Pharmacology & Therapeutics 111 (2006) 533 – 554

www.elsevier.com/locate/pharmthera

Associate editor: A.L. Morrow

Ethanol modulation of GABAergic transmission: The view from the slice

J.L. Weiner a,*, C.F. Valenzuela b
a
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard,
Winston-Salem, NC 27157-1083, USA
b
Department of Neurosciences, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

Abstract

For almost three decades now, the GABAergic synapse has been the focus of intense study for its putative role in mediating many of the
behavioral consequences associated with acute and chronic ethanol exposure. Although it was initially thought that ethanol interacted solely with
the postsynaptic GABAA receptors that mediate the majority of fast synaptic inhibition in the mammalian central nervous system (CNS), a number
of recent studies have identified novel pre- and postsynaptic mechanisms that may contribute to the acute and long-term effects of ethanol on
GABAergic synaptic inhibition. These mechanisms appear to differ in a brain region specific manner and may also be influenced by a variety of
endogenous neuromodulatory factors. This article provides a focused review of recent evidence, primarily from in vitro brain slice
electrophysiological studies, that offers new insight into the mechanisms through which acute and chronic ethanol exposures modulate the activity
of GABAergic synapses. The implications of these new mechanistic insights to our understanding of the behavioral and cognitive effects of
ethanol are also discussed.
D 2005 Elsevier Inc. All rights reserved.

Keywords: GABA; Ethanol; Electrophysiology; Presynaptic; Postsynaptic

Abbreviations: AMPA, a-amino-3-hydroxi-5-methylisoxazole-4-propionic acid; BZP, benzodiazepine; CeA, central nucleus of the amygdala; CIE, chronic
intermittent ethanol; CNS, central nervous system; CRF, corticotrophin releasing factor; EPSP, excitatory postsynaptic potential; GABA, g-aminobutyric acid; GDP,
giant depolarizing potential; IPSC, inhibitory postsynaptic current; IPSP, inhibitory postsynaptic potential; NMDA, N-methyl-d-aspartate; PKA, protein kinase A;
PKC, protein kinase C; PPD, paired-pulse depression; PPF, paired-pulse facilitation; TTX, tetrodotoxin; VGCC, voltage-gated calcium channel; VTA, ventral
tegmental area.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
2. The GABAergic synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
2.1. Ionotropic g-aminobutyric acid receptors (GABAA, GABAC) . . . . . . . . . . . . . 535
2.2. Metabotropic g-aminobutyric acid receptors (GABAB) . . . . . . . . . . . . . . . . . 535
3. Acute effects of ethanol on evoked GABAergic synaptic transmission . . . . . . . . . . . . 536
4. Synaptic mechanisms of ethanol action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
4.1. Presynaptic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
4.1.1. Studies with hippocampal slices. . . . . . . . . . . . . . . . . . . . . . . . 538
4.1.2. Studies with amygdala slices . . . . . . . . . . . . . . . . . . . . . . . . . 538
4.1.3. Studies with cerebellar slices . . . . . . . . . . . . . . . . . . . . . . . . . 538
4.1.4. Studies with slices from other brain regions . . . . . . . . . . . . . . . . . 539
4.2. Postsynaptic mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
4.2.1. Synaptic GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . 539
4.2.2. Extrasynaptic GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . 540

* Corresponding author. Tel.: 336 716 8692; fax: 336 716 8501.
E-mail address: jweiner@wfubmc.edu (J.L. Weiner).

0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2005.11.002
534 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554

5. Modulators of ethanol potentiation of GABAergic synaptic transmission . . . . . . . . . . . 541

5.1. GABAB receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
5.2. Neurosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542
5.3. Corticotrophin releasing factor and other peptides, neurotransmitters . . . . . . . . . 543
5.4. Protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
6. Effects of chronic ethanol on GABAergic synaptic transmission . . . . . . . . . . . . . . . . 544
6.1. Studies with hippocampal slices . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
6.2. Studies with amygdala slices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
7. Developmental implications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550

1. Introduction of such a mechanism proved surprisingly elusive. Although

g-Aminobutyric acid (GABA) was first identified over three
decades ago as the major inhibitory neurotransmitter in the
mammalian central nervous system (CNS) (Bloom & Iversen,
1971). Almost immediately following those pioneering studies,
reports began to emerge suggesting that the acute behavioral
and cognitive effects of ethanol were mediated in part via an
enhancement of GABAergic inhibition (e.g., Davidoff, 1973;
Nestoros, 1980). In the coming years, numerous studies,
employing a variety of behavioral, neurochemical, and
electrophysiological approaches provided additional support
for this hypothesis (for detailed, historical reviews of these
studies, see Deitrich et al., 1989; Criswell & Breese, 2005).
The first mechanistic theories centered on the idea that
ethanol enhanced synaptic inhibition primarily via a direct
allosteric facilitation of the activity of GABAA receptors,
which mediate the vast majority of fast synaptic inhibition in
the mammalian CNS. These theories were based initially on
observations from numerous behavioral studies, which noted
that ethanol shared many of the sedative, anxiolytic, and
anticonvulsant properties of drugs known to enhance GABAA
receptor function (e.g., benzodiazepines [BZPs] and barbitu-
rates; Deitrich et al., 1989) and that cross-tolerance could
develop between ethanol and drugs that enhance GABAA
receptor activity (Le et al., 1986; Mihic et al., 1992). Moreover,
GABAA receptor agonists or drugs that raise endogenous levels
of GABA were shown to potentiate the sedative, hypnotic,
ataxic, and anticonvulsant actions of ethanol (Hakkinen &
Kulonen, 1976; Frye & Breese, 1982; Liljequist & Engel,
1982; Martz et al., 1983), whereas GABAA receptor antago-
nists and certain benzodiazepine partial inverse agonists (Ro
45-1513) could reduce some of the intoxicating effects of
ethanol (Hakkinen & Kulonen, 1976; Liljequist & Engel, 1982;
Martz et al., 1983; Suzdak et al., 1986b). The findings that a
range of allosteric GABAA receptor modulators could substi-
tute for ethanol in drug discrimination studies (Kline & Young,
1986; Barry, 1991; Ator et al., 1993; Hodge & Alken, 1996)
provided further support for this theory.
While the evidence from most behavioral studies supported
the idea that ethanol enhanced GABAergic inhibition via an
allosteric facilitation of GABAA receptor function, direct
biochemical and/or electrophysiological evidence in support
initial neurochemical (Suzdak et al., 1986a; Morrow et al.,
1988) and electrophysiological (Aguayo, 1990; Reynolds &
Prasad, 1991) studies seemed to suggest that pharmacologically
relevant concentrations of ethanol could exert an allosteric
facilitation of GABAA receptor function, these effects were
often quite variable (Reynolds et al., 1992) and in some cases
not observed (Osmanovic & Shefner, 1990; White et al., 1990;
Mihic et al., 1991). Notably, evidence from in vitro studies
investigating ethanol effects on GABAergic synaptic transmis-
sion also proved highly variable (see Section 3).
Over the last decade or so, a variety of methodological
advances have greatly facilitated the study of the physiolog-
ical and pharmacological properties of synapses in in vitro
brain slice preparations. Numerous ethanol researchers have
employed these improved methods to reexamine the acute
effects of ethanol on GABAergic neurotransmission in a
number of brain regions and in a variety of species from
mouse to monkey. These recent studies have shed new light
on the complexity surrounding the interaction between ethanol
and the GABAergic synapse. While the majority of these
studies have now provided compelling evidence that ethanol
can indeed enhance GABAergic synaptic activity, these
studies also suggest that the mechanisms underlying this
enhancement are far more complex than initially appreciated
and often involve interplay between both pre- and postsyn-
aptic mechanisms.
The purpose of this article is to provide a highly focused
overview of the findings of studies from the past decade or so
that offer novel insight into specific pre- and postsynaptic
mechanisms through which ethanol enhances GABAergic
neurotransmission in the mammalian CNS. The focus will be
mainly to examine this interaction from the perspective of
native receptors in tissue slices with an emphasis on
electrophysiological studies. The implications of these new
mechanistic insights to our understanding of the behavioral and
cognitive effects of ethanol and the treatment of alcohol-related
disorders will also be discussed. Other aspects regarding the
interaction of ethanol with GABAergic transmission and
GABA receptors have been reviewed elsewhere (Macdonald,
1995; Klein & Harris, 1996; Mihic, 1999; Aguayo et al., 2002;
Boehm et al., 2004; Kumar et al., 2004; Criswell & Breese,
2005).
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
2. The GABAergic synapse
As noted in Section 1, GABA serves as the primary
inhibitory neurotransmitter in the mammalian CNS (Bloom &
Iversen, 1971). This neutral amino acid interacts with 2 classes
of ionotropic receptors (GABAA and GABAC) and 1 class of
metabotropic receptor (GABAB) to limit and shape synaptic
communication throughout the central nervous system (McKer-
nan & Whiting, 1996; Krnjevic, 1997).
2.1. Ionotropic c-aminobutyric
acid receptors (GABAA, GABAC)
GABAA receptors are the most abundant class of GABA
receptor in the brain (Sieghart, 1995). These receptors are part
of a superfamily of heteromeric ligand-gated ion channels that
includes nicotinic, glycine, and serotonin type-3 receptors
(Ortells & Lunt, 1995). All members of this superfamily share
a number of structural features in common. These receptors are
believed to be comprised of five subunits that form an intrinsic
ion channel (Sieghart, 1995). Each subunit possesses an
extracellular N-terminal ligand-binding domain that gates the
opening and closing of the ion channel pore. In the case of
ionotropic GABA receptors, this pore is an anion channel that
is primarily permeant to chloride and bicarbonate ions. To date,
eight subclasses of GABAA receptor subunits have been cloned
from the mammalian nervous system (a1 – 6, h1 – 3, g1 – 3, y, (, u,
k, and U1 – 3 (Farrant & Nusser, 2005). The expression pattern
of the various GABAA receptor subunits varies extensively
between different brain regions (Wisden et al., 1996) and even
within subcellular compartments of individual neurons (Nusser
et al., 1996). Given that the subunit composition of GABAA
receptors can profoundly influence their biophysical and
pharmacological properties (Sieghart, 1995), the large number
of subunits likely contributes to the functional diversity of
inhibitory GABAergic synapses (Hevers & Luddens, 1998;
Cherubini & Conti, 2001).
GABAA receptors mediate the majority of fast, phasic
inhibitory synaptic transmission in the mammalian CNS
(Thompson, 1994) and are known to be important targets for
a variety of therapeutic drugs such as benzodiazepines and
barbiturates, as well as endogenous modulators, like certain
neurosteroids (e.g., allopregnanolone; Macdonald & Olsen,
1994; Sieghart, 1995). These compounds interact with defined
sites on GABAA receptors and allosterically modulate receptor
function. Although the specific stoichiometry of native
GABAA receptors is not fully established, most of these
receptors are thought to be comprised of two a-, two h-, and
one of the other subunits (McKernan & Whiting, 1996).
In addition to their well-described role in mediating phasic
synaptic inhibition, GABAA receptors have also been shown to
mediate a slower form of communication between neurons,
termed tonic inhibition (for a review, see Farrant & Nusser,
2005). This sustained inhibition appears to be mediated by
extrasynaptic GABAA receptors that have a relatively low
affinity for GABA but display less desensitization than the
receptors that mediate phasic inhibition. In some brain regions,
535
these receptors contain a4hxy, a6hxy, or a5hxg2 subunits.
Although clear evidence of tonic GABAA receptor signaling
has only been demonstrated in a few brain regions to date,
these extrasynaptic receptors are increasingly being recognized
as playing significant roles in regulating neuronal excitability
and are likely important targets for a number of therapeutic and
endogenous modulators (Farrant & Nusser, 2005).
GABAC receptors make up another class of ionotropic
GABA receptors. These receptors are primarily expressed in
the adult retina and in a few other regions, like the
hippocampus and amygdala, primarily during early stages of
development (Qian & Dowling, 1993; Enz et al., 1995; Martina
et al., 1995; Yeh et al., 1996; Delaney & Sah, 1999, 2001).
These receptors are gated by GABA and certain conforma-
tionally restricted analogues of GABA (e.g., cis-4-aminocro-
tonic acid), contain an integral chloride channel, and have a
number of unique pharmacological properties that further
distinguish them from the more prevalent GABAA receptors
(Bormann, 2000). These receptors are believed to be pentamers
formed from a single class of subunits, U, of which three
variants have been cloned (Bormann, 2000).
