In agreement with previous work evaluating mIPSCs in the cerebellar slice preparation (Ming et al., 2006
; Kelm et al., 2007
), ethanol enhanced the frequency of sIPSCs, but it did not affect sIPSC amplitude or decay time. Although the mechanically dissociated neurons in the present study were tested in the absence of TTX, a similar increase in frequency of mechanically dissociated Purkinje cell mIPSCs was reported by Kelm et al. (2007)
in the presence or absence of TTX. Thus, voltage-sensitive sodium channels on the preterminal axon or on the presynaptic terminal are not necessary for the effect of ethanol on mIPSCs or sIPSCs. The decrease in PPR during ethanol administration to cerebellar Purkinje neurons is consistent with the elevated frequency of sIPSCs and supports the concept that ethanol increases both spontaneous and stimulated GABA release. Likewise, ethanol enhanced the frequency of mIPSCs from neurons in the SNR slice without affecting mIPSC amplitude or decay time. These observations at these two brain sites are consistent with ethanol increasing release of GABA from presynaptic terminals onto the cerebellar Purkinje and SNR neurons with no measurable effect of ethanol on the postsynaptic receptor. This lack of an effect of ethanol on postsynaptic GABA receptor function is in agreement with earlier conclusions where ethanol did not alter the effect of direct application of GABA to neurons from these brain sites (Criswell et al., 2003
). However, because this preparation was from young animals and was optimized for detection of presynaptic events, it may not be representative of in vivo neural function. Therefore, due to alterations in the internal environment of the postsynaptic neuron due to whole-cell patch recording, including dialysis of intracellular molecules and high chloride concentrations, these later results should be viewed with caution (Weiner and Valenzuela, 2006
). Similar to previous findings in the cerebellum and the SNR, prior studies have demonstrated increased GABA release in several other regions of brain including the brainstem (Sebe et al., 2003
), basolateral amygdala (Zhu and Lovinger, 2006
), hippocampus (2006), and central amygdala (Roberto et al., 2003
). Thus, these data collectively provide convincing data that ethanol can facilitate presynaptic release of GABA at several sites in the brain.
In contrast to the ability of ethanol to increase GABA release from presynaptic terminals in the cerebellum, SNR, and other brain sites, ethanol did not affect sIPSCs from lateral septal or cerebrocortical neurons. In addition, ethanol did not change the PPR in the lateral septum. These observations provide evidence that ethanol does not enhance spontaneous or stimulated GABA release from these sites at the ethanol concentrations tested. Furthermore, ethanol did not affect sIPSC amplitude or decay time at these latter brain sites, indicating that ethanol did not affect postsynaptic function. This latter finding in the lateral septum is also in agreement with previous data that ethanol does not affect postsynaptic receptors on neurons at this brain site (Criswell et al., 2003
). However, as noted earlier, this preparation was optimized for detection of presynaptic events, and only a sample of easily patched neurons is represented. Thus, there may be specific neurons or conditions under which either presynaptic or postsynaptic effects of ethanol can be observed at these brain sites. Similar to the lack of effect of ethanol on presynaptic terminals in the lateral septum and cerebral cortex affecting GABA release, Proctor et al. (2006)
failed to find an effect of ethanol on PPR from CA-1 hippocampal neurons in mice.
Taken together, these varying results provide evidence that ethanol is having a regionally specific effect on GABA release in brain. Whereas the mechanism underlying the effect of ethanol on presynaptic release of GABA from cerebellar Purkinje neurons has not been resolved, recent work has implicated the release of calcium from internal stores (Kelm et al., 2007
). This observation provides a potential lead to determine whether this mechanism associated with calcium release from internal stores applies to other brain sites where ethanol increases GABA release. Likewise, it will be of interest to determine whether this mechanism is absent in neurons where ethanol does not influence GABA release.
Earlier work in the cerebellar slice preparation and in mechanically dissociated neurons from the amygdala found a similar enhancement of GABA release by ethanol in the presence of TTX (Ming et al., 2006
; Zhu and Lovinger, 2006
; Kelm et al., 2007
). This indicates that an effect of ethanol on TTX-sensitive Na+
channels on the presynaptic terminal or preterminal axon is not required for an effect on GABA release. This conclusion was supported in the present investigation by the clear increase in mIPSC frequency by ethanol observed in the SNR slice in the presence of TTX ().
