www.elsevier.com/locate/pharmthera

Pharmacology & Therapeutics 101 (2004) 211–226

Associate editor: D.R. Sibley

Ethanol regulation of g-aminobutyric acidA receptors: genomic and

nongenomic mechanisms

Sandeep Kumar, Rebekah L. Fleming, A. Leslie Morrow*

Departments of Psychiatry and Pharmacology, Center For Alcohol Studies, University of Chapel Hill at North Carolina, CB#7178,

Chapel Hill, NC 27599, USA

Abstract

g-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that, predominately, mediate inhibitory synaptic transmission in

the CNS. These receptors are pentameric complexes that are comprised of subunits from several classes (a, h, g, y, q), with each class

consisting of several isoforms. Chronic ethanol consumption alters GABAA receptor function producing cellular tolerance to GABA and

ethanol, cross-tolerance to benzodiazepines and barbiturates, and sensitization to inverse agonists. Recent studies have clearly demonstrated

that GABAA receptors play an important role in ethanol dependence and functional properties of GABAA receptor are altered following

chronic ethanol administration. However, the exact mechanisms that account for alterations in GABAA receptor function following chronic

ethanol administration have not been resolved. The mechanisms responsible for adaptation of GABAA receptors to chronic ethanol exposure

may involve ethanol-induced changes in cell surface expression, subcellular localization, synaptic localization, receptor phosphorylation,

neurosteroids, and/or changes in GABAA receptor subunit composition. In this review, we provide an overview of recent data pertaining to

mechanisms that could be responsible for altered properties and expression of GABAA receptors following chronic ethanol administration.

D 2004 Elsevier Inc. All rights reserved.

Keywords: GABAA receptor expression; Subcellular localization; Synaptic localization; Receptor phosphorylation; Neurosteroids; Protein kinase C

Abbreviations: AKAP, A kinase anchoring proteins; AP2, adaptor complex-2; BDNF, brain-derived neurotrophic factor; CaMK, Ca2+/calmodulin-dependent

protein kinase; CCV, clathrin-coated vesicles; CIE, chronic intermittent ethanol exposure; DARP-32, dopamine and cyclic AMP-regulated phosphoproteins;

GABAA, g-aminobutyric acidA; 5-HT, serotonin; GST, glutathione-S transferase; mACh, muscarinic acetylcholine; mIPSC, miniature inhibitory postsynaptic

current; NMDA, N-methyl-D-aspartate; PKA, protein kinase A; PKC, protein kinase C; QT-6, quail fibroblast cells; RACK, receptor for activated kinase C;

THDOC, tetrahydroxydeoxycorticosterone or 3a,21-dihydroxy-5a-pregnan-20-one; 3a,5a-THP, 3a-hydroxy-5a-pregnan-20-one.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

2. Effects of ethanol on g-aminobutyric acidA receptors. . . . . . . . . . . . . . . . . . . . . . . 212

3. Effects of prolonged ethanol exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

4. Mechanisms of g-aminobutyric acidA receptor adaptations following ethanol exposure . . . . . 214

4.1. Alterations of g-aminobutyric acidA receptor gene expression . . . . . . . . . . . . . . 214

4.2. Post-translational modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

4.3. Subcellular localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

4.4. Synaptic localization of receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

4.5. Regulation of g-aminobutyric acidA receptors by other receptors via intracellular

signaling systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

4.6. Role of neurosteroids in ethanol tolerance and dependence . . . . . . . . . . . . . . . . 220

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

0163-7258/$ – see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.pharmthera.2003.12.001

* Corresponding author. Tel.: 919-966-7682; fax: 919-966-9099.

E-mail address: morrow@med.unc.edu (A.L. Morrow).

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226212

1. Introduction

g-Aminobutyric acidA (GABAA) receptors are a family

of chloride ion channels that mediate inhibitory neurotrans-

mission throughout the CNS. These receptors are hetero-

meric protein complexes consisting of several homologous

membrane-spanning glycoprotein subunits. Molecular clon-

ing has revealed multiple GABAA receptor subunits that

can be divided by homology into subunit classes with

several members: a1–6, h1–4, g1–3, y, q, and k (Sieghart

& Sperk, 2002). The majority of GABAA receptors are

composed of 2 a-, 2 h-, and 1 g-subunits, where the g-

subunit is located between a- and h-subunits (Tretter et al.,1997; Sieghart et al., 1999) and expressed on cell mem-

branes to inhibit neuronal signaling (Sieghart, 2000). The

large number of GABAA receptor subunits generates the

potential for various subunit compositions that may ac-

count for variable sensitivity to modulatory drugs, such as

benzodiazepines, barbiturates, neurosteroids, and possibly

alcohol and general anesthetics. GABAA receptors have

been divided into multiple subtypes based on different

receptor distributions in brain and the pharmacological

profiles of several compounds (Seeburg et al., 1990).

Recombinant expression studies have demonstrated that

subunit composition determines the functional properties

of receptor subtypes. Type I benzodiazepine receptor char-

acteristics are observed with receptors consisting of

a1hxg2-subunits. By contrast, the expression of a2hxg2,

a3hxg2, or a5hxg2-subunits results in type II benzodiaze-

pine site pharmacology (Sieghart, 1989). Other GABAA

receptors are insensitive to benzodiazepines, including a4yand a6y receptors (Sieghart & Sperk, 2002). For example,

substitution of a4 for a1-subunits in recombinant receptors

produces particularly remarkable effects on GABA and

benzodiazepine sensitivity (Wafford et al., 1996; Whitte-

more et al., 1996). Notably, these changes in receptor

Table 1

Proteins associated with GABAA receptors

Proteins Method Brain area/cell type

PKCg co-immunoprecipitation cerebral cortex

PKCq confocal microscopy brain

PKCh11 co-immunoprecipitation cortical neurons

RACKI co-immunoprecipitation HEK 293 cells/brain

cortical neurons

Serine kinase

(GTAP 34)

co-immunoprecipitation brain (calf)

Gephyrin confocal microscopy brain

Adaptin-a/clathrin co-immunoprecipitation/

confocal microscopy

cerebral cortex/

hippocampus

AKAP 150/PKA co-immunoprecipitation/

confocal microscopy

adult brain/cultured

hippocampal cells

GABARAP GST pull-down assay/

co-immunoprecipitation/

confocal microscopy

yeast cells/brain/QT-

quail fibroblast

pharmacology are mimicked in vivo following drug-in-

duced adaptations in receptor subunit gene expression

(Grobin et al., 1998). The GABAA receptor a1-subunit is

the most abundant a-subunit in adult brain, highly

expressed throughout most brain regions and is a compo-

nent of f 50% of all GABAA receptors (Kralic et al.,

2002a). The analysis of subunit composition by co-immu-

noprecipitation studies have demonstrated that 98% of a1-

subunit-containing receptors are assembled with a g- or y-subunit (Sieghart & Sperk, 2002). In contrast, a significant

fraction of a4-subunit-containing receptors are comprised

of a4- and h-subunits only (Bencsits et al., 1999). GABAA

receptors also associate with various other proteins (see

Table 1) that anchor the receptor, mediate trafficking and

post-translational modifications, and modulate receptor

function under various conditions. These protein interac-

tions are not yet well understood, but clearly influence

GABAA receptor assembly, expression on the membrane

surface, endocytosis, and channel function that ultimately

controls the inhibitory tone of the CNS.

2. Effects of ethanol on ;-aminobutyric acidA receptors

Ethanol has many sites of action in the brain, but few

have been linked as closely to its behavioral actions as

GABAA receptors. At low doses, ethanol is anxiolytic,

sedative-hypnotic, anticonvulsant, motor-incoordinating,

and impairs cognitive function (Majchrowicz, 1975; Frye

et al., 1981; Matthews et al., 1995; Givens & McMahon,

1997). These behavioral effects of acute ethanol adminis-

tration are remarkably similar to those of known GABAA

receptor modulators, including benzodiazepines, barbitu-

rates, and 3a,5a-reduced neuroactive steroids.

Many laboratories have attempted to demonstrate that

physiological concentrations of ethanol alter GABAA re-

Receptor type/subunit Reference

a1/a4-subunit-

containing receptors

Kumar et al., 2002

a1-, h2/3-, or g2-subunit Olive & Hodge, 2000

h1/h3-subunit Brandon et al., 1999

/ h1/h3 Brandon et al., 1999

h3-subunit Kannenberg et al., 1999

a1-, a2-, a3-, or g2-

subunit-containing

receptors

Sassoe-Pognetto et al., 2000

a1-subunit-containing

receptors/h- and g-subunits

Kittler et al., 2000;

Kumar et al., 2003b

h1/h3 Brandon et al., 2003

6 g (g2) Chen et al., 2000; Wang

& Olsen, 2000; Nymann-

Andersen et al., 2002

Table 2

Effects of chronic ethanol administration on GABAA receptor function in

the cerebral cortex, hippocampus, and cerebellum

Receptor property Alteration Source

GABA-mediated Cl �

channel function1,2decreased Martz et al., 1983;

Morrow et al., 1988;

Gonzalez & Czachura,

1989; Criswell et al.,

1993; Kang et al., 1998;

Cagetti et al., 2003

GABA-mediated Cl �

channel function3no change Allan & Harris, 1987;

Buck & Harris, 1990;

Frye et al., 1993

Pentobarbital-mediated

Cl� flux1decreased Morrow et al., 1988

Ethanol-enhanced

Cl� flux3abolished Allan & Harris, 1987;

Morrow et al., 1988

Benzodiazepine-

enhanced Cl � flux2,3decreased Buck & Harris, 1990;

Cagetti et al., 2003

Inverse agonist

modulation2,3increased Mehta & Ticku, 1989;

Buck & Harris, 1990;

Cagetti et al., 2003

Neuroactive steroid

modulation1,2increased Devaud et al., 1996;

Cagetti et al., 2003

1 Cerebral cortex.2 Hippocampus.3 Cerebral cortex and cerebellum.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 213

ceptor function; however, whether ethanol directly affects

GABAA receptors remains controversial. In vivo studies

examining ethanol interactions with GABAA receptors give

mixed results, suggesting that complex mechanisms are

involved in the actions of ethanol. Acute ethanol adminis-

tration potentiates GABA-mediated inhibition in vivo, but

only in specific brain regions or cell types (Celentano et al.,

1988; Givens & Breese, 1990; Proctor et al., 1992; Rey-

nolds et al., 1992; Aguayo et al., 1994; Frye et al., 1994;

Soldo et al., 1994; Sapp & Yeh, 1998). Ethanol has been

shown to alter GABAergic synaptic transmission in both the

hippocampus (Weiner et al., 1994) and the amygdala (Rob-

erto et al., 2003). Ethanol increased mean open time,

frequency of opening, and burst duration, while decreasing

mean closed time of GABAA receptors in dorsal root

ganglion cells (Takebayashi et al., 1998). Other studies

indicate that direct interaction of ethanol with neuronal

GABAA receptors using patch clamp recording techniques

is absent or minimal at pharmacologically relevant ethanol

concentrations (Frye et al., 1994; Crews et al., 1996;

Marszalec et al., 1998).