2.2. Metabotropic c-aminobutyric acid receptors (GABAB)
In addition to its well-characterized ionotropic actions,
GABA activates a class of metabotropic receptors that also
play an important inhibitory role in the mammalian nervous
system (Marshall et al., 1999; Bowery & Enna, 2000; Couve et
al., 2000). GABAB receptors are heterodimers made up of two
homologous subunits (GB1 and GB2). They belong to the
family C (class III) group of G protein-coupled receptors that
also includes metabotropic glutamate receptors and extracellu-
lar Ca2+ sensing receptors. Interestingly, only a heteromeric
GABAB receptor assembly, formed in part by a coiled-coil
association between the carboxy termini of GB1 and GB2,
displays native agonist affinity and full functional coupling
(Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998).
Both GB1 and GB2 subunits are widely expressed throughout
the central nervous system, at both pre- and postsynaptic loci
(Margeta-Mitrovic et al., 1999; Sloviter et al., 1999). Activa-
tion of postsynaptic GABAB receptors decreases excitability by
opening G-protein-coupled inwardly rectifying potassium
channels (GIRKs) (Misgeld et al., 1995). Activation of
presynaptic GABAB receptors inhibits neurotransmission,
possibly by inhibiting voltage-gated calcium channels
(VGCCs). Both pre- and postsynaptic GABAB receptor signals
are coupled by pertussis toxin-sensitive Gi/o proteins and may
involve a direct interaction between liberated Ghg subunits and
their downstream effectors (e.g., GIRKs, Ca2+ channels)
(Kajikawa et al., 2001). Activation of GABAB receptors has
also been shown to inhibit adenylyl cyclase (Bowery & Enna,
2000), possibly through Gia (Nishikawa et al., 1997), and may
also have complex effect on protein kinase C (PKC) activity
(Tremblay et al., 1995). Although early studies suggested the
existence of multiple subtypes of GABAB receptors, recent
studies with GB1 knockout mice suggest that a common
receptor class mediates all pre- and postsynaptic GABAB
536 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
receptor effects that have been characterized to date (Prosser et
al., 2001; Schuler et al., 2001). Apparent pharmacological
heterogeneity of GABAB receptor effects may arise from
differences in the downstream coupling pathways of these
receptors in different brain regions.
3. Acute effects of ethanol on
evoked GABAergic synaptic transmission
In the early 1980s, as the importance of GABAergic
neurotransmission as the major inhibitory element in the
mammalian CNS became more widely appreciated, several
brain slice electrophysiology laboratories began to examine the
acute effects of ethanol on the inhibitory component of
synaptic transmission. Most of these original studies employed
sharp electrode current clamp methods to study the effects of
ethanol on electrically evoked compound synaptic potentials
that contained both excitatory (glutamatergic) and inhibitory
(GABAergic) components. In addition, the majority of these
early studies focused on hippocampal synapses, as the
hippocampal slice was the first in vitro brain tissue preparation
to gain widespread use in electrophysiological studies.
In some of the earliest studies employing these methods,
Carlen et al. (Durand et al., 1981; Carlen et al., 1982) reported
that bath application of low concentrations of ethanol (< 20 mM)
potentiated the inhibitory GABAergic component of evoked
synaptic potentials recorded from rat hippocampal CA1
pyramidal neurons. Interestingly, using a method that allowed
for focal application of ethanol to the soma or dendritic regions
of these cells revealed that the potentiating effect of ethanol was
markedly greater when applied to the somatic region of CA1
neurons. These experiments provided the first evidence that
pharmacologically relevant concentrations of ethanol could
enhance inhibitory synaptic potentials and that, at least in the
hippocampus, perisomatic GABAergic synapses exhibited a
relatively high sensitivity to ethanol.
Unfortunately, following these initial positive reports,
subsequent studies investigating ethanol effects on hippocam-
pal GABAergic inhibition generated rather disparate results.
For example, Siggins et al. (1987), employing methods
virtually identical to those of Carlen et al., reported
predominantly depressant effects of bath-applied ethanol on
inhibitory postsynaptic potentials (IPSPs) recorded from CA1
and CA3 pyramidal neurons in rat hippocampal slices.
Similarly, Proctor et al. (1992) did not observe a significant
effect of 80 mM ethanol on the amplitude of the GABAA
receptor-mediated component of evoked synaptic potentials in
rat CA1 neurons.
In the early 1990s, the first studies were conducted that
employed the whole-cell patch clamp recording technique
along with pharmacological approaches that permitted the
definitive isolation of evoked synaptic currents mediated solely
by the activation of GABAA receptors (i.e., blocked by
GABAA receptor antagonists, reversed at the expected equi-
librium potential for chloride). Unfortunately, these technical
advances did not result in an immediate clarification of ethanol
actions on GABAergic synapses. Morrisett and Swartzwelder
(1993) used the whole-cell patch clamp method to investigate
the mechanism underlying ethanol inhibition of long-term
potentiation in rat dentate granule cells and reported no effect
of 75 mM ethanol on monosynaptic GABAA inhibitory
postsynaptic currents (IPSCs). Another group reported a
similar absence of an ethanol effect on hippocampal CA1
IPSCs (Soldo et al., 1994); however, 80 mM ethanol was
shown to potentiate GABAergic responses in cortical neurons
as well as in cells in the medial and lateral septum. In contrast,
another study reported a concentration-dependent increase in
the amplitude of GABAA IPSCs in rat CA1 pyramidal neurons
(Weiner et al., 1994). In that study, a possible modulatory role
of protein kinase C was also reported, as the observed ethanol
facilitation was significantly attenuated by inhibitors of this
kinase.
Given the disparate nature of these early findings, by the
mid-1990s many were beginning to question the importance of
GABAergic synapses as relevant targets of ethanol action.
However, perhaps because of the technical advances in the
field of slice electrophysiology and/or the recognition that
modulatory factors such as kinase activity could influence
ethanol effects on GABAergic synapses and potentially explain
some of reported disparity in the literature, this era was marked
by a seemingly renewed interest in resolving this issue.
In a landmark paper, Siggins et al. (Wan et al., 1996)
discovered that, although compound GABAA/GABAB IPSP
responses in CA1 neurons were insensitive to ethanol,
pharmacological isolation of the GABAA receptor-mediated
component using an antagonist of metabotropic GABAB
receptors, unmasked a reliable and relatively potent potentiat-
ing effect of ethanol on the remaining GABAA response.
Although the specific mechanism underlying this effect was
not clearly understood, these findings suggested that some
previously unrecognized GABAB receptor-dependent mecha-
nism effectively occluded ethanol potentiation of GABAA
IPSCs at these synapses (for further discussion, see Section
5.1).
Another advance that helped to resolve some of the disparity
regarding the ethanol sensitivity of hippocampal GABAergic
synapses came with the discovery that subpopulations of
GABAergic synapses onto individual CA1 pyramidal neurons
were differentially sensitive to ethanol. It had been known for
several years that stimulation of dendritic and perisomatic loci
of the CA1 region elicits GABAA IPSCs with distinct
physiological and pharmacological properties (Pearce, 1993;
Pearce et al., 1995). In 1997, Weiner et al. demonstrated that
somatic GABAA IPSCs onto CA1 pyramidal neurons were
reliably potentiated by ethanol at concentrations as low as
40 mM (Weiner et al., 1997a). However, concurrent record-
ings of dendritic IPSCs revealed that these distal inhibitory
synapses were significantly less sensitive to ethanol across a
broad range of concentrations (40 –160 mM). Notably, no
differences were observed between the sensitivity of these
proximal and distal IPSCs to known allosteric modulators of
GABAA receptors such as zolpidem or pentobarbital.
These two important observations, that GABAB receptor
activity may limit ethanol’s overall facilitatory effect on
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
GABAergic synaptic inhibition and that perisomatic synapses
are more sensitive to ethanol, have been replicated in a number
of subsequent studies (Kang et al., 1998a, 1998b; Poelchen et
al., 2000; Ariwodola & Weiner, 2004; Wu et al., 2005) and
have clarified that, at least in the CA1 region of the
hippocampus, pharmacologically relevant concentrations of
ethanol can potentiate the activity of at least a subset of
GABAergic synapses.
In addition to these hippocampal studies, several other
recent reports have demonstrated significant facilitatory effects
of ethanol on GABAergic synapses in other brain regions.
Thus, at concentrations below 100 mM, ethanol has been
shown to potentiate evoked GABAA IPSCs in the central
nucleus of the rat (Roberto et al., 2003) and mouse (Nie et al.,
2004) amygdala, as well as the rat nucleus accumbens (Nie et
al., 2000). Ethanol has also been shown to potentiate
perisomatic-evoked GABAA IPSCs onto dentate granule
neurons in hippocampal slices prepared from cynamolgus
macaques with a potency and efficacy indistinguishable from
that observed in recordings from rat dentate granule neurons
(Ariwodola et al., 2003).
In addition to these recent studies indicating that ethanol can
indeed potentiate evoked GABAergic synaptic transmission in
a variety of brain regions in rodent and non-human primate
brain slices, several reports have also provided initial evidence
that may indicate a possible linkage between these synaptic
effects of ethanol and the behavioral and cognitive effects of
this drug.
For example, ethanol has been shown to impair performance
in a number of memory tasks and particularly spatial memory,
which is known to involve the hippocampus (Hartley &
Burgess, 2005). Long-term potentiation of the Shaffer collat-
eral input to hippocampal CA1 pyramidal neurons is thought to
represent an electrophysiological substrate for the synaptic
strengthening that underlies the formation of spatial memory.
Notably, Schummers and Browning (2001) have demonstrated
that ethanol reliably blocks this form of synaptic plasticity and
that this effect likely involves both an inhibition of N-methyl-
d-aspartate (NMDA) receptor-mediated synaptic activation as
well as a facilitatory effect on GABAA receptor-mediated
synaptic inhibition.
Poelchen et al. (2000) have taken advantage of several
inbred lines of rats and mice that were selected for differences
in their acute sensitivity to the sedating effects of ethanol.
Using these inbred strains of rodents, they have demonstrated
that the ethanol sensitivity of the perisomatic GABAergic
synapses onto CA1 neurons in each of these selected lines
correlates with behavioral sensitivity to ethanol’s sedative
effects. For example, 80 mM ethanol significantly potentiated
the amplitude of proximal GABAA IPSCs in recordings from
HAS rats, which exhibit a relatively high sensitivity to ethanol-
induced loss of righting reflex. In contrast, proximal IPSCs
recorded from LAS rats, which show minimal ethanol-
associated loss of righting reflex, were insensitive to this
concentration of ethanol. More recently, these investigators
have demonstrated the same relationship between the in vitro
ethanol sensitivity of proximal GABAA IPSCs recorded from
537
CA1 pyramidal neurons and in vivo sensitivity to ethanol’s
sedative effects in transgenic mice with targeted deletions of
PKCg and ( (Proctor et al., 2003). These studies provide the
first evidence, albeit correlational, of a relationship between the
ethanol sensitivity of a GABAergic synapse and behavioral
sensitivity to ethanol. Although proximal hippocampal
GABAergic synapses may not be causally related to the
specific behavioral phenotype of these selected lines (decrease
in sensitivity to ethanol-induced loss of righting reflex), the
strong correlation observed suggests that the genes that
regulate the ethanol sensitivity of proximal GABAergic
synapses in the rat hippocampus are likely to affect the ethanol
sensitivity of GABAergic synapses in other brain regions that
may play such a role. Therefore, proximal IPSCs may provide
a useful assay with which to study the physiological mechan-
isms that regulate the ethanol sensitivity of GABAergic
synapses.
4. Synaptic mechanisms of ethanol action
From the preceding review, it is evident that pharmacolog-
ically relevant concentrations of ethanol can potentiate evoked
GABAergic neurotransmission in a number of different brain
regions in rodent and non-human primate brain slice prepara-
tions. In addition, there is emerging evidence that the effects of
ethanol on GABAergic synapses may indeed be related to
some of the acute behavioral consequences associated with
ethanol exposure.
As a result of these studies, attention has turned away from
the question of ‘‘Does ethanol potentiate GABAergic neuro-
transmission?’’ to the more important and interesting question
of ‘‘How does ethanol potentiate GABAergic synaptic activity?
Alterations in the shape of evoked IPSCs can arise from a
variety of pre- and postsynaptic mechanisms. Most of the early
studies that observed facilitatory effects of ethanol on evoked
GABAergic synaptic responses interpreted these changes as
postsynaptic allosteric effects of ethanol on GABAA receptors.
