probe membrane because of dilution in the interstitial space. With this protocol, we observed that local ethanol administration induced a significant and dose-dependent (0.1–1.0 M) increase in dialysate GABA levels to maximum of approximately 200% of baseline (see example in ). Interestingly, there was no effect of ethanol on glutamate, glycine, or taurine levels in these same dialysate samples indicating a selective effect of ethanol on GABA release under these conditions. Consequently, these findings provide important in vivo support for the electrophysiological observations made in the slice preparation, which collectively indicate that acutely administered ethanol increases GABA release in the CeA of ethanol-naïve rats.
Fig. 2 Effect of local ethanol (EtOH) administration on dialysate γ-aminobutyric acid (GABA) levels collected from the central nucleus of the amygdala (CeA) of EtOH-naive and chronic EtOH treatment (CET) rats. Stimulatory effect of acute and chronic (more ...)
Chronic ethanol exposure is known to induce many long-term adaptations in CNS function and some data suggest that chronic ethanol consumption reduces GABA ergic function thereby producing tolerance to many of the presumed GABA-mediated behavioral effects of ethanol. Based on these observations, we evaluated alterations in CeA GABA function in animals given chronic ethanol treatment (CET) through long-term ethanol vapor inhalation. Ethanol vapor concentrations were titrated to maintain blood alcohol levels of 150 to 200 mg% for more than 14 days in male Sprague–Dawley rats. Subsequent electrophysiological and microdialysis measures were made in the CeA 2 to 8 hours after removal from the ethanol vapor, a time frame during which significant signs of ethanol withdrawal have been observed using this paradigm (Macy et al., 1996
). We found that the amplitude of evoked GABAA
-mediated IPSP/Cs was significantly enhanced in the CeA of rats previously exposed to CET relative to ethanol-naïve controls. Moreover, both the frequency and amplitude of spontaneously occurring mIPSCs were robustly enhanced in CET rats relative to controls and collectively these observations suggest that basal GABAergic transmission is enhanced following CET. This was supported by the observation of significantly higher baseline dialysate GABA levels collected from the CeA of CET rats compared with ethanol naïve rats. Baseline dialysate levels of glycine and taurine were unaltered by CET, though baseline glutamate levels were significantly increased in CeA microdialysates collected from CET rats. Interestingly, acute ethanol exposure enhanced the amplitude of evoked GABAA
-mediated IPSCs to a similar extent in CeA slices from CET and ethanol-naïve rats, and this similarity was observed across a range of stimulus intensities. In fact, estimated EC50
s for the ethanol-induced enhancement of evoked IPSCs amplitude was 18 and 20 mM for the CET and naïve groups, respectively. Ethanol was also found to produce a similar decrease in paired-pulse facilitation ratios and a similar increase in the frequency of spontaneous mIPSCs in the CeA of CET and naïve rats. These observations suggest that CET does not induce tolerance to ethanol-induced increases in GABA release in the CeA. This was supported by in vivo microdialysis measures in which local ethanol administration significantly enhanced GABA levels in CeA dialysates collected from CET rats, despite the nearly 5-fold increase in baseline dialysate GABA levels in these animals. As in ethanol-naïve rats, local ethanol administration did not alter glycine or taurine levels in CeA dialysates from CET rats, though a significant and ethanol concentration–dependent increase in dialysate glutamate levels was observed.
Collectively these findings indicate that ethanol increases GABA transmission in CeA neurons. The consistent observations of ethanol-induced decreases in paired-pulse facilitation ratios, increases in mIPSC frequencies and increased GABA levels in CeA dialysates indicate that acute ethanol increases presynaptic GABA release. In addition, ethanol increased the amplitude of mIPSCs in approximately half the neurons evaluated, suggesting an influence of ethanol on postsynaptic mechanisms of some, but not all, neurons. The effects of acute ethanol on GABA transmission were preserved in CET rats, indicating a lack of tolerance to the stimulatory effects of ethanol. However, the robust increase in basal IPSCs, mIPSC frequency and baseline dialysate GABA levels following CET suggests a novel neuroadaptive mechanism that may be involved in the development of ethanol dependence.