Ethanol potentiation of GABA responses at remarkably

low concentrations (1–5 mM) has recently been demon-

strated in recombinant GABAA receptors containing a4y-and a4h3y-subunits in oocytes (Sundstrom-Poromaa et al.,

2002; Wallner et al., 2003). Furthermore, this high ethanol

sensitivity is recapitulated in vivo in progesterone-with-

drawn animals that exhibit an increase in the expression

of this receptor subtype in the hippocampus (Sundstrom-

Poromaa et al., 2002). Notably, many other recombinant

GABAA receptors are insensitive to low ethanol concen-

trations ( < 50 mM) in any expression system (Harris et al.,

1998), including chimeric GABA/glycine receptors that

respond to alcohols at high mM concentrations (Mihic et

al., 1997).

Multiple mechanisms may influence the sensitivity of

GABAA receptors to ethanol. In some studies, certain exper-

imental conditions have been manipulated to uncover an

effect of ethanol (Weiner et al., 1994, 1997a; Wan et al.,

1996). h-Adrenergic receptor stimulation and subsequent

elevation of cAMP and protein kinase A (PKA) activity are

required to detect ethanol potentiation of GABA in cerebellar

Purkinje cells (Freund & Palmer, 1997; Wang et al., 1999).

Furthermore, elevation of protein kinase C (PKC) in hippo-

campal slices increased the sensitivity of CA1 neurons to

ethanol (Weiner et al., 1997b). Endogenous neurosteroids

may also play an important role in the complex interactions

between ethanol and GABAA receptors. Systemic ethanol

administration dramatically elevates both plasma and cere-

bral cortical 3a-hydroxy-5a-pregnan-20-one (3a,5a-THP)

and 3a,21-dihydroxy-5a-pregnan-20-one (THDOC) to phar-

macologically active levels (Morrow et al., 1998; Barbaccia

et al., 1999). A growing body of evidence suggests that brain

concentrations of endogenous neurosteroids could modulate

the behavioral and/or electrophysiological effects of ethanol

discussed above (see Morrow et al., 2003, for a review).

3. Effects of prolonged ethanol exposure

Prolonged ethanol consumption results in the develop-

ment of tolerance to many of the GABAergic effects of

ethanol including the anxiolytic, sedative, motor-in-coordi-

nating, and positive reinforcing effects of ethanol. With-

drawal from ethanol and, particularly, repeated withdrawals

from ethanol produce marked increases in CNS excitability

that form a criterion for ethanol dependence. Substantial

evidence suggests that these behavioral and neural adapta-

tions involve marked adaptations in the pharmacological

properties of GABAA receptors. The molecular determi-

nants of these alterations in GABAA receptor function are

complex, probably due to the heterogeneity of GABAA

receptor subtypes and the diversity of biochemical mecha-

nisms that regulate the function of these receptors.

The development of ethanol tolerance and dependence is

associated with alterations in many of the functional prop-

erties of GABAA receptors throughout brain (Table 2). In

the cerebral cortex, muscimol- and pentobarbital-stimulated

Cl � uptake is decreased following chronic ethanol expo-

sure. The ability of ethanol to potentiate GABA- or musci-

mol-stimulated Cl� uptake is lost following chronic ethanol

administration in both cortex and cerebellum. Benzodiaze-

pine enhancement of muscimol-stimulated chloride flux is

reduced in the cerebral cortex of mouse microsacs, while the

functional efficacy of inverse agonists is enhanced. Poten-

tiation of Cl � uptake by the neuroactive steroids, 3a,5a-

THP and THDOC, is enhanced in ethanol-dependent rats

(Devaud et al., 1996). Furthermore, chronic intermittent

ethanol exposure (CIE) produces many changes in hippo-

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226214

campal GABAA receptor function and pharmacology.

GABA-activated Cl � flux in hippocampal slices is reduced

following CIE (Kang et al., 1998). Hippocampal cells

become insensitive to alphaxalone and diazepam but re-

spond to bretazenil as an agonist and Ro15-4513 binding is

increased (Cagetti et al., 2003). Therefore, it is clear that

chronic ethanol exposure leads to altered properties of

GABAA receptors.

4. Mechanisms of ;-aminobutyric acidAreceptor adaptations following ethanol exposure

The precise mechanisms that account for alterations in

GABAA receptor function and increased CNS excitability

following chronic ethanol administration are becoming clear.

Several mechanisms for GABAA receptor adaptation follow-

ing chronic ethanol exposure have been proposed, which

include alterations in gene expression, post-translational

modification, subcellular localization, synaptic localization,

regulation by other receptor interactions, intracellular sig-

naling, and neurosteroid responses to ethanol (Fig. 1). These

mechanisms include both genomic and nongenomic alter-

ations that are likely to interact to explain the complex

adaptations of GABAA receptors to ethanol throughout

brain. The purpose of this review is to discuss evidence that

these adaptations underlie ethanol regulation of GABAA

Fig. 1. Potential mechanisms of GABAA receptor regulation following chronic et

receptor assembly: Chronic ethanol administration results in altered mRNA and pe

regulation of subunit expression varies across brain regions. Altered subunit expr

composition and function of cell surface GABAA receptors. Internalization: Chron

receptors internalized in CCV (see Section 4.3). Internalized receptor can be degrad

processes. Internalized receptor subunits could also provide signals that contribute

(see Section 4.2): Chronic ethanol exposure alters receptor function without altering

is exogenously controlled. Chronic ethanol exposure reduces the level of phosphor

translational modification of receptor subunits could alter receptor function by a

translational modification of receptor subunits and/or associated proteins could als

of GABAA receptors and regulate their expression in vivo (see Section 4.6). NM

modulated by ethanol and can indirectly modulate GABAA receptor function v

proteins involved in these actions include PKC, PKA, CaMK, Ca2+, and DARP-

receptor function and highlight recently discovered mecha-

nisms that may play an important role in pharmacological

adaptations to ethanol.

4.1. Alterations of c-aminobutyric acidA receptor gene

expression

Recombinant expression and homologous gene deletion

studies have shown that functional properties of receptors

are regulated by the subunit composition of receptors. For

example, recombinant GABAA receptors with a4h2g2-sub-

units respond to GABA and benzodiazepine agonists with

lower efficacy than a1h2g2 receptors (Whittemore et al.,

1996). Moreover, homologous genetic deletion of a1-sub-

units dramatically alters the pharmacological properties of

GABAA receptors, reducing the potency and efficacy of

GABA and the benzodiazepine, diazepam. Furthermore,

these mice exhibit increased seizure susceptibility and a

reduction in the anticonvulsant actions of benzodiazepines

(Kralic et al., 2002a, 2002b).

Chronic ethanol administration differentially alters the

expression of distinct GABAA receptor subunit mRNA and

peptides in various brain regions. The level of GABAA

receptor a1-, a2-, and a3-subunit mRNA and peptides are

reduced in the cerebral cortex (Mhatre & Ticku, 1992;

Devaud et al., 1997), while a4-, h1-, h2-, h3-, g2-, and g2-

subunit peptide or mRNA levels are increased in the

hanol administration. From right to left: Altered mRNA, peptide levels and

ptide levels of various GABAA receptor subunits (see Section 4.1). Ethanol

ession could modify receptor assembly resulting in a change in the subunit

ic ethanol administration causes an increase in GABAA receptor a1-subunit

ed or recycled back to the cell surface and PKC has been implicated in both

to the regulation of receptor gene expression. Post-translational modification

receptor subunit expression in recombinant systems where gene expression

ylated a1-subunit containing GABAA receptors or associated proteins. Post-

ltering receptor conformation and/or channel conductance. However, post-

o alter receptor trafficking. Neurosteroids: Neurosteroids alter the sensitivity

DA, mACh, 5-HT, h-adrenergic receptor cross-talk: These receptors are

ia various intracellular signaling proteins (see Section 4.5). The signaling

32.

Table 3

Consensus sites for phosphorylation in GABAA receptor subunits

h1 – 4 PKC, PKA, CaMKII Wisden et al., 1991;

g2 S/L PKC, CaMKII Moss et al., 1992a;

a4 PKC, PKA Macdonald, 1995;

a6 PKC, PKA Mohler et al., 1996

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 215

cerebral cortex following chronic ethanol exposure (Devaud

et al., 1997; Mahmoudi et al., 1997; Matthews et al., 1998a).

Since a1-subunits are the most abundant subunit in the

cerebral cortex, the reduction in the expression of this

subunit is likely to have significant functional consequen-

ces. GABAA receptor a4-subunit expression measured by

immunohistochemical labeling in brain sections is sparse

across most brain regions except the thalamus (Bencsits et

al., 1999; Pirker et al., 2000; Peng et al., 2002). However,

selective modulation of a4-subunit expression in the hippo-

campus using antisense oligonucleotides has been shown to

significantly alter the functional properties of GABAA

receptors and modulate steroid withdrawal excitability

(Moran et al., 1998). Therefore, despite the relatively low

expression of a4-subunit peptide, modulation of a4-subunit

expression clearly influences GABAA receptor function and

GABA-mediated behaviors in vivo. Hence, alterations in

GABAA receptor subunit expression appear critically im-

portant in regulation of GABAA receptor function following

chronic ethanol administration.