As noted earlier, these interpretations were based largely on the
many observed similarities between the in vitro and in vivo
effects of ethanol and those of well-known allosteric mod-
ulators of GABAA receptors, such as benzodizapines and
barbiturates. Interestingly, in the last few years, a number of
studies have identified novel pre- and postsynaptic actions of
ethanol that likely contribute to its facilitatory effects on
GABAergic transmission. Collectively, the results of these
recent studies indicate that ethanol acts in a manner far more
complex than that of simple, allosteric modulators of synaptic
GABAA receptors. Although many of these studies examined
both pre- and postsynaptic mechanisms, these will be
considered in separate sections for clarity.
4.1. Presynaptic mechanisms
Although it has long been recognized that presynaptic
alterations can profoundly influence the shape and size of
evoked synaptic responses, relatively few studies had consid-
ered the possibility that ethanol may enhance GABAergic
538 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
synaptic inhibition, in part, via a presynaptic facilitation of
GABA release. In vitro brain slice preparations provide a
number of highly sensitive experimental strategies that can be
employed to detect presynaptic changes in transmitter release
(for reviews of these approaches, see Weiner, 2002; Siggins et
al., 2005). These methods include direct measures such as the
frequency of spontaneous [tetrodotoxin (TTX)-sensitive] or
quantal, miniature (TTX-insensitive) IPSCs, or indirect
measures such as alterations in the ratio of pairs of IPSCs
evoked at short inter-event intervals (25 –50 msec) (paired-
pulse plasticity).
The first clear evidence of presynaptic effects of ethanol on
inhibitory synaptic transmission came not from studies on
brain slices but rather from some elegant studies in neonatal
and juvenile spinal cord and brain stem preparations. Synaptic
inhibition in the brain stem and spinal cord is mediated by
both glycine and GABA, which activate distinct populations
of ligand-gated anionic channels. Cheng et al. characterized
the ability of ethanol to reduce neuronal excitability in the
developing spinal cord and noted that bath application of 70
mM ethanol significantly increased the ratio of spontaneously
occurring inhibitory-to-excitatory synaptic currents in record-
ings from ventral horn motoneurons (Cheng et al., 1999).
From a careful analysis of their data, they noted that this shift
arose from a reduction in the frequency of spontaneous
excitatory postsynaptic potentials (EPSPs) and an increase in
the occurrence of spontaneous GABA/glycine IPSPs. Subse-
quently, another group provided additional evidence of
presynaptic effects of ethanol at inhibitory synapses by
demonstrating that ethanol (30 – 100 mM) could significantly
increase the frequency of TTX-resistant miniature glycine
(Eggers et al., 2000; Sebe et al., 2003) and GABAA (Sebe et
al., 2003) IPSCs in recordings from neurons in neonatal and
juvenile brain stem slices.
4.1.1. Studies with hippocampal slices
The presynaptic effects of ethanol on inhibitory neurotrans-
mission in the brain were assessed in a slice study investigating
ethanol effects on presynaptic kainate receptors that regulate
GABA release in that brain region (Carta et al., 2003). In that
study, 50 mM ethanol was shown to increase the frequency of
spontaneous GABAA IPSCs recorded from rat CA1 pyramidal
neurons, indicating that ethanol either increased interneuronal
excitability or GABA release at the terminals. A similar effect
of 80 mM ethanol was also observed in a recent study that
characterized GABAB receptor modulation of ethanol effects
on hippocampal GABAergic synapses (Ariwodola & Weiner,
2004).
Sanna et al. (2004) have also noted a similar effect of
ethanol on the frequency of TTX-resistant mIPSCs in rat CA1
pyramidal neurons. In their experiments, this facilitation of
GABA release required up to 10 min to develop and then
persisted throughout the duration of a 30-min ethanol exposure
(Sanna et al., 2004). This ethanol treatment was also associated
with some complex postsynaptic alterations in GABAA IPSCs
that were shown to be mediated, in part, by the release of an
endogenous neurosteroid (see Section 5.2). Other preliminary
evidence also suggests that the predominant effect of ethanol
on mIPSCs recorded from rat and monkey hippocampal
neurons is an increase in the frequency of mIPSCs (Weiner
et al., 2005).
4.1.2. Studies with amygdala slices
Another demonstration of a presynaptic effect of ethanol on
a GABAergic synapse was reported by Roberto et al. (2003) in
neurons within the rat central nucleus of the amygdala (CeA).
Following up on the observation that ethanol potently
increased the amplitude of pharmacologically isolated GABAA
IPCSs (apparent EC50 = 20 mM), they demonstrated that 44 and
66 mM ethanol significantly decreased paired-pulse facilitation
(PPF) of evoked IPSCs in these cells, consistent with an
increase in GABA release probability. More direct evidence
came from an analysis of TTX-resistant GABAA mIPSCs
where 44 mM ethanol significantly increased mIPSC frequency
in all cells tested.
4.1.3. Studies with cerebellar slices
Carta et al. (2004) have recently conducted the first whole-
cell patch clamp studies on the acute effects of ethanol on
GABAergic neurotransmission in rat cerebellar slices. This
brain region is known to play an integral role in the control of
motor systems and likely contributes to ethanol’s well-known
ataxic effects. In their first study, they characterized the effects
of ethanol on GABAergic input to granule cells that serve as
the predominant relay of sensory information to Purkinje
neurons (Carta et al., 2004). They found that ethanol potently
increases the frequency of spontaneous GABAA IPSCs onto
granule cells (threshold: 20 mM) without altering the
amplitude of these currents, even at concentrations as high
as 100 mM. This finding was recently replicated by Hanchar
et al. (2005) who also reported that a mutation in the a6-
subunit contained in extrasynaptic receptors expressed in
granule cells increases the effect of ethanol on sIPSC
frequency. Ethanol also increased a tonic current in these
experiments and this issue is discussed in detail in Section
4.2.2. Interestingly, ethanol did not alter the amplitude of
evoked GABAA IPSCs nor did it increase the frequency of
TTX-resistant miniature IPSCs (Carta et al., 2004). Notably,
ethanol had no effect on glutamate release at excitatory
synapses onto granule cells. By directly recording the
occurrence of spontaneous action potentials from the Golgi
interneurons that provide GABAergic input to the granule
cells, they found that ethanol actually increased the firing rate
of these GABAergic interneurons. Thus, at least at GABAer-
gic synapses onto cerebellar granule neurons, ethanol appears
to facilitate GABAergic inhibition via an increase in
interneuronal excitability with no change in GABA release
properties at the terminals.
More recent work by this group has begun to characterize
the effects of ethanol on other GABAergic synapses that are
involved in the inhibitory control of cerebellar circuits. It was
found that 50 mM ethanol transiently increases sIPSC and
mIPSC frequency at GABAergic synapses onto Purkinje
neurons (Mameli et al., 2005a).
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
4.1.4. Studies with slices from other brain regions
Several additional preliminary reports have emerged
recently, which provide further evidence that ethanol can
facilitate GABA release at inhibitory synapses throughout
the CNS. Thus, ethanol has been shown to increase sIPSCs
onto medium spiny neurons in the nucleus accumbens of rat
ventral striatal slices (Crowder & Weiner, 2002). In addition,
two recent reports have demonstrated that ethanol can also
increase the frequency of mIPSCs recorded from mechan-
ically isolated nerve bouton preparations from cells in the
rat basolateral nucleus of the amygdala (Zhu & Lovinger,
2005) and cerebellar Purkinje neurons (Criswell & Breese,
2005). These preparations include only the terminals of
GABAergic inputs thus localizing the possible presynaptic
locus of ethanol action solely to the axon terminals.
Interestingly, Criswell and Breese reported no effects of
ethanol on GABAergic synapses in isolated cortical neurons,
suggesting that there may be fundamental differences in the
release machinery of GABAergic terminals within the cortex
and cerebellum.
4.2. Postsynaptic mechanisms
4.2.1. Synaptic GABAA receptors
4.2.1.1. Studies with hippocampal slices. A number of recent
studies with hippocampal slices have failed to demonstrate
effects of ethanol on the function of postsynaptic GABAA
receptors. Carta et al. (2003) did not detect effects of ethanol
(10 –50 mM) on sIPSC amplitude in CA1 pyramidal neurons.
Ethanol (50 and 100 mM) did not alter the kinetics of mIPSCs
in CA1 pyramidal neurons (Spigelman et al., 2004). Wei et al.
(2004) reported that 30 mM ethanol did not affect either the
decay or amplitude of sIPSCs in both dentate gyrus granule
cells and CA1 pyramidal neurons. Galindo et al. (2005)
showed that acute exposure of neonatal slices to 50 mM
ethanol does not affect the amplitude or kinetics of GABAA-
mediated sPSCs or mPSCs both in pyramidal neurons and
interneurons of the CA3 region.
In contrast to these studies, experimental evidence from
several laboratories indicates that hippocampal postsynaptic
GABAA receptors can be modulated by acute ethanol
exposure. In an elegant series of studies, Sanna et al. (2004)
discovered that ethanol increases mIPSC amplitude in CA1
pyramidal neurons in a biphasic manner. During the initial
(3 min) phase of ethanol (50 – 100 mM) application, mIPSC
amplitude is increased and this effect partially reverses after
10 min of continuous ethanol administration. After 30 min of
continuous ethanol exposure, a late effect of ethanol is
evident in which mIPSC amplitude increases again and decay
is prolonged. Importantly, the delayed effect of ethanol cannot
be observed in slices pretreated with finasteride, which
inhibits synthesis of several neurosteroids, including allopreg-
nanolone. These results suggest that ethanol modulates
GABAA receptors in CA1 pyramidal neurons via a direct
mechanism and also an indirect process that involves an
increase in brain steroidogenesis (see Section 5.2).
539
Wu et al. (2005) studied the effects of ethanol on GABAA
receptor-mediated currents evoked by local application of
exogenous GABA in CA1 pyramidal neurons. Acute exposure
to 80 mM ethanol increased the amplitude of currents evoked
at the soma by ¨ 15%, and this effect was not modified by
application of a GABAB receptor antagonist. Conversely,
ethanol did not enhance the amplitude of currents evoked at
distal dendrites under control conditions. However, potentia-
tion by ethanol of these distally evoked GABAA responses
could be observed under conditions of GABAB receptor
inhibition. Taken together, these results suggest that ethanol
directly modulates the function of postsynaptic GABAA
receptors and that the sensitivity of distal GABAA receptors
to ethanol is regulated by postsynaptic GABAB receptors (for
further discussion, see Section 5.1).
4.2.1.2. Studies with cortical slices. Soldo et al. (1998)
examined the effect of ethanol on GABAA receptor function in
cortical slices from 7- to 8-week-old rats. Local pressure
ejection of GABA onto layer V – VI neocortical neurons
activated biphasic responses that consisted of an early
hyperpolarizing phase (mediated by Cl influx) and a delayed
depolarizing phase (primarily mediated by HCO3 efflux)
(Staley et al., 1995). Both of these phases were blocked by
GABAA receptor antagonists. Ethanol (80 mM) significantly
enhanced the hyperpolarizing phase of these responses without
affecting the depolarizing phase. Effects of ethanol on
postsynaptic GABAA receptor-mediated responses were also
observed in another study with layer II pyramidal neurons of
the piriform cortex in which currents were evoked by local
pressure application of GABA. Signore and Yeh (2000)
detected a clear potentiating effect of 25 mM ethanol in 20%
of the neurons studied. However, they also detected a clear
inhibitory effect of ethanol in 17% of the neurons and GABAA
receptor-mediated responses were insensitive to ethanol in the
majority of neurons. Taken together, these studies clearly
indicate that the sensitivity of cortical GABAA receptors to
ethanol is heterogeneous.
4.2.1.3. Studies with cerebellar slices. Using parasagittal
cerebellar slices and patch clamp electrophysiological techni-
ques, it was found that ethanol (20 – 100 mM) did not affect the
amplitude of sIPSCs or mIPSCs, or the peak amplitude of
IPSCs evoked in cerebellar granule neurons by stimulation of
Golgi cells (Carta et al., 2004). Lack of an effect of ethanol on
sIPSC amplitude in cerebellar granule neurons was also
observed by Hanchar et al. (2005). These findings suggest
that postsynaptic GABAA receptors presumably containing
a1hxg subunits in cerebellar granule cells are not acutely
modulated by ethanol. In a related study, the sensitivity of
postsynaptic GABAA receptors to ethanol in Purkinje neurons
was evaluated using acute cerebellar slices (Mameli et al.,
2005a). These receptors are thought to contain a1hxg subunits
as well. Ethanol (50 mM) did not affect the amplitude of
sIPSCs or mIPSCs, suggesting that it does not modulate
postsynaptic GABAA receptors in these neurons. These results
are in agreement with those of Criswell and Breese (2005) who
540 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
did not find an effect of 50 mM ethanol on mIPSC amplitude or
decay time in mechanically dissociated Purkinje neurons.