It is conceivable that ethanol could regulate GABA release by acting on the postsynaptic neuron to release a retrograde messenger such as nitric oxide (Shin and Linden, 2005
) or an endogenous cannabinoid (Galante and Diana, 2004
). However, the lack of NMDA receptors on cerebellar Purkinje neurons (Shin and Linden, 2005
) argues against NMDA receptor-mediated nitric oxide release contributing to the effect of ethanol on GABA release at that site. Likewise, because the dendritic tree of the Purkinje neuron is removed during the mechanical dissociation, thereby eliminating the usual space-clamp problems, the voltage-clamp recording should minimize depolarization-induced cannabinoid release from mechanically dissociated neurons in the cerebellum or other brain sites (Galante and Diana, 2004
). In support of this view, Kelm et al., (2007)
demonstrated a similar increase in mIPSC frequency from cerebellar Purkinje neurons by ethanol in the presence of a CB1 receptor antagonist.
receptors can provide feedback inhibition of GABA release, and the activation of these receptors by endogenous GABA in some brain regions could decrease the ability of ethanol to elicit GABA release (Ariwodola and Weiner, 2004
; Zhu and Lovinger, 2006
). Therefore, differential GABAB
receptor inhibition of GABA release represents a potential mechanism for the lack of effect of ethanol in some brain regions; moreover, we observed this effect in the SNR (). However, in the lateral septum, the observed lack of an effect of ethanol on GABA release in the presence of a GABAB
antagonist argues against this explanation. In addition, GABAB
-mediated feedback inhibition of increased GABA release would only be expected if the ethanol initially caused at least some increase in GABA release. Thus, a mechanism distinct from GABAB
receptor inhibition must be sought to elucidate the mechanism of the regional specificity associated with the action of ethanol to release GABA from presynaptic neurons.
The noted regional specificity of ethanol to release GABA has considerable relevance to actions of ethanol on the CNS and to drugs that influence GABA function. The ability of ethanol to release GABA could account for a part of the behavioral GABAmimetic profile of ethanol resembling that of benzodiazepines and barbiturates (Frye and Breese, 1982
), both of which are known to act primarily by a GABAergic mechanism (Martz et al., 1983
). In addition, behavioral actions of ethanol are additive or superadditive with these GABAergic compounds, and they substitute for ethanol in discrimination studies (Frye and Breese, 1982
; Frye et al., 1983
; Martz et al., 1983
; Grant et al., 2000
). Although met with controversy (Wallner et al., 2003
; Borghese et al., 2006
), the ability of ethanol to enhance the action of GABA on receptors containing α6β3δ and α4β3δ subunits could be enhanced by the increased release of GABA by ethanol. Thus, in this case, “spill over” from increased GABA release by ethanol could have a direct effect on these extrasynaptic GABAA
receptors to increase inhibitory tone (Wei et al., 2003
). This scenario would be dependent upon a brain site where ethanol released GABA and where these ethanol-sensitive GABAA
receptors are localized. Previous work has shown that substantia nigra and cerebellar Purkinje neurons are highly sensitive to enhancement of currents gated by exogenously applied GABA by neuroactive steroids (Criswell et al., 2003
). Thus, there are receptors present that could respond to the increased GABA release by ethanol.
The regional differences in the ability of ethanol to release GABA could offer an explanation of earlier studies showing the regionally specific effects of ethanol on GABA function using in vivo extracellular recording (Bloom and Siggins, 1987
; Givens and Breese, 1990
; Criswell et al., 1993
). Furthermore, this regional specificity for GABA release by ethanol may account for the selective effect of ethanol on specific behaviors. In other words, only behaviors mediated by a brain region sensitive to this action of ethanol would be modified by ethanol. A caution for this interpretation is that concentrations of ethanol below 50 mM did not induce a reliable increase in mIPSC frequency in vitro—a finding that might be expected for this action of ethanol to contribute to all aspects of its GABAmimetic profile (Frye et al., 1979
). Future studies should be able to define the accuracy of the view that release of GABA from presynaptic terminals in specific brain regions is a critical contributor to the GABAmimetic profile of ethanol.