The regulation of various GABAA receptor subunits by

ethanol differs across brain regions (Grobin et al., 2000).

For example, chronic ethanol consumption for 14 days

increases GABAA receptor a4-subunit expression in the

cerebral cortex and hypothalamus (Devaud et al., 1997),

decreases a4-subunit levels in that amygdala and nucleus

accumbens (Papadeas et al., 2001), and does not alter a4-

subunit levels in the hippocampus or ventral tegmental

area (Matthews et al., 1998a; Papadeas et al., 2001).

However, repeated ethanol withdrawals or longer ethanol

exposure increases a4-subunit expression in the hippo-

campus (Matthews et al., 1998a; Cagetti et al., 2003), and

these effects are associated with alterations in the phar-

macological responses of GABAA receptors to benzodi-

azepine agonists and inverse agonists (Cagetti et al.,

2003). Furthermore, in the cerebellum, a6-subunit mRNA

and peptide levels are increased while a1-subunit peptide

and mRNA are decreased following chronic ethanol

administration (Morrow et al., 1992; Mhatre & Ticku,

1993). Therefore, it is clear that adaptation in GABAA

receptor subunit expression caused by chronic ethanol

administration is not universal across brain regions. The

mechanisms that underlie regional differences in the

regulation of GABAA receptors by ethanol are unclear

but may involve mechanisms to be described in the

following sections.

4.2. Post-translational modification

Protein phosphorylation is a common regulatory mech-

anism for maintaining receptor structure and function and

has been implicated in modulation of GABAA receptor

function under numerous physiologic conditions (Kellen-

berger et al., 1992; Leidenheimer et al., 1992; Krishek et

al., 1994; Poisbeau et al., 1999; Brandon et al., 2000).

GABAA receptor phosphorylation can cause diverse func-

tional effects, ranging from enhancement to inhibition

depending upon the sites phosphorylated (Lin et al.,

1996; McDonald et al., 1998). Several protein kinases

including PKC, PKA, and Ca2 + /calmodulin-dependent

protein kinase-II (CaMKII) have consensus sites on

GABAA receptor subunits and can phosphorylate these

subunits (McDonald & Moss, 1997; Brandon et al., 2000)

(see Table 3). Enzyme substrate co-localization is important

for substrate activation and provides substrate specificity

for kinases. Targeting of protein kinases to GABAA recep-

tors can be a critical factor in controlling the functional

modulation of receptors by phosphorylation and several

intermediary proteins can regulate this process. Intermedi-

ary proteins that are important for transportation and

anchoring of PKC and PKA are receptor for activated

kinase C (RACK) and A kinase anchoring proteins

(AKAP), respectively. Recent studies have shown that

GABAA receptors are closely associated with several sig-

naling proteins (see Table 1), including PKC and PKA, that

can alter receptor function.

PKC phosphorylation of GABAA receptor subunits reg-

ulates GABAA receptor function (Poisbeau et al., 1999;

Brandon et al., 2000), expression (Chapell et al., 1998;

Connolly et al., 1999), and adaptation following ethanol

exposure (Kumar et al., 2002). There are 3 major classes of

PKC: cPKC (Ca2 + -dependent PKCa, h, and g), nPKC

(Ca2 + -independent y and q), and aPKC (atypical or Ca2 + -

and phorbol ester-independent ~). Despite the existence of

several isoforms of PKC, previous studies have demonstrat-

ed that only h, g, and q isoforms interact with GABAA

receptors and modulate receptor function (Connolly et al.,

1999; Hodge et al., 1999; Brandon et al., 2000; Olive &

Hodge, 2000; Kumar et al., 2002). PKCg co-immunopreci-

pitates with GABAA receptors isolated with both a1- and

a4-subunit antibodies in the cerebral cortex (Kumar et al.,

2002) and PKCq co-localizes with various GABAA receptor

subunits across brain (Olive & Hodge, 2000). Genetic

deletion of PKCg and q has clearly demonstrated that these

isoforms of PKC modulate the effects of ethanol on GABAA

receptors both in vitro and in vivo (Proctor et al., 2003). For

example, PKCg null mice show reduced sensitivity to the

anxiolytic effects of both intoxicating and sedative doses of

ethanol when compared with wild-type mice (Bowers et al.,

2001), while PKCq null mice show increased ethanol

sensitivity (Hodge et al., 1999). Furthermore, mice lacking

the gene for PKCg show a significant reduction in ethanol

potentiation of muscimol-stimulated Cl � influx compared

with responses in wild-type mice (Harris et al., 1995). In

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226216

contrast, ethanol and flunitrazepam potentiation of musci-

mol-stimulated Cl � uptake is greater in microsacs from

PKCq null mutant mice compared with wild-type controls

(Hodge et al., 1999). Therefore, it appears that q and g

isoforms of PKC have opposing effects on GABAA receptor

function. Furthermore, there are quantitative and qualitative

differences in expression of PKC isoforms in brain and

nonneuronal cells. For example, PKCg is only present in the

CNS, and its expression varies across brain regions (Kik-

kawa et al., 1989; Wetsel et al., 1992). In the cerebellum,

PKCg is highly expressed in Purkinje cells but not in other

neurons (Yoshida et al., 1988; Kikkawa et al., 1989;

Barmack et al., 2000). Taken together, it is likely that both

PKCg and q mediate phosphorylation of GABAA receptors

and/or associated proteins and regulate GABAA receptor

function in a cell-specific manner.

Studies have suggested that altered phosphorylation of

GABAA receptor subunits mediates changes in receptor

function following ethanol exposure, since ethanol can alter

GABAA receptor function under conditions where subunit

expression is fixed (Klein et al., 1995). Direct phosphory-

lation of receptor subunits can alter function by altering the

conformational state of the receptor that could either alter

the binding of GABA or alter channel conductance inde-

pendently (Churn & DeLorenzo, 1998; Oh et al., 1999;

Churn et al., 2002). Studies using recombinant GABAA

receptor subunits have shown that PKC phosphorylation of

a1-, h2-, and g2-subunits produces inhibition of GABAA

receptor-stimulated current (Kellenberger et al., 1992;

Krishek et al., 1994). However, the evidence for a role of

phoshorylation of native GABAA receptors is controversial

(Heuschneider & Schwartz, 1989; Leidenheimer et al.,

1990, 1992; Ticku & Mehta, 1990; Ghansah & Weiss,

2001). We have recently found preliminary evidence that

adenosine triphosphate and the phorbol ester phorbol 12,13-

dibutyrate enhance the phosphorylation of type 1 GABAA

receptors and reduce muscimol-stimulated Cl � uptake in

rat cerebral cortical synaptoneurosomes when synaptoneur-

osomes are prepared in the presence of co-factor and/or

activator (Kumar et al., 2003a). Similarly, PKCq-mediated

phosphorylation inhibits ethanol and benzodiazepine

responses, since PKCq inhibition or deletion enhances

benzodiazepine and ethanol potentiation of GABA

responses in synaptoneurosomes (Hodge et al., 1999). In

contrast, PKC activation enables the allosteric enhancement

of synaptic GABA currents by neurosteroids (Leidenheimer

& Chapell, 1997; Fancsik et al., 2000).

Chronic ethanol exposure diminishes GABA and benzo-

diazepine responses, yet it enhances neurosteroid potentia-

tion of GABAA receptors in cortical synaptoneurosomes

(Devaud et al., 1996). Similar responses are observed in

hippocampal neurons following chronic ethanol administra-

tion (Cagetti et al., 2003). Therefore, it might be predicted

that chronic ethanol administration would increase PKC

phosphorylation of GABAA receptors. However, the lack

of phospho-specific antibodies against GABAA receptor

subunits has precluded study of phosphorylation state of

receptor subunits in vivo. To address this question, we

investigated the effect of chronic ethanol administration on

the association of PKCg with a1-subunit-containing

GABAA receptors in cerebral cortical membranes. Chronic

ethanol exposure reduced co-immunoprecipitation of PKCg

with these receptors (Kumar et al., 2002). To measure the

level of phosphorylated a1-subunit-containing GABAA

receptors in the synaptic membrane fraction, phosphopro-

teins were immunoprecipitated and GABAA a1-subunits

were identified in the denatured phospho-immunoprecipi-

tate. Preliminary data suggest that chronic ethanol exposure

reduces the levels of phosphorylated a1-subunit-containing

GABAA receptors and/or associated proteins in the mem-

brane fraction of cerebral cortex (Fig. 2). Studies on the

phosphorylation state of a4-subunit-containing receptors are

underway. These studies will address the possibility that

altered phosphorylation of receptors may contribute to

altered function of GABAA receptors either via direct effects

on receptor function or effects on receptor trafficking (see

Section 4.3).

Other protein kinases can also modulate GABAA recep-

tor function by phosphorylation of GABAA receptor sub-

units. The activation of PKA can increase or decrease in

GABA-activated currents depending upon the cell type. For

example, PKA activation causes a decrease in GABA-

activated currents in cultured cerebellar granule cells,

hippocampal pyramidal cells, and spinal cord neurons

(Porter et al., 1990; Moss et al., 1992b; Robello et al.,

1993; Poisbeau et al., 1999). In contrast, PKA activation

increases GABAA receptor response in hippocampal den-

tate granule neurons and cerebellar Purkinje neurons (Kano

& Konnerth, 1992; Kano et al., 1992; Nusser et al., 1999).

These contradictory responses following PKA activation

could be due to the presence of different GABAA receptor

subtypes in various cells. In addition, there are cell-specific

differences in the expression of PKA isozymes and AKAP

(Ulfig & Setzer, 1999; Sik et al., 2000). PKA modulation

of GABAA receptors may be relevant to ethanol-induced

receptor adaptations. PKA-mutant mice consume more

ethanol and show low sensitivity to ethanol-induced seda-

tion compared with wild-type mice (Thiele et al., 2000).

Furthermore, chronic ethanol administration alters translo-

cation of PKA (Dohrman et al., 2002). Hence, PKA can

modulate GABAA receptor function and may contribute to

development of ethanol dependence following chronic

ethanol administration.