4.2.1.4. Studies with slices from other brain regions. Nie et
al. (2000) studied the effects of ethanol on currents evoked by
local application of GABA onto nucleus accumbens core
neurons in rat ventral striatal slices. They reported that ethanol
significantly potentiated GABA currents in ¨ 50% of nucleus
accumbens neurons at concentrations of 44– 100 mM. Inter-
estingly, in those cells displaying ethanol-sensitive GABA
currents, this potentiation could be blocked by MCPG, a group
1/group 2 metabotropic glutamate receptor antagonist.
Roberto et al. (2003) found a significant effect of 44 mM
ethanol on mIPSC amplitude in 4 out of 6 neurons tested in the
central amygdala of rats (Roberto et al., 2003). These
investigators also tested the effect of ethanol on GABAA
receptor-mediated potentials evoked by local application of
GABA and found that 44 mM ethanol enhanced these events in
11 out of 16 neurons tested. Interestingly, there was a slight
inhibitory effect of ethanol in the other 5 neurons. Thus, as for
neurons in other brain regions, ethanol exerts inconsistent
effects on amygdalar postsynaptic GABAA receptor-mediated
responses evoked by exogenous agonist application.
The acute effects of ethanol on motoneuron functioning
were examined in two independent studies. Sebe et al. (2003)
investigated the effect of ethanol on GABAA receptor-mediated
synaptic currents in motoneurons in brain stem slices. They
found that 30 mM ethanol did not affect mIPSCs amplitude.
However, 100 mM ethanol significantly decreased mIPSC
amplitude by ¨ 6% in neonatal neurons and ¨16% in neurons
from juvenile animals. Ziskind-Conhaim et al. (2003) showed
that 70 mM ethanol did not affect the amplitude or decay of
mIPSCs in motoneurons in spinal cord slices from newborn
rats. Thus, it appears as if postsynaptic GABAA receptors in
motoneurons are relatively insensitive to subanesthetic con-
centrations of ethanol.
4.2.2. Extrasynaptic GABAA receptors
Recent studies with recombinant receptors revealed subunit
configurations with high sensitivity to ethanol (reviewed in
Boehm et al., 2004) and these findings have prompted
investigations both with acute hippocampal and cerebellar
slices, as discussed below. Sundstrom-Poromaa et al. (2002)
reported that low concentrations of ethanol (1 – 3 mM)
potentiated currents mediated by a4h2y recombinant receptors
expressed in Xenopus oocytes. It was also shown that
progesterone withdrawal increases hippocampal expression of
the a4- and y-subunits and that this was associated with
enhanced sensitivity of currents evoked by exogenous GABA
to acute ethanol exposure. More recently, Wallner et al. (2003)
reported findings that are in general agreement with those of
Sundstrom-Poromaa et al. Specifically, they demonstrated that
receptors containing a4h3y and a6h3y subunits were potenti-
ated by concentrations of ethanol as low as 3 mM. Receptors
comprised of a4h2y subunits were also potentiated by ethanol,
although higher concentrations ( 30 mM) were required to
observe significant effects. Notably, it was discovered that a
single amino acid change in the a6-subunit dramatically
increases the sensitivity to ethanol of a6h3y GABAA receptors
expressed in Xenopus oocytes (Hanchar et al., 2005). Receptors
containing the a4- and y-subunits are present in several brain
regions, including the dentate gyrus, whereas those containing
the a6- and y-subunits are abundantly expressed in cerebellar
granule neurons (Farrant & Nusser, 2005). Importantly,
receptors containing these subunits are mainly localized in
extrasynaptic compartments and it has been postulated that
these receptors may be the primary targets of ethanol in the
brain (Wallner et al., 2003). However, recent findings have
raised questions regarding this postulate by demonstrating that
recombinant a4h3y GABAA receptor are only sensitive to high
concentrations of ethanol (Borghese et al., in press).
4.2.2.1. Studies with hippocampal slices. Wei et al. (2004)
assessed the acute effects of ethanol on tonic inhibition
mediated by a4- and y-subunit-containing extrasynaptic
GABAA receptors in mouse dentate gyrus granule neurons.
Experiments were performed in acute hippocampal slices from
C57Bl/6J mice (40 – 60 days old) using whole-cell patch clamp
recordings and a CsCl-based internal solution. It was found that
ethanol (30 mM) increased the tonic current by ¨ 80%. This
effect could not be observed either in dentate gyrus granule
neurons from y-subunit knockout mice or in CA1 pyramidal
neurons where tonic currents are mediated by a5- and g-
subunit-containing receptors. Importantly, 30 mM ethanol
reduced the excitability of dentate gyrus granule cells but not
CA1 pyramidal neurons. These results are in agreement with
those of Wallner et al. (2003) and indicate that native
extrasynaptic GABAA receptors containing a4- and y-subunits
are sensitive to pharmacologically relevant concentrations of
ethanol. It should however be noted that another recent study
reported no effect of 30 mM ethanol on a similar tonic current
in slices prepared from P20 to P26 mice with a mixed C57BL/6
and 129/SvJae genetic background, suggesting that factors
other than just the subunit composition of extrasynaptic
GABAA receptors may influence their ethanol sensitivity
(Borghese et al., in press). In addition, Spigelman et al.
(2004) reported that 100 mM ethanol potentiates tonic currents
in rat CA1 pyramidal neurons. Thus, a5- and g-subunit-
containing receptors in these neurons may also be important
targets of ethanol, at least at relatively high concentrations.
4.2.2.2. Studies with cerebellar slices. Carta et al. (2004)
examined the acute effects of ethanol on tonic GABAergic
currents in rat cerebellar granule neurons. These currents are
mediated by a6- and y-subunit-containing extrasynaptic
GABAA receptors, which can be activated both by ambient
levels of GABA and also by spillover of synaptically released
GABA (Kaneda et al., 1995; Wall & Usowicz, 1997; Hamann
et al., 2002). Using acute cerebellar parasagittal slices, it was
found that ethanol (20 –100 mM) significantly increased the
tonic current; however, this effect was not observed in the
presence of TTX. As discussed in the Section 4.1, experimental
evidence suggests that ethanol actually modulates the tonic
current indirectly by increasing spillover of GABA released
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
from Golgi cells in an action potential-dependent manner.
These findings suggest that, unlike recombinant a6h2/3y
receptors expressed in Xenopus oocytes, native extrasynaptic
receptors are not sensitive to subanesthetic concentrations of
ethanol in cerebellar granule neurons. The reason for this could
be that the subunit composition of native extrasynaptic
receptors is different from that of the recombinant receptors
that display high sensitivity to ethanol. For instance, extra-
synaptic receptors could also contain a1- and h1-subunits (Poltl
et al., 2003) and this may decrease sensitivity to ethanol.
Differences in post-translational modifications and association
with other proteins could also be responsible for the lack of
sensitivity of extrasynaptic native receptors to ethanol with
respect to their recombinant counterparts.
Experimental factors could be another reason for the
apparent lack of ethanol sensitivity of native extrasynaptic
receptors in cerebellar slices. Carta et al. (2004) recorded tonic
currents in the whole-cell patch clamp configuration evoked by
endogenous GABA at 31 -C using a high Cl containing
internal solution in the absence of GABA transporter inhibitors.
Under these experimental conditions, a direct effect of ethanol
on extrasynaptic receptors could not be observed. However,
Hanchar et al. (2005) recently demonstrated direct potentiation
of tonic currents by ethanol using a different experimental
paradigm. These investigators recorded tonic currents in the
whole-cell patch clamp configuration evoked by endogenous
GABA at 20– 23 -C using a high Cl containing internal
solution in the presence of exogenous GABA and a GABA
transporter inhibitor (N0-711), which apparently had to be
added to elicit tonic currents in their slices in the presence of
TTX. Under these conditions, an effect of 10 mM ethanol on
tonic currents was observed, indicating that ethanol directly
potentiates extrasynaptic receptors. Taken together with the
findings of Carta et al. (2004), these findings suggest that
ethanol can directly potentiate cerebellar extrasynaptic recep-
tors under certain experimental conditions. Further studies will
be needed to conclusively determine the physiological rele-
vance of these effects.
The effects of ethanol on GABAA receptors containing the
a6-subunit have been the focus of much recent research
interest. This interest was generated by the initial finding that
alcohol non-tolerant (ANT) rats carried a substitution from
arginine (R) to glutamine (Q) at position 100 of the a6-subunit
(Korpi et al., 1993). Alcohol-tolerant (AT) rats carried an
arginine at this position. These rats were selectively bred for
ethanol-induced ataxia on the inclined plane, where AT rats
tolerate a greater inclination angle than ANT rats (Eriksson &
Sarviharju, 1984). Subsequently, this mutation has been found
in other selected lines such as Sardinian non-preferring rats
(Saba et al., 2001) and Alko alcohol (AA) and Alko non-
alcohol (ANA) rats (Carr et al., 2003). As discussed above and
in Section 4.1, Hanchar et al. (2005) discovered that the a6-
R100Q polymorphism is present in outbred Sprague – Dawley
rats. They found that 10 mM ethanol increases tonic currents
(evoked by exogenous GABA in the presence of TTX and
GABA transporter inhibition) in cerebellar granule neurons to a
greater extent in slices prepared from rats homozygous for the
541
100R than the 100Q genotypes. On the basis of these results,
Hanchar et al. proposed that this single amino acid difference
contributes to the phenotype of ANT rats. This issue was
studied by Valenzuela et al. (2005) in acute cerebellar slices
from inbred AT and ANT rats homozygous for the 100R and
the 100Q genotypes, respectively (Radcliffe et al., 2004), and it
was found that ethanol modulation of tonic currents was not
significantly different in slices from these rats. This result
suggests that the effect of the a6-R100Q polymorphism on the
sensitivity to ethanol of extrasynaptic GABAA receptors in
cerebellar granule neuron does not apply to all rat lines and that
it depends on genetic background.
5. Modulators of ethanol
potentiation of GABAergic synaptic transmission
5.1. GABAB receptors
As noted earlier, Wan et al. (1996) were the first to report
that blockade of metabotropic GABAB receptors enhances
ethanol potentiation of evoked GABAA IPSCs in rat hippo-
campal CA1 pyramidal neurons. Since then, several other
reports have also noted that blockade of GABAB receptors
facilitates the detection of ethanol’s potentiating effects on
GABAergic synapses in the hippocampus (Kang et al., 1998b;
Wu et al., 2005) and nucleus accumbens (Siggins et al., 1999).
These findings suggest that some GABAB receptor-dependent
mechanism may actively limit ethanol enhancement of
hippocampal GABAergic inhibition. Given the recent observa-
tions that acute ethanol exposure increases GABA release at
these synapses (Carta et al., 2003; Sanna et al., 2004), one
possibility is that this presynaptic effect of ethanol may elevate
ambient GABA levels sufficiently to enhance presynaptic
GABAB receptor activity, thus reducing ethanol’s overall
facilitatory effect on evoked GABAergic responses (Siggins
et al., 1999). Ariwodola and Weiner (2004) recently investi-
gated this hypothesis using whole-cell patch recordings in rat
CA1 pyramidal neurons. They examined the perisomatic
GABAA IPSCs that are sensitive to ethanol, even in the
absence of GABAB receptor blockade (Weiner et al., 1997a;
Poelchen et al., 2000). Although proximal IPSCs were
potentiated by 80 mM ethanol, pretreatment with a GABAB
receptor antagonist significantly facilitated this enhancement.
As previously demonstrated by others (Frye & Fincher, 1996;
Wan et al., 1996), ethanol had no direct effects on postsynaptic
GABAB receptor activity. In contrast, ethanol did significantly
enhance the presynaptic inhibitory effect of a GABAB receptor
agonist on the amplitude of evoked IPSCs and this effect was
significant across the same concentration range over which
ethanol potentiated proximal IPSCs (40 – 80 mM). Taken
together, these findings are consistent with the hypothesis that
ethanol facilitation of GABA release may, in effect, limit this
drug’s overall potentiating effect on evoked IPSCs via
activation of presynaptic GABAB receptors. To more directly
assess this hypothesis, these investigators also examined the
effect of 80 mM ethanol on sIPSCs in the absence and presence
of a GABAB receptor antagonist. Ethanol significantly
542 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
increased the frequency of sIPSCs as well as sIPSC amplitude
and area. Bath application of the GABAB receptor antagonist
SCH 50911 alone had no effect on sIPSCs; however,
pretreatment with this drug selectively increased the ethanol-
mediated facilitation of sIPSC frequency without altering
ethanol potentiation of sIPSC amplitude or area. Although the
synaptic mechanisms underlying changes in sIPSC parameters
can be complex, typically alterations in the frequency of these
events are typically mediated presynaptically whereas changes
in the shape of sIPSCs are attributed to postsynaptic mechan-
isms. Thus, these results suggest that, at least in the hippocam-
pus, ethanol may enhance evoked GABAA IPSCs via distinct
pre- and postsynaptic mechanisms and that the presynaptic
element of this enhancement may limit ethanol’s overall effect
at these synapses via activation of GABAB autoreceptors.