The phosphorylation state of a given protein is governed

by the balance between protein kinases that transfer phos-

phate from adenosine triphosphate to the protein (phos-

phorylation) and protein phosphatases that catalyze the

reverse reaction (dephosphorylation). It is now widely

acknowledged that the regulation of protein phosphoryla-

tion requires coordinated control of both kinases and

phosphatases. Chronic ethanol exposure alters PKC levels

in PC-12 cells (Messing et al., 1991) and membrane

Fig. 2. Chronic ethanol administration decreases phosphorylated a1-subunit-containing GABAA receptors. Ethanol was administered to male Sprague-Dawley

rats for 2 weeks using a pair-fed design as previously described (Devaud et al., 1997). On day 15, animals were sacrificed and cerebral cortex was dissected and

stored at � 80 jC. The synaptic fraction of cortex was solubilized and immunoprecipitated using a Phosphoprotein Purification Kit (Qiagen, Valencia, CA) that

immunoprecipitates any proteins phosphorylated at serine, threonine, and/or tyrosine residues. The phosphoprotein immunoprecipitate was then denatured and

a1-subunit protein was detected by western blot analysis. (A) Chronic ethanol administration decreased the level a1-subunit peptide in the phosphoprotein

immunoprecipitate by 50.98 F 15.38 % ( P = 0.0106, n = 5). (B) To confirm the specificity of the phosphoprotein purification, we dephosphorylated proteins

in the synaptic fraction by incubating synaptic fraction with protein phosphatase enzyme (k-PPase, 40,000 units, Biolabs, New England) in a water bath at 30

jC for 90 min and purified the phosphorylated proteins. No a1-subunit peptide was detected in the phosphoprotein immunoprecipitate of dephosphorylated

proteins following western blot analysis. Therefore, the immunoprecipitation of phosphorylated a1-subunit-containing GABAA receptors was specific and

nonspecific binding of a1-subunits to the phosphoprotein column was undetectable.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 217

fractions of the frontal and limbic cortices in mice (Narita

et al., 2001), but not in P2 fraction of rat cerebral cortex

(Kumar et al., 2002). The effect of chronic ethanol expo-

sure on protein phosphatase expression and activity is still

not known. However, phosphatase inhibitors alter the

function of GABAA receptors (Huang & Dillon, 1998).

In addition, we recently found that blockade of phosphatase

activity throughout the preparation of cerebral cortical

synaptoneurosomes decreases muscimol-induced chloride

uptake (Kumar et al., 2003a). Hence, the balance between

phosphatase and kinase activity is important for GABAA

receptor function. Chronic ethanol administration could

alter this balance to regulate receptor function and or

trafficking. Further studies on the effects of ethanol on

protein phosphatase activity are warranted.

Alterations in the phosphorylation state of GABAA

receptors could alter receptor function directly or indirectly.

Phosphorylation may directly alter receptor conformation

and channel conductance while indirect actions may pro-

duce alterations in receptor subunit composition at the

membrane surface by altering the normal trafficking of

receptors. This possibility is discussed in Section 4.3.

4.3. Subcellular localization

One mechanism that regulates the strength of synaptic

inhibition involves the density and/or composition of

GABAA receptors on the cell surface. Functional receptors

are expressed on cell membranes, and therefore, cell surface

expression of receptor peptides represents a useful tool for

identification of receptors that are likely functional. The

expression of GABAA receptors involves a highly regulated

process of synthesis, assembly, endocytosis, and recycling

to the membrane or degradation (see Fig. 3). Golgi-derived

vesicles provide newly synthesized receptors to the cell

surface, whereas clathrin-coated vesicles (CCV) mediate

endocytosis of surface receptors that are ultimately degraded

or recycled back to the cell surface (Lodish et al., 1996).

Alterations in the expression and composition of various

GABAA receptors could result from selective endocytosis

and/or recycling to the cell surface.

Chronic ethanol exposure selectively increases the inter-

nalization of a1-subunit-containing GABAA receptors into

CCVof the cerebral cortex with a corresponding decrease in

these receptors in the synaptic fraction (Kumar et al.,

2003b). In contrast, there is no change in the internalization

of a4-subunit-containing receptors into CCV, although there

is a significant increase in a4-subunit peptide in the synaptic

fraction following chronic ethanol exposure. Furthermore,

acute ethanol administration has no effect on the subcellular

localization of either receptor subtype. Hence, the regulation

of intracellular trafficking following chronic ethanol expo-

sure appears to alter the subtypes of GABAA receptors on

the cell surface and this may account for alterations in the

pharmacological properties of GABAA receptors that have

been observed.

Fig. 3. Mechanisms of GABAA receptor endocytosis and recycling.

Endocytosis by CCV requires AP2 and clathrin binding to the receptor. AP2

is specific for endocytosis of surface proteins and it consists of 4 subunits:

h, A, a, and j (Hirst & Robinson, 1998). First, h- and A-subunits of AP2recognize and bind to GABAA receptors, then clathrin binds the complex

and the receptor is internalized (see Section 4.3). Internalized receptors may

be recycled back to the cell surface or degraded depending upon undefined

intracellular signals. These signals appear to involve post-translational

modifications of receptors or associated proteins mediated by PKC. AP1

transports newly synthesized receptors from trans-Golgi reticulum to the

cell surface.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226218

Internalization of GABAA receptors following chronic

ethanol administration is probably mediated by clathrin and

adaptor complex since the association of adaptin-a and

clathrin with a1-subunit-containing GABAA receptors in

the intracellular fraction was increased following chronic

ethanol administration (Kumar et al., 2003b). Recent studies

show that the dileucine motif found in h2-subunits of

GABAA receptors is required for recognition by the adaptor

complex-2 (AP2) that precedes clathrin-dependent endocy-

tosis (Herring et al., 2003). Therefore, it is possible that

PKC-mediated post-translational modification of GABAA

receptor subunits, receptor-associated proteins, and/or AP2

following chronic ethanol administration alters the recogni-

tion and endocytosis of GABAA receptors.

Recent studies have suggested that trafficking of GABAA

receptors can be regulated by PKC. Flunitrazepam-induced

reduction in expression of GABAA receptor a1-subunit is

prevented by PKC inhibitors in cultured cerebellar granule

cells (Johnston et al., 1998). Several studies in A293 cells

suggest that PKC activation leads to increased internaliza-

tion of a1h2g2 GABAA receptors (Connolly et al., 1999;

Cinar & Barnes, 2001). PKC may also play a role in

ethanol-induced changes in GABAA receptor trafficking.

Chronic ethanol consumption results in decreased associa-

tion of PKCg with a1-subunit-containing GABAA receptors

and decreased expression of a1-subunit at the cell surface.

In contrast, chronic ethanol exposure results in increased

association of PKCg with a4-subunit-containing GABAA

receptors and increased expression of a4 at the cell surface

(Kumar et al., 2002). Furthermore, in the hippocampus, the

association of PKCg with GABAA receptors is not altered

(Kumar et al., unpublished observation), and there is no

alteration of a1-subunit expression following chronic etha-

nol exposure (Matthews et al., 1998a; Morrow et al.,

2001a). Therefore, it appears that association of PKCg with

GABAA receptors may influence the trafficking (decreasing

internalization and/or increasing recycling) of receptors in

vivo following chronic ethanol administration. However, it

is still unknown whether PKCg phosphorylates GABAA

receptor subunits or associated proteins to alter trafficking

of receptors in vivo. Moreover, various isoforms of both

receptor and kinase are present in different levels in various

brain regions that complicate the interaction between recep-

tors and PKC. Therefore, region specific or cell-specific

effects of ethanol on GABAA receptors could be due to cell-

specific differences in interaction between the receptor and

receptor-associated signaling proteins, such as PKC.

Chronic ethanol consumption results in increased expres-

sion of a4, h2, and h3 GABAA receptor subunits in the

cerebral cortex and all contain consensus phosphorylation

sites for PKC (Wisden et al., 1991; Macdonald, 1995;

Mohler et al., 1996). In contrast, a1-, a2-, and a3-subunits

are decreased in the cortex and these subunits do not contain

consensus phosphorylation sites for PKC. The correlation

between the presence of PKC phosphorylation sites and

their increased cell surface expression following chronic

ethanol exposure could be a coincidence or it might indicate

that PKC regulates subunit expression. Differential expres-

sion of these subunits on the cell surface following chronic

ethanol exposure could be a result of selective PKC phos-

phorylation, endocytosis, and/or recycling. Other studies

have shown that threonine phosphorylation diverts internal-

ized epidermal growth factor receptors from degradative to

recycling pathways (Bao et al., 2000). Thus, differential

effects of ethanol on the association of PKCg with these

receptors and phosphorylation may influence endocytosis

and/or recycling of these receptors in vivo. We have recently

detected PKCg and PKCq peptides in the CCV fraction of

the cerebral cortex (Fig. 4). Furthermore, chronic ethanol

exposure increases PKCq peptide levels while PKCg levels

remain unchanged in CCV fraction (Fig. 4). However, the

functional significance of these findings remains to be

established.

4.4. Synaptic localization of receptors

Application of GABAA agonist in most pharmacological

studies activates both synaptic and extrasynaptic receptors.

However, synaptic and extrasynaptic receptors are thought

to play different roles in the regulation of neuronal excit-

ability. Some studies have shown that the a1-subunit is

usually expressed in synaptic GABAA receptors while the

a4-subunit predominantly occurs in extrasynaptic receptors

(Mody, 2001). GABAA receptors containing a1- and a4-

subunits have different pharmacological characteristics, in-

Fig. 4. Chronic ethanol administration increases peptide levels of PKCq but not of PKCg in the CCV fraction of cerebral cortex. Ethanol was administered to

rats as described in Fig. 2. Animals were sacrificed on day 15 and cerebral cortex was dissected and stored at � 80 jC. The CCV fraction was isolated (Kumar

et al., 2003b), analyzed by western blot analysis, and probed using anti-PKCg, PKCq, and h-actin antibodies. Representative western blot analysis showing (A)PKCq and (B) PKCg peptides in the CCV fraction of cerebral cortex from control (lane 1) and ethanol-dependent (lane 2) rats. Peptides levels of PKCq andPKCg were normalized to h-actin for equal gel loading and transfer. Chronic ethanol administration (A) increased the peptide levels of PKCq by 47.44 F18.5% ( P < 0.05, n = 5) in the CCV fraction while (B) peptides levels of PKCg were not altered (n = 5). Each independent experiment was conducted in

duplicate using CCV fractions prepared from pooled cerebral cortices of 8 rats per group.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 219

cluding different sensitivities to zolpidem, Ro15-4513, flu-

mazenil, and bretanzenil (Wafford et al., 1996; Whittemore

et al., 1996). Ethanol has been shown to alter GABAergic

synaptic transmission in both the hippocampus (Weiner et

al., 1994) and the amygdala (Roberto et al., 2003). Chronic

ethanol exposure in vivo has been shown to alter the

function of GABAA receptors measured by Cl� flux in

synaptoneurosomes and single-unit recording (Grobin et al.,

1998). However, neither Cl � flux assays nor assays of

single-unit activity in vivo can discriminate between

changes in synaptic versus extrasynaptic GABAA receptor

function. To date, only one study has specifically addressed

the effects of chronic ethanol exposure on synaptic GABAA

receptors by investigation of miniature inhibitory postsyn-

aptic current (mIPSC). mIPSC are postsynaptic currents that

occur in response to spontaneous fusion of a single vesicle

of GABA. Because the synaptic machinery precisely con-

trols the application and removal of agonist, changes in the

kinetics of the postsynaptic receptor response to GABA are

reflected in the changes in the shape of the mIPSC. Cagetti

et al. (2003) found changes in synaptic GABAA receptor

function in the hippocampus of rats that had undergone CIE

treatment followed by 2 days of withdrawal. CIE treatment

decreased the frequency, rise time, amplitude, and decay

time of mIPSCs in pyramidal neurons from CA1, an overall

change in GABAA receptor function that is consistent with

increased neuronal excitability. Interestingly, CIE eliminated

the effects of diazepam on the mIPSC and increased mIPSC

sensitivity to the a4-subunit-selective benzodiazepine Ro15-

4513. These pharmacological changes are consistent with a

decreased expression of the a1-subunit-containing (type 1)

receptors and an increased expression of the a4-subunit-

containing receptors in the synapse. In another study, Hsu et

al. (2003) demonstrated that brief (48- to 72-hr) exposure to

the neurosteroid 3a,5a-THP decreases the decay time of

mIPSCs recorded in hippocampal CA1 pyramidal cells.

mIPSCs from 3a,5a-THP-treated rats were also insensitive

to diazepam and more sensitive to Ro15-4513. The decrease

in mIPSC decay time was blocked by infusion of a4-subunit

antisense oligonucleotides into the hippocampus, suggesting

that this change in synaptic GABAA receptor kinetics is due

to an increase in synaptic a4-subunit expression. Taken

together, these studies suggest that translocation of a4-

subunit receptors from an extrasynaptic to synaptic locali-

zation may represent a mechanism of GABAA receptor

adaptation to both ethanol and neurosteroid exposure. Since

systemic ethanol administration increases GABAergic neu-

rosteroid concentrations in brain (Morrow et al., 1999;

VanDoren et al., 2000; Morrow et al., 2003), this adaptation

to ethanol may involve a neurosteroid intermediary.

4.5. Regulation of c-aminobutyric acidA receptors

by other receptors via intracellular signaling systems

As our understanding of cellular signaling pathways has

advanced, it has become increasingly apparent that, intracel-

lular signal transduction pathways are activated by many

postsynaptic receptors that, in turn, alter the function of other

postsynaptic receptors. Furthermore, it is known that differ-

ent intracellular signal transduction pathways can interact

through the mutual regulation of kinases and phosphatases to

provide an additional layer of complexity to their regulation

(Dombradi et al., 2002). Intracellular communication be-

tween receptors is critical for the coordinated function of

cells. The cross-talk between receptors might result from

intracellular signaling pathways involving inositol triphos-

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226220

phate (IP3), Ca2+ , PKC, PKA, and/or protein phosphatases.

There are several examples of interactions among muscarinic

acetylcholine (mACh), N-methyl-D-aspartate (NMDA), se-

rotonin (5-HT), and h-adrenergic receptors with GABAA

receptors. For example, mACh receptor activation decreases

GABA-mediated currents in cultured superior cervical gan-

glion neurons, and this effect is blocked by PKC inhibitors

(Brandon et al., 2002). Furthermore, chronic ethanol admin-

istration alters the function and/or expression of many of the

receptors and kinases involved in cross-talk with GABAA

receptors. Therefore, it is essential to understand the inter-

actions of GABAA receptors with other receptors for a clear

understanding of GABAA receptor adaptations following

chronic ethanol administration.

Chronic ethanol exposure up-regulates NMDA receptor

function and down-regulates GABAA receptor function (see

Crews et al., 1996, for a review). Since many neurons

contain both NMDA and GABAA receptors and ethanol

modulates both NMDA and GABAA receptors, interactions

between these receptors may play an important role in

GABAA receptor adaptation following ethanol administra-

tion. For example, MK-801 administration to rats, like

ethanol, increases the membrane expression of GABAA

receptor a4-subunits in the hippocampus (Matthews et al.,

1998b). The effect on GABAA receptors following NMDA

receptor activation is mediated by intracellular Ca2+ , since

the Ca2+ chelator (BAPTA) blocks the down-regulation of

GABAA receptors following NMDA receptor activation

(Robello et al., 1997). In addition, the application of

glutamate in hippocampal cultures leads to translocation

of PKCa and g to the plasma membrane and cytoplasmic

organelles, respectively, while PKCq localization remains

unaltered (Etoh et al., 1991; Buchner et al., 1999). There-

fore, interactions of NMDA and GABAA receptors via

intracellular signaling pathways may play a vital role in

GABAA receptor adaptation following chronic ethanol

administration. In addition, both NMDA and GABAA

receptor activation, as well as ethanol administration alter

brain-derived neurotrophic factor (BDNF) expression in

neurons (Zafra et al., 1991; MacLennan et al., 1995;

Tapia-Arancibia et al., 2001; Obrietan et al., 2002). BDNF

has been shown to alter GABAA receptor function and

expression. For example, addition of BDNF in cortical cell

cultures increases phosphorylation of GABAA receptor h3-

subunits (Jovanovic et al., 2000) and increases internaliza-

tion of GABAA receptor a2-, h2/3-, and g2-subunits (Brunig

et al., 2001). In contrast, chronic application of BDNF

increases expression of GABAA receptor a6-subunit in

cerebellar granular cells that is blocked by concomitant

application of ethanol (Ericson et al., 2003). Therefore, it is

possible that ethanol-induced altered trafficking and ex-

pression of GABAA receptors are partially mediated by

BDNF. Future studies on interactions of BDNF and

GABAA receptor following chronic ethanol exposure are

needed to clarify the role of BDNF on ethanol-induced

adaptation of GABAA receptors.

Likewise, 5-HT receptors can modulate GABAA receptor

function and expression via PKC-mediated pathways. Acti-

vation of 5-HT2 receptor results in inhibition of GABAA

receptor-mediated currents that can be blocked by the Ca2 +

chelator (BAPTA) and the RACK inhibitory peptide

(RACKI-rVI) (Feng et al., 2001). In addition, 5-HT1A

receptor knock-out mice exhibit down-regulation of

GABAA receptor a2-subunit mRNA levels and up-regula-

tion of a1-subunit mRNAs in both the cortex and the

amygdala, while a4-subunit mRNAs are decreased in the

hippocampus (Sibille et al., 2000). Hence, it is clear that

activation of 5-HT receptors can alter GABAA receptor

function and expression. In addition, recent studies have

concluded that dopamine and h-adrenergic (Flores-Hernan-

dez et al., 2000) receptors can also modulate GABAA

receptors via PKA-mediated pathways. Taken together, it

is obvious that receptor cross-talk plays a vital role in

modulation of GABAA receptor function and expression.

Therefore, a clear understanding of these interactions be-

tween receptors is critical for understanding the mechanisms

that underlie ethanol tolerance and dependence.

4.6. Role of neurosteroids in ethanol tolerance and

dependence

Ethanol tolerance results in a reduction in the anxiolytic,

motor-incoordinating, and sedative-hypnotic effects of eth-

anol and a requirement for higher ethanol doses to elicit

pharmacologic effects (Boisse & Okamoto, 1980; Le et al.,

1986; Brown et al., 1988). There is some evidence indicat-

ing that alterations in the effects of ethanol on neurosteroid

levels may play a role in the development or maintenance of

ethanol tolerance. Although acute systemic ethanol admin-

istration elevates 3a,5a-THP levels in the brain, chronic

ethanol consumption does not alter steady-state 3a,5a-THP

levels (Janis et al., 1998). Indeed, ethanol-dependent male

rats show decreased levels of 3a,5a-THP in the cerebral

cortex as compared with pair-fed controls, while ethanol-

withdrawn animals showed a return of cortical 3a,5a-THP

to baseline levels (Janis et al., 1998). Furthermore, ethanol-

dependent and -withdrawn animals given a moderate etha-

nol challenge showed reduced levels of ethanol-induced

3a,5a-THP in the cerebral cortex as compared with etha-

nol-naive rats (Morrow et al., 2001b). Hence, tolerance to

ethanol may involve a loss of the ability of ethanol to

increase 3a,5a-THP levels and a corresponding reduction in

related pharmacological effects of ethanol. The loss of the

neurosteroid response to ethanol may contribute to the

changes in GABAA receptor function, expression, and

subcellular localization that are associated with the devel-

opment of ethanol tolerance (Grobin et al., 1998). There-

fore, the interplay of the neuromodulating and genomic

effects of 3a,5a-THP may play a key role in adaptations

to ethanol.