Interestingly, a recent paper by Wu et al. (2005) challenges
the above conclusions, suggesting that postsynaptic GABAB
receptors are primarily responsible for reducing ethanol’s
effects on hippocampal GABAergic synapses. Under their
recording conditions, pretreatment with the GABAB receptor
antagonist CGP-52432 had no effect on ethanol potentiation of
proximally evoked IPSCs in CA1 neurons. However, CGP
pretreatment significantly increased ethanol enhancement of
distally evoked IPSCs, which, under standard recording
conditions, are markedly less sensitive to ethanol (Weiner et
al., 1997a). Notably, these investigators reported that a
component of the current underlying both proximally and
distally evoked IPSCs was mediated by the activation of
postsynaptic GABAB receptors and that the magnitude of
ethanol’s potentiating effect on proximal and distal responses
was inversely correlated with the percentage of the total IPSC
current mediated by GABAB receptors. In addition, these
investigators demonstrated that blockade of GABAB receptors
significantly enhanced ethanol potentiation of currents evoked
by exogenous GABA application to dendritic regions of CA1
neurons. Taken together, these findings suggest that activation
of postsynaptic GABAB receptor activity decreases the ethanol
sensitivity of postsynaptic GABAA receptors in CA1 neurons.
Thus, the results of several recent studies clearly suggest an
important role for GABAB receptors in regulating the ethanol
sensitivity of GABAergic synapses in hippocampus and
accumbens. However, additional studies will be needed to
clarify the role of pre- and postsynaptic GABAB receptors in
mediating this regulatory effect.
Two additional studies suggest additional indirect roles for
GABAB receptors in regulating ethanol effects on synaptic
transmission. Melis et al. (2002) reported that, in mice, a single
injection of ethanol (2 g/kg ip), which produced a modest, but
significant increase in ethanol consumption and preference also
resulted in an increase in GABA release at GABAergic
synapses onto ventral tegmental area (VTA) DA neurons.
Thus, in recordings from slices prepared from mice that had
received a saline injection 24 hr earlier, paired-pulse stimula-
tion at an inter-event interval of 25 msec generated PPF, as seen
in many brain regions. In contrast, this stimulation protocol
generated pronounced paired-pulse depression (PPD) in slices
prepared from animals 24 hr following a single ethanol
injection and this alteration in short-term plasticity lasted at
least one week following the ethanol treatment. Interestingly,
bath application of a GABAB receptor antagonist had no effect
on PPF in slices from saline-treated animals; however,
blockade of GABAB receptors converted PPD to PPF in
ethanol-treated slices. From these and other experiments, the
authors concluded that, in the mouse VTA, a single injection of
ethanol can result in a sustained increase in GABA release at
GABAergic synapses that is sufficient to engage presynaptic
GABAB receptors and thus alter short-term plasticity.
Finally, Steffensen et al. (2000) have demonstrated that, in
the accumbens core, GABAB receptor antagonist pretreatment
blocks ethanol inhibition of NMDA EPSPs but not currents
evoked by exogenous application of NMDA. Although there
are numerous interpretations of these findings, 1 possibility is
that ethanol inhibition of NMDA EPSCs in their accumbal
preparation is mediated in part by GABA release from
neighboring GABAergic synapses onto presynaptic GABAB
receptors.
5.2. Neurosteroids
There is a growing body of literature suggesting an
important interaction between ethanol and certain neuroactive
steroid products of progesterone metabolism (see Morrow et
al., 1999). Some of these neurosteroids, like allopregnanolone,
are potent, allosteric enhancers of GABAA receptor activity
(Lambert et al., 2001) and have also been shown to exert
presynaptic facilitatory effects on GABA release in some brain
regions (Poisbeau et al., 1997). Moreover, acute ethanol
administration significantly elevates levels of allopregnanolone
in plasma and the CNS (Barbaccia et al., 1999; Morrow et al.,
2001) and pretreating rats with finasteride, an inhibitor of
allopregnanolone biosynthesis has been shown to attenuate
some of ethanol’s behavioral effects (e.g., hypnosis) (VanDoren
et al., 2000). These observations have led to the hypothesis that
ethanol may exert some of its behavioral and cognitive effects
indirectly via an increase in the levels of neurosteroids that
enhance GABAergic neurotransmission.
As noted earlier, the first direct evidence that neurosteroids
may contribute to ethanol potentiation of GABAergic synapses
was recently published by Sanna et al. (2004). In an elegant in
vitro study in acutely prepared rat hippocampal slices, they
demonstrated that bath application of ethanol, at concentrations
of 50 and 100 mM, significantly elevated levels of allopregna-
nolone within 20 min. Using whole-cell patch clamp recording
methods, they then demonstrated that prolonged application of
100 mM ethanol resulted in a significant increase in the
amplitude of TTX-resistant mIPSCs. This effect was detected
within 3 min, decreased slightly by 10 min, but remained
significant for the duration of a 30-min ethanol application.
Ethanol also resulted in a significant and sustained increase in
the frequency of mIPSCs; however, this presynaptic change
was not apparent until 10 min into the application. Most
notably, pretreating slices with finasteride, a broad-spectrum
inhibitor of neurosteroid biosynthesis, abolished the sustained
potentiating effect of ethanol on mIPSC amplitude after the
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
first 3 min but had no effect on ethanol enhancement of mIPSC
frequency at any time point. Similar results were also observed
with evoked IPSCs. Bath application of ethanol transiently
increased the amplitude of eIPSCs within the first few minutes
of the ethanol application. This potentiation decreased mark-
edly within the first 10 min and then reappeared for the
duration of the ethanol application. Monitoring of paired-pulse
facilitation of evoked IPSCs revealed a slowly developing
increase in release probability that was also sustained,
consistent with the observed increase in mIPSC frequency.
Interestingly, pretreatment with finasteride had no effect on the
initial potentiation of evoked IPSCs but completely blocked the
sustained effect of ethanol on these responses.
Taken together, these findings suggest a complex interaction
between ethanol, neurosteroid biosynthesis, and the regulation
of GABAergic transmission. Ethanol appears to have a direct,
albeit transient, postsynaptic effect on GABAA receptor
function at these synapses that displays marked acute tolerance
(within 10 min). However, a slowly developing postsynaptic
enhancement, mediated by the release of a neurosteroid, results
in the reemergence of a postsynaptic increase in GABAergic
neurotransmission. In addition, ethanol also exerts a sustained,
presynaptic facilitatory effect on GABA release at these
synapses that is independent of any actions of neurosteroids.
These intriguing findings suggest an important role for
neurosteroid biosynthesis in mediating long-lasting effects of
ethanol on GABAergic synapses. However, these findings also
raise several important questions. It is not clear why the onset
of the presynaptic effects of ethanol observed in this study was
much slower than that reported in other investigations of
ethanol effects on GABA release (Siggins et al., 2005). It will
also be important to determine why this neurosteroid-indepen-
dent, sustained presynaptic increase in GABA release did not
contribute to the observed increase in evoked IPSCs. Never-
theless, these important data will surely stimulate additional
studies directed at investigating the contribution of neuroster-
oid release to ethanol actions on GABAergic synapses in the
hippocampus and other brain regions.
5.3. Corticotrophin releasing factor
and other peptides, neurotransmitters
In a very important paper, it was recently reported that the
presynaptic effect of ethanol on GABA release in mouse central
amygdala neurons involves corticotrophin releasing factor
(CRF) receptors (Nie et al., 2004). Ethanol decreased PPF in
these neurons and this effect was mimicked by exogenous
application of CRF. Importantly, the effects of both ethanol and
CRF were blocked by antagonists of type 1 CRF receptors
(CRFR1) and absent in CRFR1 knockout mice. More recently,
the same group of investigators reported that the concomitant
application of ethanol (44 mM) and CRF (200 nM) additively
decreased PPF in central amygdala neurons (Roberto et al.,
2005). Based on these studies, it has been hypothesized that
ethanol could enhance the release of GABA indirectly by
increasing CRF release that then acts in an autocrine fashion on
presynaptic CRFR1. Alternatively, ethanol could directly
543
activate CRFR1 and increase GABA release. The increase in
GABA release could lead to inhibition of GABAergic
interneurons and subsequently to downstream disinhibition of
excitatory neurons (Siggins et al., 2005).
Very interesting preliminary evidence has been recently put
forward indicating that opioid peptides also regulate the effects
of ethanol on GABA release in central amygdala neurons.
Moore et al. (2005) found that blockade of delta opioid
receptors enhances ethanol-induced GABA release in these
neurons. Importantly, similar effects have been observed with
delta, but not mu, opioid receptor knockout mice. These
findings indicate that activation of delta opioid receptors exert
an opposite modulatory role to that of CRFR1 on ethanol’s
effects on GABA release in the central amygdala.
It has been demonstrated that effects of ethanol on fast
synaptic transmission at GABAergic interneurons can also
regulate the inhibitory output provided by these neurons.
Studies with hippocampal slices have shown that the kainate
subtype of glutamate receptors modulates the excitability of
GABAergic interneurons in the CA1 region. Kainate receptors
are part of the superfamily of glutamate-gated ion channels
along with NMDA and a-amino-3-hydroxi-5-methylisoxazole-
4-propionic acid (AMPA) receptors. The first evidence that
ethanol modulated interneuronal kainate receptors was provided
by Crowder et al. (2002), who reported that 20– 80 mM ethanol
reduces kainate receptor-mediated inhibition of evoked IPSCs
in CA1 pyramidal neurons. Subsequently, Carta et al. (2003)
discovered that ethanol potently inhibits (IC50 = 4.4 mM)
kainate receptor-mediated increase in sIPSC frequency in
CA1 pyramidal neurons. This action was due to an ethanol-
induced inhibition of interneuronal firing in response to kainate
receptor activation by either exogenous agonist or synaptically
released glutamate. Interneuronal firing in response to activa-
tion of AMPA receptors or a voltage step was not affected by
ethanol. Thus, ethanol increases excitability of CA1 pyramidal
neurons indirectly by reducing the kainate receptor-dependent
drive of GABAergic interneurons. This effect may explain some
of the paradoxical excitatory actions of ethanol, which can be
observed during the initial phases of intoxication and may
contribute to alcohol abuse.
5.4. Protein kinases
Several studies have reported modulatory influences of
protein kinase activity, particularly PKC, on ethanol potenti-
ation of GABAergic synaptic transmission. Weiner et al. (1994)
reported a possible role for PKC in modulating ethanol
potentiation of GABAA IPSCs in rat CA1 pyramidal neurons.
Under their recording conditions, ethanol significantly in-
creased the amplitude of IPSCs at concentrations as low as 20
mM. However, this potentiation was significantly reduced by
pretreating slices with a PKC inhibitor or by intracellular
dialysis of the cell being recorded with a peptide inhibitor of
PKC.
In a subsequent study, it was noted that a slice incubation
protocol in which hippocampal tissue was gradually warmed to
ambient temperature over a 2-hr period following dissection
544 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
(Cold Shock; Watson et al., 1997) resulted in a significant
elevation of basal PKC activity that was not observed if slices
were maintained at ambient temperature (Weiner et al., 1997b).
Notably, ethanol potentiation of GABAA IPSCs was signifi-
cantly greater in recordings from ‘‘Cold Shocked’’ slices and
this difference was completely blocked by pretreatment with a
PKC inhibitor. In addition, the facilitatory effects of pentobar-
bital and flunitrazepam were unaffected by the Cold Shock
incubation. Siggins et al. (1999) have also reported a facilitatory
effect of PKC on ethanol potentiation of currents evoked by
exogenous application of GABA onto rat accumbal core
neurons in rat ventral striatal slices. In their experiments, bath
application of the phorbol ester, phorbol 12-myristate 13-acetate
(PMA), had no effect on GABA currents itself but significantly
increased the percentage of cells displaying ethanol-sensitive
GABA currents as well as the absolute magnitude of the
potentiating effect of 44 mM ethanol. The effects of PMA were
blocked by pretreating slices with the kinase inhibitor sphingo-
sine; however, another PKC activator, phorbol 12, 13-diacetate,
did not facilitate ethanol enhancement of GABA currents.