Changes in the sensitivity to neurosteroids are also

associated with the development of ethanol dependence.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 221

Administration of 3a,5a-THP blocks the decrease in bicu-

culline-induced seizure threshold seen in ethanol-withdrawn

rats at doses of 3a,5a-THP that do not increase seizure

threshold in pair-fed control rats (Devaud et al., 1995a).

Indeed, there is a dramatic sensitization to the anticonvul-

sant effects of 3a,5a-THP and 3a,5a-THDOC in ethanol-

dependent rats (Devaud et al., 1996, 1998). This behavioral

sensitization to neurosteroids is accompanied by an increase

in sensitivity of GABAA receptors to 3a,5a-THP and

3a,5a-THDOC in cerebral cortical synaptoneurosomes us-

ing a Cl � flux assay (Devaud et al., 1996) and is not due to

different endogenous levels of 3a,5a-THP in withdrawn

versus control rats (Janis et al., 1998). Furthermore, neuro-

steroid sensitization during withdrawal is accompanied by

an increase in GABAA receptor a4-, g1-, h2-, and h3-subunit

mRNA and peptides. These subunits have been shown to

maintain or increase sensitivity of GABAA receptors to

neurosteroid in recombinant expression systems (Puia et

al., 1990, 1991).

The relationship between neurosteroid levels and the

sensitivity of GABAA receptors to neurosteroids in etha-

nol-dependent rats remains controversial. Although one

study found no change in endogenous 3a,5a-THP levels

in the cortex or plasma of ethanol-withdrawn rats (Janis et

al., 1998), other studies have reported decreases in neuro-

steroid levels in ethanol-withdrawing mice and humans.

Plasma 3a,5a-THP levels were decreased in ethanol-with-

drawn B6 and D2 mice, relative to controls (Finn et al.,

2000). Interestingly, the B6 mice showed a decrease in

3a,5a-THP levels of 15%, whereas there was a 50%

decrease in D2 mice. The greater reduction in endogenous

neurosteroid in withdrawn D2 mice could contribute to the

greater withdrawal severity experienced by those animals. In

alcoholics, decreased levels of both 3a,5a-THP and 3a,5a-

THDOC have been reported during the early phase of

ethanol withdrawal (Romeo et al., 1996). This early phase

of ethanol withdrawal is characterized by increases in

anxiety and depression that can be measured by psycholog-

ical tests. Later in withdrawal, when the anxiety and

depression disappear, neurosteroid levels also return to

normal. This study suggests that reduced levels of endog-

enous GABAergic neurosteroids contribute to the negative

consequences of ethanol withdrawal.

Withdrawal from progesterone and associated decreases

in 3a,5a-THP result in behavioral changes that mimic the

signs of ethanol withdrawal. Specifically, progesterone-

withdrawn female rats exhibit increased anxiety (Gallo &

Smith, 1993), increased sensitivity to convulsant drugs that

act on GABAA receptors (Smith et al., 1998a), and de-

creased sensitivity to the sedative effects of benzodiazepines

(Moran & Smith, 1998). Whole-cell patch clamp electro-

physiology in dissociated neurons from CA1 of the hippo-

campus showed that the behavioral changes were

accompanied by changes in the function of GABAA recep-

tors associated with increases in a4-subunit peptide and

mRNA during progesterone withdrawal (Smith et al., 1998a,

1998b). Furthermore, blocking this increase by infusion of

antisense a4 oligonucleotides into the hippocampus during

progesterone withdrawal eliminates the increase in seizure

sensitivity (Smith et al., 1998a), behavioral tolerance to

benzodiazepines (Moran et al., 1998), and the change in

electrophysiological properties of GABAA receptors (Smith

et al., 1998a). Therefore, in this model, it seems that

withdrawal from exposure to progesterone and 3a,5a-THP

causes a change in GABAA receptor subunit expression that

results in the changes in GABAA receptor function that are

responsible for the behavioral signs of withdrawal.

Ethanol- and progesterone-withdrawing animals exhibit

similar withdrawal signs, such as increased anxiety, in-

creased seizure susceptibility, and decreased sensitivity to

benzodiazepines. These changes occur at the same time as

increases in a4-subunit mRNA (Devaud et al., 1995b) and

peptide levels (Devaud et al., 1997) in the cerebral cortices

and hippocampus (Cagetti et al., 2003) of ethanol-dependent

rats. These adaptations are associated with alterations in

GABAA receptor function, including reduced sensitivity to

GABA and benzodiazepines and increased sensitivity to

inverse agonists (Table 2). Therefore, it is possible that

changes in endogenous neurosteroid levels that occur during

chronic ethanol exposure and/or withdrawal contribute to

the changes in GABAA receptor function that underlie

ethanol dependence. However, ethanol withdrawal is asso-

ciated with different changes in GABAA receptor expression

and function that were not reported following progesterone

withdrawal, including changes in a1–3-, h2/3-, and g1–2-

subunit expression and sensitization to 3a,5a-THP (Devaud

et al., 1996, 1997). Moreover, ethanol-dependent and with-

drawn animals are tolerant to ethanol, and progesterone-

withdrawn rats are more sensitive to ethanol (Sundstrom-

Poromaa et al., 2002). Therefore, further studies are needed

to determine the role of neurosteroids in ethanol-induced

adaptations of GABAA receptors.

5. Conclusions

Recent studies have clearly demonstrated that adapta-

tions in GABAA receptors play an important role in ethanol

dependence. However, the exact mechanisms that account

for alterations in GABAA receptor function following

chronic ethanol administration have not been resolved.

The molecular determinants of these alterations in GABAA

receptor function are complex, probably due to the hetero-

geneity of GABAA receptor subtypes and the diversity of

biochemical mechanisms that regulate the function of these

receptors. GABAA receptors are a primary target for the

actions of ethanol in the brain. Chronic ethanol administra-

tion consistently alters the mRNA levels and membrane

expression of various GABAA receptor subunits, suggesting

that alterations in GABAA receptor expression may account

for alterations in GABAA receptor function. In addition,

chronic ethanol can modulate GABAA receptor functions by

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226222

altering post-transcriptional modifications of receptor sub-

units, subcellular localization of receptors, and/or interac-

tions with other neurotransmitter systems. Endogenous

regulators like neurosteroids and signaling proteins also

play a role in modulating GABAA receptor function follow-

ing chronic ethanol administration. Therefore, it is likely

that multiple mechanisms are responsible for adaptation of

GABAA receptors following chronic ethanol exposure.

Acute ethanol administration potentiates GABA-mediat-

ed inhibition both in vitro and in vivo, but does not alter

GABAA receptor subunit expression. Hence, the effect of

acute ethanol exposure on GABAA receptors is probably

primarily due to intracellular signaling systems that may

include neurosteroids and/or post-translational receptor

modifications. Since post-translational modification is a

short-lived effect, receptors can revert to normal immedi-

ately and the whole process requires less energy and time

compared with long-lasting changes in subunit expression

following prolonged ethanol exposure. Prolonged ethanol

exposure results in long-lasting changes in GABAA receptor

subunit expression on the cell surface that could be due to

altered receptor trafficking, subcellular or synaptic localiza-

tion of receptors, altered steroid responses to ethanol, and

possibly altered composition of GABAA receptors. These

changes clearly involve intracellular signaling cascades,

although the exact sequence of adaptations is yet unknown.

Taken together, it is clear that ethanol regulates GABAA

receptor function via both genomic and nongenomic mech-

anisms that interact to produce the complex adaptations in

brain resulting in tolerance and dependence upon ethanol.

Acknowledgments

This work was supported by NIH grants AA 11605 and

AA 09013. We thank Brooke Schwildwacter and Todd

O’Buckley for excellent laboratory assistance.

References

Aguayo, L. G., Pancetti, F. C., Klein, R. L., & Harris, R. A. (1994).

Differential effects of GABAergic ligands in mouse and rat hippocam-

pal neurons. Brain Res 647, 97–105.

Allan, A. M., & Harris, R. A. (1987). Acute and chronic ethanol treatments

alter GABA receptor-operated chloride channels. Pharmacol Biochem

Behav 27, 665–670.

Bao, J., Alroy, I., Waterman, H., Schejter, E. D., Brodie, C., Gruenberg, J.,

& Yarden, Y. (2000). Threonine phosphorylation diverts internalized

epidermal growth factor receptors from a degradative pathway to the

recycling endosome. J Biol Chem 275, 26178–26186.

Barbaccia, M. L., Affricano, D., Trabucchi, M., Purdy, R. H., Colombo, G.,

Agabio, R., & Gessa, G. L. (1999). Ethanol markedly increases

‘‘GABAergic’’ neurosteroids in alcohol-preferring rats. Eur J Pharma-

col 384, R1–R2.

Barmack, N. H., Qian, Z., & Yoshimura, J. (2000). Regional and cellular

distribution of protein kinase C in rat cerebellar Purkinje cells. J Comp

Neurol 427, 235–254.

Bencsits, E., Ebert, V., Tretter, V., & Sieghart, W. (1999). A significant

part of native gamma-aminobutyric acidA receptors containing alpha4

subunits do not contain gamma or delta subunits. J Biol Chem 274,

19613–19616.

Boisse, N. N., & Okamoto, M. (1980). Ethanol as a sedative-hypnotic:

comparison with barbiturate and non-barbiturate sedative-hypnotics.

In H. Rigter, & J. C. Crabbe (Eds.), Alcohol Tolerance and Dependence

( pp. 265–292). Amsterdam: Elsevier.

Bowers, B. J., Elliott, K. J., & Wehner, J. M. (2001). Differential sensitivity

to the anxiolytic effects of ethanol and flunitrazepam in PKCgamma

null mutant mice. Pharmacol Biochem Behav 69, 99–110.

Brandon, N. J., Uren, J. M., Kittler, J. T., Wang, H., Olsen, R., Parker, P.

J., & Moss, S. J. (1999). Subunit specific association of protein kinase

C and the receptor for activated C kinase with GABA type A receptors.

J Neurosci 19, 9228–9234.

Brandon, N. J., Delmas, P., Kittler, J. T., McDonald, B. J., Sieghart, W.,

Brown, D. A., Smart, T. G., & Moss, S. J. (2000). GABAA receptor

phosphorylation and functional modulation in cortical neurons by a

protein kinase C-dependent pathway. J Biol Chem 275, 38856–38862.