In addition to these reports on ethanol/PKC interactions, a
number of studies, using extracellular single unit recordings in
anesthetized whole animal preparations, have demonstrated
that noradrenaline receptor-mediated stimulation of protein
kinase A (PKA) can increase the ethanol sensitivity of GABAA
receptors in cerebellar Purkinje neurons. For example, although
ethanol generally has been shown to have little effect on
GABA-mediated inhibition of Purkinje cell firing, these
GABAA receptor-mediated depressions of neuronal activity
are potentiated by ethanol following pretreatment with the h
receptor agonist isoproterenol, cAMP analogues, or selective
activators of PKA (Lin et al., 1991; Lin et al., 1994; Freund &
Palmer, 1996). In addition, pretreatment with the inhibitor of
cAMP signaling, Rp-cAMPs, blocks the ethanol-sensitizing
effect of isoproterenol on Purkinje cell GABAA receptors
(Freund & Palmer, 1997). Thus, these studies suggest a potent
modulatory role for noradrenaline in regulating the postsynap-
tic sensitivity of GABAA receptor in cerebellar Purkinje cells.
To our knowledge, no studies have characterized this interac-
tion at the level of the GABAergic synapse in cerebellar slices
or in any other brain region.
Finally, one study has provided some correlational evidence
regarding the possible behavioral relevance of protein kinase
regulation of ethanol effects on GABA synapses. As discussed
earlier, Proctor et al. (2003) examined loss of righting reflex and
the ethanol sensitivity of proximal GABAergic synapses in the
CA1 region of transgenic mice with deletions of PKCg and
PKC(. Mice with a deletion of PKCg were less sensitive to the
soporific effect of ethanol (3.5 g/kg ip) than their wild-type
controls, whereas PKC( mice were more sensitive and these
differences were not related to differences in ethanol metabo-
lism. Interestingly, the ethanol sensitivity of proximal GABAer-
gic synapses in these lines correlated with behavioral sensitivity
to loss of righting reflex. Thus, 80 mM potentiated IPSCs in
PKC( knockout mice but not their wild-type controls and
conversely this concentration of ethanol had no effect on IPSCs
in PKCg knockout mice but did potentiate these responses in
their wild-type littermates. Taken together, these experiments
seem to suggest that specific isozymes of PKC have differential
effects on regulating the ethanol sensitivity of GABAergic
synapses and possibly behavioral sensitivity to this drug.
6. Effects of chronic ethanol on
GABAergic synaptic transmission
From the preceding review, it is becoming increasingly clear
that acute ethanol exposure has complex effects on pre- and
postsynaptic elements of GABAergic synapses. Collectively,
these synaptic actions of ethanol lead to an enhancement of
GABAergic inhibition. Many investigators have also examined
the long-term consequences of ethanol exposure on GABAer-
gic signaling. It is well known that chronic ethanol consump-
tion can lead to tolerance and physical dependence (Chandler et
al., 1998) and that withdrawal following long-term ethanol
treatment is associated with an increase in neuronal excitability
that is often associated with anxiogenesis, hyperexcitability,
and even seizure activity (Littleton, 1998; Kliethermes, 2005).
Many studies have evaluated the hypothesis that these
alterations result, in part, from compensatory adaptation to
the acute facilitatory effects of ethanol on GABAergic
synapses. These adaptive changes are thought to lead to a
pronounced hypofunction of GABAergic neurotransmission
and possibly the development of tolerance to the acute effects
of ethanol on these synapses. As with many of the initial
studies on acute effects of ethanol on GABAergic signaling, the
vast majority of early studies characterizing chronic effects of
ethanol on GABAergic transmission focused primarily on
postsynaptic properties and mainly the subunit composition of
the GABAA receptors themselves. Although some disparity
was noted in these early studies (possibly reflecting differences
in the chronic ethanol treatment duration and protocol, brain
region examined, and methods of assessing receptor function),
in general most studies were generally in agreement that
chronic ethanol exposure and withdrawal did not result in
dramatic decreases in the number of GABAA receptors in most
brain regions. However, many of these studies reported marked
changes in the expression of specific GABAA receptor subunits
and hypothesized that alterations in the subunit composition of
these receptors were primarily responsible for the physiological
and pharmacological alterations in GABAergic signaling
associated with chronic ethanol exposure. For an excellent
review of these studies, please refer to Grobin et al. (1998).
Relatively few studies have employed in vitro brain slice
methods to examine the consequences of long-term ethanol
exposure on GABAergic synaptic transmission. It is important
to consider that, regardless of the acute mechanisms through
which ethanol alters inhibitory synaptic communication, it is
possible that both pre- and postsynaptic mechanisms may
contribute to adaptation at these synapses. In this section, we
will review recent studies with brain slices that have addressed
the effects of long-term ethanol exposure on pre- and
postsynaptic elements of GABAergic synaptic transmission
and have also examined whether these synapses develop
tolerance to the acute effects of ethanol.
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
6.1. Studies with hippocampal slices
One of the most extensive characterizations of GABAergic
synaptic adaptation following chronic ethanol exposure has
been carried out in an elegant series of studies by Olsen,
Spigelman, and colleagues. These investigators have developed
a chronic intermittent ethanol (CIE) treatment paradigm in
which rats are given a 5- to 6-g/kg dose of ethanol on alternate
days for 60 treatments (120 days). In their initial studies, these
investigators demonstrated that this protocol resulted in a
decrease in the threshold for seizure induction by the GABAA
receptor antagonist, pentelynetetrazol (Kokka et al., 1993) that
persisted for at least 40 days after the last ethanol dose. They
also demonstrated, using biochemical and extracellular record-
ing methods, that CIE resulted in a decrease in GABAA
receptor activity in hippocampal slices that also lasted for at
least 40 days (Kang et al., 1996).
More recently, these investigators have employed immuno-
cytochemisty as well as patch clamp recording techniques to
more directly examine the effects of the CIE protocol on
GABAergic synapses in the rat hippocampus. Using antibodies
to specific GABAA receptor subunits, they have observed a
significant increase in a4- and g2-subunits and a decrease in a1-
and y-subunits in hippocampal homogenates from CIE-treated
animals withdrawn for two days (Cagetti et al., 2003). The
reciprocal changes in a1- and a4-subunits are some of the most
consistent subunit changes observed in chronic ethanol studies
(Grobin et al., 2000b).
Using analysis of TTX-resistant mIPSCs recorded from
CA1 pyramidal neurons of CIE-treated and control rats, they
reported modest but statistically significant decreases in the
amplitude and decay of these responses (Cagetti et al., 2003),
changes that may be consistent with the observed alterations in
the expression of a1- and a4-subunits. They also reported a
small but significant decrease in mIPSC frequency, suggesting
that chronic ethanol exposure may also be associated with a
presynaptic decrease in GABA release at these synapses.
A number of pharmacological alterations in the properties of
GABAergic synapses were also observed and, for the most
part, these changes were consistent with the observed altera-
tions in subunit expression. For example, they noted a dramatic
loss of the acute potentiating effect of diazepam and the
neurosteroid alphaxalone on mIPSCs in slices from CIE-treated
rats (Cagetti et al., 2003), possibly reflecting the loss of a1- and
y-subunits, respectively. Interestingly, evoked IPSCs retained
their sensitivity to alphaxalone (Kang et al., 1998b), possibly
reflecting differences in the populations of GABAA receptors
that underlie evoked and mIPSCs. Modulation of mIPSCs by
drugs with some selectivity for a4-subunits, which were
elevated in CIE tissue, were either unchanged (bretazenil) or
increased (RO 15-4513, DMCM) (Kang et al., 1998a, 1998b;
Cagetti et al., 2003). Most notably, although the acute effects of
ethanol on mIPSCs were not examined in these studies, the
ethanol sensitivity of evoked IPSCs was significantly enhanced
in CIE slices (Kang et al., 1998a, 1998b).
These investigators also carried out several behavioral
studies in CIE-treated animals that lend added significance to
545
some of the observed changes in the properties of hippocampal
GABAergic synapses. For example, they observed a significant
increase in anxiety-like behavior, assessed in a light-dark box
assay, two days after CIE, at the same time point where pre-
and postsynaptic decreases in mIPSC signaling were noted
(Cagetti et al., 2003). In addition, marked tolerance to the
sedating effects of alphaxalone and diazepam were observed at
this same time point, consistent with the loss of the acute
potentiating effects of these compounds on mIPSCs. Interest-
ingly, the anxiolytic effects of diazepam were still observed in
CIE-treated rats, suggesting that the subunit composition of
GABAA receptors underlying diazepam’s anxiolytic effects
may not be perturbed by long-term ethanol exposure. Given
that sensitization rather than tolerance to the acute potentiating
effects of ethanol on evoked GABAA IPSCs was observed in
slices obtained from CIE-treated rats, it will be important in
future studies to examine the behavioral effects of acute ethanol
exposure in CIE-treated rats.
More recently, these investigators have carried out the first
comparison of the effects of chronic ethanol exposure on
synaptic and extrasynaptic receptors recorded from CA1
neurons (Liang et al., 2004). Several similarities were noted
in the adaptation of synaptic mIPSCs and a tonic extrasynaptic
GABAA receptor-mediated conductance associated with CIE.
For example, both mIPSCs (Cagetti et al., 2003) and the tonic
current displayed marked tolerance to diazepam as well as
zolpidem, at a concentration thought to be selective for a1-
containing GABAA receptors (0.3 AM). In contrast, experi-
ments with THIP revealed significant differences in the
adaptive changes associated with CIE in these 2 GABAergic
synaptic currents. THIP is a high affinity, high efficacy agonist
at a4-containing GABAA receptors and a partial agonist at
most other GABAA receptor assemblies. Bath application of
THIP potently activated the tonic GABA current in slices from
untreated and saline-treated rats; however, this effect was
significantly attenuated in slices from CIE-treated animals
(Liang et al., 2004). In contrast, THIP, which depressed
mIPSCs in untreated and saline-treated slices, actually resulted
in a robust potentiation of mIPSCs in CIE-treated rats. These
complex changes were associated with modest tolerance to the
soporific effects of THIP in CIE-treated rats but no change in
the anxiolytic effects of this drug (Liang et al., 2004). These
recent findings are generally consistent with previous studies
suggesting that chronic ethanol exposure results in a decrease
in BZP-sensitive a1-subunits and an increase in BZP-insensi-
tive a4-subunits at synaptic receptors (Grobin et al., 2000a).
These studies further suggest that although CIE may result in a
similar decrease in a1-subunit expression at extrasynaptic sites,
this change may not be accompanied by an increase in a4-
subunit expression.
A recent preliminary study has provided the first evidence
of adaptive changes in GABAergic synapses in monkey
hippocampus using a non-human primate model of ethanol
self-administration (Weiner et al., 2005). In this paradigm,
cynamolgus macaques are trained on operant panels to self-
administer a 4% ethanol solution. Once trained, monkeys are
then given almost continuous access to the panels (22 hr) every
546 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
day and many subjects routinely consume ethanol in doses
exceeding 2 g/kg/day (Vivian et al., 2001). The first study
characterized the GABAergic synapses onto dentate granule
cells in slices prepared immediately following the last day of
18 months of daily ethanol drinking. Control subjects were
age- and sex-matched animals that had free access to food and
water but were not exposed to the operant panels. Initial
electrophysiological findings revealed a significant increase in
paired-pulse facilitation of GABAA IPSCs. This finding is
consistent with a decrease in release probability and is in
agreement with the decrease in mIPSC frequency observed in
rats following CIE (Cagetti et al., 2003). Also in agreement
with the rat CIE studies, no tolerance was observed to the acute
potentiating effect of ethanol on evoked GABAA IPSCs.
However, the acute potentiating effect of flunitrazepam was
also unaffected by a prolonged history of ethanol drinking,
unlike the dramatic tolerance to benzodiazepine enhancement
of evoked and mIPSCs observed in the rat CIE model.