Brandon, N. J., Jovanovic, J. N., Smart, T. G., & Moss, S. J. (2002).

Receptor for activated C kinase-1 facilitates protein kinase C-depen-

dent phosphorylation and functional modulation of GABA(A) recep-

tors with the activation of G-protein-coupled receptors. J Neurosci 22,

6353–6361.

Brandon, N. J., Jovanovic, J. N., Colledge, M., Kittler, J. T., Brandon, J. M.,

Scott, J. D., & Moss, S. J. (2003). A-kinase anchoring protein 79/150

facilitates the phosphorylation of GABA(A) receptors by cAMP-depen-

dent protein kinase via selective interaction with receptor beta subunits.

Mol Cell Neurosci 22, 87–97.

Brown, M. E., Anton, R. F., Malcolm, R., & Ballenger, J. C. (1988).

Alcohol detoxification and withdrawal seizures: clinical support for a

kindling hypothesis. Biol Psychiatry 23, 507–514.

Brunig, I., Penschuck, S., Berninger, B., Benson, J., & Fritschy, J. M.

(2001). BDNF reduces miniature inhibitory postsynaptic currents by

rapid downregulation of GABAA receptor surface expression. Eur J

Neurosci 13, 1320–1328.

Buchner, K., Adamec, E., Beermann, M. L., & Nixon, R. A. (1999). Iso-

form-specific translocation of protein kinase C following glutamate

administration in primary hippocampal neurons. Brain Res Mol Brain

Res 64, 222–235.

Buck, K. J., & Harris, R. A. (1990). Benzodiazepine agonist and inverse

agonist actions on GABAA receptor-operated chloride channels: II.

Chronic effects of ethanol. J Pharmacol Exp Ther 253, 713–719.

Cagetti, E., Liang, J., Spigelman, I., & Olsen, R. W. (2003). Withdrawal

from chronic intermittent ethanol treatment changes subunit composi-

tion, reduces synaptic function, and decreases behavioral responses to

positive allosteric modulators of GABAA receptors. Mol Pharmacol 63,

53–64.

Celentano, J. J., Gibbs, T. T., & Farb, D. H. (1988). Ethanol potentiates

GABA- and glycine-induced chloride currents in chick spinal cord

neurons. Brain Res 455, 377–380.

Chapell, R., Bueno, O. F., Alvarez-Hernandez, X., Robinson, L. C., &

Leidenheimer, N. J. (1998). Activation of protein kinase C induces

gamma-aminobutyric acid type A receptor internalization in Xenopus

oocytes. J Biol Chem 273, 32595–32601.

Chen, L., Wang, H., Vicini, S., & Olsen, R. W. (2000). The gamma-amino-

butyric acid type A (GABAA) receptor-associated protein (GABARAP)

promotes GABAA receptor clustering and modulates the channel kinet-

ics. Proc Natl Acad Sci USA 97, 11557–11562.

Churn, S. B., & DeLorenzo, R. J. (1998). Modulation of GABAergic re-

ceptor binding by activation of calcium and calmodulin-dependent ki-

nase II membrane phosphorylation. Brain Res 809, 68–76.

Churn, S. B., Rana, A., Lee, K., Parsons, J. T., De Blas, A., & Delorenzo,

R. J. (2002). Calcium/calmodulin-dependent kinase II phosphorylation

of the GABAA receptor alpha1 subunit modulates benzodiazepine bind-

ing. J Neurochem 82, 1065–1076.

Cinar, H., & Barnes Jr., E. M. (2001). Clathrin-independent endocytosis of

GABA(A) receptors in HEK 293 cells. Biochemistry 40, 14030–14036.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 223

Connolly, C. N., Kittler, J. T., Thomas, P., Uren, J. M., Brandon, N. J.,

Smart, T. G., & Moss, S. J. (1999). Cell surface stability of gam-

ma-aminobutyric acid type A receptors: dependence on protein

kinase C activity and subunit composition. J Biol Chem 274,

36565–36572.

Crews, F., Morrow, A. L., Criswell, H., & Breese, G. (1996). Effects of

ethanol on ion channels. In R. J. Bradley, & R. A. Harris (Eds.), Inter-

national Review of Neurobiology, vol. 39 (pp. 283–367). New York:

Academic Press.

Criswell, H. E., Simson, P. E., Johnson, K. B., & Breese, G. R. (1993).

Chronic ethanol decreases the ability of GABA to inhibit ethanol-sen-

sitive neurons in the medial septum. Alcohol Clin Exp Res 17, 477.

Devaud, L. L., Purdy, R. H., & Morrow, A. L. (1995a). The neurosteroid,

3a-hydroxy-5a-pregnan-20-one, protects against bicuculline-induced

seizures during ethanol withdrawal in rats. Alcohol Clin Exp Res 19,

350–355.

Devaud, L. L., Smith, F. D., Grayson, D. R., & Morrow, A. L. (1995b).

Chronic ethanol consumption differentially alters the expression of g-

aminobutyric acid type A receptor subunit mRNAs in rat cerebral cor-

tex: competitive, quantitative reverse transcriptase-polymerase chain

reaction analysis. Mol Pharmacol 48, 861–868.

Devaud, L. L., Purdy, R. H., Finn, D. A., & Morrow, A. L. (1996). Sen-

sitization of g-aminobutyric acidA receptors to neuroactive steroids in

rats during ethanol withdrawal. J Pharmacol Exp Ther 278, 510–517.

Devaud, L. L., Fritschy, J. -M., Sieghart, W., & Morrow, A. L. (1997).

Bidirectional alterations of GABAA receptor subunit peptide levels in

rat cortex during chronic ethanol consumption and withdrawal. J Neuro-

chem 69, 126–130.

Devaud, L. L., Fritschy, J. -M., & Morrow, A. L. (1998). Influence of

gender on chronic ethanol-induced alternations of GABAA receptors

in rats. Brain Res 796, 222–230.

Dohrman, D. P., Chen, H. M., Gordon, A. S., & Diamond, I. (2002).

Ethanol-induced translocation of protein kinase A occurs in two phases:

control by different molecular mechanisms. Alcohol Clin Exp Res 26,

407–415.

Dombradi, V., Krieglstein, J., & Klumpp, S. (2002). Regulating the regu-

lators. Conference on protein phosphorylation and protein phospha-

tases. EMBO Rep 3, 120–124.

Ericson, M., Haythornthwaite, A. R., Yeh, P. W., & Yeh, H. H. (2003).

Brain-derived neurotrophic factor mitigates chronic ethanol-induced at-

tenuation of gamma-aminobutyric acid responses in cultured cerebellar

granule cells. J Neurosci Res 73, 722–730.

Etoh, S., Baba, A., & Iwata, H. (1991). NMDA induces protein kinase C

translocation in hippocampal slices of immature rat brain. Neurosci Lett

126, 119–122.

Fancsik, A., Linn, D. M., & Tasker, J. G. (2000). Neurosteroid modula-

tion of GABA IPSCs is phosphorylation dependent. J Neurosci 20,

3067–3075.

Feng, J., Cai, X., Zhao, J., & Yan, Z. (2001). Serotonin receptors modulate

GABA(A) receptor channels through activation of anchored protein

kinase C in prefrontal cortical neurons. J Neurosci 21, 6502–6511.

Finn, D. A., Gallaher, E. J., & Crabbe, J. C. (2000). Differential change in

neuroactive steroid sensitivity during ethanol withdrawal. J Pharmacol

Exp Ther 292, 394–405.

Flores-Hernandez, J., Hernandez, S., Snyder, G. L., Yan, Z., Fienberg,

A. A., Moss, S. J., Greengard, P., & Surmeier, D. J. (2000). D(1)

dopamine receptor activation reduces GABA(A) receptor currents in

neostriatal neurons through a PKA/DARPP-32/PP1 signaling cas-

cade. J Neurophysiol 83, 2996–3004.

Freund, R. K., & Palmer, M. R. (1997). Beta adrenergic sensitization of

gamma-aminobutyric acid receptors to ethanol involves a cyclic AMP/

protein kinase A second-messenger mechanism. J Pharmacol Exp Ther

280, 1192–1200.

Frye, G. D., Chapin, R. E., Vogel, R. A., Mailman, R. B., Kilts, C. D.,

Mueller, R. A., & Breese, G. R. (1981). Effects of acute and chronic

1,3-butanediol treatment on central nervous system function: a compar-

ison with ethanol. J Pharmacol Exp Ther 216, 306–314.

Frye, G. D., Mathew, J., & Trezciakowski, J. (1993). Effect of ethanol

dependence on GABAA antagonist-induced seizures and agonist-stim-

ulated chloride uptake. Alcohol 8, 453–459.

Frye, G. D., Fincher, A. S., Grover, C. A., & Griffith, W. H. (1994).

Interaction of ethanol and allosteric modulators with GABAA-activated

currents in adult medial septum/diagonal band neurons. Brain Res 635,

283–292.

Gallo, M. A., & Smith, S. S. (1993). Progesterone withdrawal decreases

latency to and increases duration of electrified prod burial: a possible rat

model of PMS anxiety. Pharmacol Biochem Behav 46, 897–904.

Ghansah, E., & Weiss, D. S. (2001). Modulation of GABA(A) receptors by

benzodiazepines and barbiturates is autonomous of PKC activation.

Neuropharmacology 40, 327–333.

Givens, B. S., & Breese, G. R. (1990). Site-specific enhancement of g-

aminobutyric acid-mediated inhibition of neural activity by ethanol in

the rat medial septum. J Pharmacol Exp Ther 254, 528–538.

Givens, B. S., & McMahon, K. (1997). Effects of ethanol on nonspatial

working memory and attention in rats. Behav Neurosci 111, 275–282.

Gonzalez, L. P., & Czachura, J. F. (1989). Reduced behavioral responses to

intranigral muscimol following chronic ethanol. Physiol Behav 46,

473–477.

Grobin, A. C., Matthews, D. B., Devaud, L. L., & Morrow, A. L. (1998).