6.2. Studies with amygdala slices
Roberto et al. (2004) have recently employed an inhalational
model of chronic ethanol treatment to study adaptation of
GABAergic signaling in the central nucleus of the amygdala
(CeA). In their model, male Sprague –Dawley rats are exposed
to a continuous ethanol vapor for two weeks with a targeted
blood alcohol level of 150 – 200 mg/dL. Control rats are
maintained in similar chambers without ethanol vapor and all
animals are sacrificed immediately following the last exposure
day. Using this paradigm, they found an increase in the input/
output curve for evoked IPSCs along with a decrease in paired-
pulse facilitation. Analysis of mIPSCs revealed a significant
increase in frequency with no change in amplitude. From these
studies they conclude that chronic ethanol exposure results in
an increase in GABAergic signaling and unlike the hippocam-
pus, this appears to be due to a presynaptic increase in GABA
release. This conclusion was further supported by in vivo
microdialysis measures showing a 4-fold increase in baseline
GABA levels in the CeA of CET animals. Notably, in
agreement with the chronic ethanol studies in rat (Kang et
al., 1998b) and monkey (Weiner et al., 2005) hippocampus, no
tolerance to the acute potentiating effect of ethanol on evoked
or mIPSCs was observed in the CeA.
Taken together, these studies demonstrate that GABAergic
synapses undergo complex alterations following chronic
ethanol exposure that, for the most part, would lead to a
decrease in inhibitory tone. These data also suggest that, as
with the acute effects of ethanol, long-term exposure to this
drug results in both pre- and postsynaptic alterations and these
changes may differ between brain regions. Moreover, recent
evidence has demonstrated a temporal relationship between
these neuroadaptive changes in GABAergic synaptic activity
and some of the behavioral alterations associated with chronic
ethanol exposure and withdrawal (e.g., reduced seizure
threshold, anxiogenesis, hyperactivity).
Additional studies will be needed to more carefully examine
the specific exposure durations required to elicit these changes
in GABAergic synapses, the molecular mechanisms responsi-
ble for these adaptive changes, as well as their behavioral
consequences with respect to withdrawal and dependence.
Interestingly, one of the most consistent findings to emerge
from these recent studies is that tolerance does not appear to
develop to the acute potentiating effect of ethanol on
GABAergic synapses. These finding suggest that the synaptic
mechanisms associated with the tolerance that is known to
develop to some of ethanol’s behavioral effects (e.g., ataxia,
sedation) may not involve GABAergic mechanisms. In fact, the
persistence of (or even sensitization to) ethanol’s facilitatory
effects on GABAergic synapses may suggest that these effects
play an important role in the maintenance of addictive drinking
behavior.
7. Developmental implications
Exposure to ethanol can have devastating effects on the
developing brain that persist into adulthood. Neuropsycholog-
ical studies have demonstrated that fetal ethanol exposure leads
to deficits in learning and memory, and animal and human
studies suggest that this is due, in part, to permanent damage of
the hippocampal formation (Berman & Hannigan, 2000;
Bonthius et al., 2001a, 2001b; Culmsee et al., 2001; Hamilton
et al., 2003). However, the mechanism(s) through which
developmental exposure to ethanol produces these effects is
not fully understood. A potential mechanism that could explain
some of the neuroteratogenic actions of ethanol that has been
recently identified is the synchronized activity that is present in
immature hippocampal neuronal networks (Galindo et al.,
2005). This activity is driven, in part, by the excitatory actions
of GABAA-Rs.
In the mature vertebrate brain, GABAA-Rs mediate the
majority of fast inhibitory neurotransmission. Through these
receptors, Cl flows into mature neurons and causes a
hyperpolarization, contributing to a decrease in the ability of
these neurons to reach action potential threshold. In contrast,
activation of GABAA-Rs in immature neurons has the opposite
effect; that is, it causes a depolarization and increases neuronal
excitability. GABA is excitatory in immature neurons because
these cells have a relatively high intracellular concentration of
Cl ; [Cl ]i is 20– 40 mM higher in immature than in mature
neurons (Ben-Ari, 2002). Higher [Cl ]i results in a shift of the
Cl equilibrium potential to more depolarized levels than the
resting membrane potential. Therefore, activation of GABAA-
Rs causes inward currents (i.e., Cl efflux) at the resting
membrane potential. Immature neurons have higher [Cl ]i
primarily because they express low levels of the transporter
KCC2 (Hubner et al., 2001). This transporter is more
abundantly expressed in mature neurons, where it extrudes
Cl and keeps [Cl ]i relatively low. The depolarizing action of
GABA in immature neurons contributes to the generation of
giant depolarizing potentials (GDPs), which are the first pattern
of synchronized activity to appear in the brain. In the rodent
hippocampus, GDPs can be detected during the first 2 weeks of
life and are characterized as long duration (400 –800 ms),
depolarizing potentials or inward currents with a frequency of
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
¨ 0.005 –0.2 Hz. GDPs are generated by a complex mechanism
in which the excitatory actions of GABA play a central role
(Sipila et al., 2005).
Galindo et al. (2005) found that acute ethanol exposure
potently increases GABAA-sPSC frequency at pyramidal
neurons and interneurons in the CA3 region of neonatal rats.
It also elevates GABAA-mPSC frequency at interneurons.
These effects result in a dramatic increase in GDP frequency
and the Ca2+ transients associated with these events. It has long
been recognized that GABA can elevate [Ca2+]i in developing
neurons. Gaiarsa et al. (1995) showed that synaptic GABAA-R
activation induces elevations in [Ca2+]i both in CA3 pyramidal
neurons and interneurons and that this effect was blocked by an
antagonist of VGCCs. The same team of investigators
subsequently demonstrated that increases in [Ca2+]i are
synchronized with GDPs in CA3 pyramidal neurons (Leine-
kugel et al., 1997). More recently, it was demonstrated that
these synchronized Ca2+ elevations emerge at late embryonic
stages in the CA1 and CA3 region of the hippocampus and it
has been postulated that they lead to correlated increases in
gene expression and synaptogenesis in large numbers of
neurons (Aguado et al., 2003). These transients likely
contribute to activity-dependent refinement of developing
neuronal circuits. For instance, pairing of GDPs with stimula-
tion of mossy fibers induces a long-lasting enhancement of
synaptic transmission at mossy fiber-to-CA3 pyramidal neuron
synapses (Kasyanov et al., 2004). Moreover, these network-
driven synchronized Ca2+ transients may play a role in
dendritic growth and synapse remodeling (Lauri et al., 2003;
Colin-Le Brun et al., 2004). Therefore, ethanol-induced
alterations in this GABA-driven pattern of synaptic activity
could have profound effects on the normal maturation of
hippocampal circuits and contribute to the pathophysiology of
fetal alcohol spectrum disorder.
Other recent evidence suggests that there may be develop-
mental changes in the acute ethanol sensitivity of GABAergic
synapses. Li et al. (2003) found that the potentiating effect of
ethanol (30 and 60 mM) on evoked GABAA IPSCs in rat CA1
pyramidal neurons was significantly greater in slices from adult
rats (16 weeks of age) than younger rats (2 –4 weeks old).
More recently, these investigators reported that acute ethanol
exposure causes a greater increase in sIPSC frequency in CA1
pyramidal neurons from adult rats compared to those from 4-
week-old rats (Li et al., 2005). Ethanol also increased the
frequency of TTX-resistant mIPSCs but the magnitude of this
effect did not differ between the 2 age groups. These important
findings indicate that, at least in the hippocampus, the acute
ethanol sensitivity of GABAergic synapses may increase with
age. Moreover, this developmental change appears to involve
an increase in the ethanol sensitivity of some presynaptic
mechanism associated with action-potential-dependent GABA
release.
8. Conclusions
Collectively, the results of the studies described in this
review provide compelling evidence that pharmacologically
547
relevant concentrations of ethanol (< 100 mM) significantly
potentiate GABAergic synaptic transmission in a number of
brain regions. As summarized in Fig. 1, many of these recent
studies have shown that acute ethanol exposure can result in a
significant modulation of GABA release. Depending on the
brain region or even the type of synapse examined, this
presynaptic effect may be mediated via a decrease in
glutamate-driven somatic/axonal excitability (Carta et al.,
2003), an increase in release probability at the axon terminals
(Criswell & Breese, 2005; Zhu & Lovinger, 2005), or possibly
some combination of the two (Ariwodola & Weiner, 2004). In
addition, this presynaptic facilitation of GABA release may
(Roberto et al., 2003; Sanna et al., 2004; Wu et al., 2005) or
may not (Carta et al., 2003) be accompanied by a postsynaptic
facilitation of GABAA receptor function. Recent studies have
also demonstrated that ethanol potentiation of some GABAer-
1 AP-dependent
Release
2 Release Machinery
G
3
4
8
Neurosteroid
4
G
5 Synaptic
7 Phosphorylation
6 Extrasynaptic
Fig. 1. Potential sites of action of acute ethanol exposure ethanol at GABAergic
synapses. (1) Ethanol has been shown to increase spontaneous action potential
firing of GABAergic interneurons (for instance, Golgi cells; Carta et al., 2004)
or decrease the kainate-driven firing of these neurons (for instance, CA1
interneurons; Carta et al., 2003). (2) Ethanol has been shown to increase
quantal GABA release in some neuronal populations (for example, central
amygdala neurons; Roberto et al., 2003). Ethanol modulation of presynaptic
protein phosphorylation pathways or release machinery components could also
play an important role. (3) It could also act by enhancing the function of
presynaptic voltage-gated Ca2+ channels or inhibiting the function of voltage-
gated K+ channels. (4) Ethanol could indirectly act via activation of pre- and/or
postsynaptic G protein-coupled receptors (for instance, CRF or GABAB
receptors; Ariwodola & Weiner, 2004; Nie et al., 2004). (5) Studies suggest that
ethanol also acts by potentiating postsynaptic GABAA receptor function in
some neuronal populations (for instance, CA1 pyramidal neurons; Wu et al.,
2005). (6) Extrasynaptic GABAA receptors have been shown to be modulated
by ethanol (for example, in cerebellar (Carta et al., 2004; Hanchar et al., 2005)
and dentate gyrus granule neurons (Wei et al., 2004). (7) Ethanol can modulate
these receptors indirectly through changes in their phosphorylation state.
Alternatively, the phosphorylation state could affect their sensitivity to ethanol
(for instance, see Harris et al., 1995; Weiner et al., 1997b; Proctor et al., 2003).
(8) Ethanol could indirectly modulate GABAA receptors via an increase in
neurosteroid biosynthesis (for instance, see Sanna et al., 2004).
548 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
gic synapses may be influenced by additional factors such as
endogenous neuromodulators (Sanna et al., 2004), G protein-
coupled receptors (Wan et al., 1996; Moore et al., 2005), and/or
protein kinases (Weiner et al., 1997b). Long-term ethanol
exposure also appears to result in complex pre- and postsyn-
aptic alterations in GABAergic synapses and these changes too
may vary between brain regions.
The ‘‘good news’’ from all of these recent studies is that
together they provide consistent support for the hypothesis that
GABAergic synapses are indeed one of the most sensitive CNS
targets of acute and chronic ethanol exposure. The ‘‘bad news’’
is that the dizzying array of reported mechanisms of ethanol
action raise almost as many questions as they answer. For
example: what are the specific mechanisms underlying
ethanol’s presynaptic effects? What determines the ethanol
sensitivity of postsynaptic GABAA receptors? And do the pre-
and postsynaptic effects of ethanol contribute equally to
ethanol’s overall enhancement of GABAergic inhibition? Even
once these questions are addressed, it will also be important to
determine the specific behavioral sequelae associated with the
acute and chronic effects of ethanol on GABAergic synaptic
inhibition. Clearly, answering these questions represents a
formidable challenge for alcohol researchers in the years to
come.
What might account for the diverse array of mechanisms
that appear to contribute to ethanol potentiation of GABAergic
synaptic transmission? While brain region differences may
contribute to much of the observed variability, there are several
important methodological issues that should also be consid-
ered. For example, it would appear from this survey of the
recent slice electrophysiology literature that the presynaptic
effects of ethanol on GABA release are more consistently
observed than its postsynaptic effects on GABAA receptor
function. While these data may indicate that ethanol enhances
GABAergic synaptic transmission primarily via presynaptic
mechanisms, these findings may also reflect a technical bias of
the electrophysiological methods used in these studies. All of
the recent reports of presynaptic effects of ethanol on GABA
release have employed some variation of the whole-cell patch
clamp recording configuration. While the excellent signal-to-
noise properties of this method greatly facilitate the detection
of increases in the frequency of small, miniature synaptic
currents (the ‘‘gold standard’’ for detecting presynaptic effects),
this recording configuration may potentially result in a
significant perturbation of the cytosolic contents of the cell
being recorded. In fact, a number of studies have reported that
the composition of patch pipette recording solutions can
influence the activity of a variety of postsynaptic ionic
conductances (Zhang et al., 1994; Velumian et al., 1997;
Chung et al., 1998; Robinson & Cameron, 2000) and signal
transduction cascades (Lenz et al., 1997; Vargas et al., 1999).
Thus, the disparity in the number of recent reports of pre- and
postsynaptic effects of ethanol on GABAergic synapses may be
due to the fact that presynaptic mechanisms are likely to be less
influenced by the disruption of the intracellular contents of the
postsynaptic cell associated with conventional whole-cell
recordings.
There is an alternative whole-cell recording method, termed
‘‘perforated patch’’, that results in significantly less disruption
of the intracellular milieu. In the ‘‘perforated patch’’ configu-
ration, an antibiotic such as nystatin or gramicidin is included
in the patch pipette solution. After forming a GigaOhm seal
with a target neuronal membrane, the antibiotic is allowed to
diffuse to the membrane surface where it embeds, forming
small ion channels. This configuration permits electrical access
to the target cell but significantly minimizes the disruption of
cytosolic contents. While this technique is technically more
difficult than conventional whole-cell recordings, it has been
successfully applied to studies of evoked (Mouginot et al.,
1998; Vale & Sanes, 2000) and even miniature GABAA IPSCs
(Zhan et al., 2005) in brain slice preparations. It may thus be
important in future studies to reexamine some of the possible
postsynaptic effects of ethanol on GABAergic synapses using
this technique.
Another significant limitation of the in vitro brain slice
preparation is that, by necessity, many afferent and efferent
projections are not preserved. This may significantly hinder the
detection of presynaptic effects of ethanol, particularly those
related to increases in presynaptic somatic excitability. There
are also numerous methodological differences in the various
recent reports on ethanol modulation of GABAergic synapses
that may have potentially influenced experimental results, such
as the temperature of slices during incubation and recording,
the composition of the patch pipette solution, the aCSF
perfusion rate, etc. With respect to the chronic ethanol studies,
additional methodological factors such as the duration and
method of ethanol exposure (e.g., vapor chamber, intragastric
intubation), withdrawal duration, and whether the ethanol
exposure was voluntary or experimenter-controlled must also
be considered. Where conflicting data have been reported, it
will be important to determine if these or other technical issues
contributed to the disparate findings.
Clearly, the most convincing evidence will come from
studies that employ multidisciplinary approaches to demon-
strate effects of ethanol on GABAergic neurotransmission. For
example, in the recent studies in the rat CeA, in which
electrophysiological increases in GABA release were detected
following acute and chronic ethanol exposure, parallel experi-
ments, using in vivo microdialysis, also demonstrated signif-
icant ethanol-associated increases in extracellular GABA
release (Roberto et al., 2003, 2004). This use of complementary
in vitro and in vivo approaches is exemplary and these or
similar approaches should be employed, wherever technically
feasible, to provide the strongest possible insight into ethanol’s
complex synaptic effects.
It is likely that multidisciplinary approaches will also be
needed to delineate the specific mechanisms responsible for
ethanol’s pre- and postsynaptic effects on GABAergic synapses
as well as the modulatory mechanisms that influence the
ethanol sensitivity of these processes. While slice electrophys-
iology assays are well suited to distinguish between pre- and
postsynaptic drug effects, these preparations may not be ideal
for more detailed investigations of subsynaptic mechanisms of
ethanol action. For example, a number of studies have
 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
demonstrated that, at some synapses, acute ethanol exposure
increases GABA release via a localized effect at the level of the
presynaptic GABAergic terminals. As reviewed recently, these
increases in terminal GABA release may result from any
number of interactions between ethanol and the many elements
that participate in the cascade linking the rapid rise in intra-
terminal calcium levels to the fusion, docking, and release of
GABA-containing vesicles from presynaptic terminals (Siggins
et al., 2005). Unfortunately, presynaptic terminals at most CNS
synapses are generally inaccessible to standard electrophysio-
logical approaches. This technical limitation, coupled with the
paucity of available pharmacological tools with which to
manipulate the various steps along the ‘‘release cascade’’, make
it unlikely that a definitive resolution of the mechanism(s) of
ethanol action on terminal GABA release will be obtained
solely from electrophysiological studies in brain slice prepara-
tions. Additional experimental strategies that may include the
integration of electrophysiological methods with confocal
imaging, neuronal cultures, and possibly the study of trans-
genic mice with altered expression of specific proteins
involved in the release process may be needed. One important
clue, noted by Siggins et al. (2005), is that although ethanol has
been shown to enhance GABA release from presynaptic
terminals in several brain regions, ethanol either inhibits or
has no effect on glutamate release at excitatory synapses in
many of these same areas (Carta et al., 2003; Hendricson et al.,
2004; Mameli et al., 2005b). Identification of the factors
responsible for these divergent effects of ethanol on GABA and
glutamate release may shed light on the ethanol-sensitive
targets within GABAergic terminals.
Perhaps the most significant challenge going forward will
be to devise experimental strategies to assess the behavioral
relevance of the reported effects of ethanol on GABAergic
transmission throughout the CNS. Where both pre- and
postsynaptic mechanisms are present, it will also be necessary
to assess the relative contribution of these localized synaptic
effects of ethanol to this drug’s behavioral actions. Again, it
would appear that relying solely on electrophysiological
approaches will not be sufficient to resolve such complex
issues. To date, electrophysiological studies have provided
exciting, albeit correlational, evidence linking ethanol modu-
lation of GABAergic synapses to behavior by demonstrating a
strong relationship between the acute ethanol sensitivity of
hippocampal GABAergic synapses and ethanol-induced sleep
time in selected rodent lines and transgenic mice (Poelchen et
al., 2000; Proctor et al., 2003). More causal evidence linking
ethanol’s synaptic effects with behavior may be gleaned by
borrowing from a strategy that has proven successful in
mapping out the specific synaptic mechanisms responsible
for the diverse behavioral effects of benzodiazepines. Benzo-
diazepines are some of the most selective allosteric modulators
of GABAA receptors function (Sieghart, 1995). These com-
pounds have long been thought to exert their anxiolytic,
sedative, hypnotic, and amnestic effects via a potentiation of
the activity of GABAA receptors comprised of a1,2,3, or 5-
subunits. By creating lines of mice in which a specific a-
subunit of the GABAA receptor was rendered insensitive to
549
benzodiazepines, several recent studies have provided compel-
ling evidence that enhancement of specific a-subunit-contain-
ing GABAA receptors are responsible for specific behavioral
effects of benzodiazepines (e.g., a2, anxiolysis) (for a review,
see Rudolph & Mohler, 2004).
Although the effects of ethanol on GABAergic neurotrans-
mission appear to be more complex than those of benzodia-
zepines, there is at least some preliminary evidence that a
similar experimental strategy may prove successful for ethanol.
Several years ago, Mihic et al. identified specific amino acid
residues on a- and h-subunits that are responsible for the
postsynaptic potentiating effect of ethanol on GABAA recep-
tors (Mihic et al., 1997). More recently, Homanics et al. have
engineered a line of mice with mutations in the a1-subunit that
render receptors containing this subunit insensitive to ethanol,
thus eliminating at least one of ethanol’s reported postsynaptic
effects. Preliminary characterization of these mice revealed a
decrease in the ethanol sensitivity of hippocampal GABAergic
synapses as well as a modest reduction in some behavioral
effects of ethanol (Boehm et al., 2004; Werner et al., 2004).
Similar experimental strategies may prove successful in
resolving the behavioral significance of other pre- and
postsynaptic effects of ethanol (e.g., potentiation of extra-
synaptic GABAA receptors, enhancement of GABA release) as
our understanding of the molecular mechanisms that mediate
these other synaptic actions is clarified.
Boehm et al. (2004) have recently provided an excellent
review of behavioral studies on the ethanol sensitivity of
established lines of GABAA receptor knockout and transgenic
mice. This review notes that an integration of the findings of
these behavioral studies with existing evidence from quantita-
tive trait loci analysis and gene expression assays may also
provide insight into specific GABAA receptor subunit assem-
blies that may underlie specific behavioral effects of ethanol.
For example, a QTL associated with sensitivity to ethanol-
induced loss of righting reflex has been localized to a region of
mouse chromosome 11 that contains the a1 gene. This finding,
coupled with the observation that a1-subunit knockout mice
exhibit a reduced sensitivity to this behavioral effect of ethanol,
provides convergent evidence in support of a role for a1-
subunit-containing GABAA receptors in mediating ethanol’s
hypnotic effects. While these approaches do not necessarily
provide insight into the specific mechanisms of ethanol action,
they can provide compelling additional support for a causative
role of specific GABAA receptor subunit assemblies in
mediating specific behavioral effects of ethanol. Of course,
the usual caveats associated with studies of transgenic animals,
such as compensation and developmental alterations, must be
headed with any of these transgenic approaches. However,
combining these molecular strategies with more traditional
electrophysiological, behavioral, and genetic approaches holds
much promise as a means of unraveling the behavioral
significance of ethanol’s complex effects on GABAergic
synaptic inhibition.
There are numerous clinical implications associated with the
successful resolution of these mechanistic issues related to the
interaction between ethanol and the GABAergic synapse. For
550 J.L. Weiner, C.F. Valenzuela / Pharmacology & Therapeutics 111 (2006) 533 – 554
example, human epidemiological studies have suggested that a
lower initial sensitivity to the motor impairing effects of
ethanol is associated with an increased risk of developing
alcohol-related problems later in life (Schuckit, 1994; Schuckit
& Smith, 1996). While many genetic studies have hypothe-
sized that polymorphisms in the genes encoding for GABAA
receptors may contribute to individual differences in behavioral
sensitivity to ethanol, the recent studies reviewed here suggest
that genes encoding for presynaptic targets of ethanol action
may also play an important role in determining behavioral
sensitivity to ethanol. In addition, the many studies demon-
strating important modulatory influences of endogenous
factors, such as neurosteroids and neuropeptides, provide yet
another list of gene products that could potentially influence
individual variations in behavioral sensitivity to ethanol
(Pierucci-Lagha et al., 2005). Thus genetic and proteomic
studies focusing on GABAA receptor genes as possible
candidate markers associated with risk of alcohol abuse may
also want to consider other pre- and postsynaptic proteins that
influence signaling through this neurotransmitter system.
Establishing the behavioral relevance and mechanisms
responsible for acute and chronic ethanol effects on GABAer-
gic synapses may also reveal new targets for the development
of more effective pharmacotherapies for the treatment of
alcohol addiction. For example, if the presynaptic effects of
ethanol on GABA release are shown to play an important role
in mediating the reinforcing effects of this drug, it may be
possible to develop pharmacological treatments to selectively
block these effects. The pathways that have been shown to
modulate the ethanol sensitivity of GABAergic synapses may
also reveal novel pharmacological approaches to treat alcohol-
related disorders. For example, very low concentrations of the
GABAB receptor agonist baclofen can completely suppress
ethanol potentiation of GABAergic synapses in the rat
hippocampus (Ariwodola & Weiner, 2004). Although the
behavioral relevance of this effect has yet to be established,
numerous clinical and preclinical studies have demonstrated
that baclofen significantly reduces ethanol drinking, craving,
and withdrawal severity (for a review, see Colombo et al.,
2004). Clearly, further studies are needed to characterize the
synaptic mechanisms responsible for these clinical findings.
However, further clarification of the specific synaptic elements
that mediate and modulate ethanol enhancement of GABAergic
synapses will likely reveal numerous potential targets for the
development of new drug treatments for alcohol addiction.
In summary, over the past decade or so, a number of
electrophysiological studies have shed new light on the
complex interaction between ethanol and the GABAergic
synapse. These studies have identified novel pre- and
postsynaptic mechanisms that mediate both acute and chronic
effects of ethanol on synaptic inhibition. In addition, several
modulatory pathways have been described which regulate (and
may even mediate) some of ethanol’s effects on these synapses.
Clearly the challenge going forward will be to take a more
multidisciplinary approach, combining electrophysiological
approaches with other in vitro and in vivo methods, in an
effort to resolve the molecular mechanisms underlying
ethanol’s complex effects on GABAergic synapses. Strategies
must also be developed to evaluate the behavioral relevance of
the many reported effects of ethanol on GABAergic neuro-
transmission. Such efforts are likely to reveal novel synaptic
elements that may serve as biomarkers for identifying
individuals at risk for abusing alcohol or as targets for the
development of more effective pharmacotherapies for the
treatment of alcohol-related disorders.
Acknowledgments
This work was supported by NIH grants AA13960,
AA11997, AA10422 (JLW) and AA14973, and AA15614
(CFV).
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