The role of GABAA receptors in the acute and chronic effects of eth-

anol. Psychopharmacology 139, 2–19.

Grobin, A. C., Fritschy, J. -M., & Morrow, A. L. (2000). Chronic ethanol

administration alters immunoreactivity for GABAA receptor subunits

in rat cortex in a region-specific manner. Alcohol Clin Exp Res 24,

1137–1144.

Harris, R. A., McQuilkin, S. J., Paylor, R., Abeliovich, A., Tonegawa, S., &

Wehner, J. M. (1995). Mutant mice lacking the gamma isoform of

protein kinase C show decreased behavioral actions of ethanol and

altered functions of gamma-aminobutyrate type A receptors. Proc Natl

Acad Sci USA 92, 3658–3662.

Harris, R. A., Mihic, S. J., & Valenzuela, C. F. (1998). Alcohol and

benzodiazepines: recent mechanistic studies. Drug Alcohol Depend

51, 155–164.

Herring, D., Huang, R., Singh, M., Robinson, L. C., Dillon, G. H., &

Leidenheimer, N. J. (2003). Constitutive GABAA receptor endocytosis

is dynamin-mediated and dependent on a dileucine AP2 adaptin-bind-

ing motif within the h2 subunit of the receptor. J Biol Chem 278,

24046–24052.

Heuschneider, G., & Schwartz, R. D. (1989). cAMP and forskolin decrease

g-aminobutyric acid-gated chloride flux in rat brain synaptoneuro-

somes. Proc Natl Acad Sci USA 86, 2938–2942.

Hirst, J., & Robinson, M. S. (1998). Clathrin and adaptors. Biochim Bio-

phys Acta 1404, 173–193.

Hodge, C. W., Mehmert, K. K., Kelley, S. P., McMahon, T., Haywood, A.,

Olive, M. F., Wang, D., Sanchez-Perez, A. M., & Messing, R. O.

(1999). Supersensitivity to allosteric GABAA receptor modulators and

alcohol in mice lacking PKCq. Nat Neurosci 2, 997–1002.Hsu, F. C., Waldeck, R., Faber, D. S., & Smith, S. S. (2003). Neurosteroid

effects on GABAergic synaptic plasticity in hippocampus. J Neurophy-

siol 89, 1929–1940.

Huang, R. Q., & Dillon, G. H. (1998). Maintenance of recombinant type A

gamma-aminobutyric acid receptor function: role of protein tyrosine

phosphorylation and calcineurin. J Pharmacol Exp Ther 286, 243–255.

Janis, G. C., Devaud, L. L., Mitsuyama, H., & Morrow, A. L. (1998).

Effects of chronic ethanol consumption and withdrawal on the neuro-

active steroid 3a-hydroxy-5a-pregnan-20-one in male and female rats.

Alcohol Clin Exp Res 22, 2055–2061.

Johnston, J. D., Price, S. A., & Bristow, D. R. (1998). Flunitrazepam

rapidly reduces GABAA receptor subunit protein expression via a

protein kinase C-dependent mechanism. Br J Pharmacol 124,

1338–1340.

Jovanovic, J. N., Kittler, J. T., Brandon, N. J., & Moss, S. J. (2000).

Characterization of the specificity in situ of the phosphorylation state-

specific antibody for GABAA receptor beta3 subunit (Program Number

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226224

522.1). 2000 Abstract Viewer/Itinerary Planner. Washington, DC:

Society for Neuroscience.

Kang, M. H., Spigelman, I., & Olsen, R. W. (1998). Alteration in the

sensitivity of GABAA receptors to allosteric modulatory drugs in rat

hippocampus after chronic intermittent ethanol treatment. Alcohol Clin

Exp Res 22, 2165–2173.

Kannenberg, K., Schaerer, M. T., Fuchs, K., Sieghart, W., & Sigel, E.

(1999). A novel serine kinase with specificity for h3-subunits is tightlyassociated with GABAA receptors. J Biol Chem 274, 21257–21264.

Kano, M., & Konnerth, A. (1992). Potentiation of GABA-mediated currents

by cAMP-dependent protein kinase. NeuroReport 3, 563–566.

Kano, M., Rexhausen, U., Dreessen, J., & Konnerth, A. (1992). Synaptic

excitation produces a long-lasting rebound potentiation of inhibitory

synaptic signals in cerebellar Purkinje cells. Nature 356, 601–604.

Kellenberger, S., Malherbe, P., & Sigel, E. (1992). Function of the a1h2g2sg-aminobutyric acid typeA receptor is modulated by protein kinase C

via multiple phosphorylation sites. J Biol Chem 267, 25660–25663.

Kikkawa, U., Kishimoto, A., & Nishizuka, Y. (1989). The protein kinase C

family: heterogeneity and its implications.AnnuRev Biochem58, 31–44.

Kittler, J. T., Delmas, P., Jovanovic, J. N., Brown, D. A., Smart, T. G., &

Moss, S. J. (2000). Constitutive endocytosis of GABAA receptors by an

association with the adaptin AP2 complex modulates inhibitory synap-

tic currents in hippocampal neurons. J Neurosci 20, 7972–7977.

Klein, R. L., Mascia, M. P., Whiting, P. J., & Harris, R. A. (1995). GABAA

receptor function and binding in stably transfected cells: chronic ethanol

treatment. Alcohol Clin Exp Res 19, 1338–1344.

Kralic, J. E., Korpi, E. R., O’Buckley, T. K., Homanics, G. E., & Morrow,

A. L. (2002a). Molecular and pharmacological characterization of

GABAA receptor a1 subunit knockout mice. J Pharmacol Exp Ther

302, 1037–1045.

Kralic, J. E., O’Buckley, T. K., Khisti, R. T., Hodge, C. W., Homanics,

G. E., & Morrow, A. L. (2002b). GABAA receptor a1 subunit

deletion alters receptor subtype assembly, pharmacological and be-

havioral responses to benzodiazepines and zolpidem. Neuropharma-

cology 43, 685–694.

Krishek, B. J., Xie, X., Blackstone, C., Huganir, R. L., Moss, S. J., &

Smart, T. G. (1994). Regulation of GABAA receptor function by protein

kinase C phosphorylation. Neuron 12, 1081–1095.

Kumar, S., Sieghart, W., & Morrow, A. L. (2002). Association of protein

kinase C with GABAA receptors containing a1 and a4 subunits in the

cerebral cortex: selective effects of chronic ethanol consumption.

J Neurochem 82, 110–117.

Kumar, S., Khisti, R. T., & Morrow, A. L. (2003a). PKC activity reduces

muscimol-stimulated 36Cl� uptake in cerebral cortical synaptoneuro-

somes and modulates the hypnotic effect of muscimol in rats (Program

Number 569.1). 2003 Abstract Viewer/Itinerary Planner. Washington,

DC: Society for Neuroscience.

Kumar, S., Kralic, J. E., O’Buckley, T. K., Grobin, A. C., & Morrow, A. L.

(2003b). Chronic ethanol consumption enhances internalization of al-

pha1 subunit-containing GABAA receptors in cerebral cortex. J Neuro-

chem 86, 700–708.

Le, A. D., Khanna, J. M., Kalant, H., & Grossi, F. (1986). Tolerance to and

cross-tolerance among ethanol, pentobarbital and chlordiazepoxide.

Pharmacol Biochem Behav 24, 93–98.

Leidenheimer, N. J., & Chapell, R. (1997). Effects of PKC activation and

receptor desensitization on neurosteroid modulation of GABAA recep-

tors. Brain Res Mol Brain Res 52, 173–181.

Leidenheimer, N. J., Browning, M. D., Dunwiddie, T. V., Hahner, L. D., &

Harris, R. A. (1990). Phosphorylation-independent effects of second

messenger system modulators on gamma-aminobutyric acidA receptor

complex function. Mol Pharmacol 38, 823–828.

Leidenheimer, N. J., McQuiklin, S. J., Hahner, L. D., Whiting, P., & Harris,

R. A. (1992). Activation of protein kinase C selectively inhibits the g-

aminobutyric acidA receptor: role of desensitization.Mol Pharmacol 41,

1116–1123.

Lin, Y. F., Angelotti, T. P., Dudek, E. M., Browning, M. D., & MacDon-

ald, R. L. (1996). Enhancement of recombinant a1h1g2L g- amino-

butyric acidA receptor whole-cell currents by protein kinase C is

mediated through phosphorylation of both h1 and g2L subunits.

Mol Pharmacol 50, 184–195.

Lodish, H., Baltimore, D., Berk, A., Zipursky, S. L., Matsuaira, P., &

Darnell, J. (1996). Synthesis and sorting of plasma membrane, se-

cretory, and lysosomal proteins. Molecular Cell Biology (3rd ed.)

(pp. 711–715). New York: Freeman.

Macdonald, R. L. (1995). Ethanol, g-aminobutyrate type A receptors,

and protein kinase C phosphorylation. Proc Natl Acad Sci USA 92,

3633–3635.

MacLennan, A. J., Lee, N., & Walker, D. W. (1995). Chronic ethanol

administration decreases brain-derived neurotrophic factor gene expres-

sion in the rat hippocampus. Neurosci Lett 197, 105–108.

Mahmoudi, M., Kang, M. H., Tillakarartne, N., Tobin, A. J., & Olsen, R.

W. (1997). Chronic intermittent ethanol treatment in rats increases

GABAA receptor a4-subunit expression: possible relevance to alcohol

dependence. J Neurochem 68, 2485–2492.

Majchrowicz, E. (1975). Induction of physical dependence upon ethanol and

the associated behavioral changes. Psychopharmacologia 43, 245–254.

Marszalec, W., Aistrup, G. L., & Narahashi, T. (1998). Ethanol modulation

of excitatory and inhibitory synaptic interactions in cultured cortical

neurons. Alcohol Clin Exp Res 22(7), 1516–1524.

Martz, A., Deitrich, R. A., & Harris, R. A. (1983). Behavioral evidence for

the involvement of gamma-aminobutyric acid in the actions of ethanol.

Eur J Pharmacol 89, 53–62.

Matthews, D. B., Simson, P. E., & Best, P. J. (1995). Acute ethanol impairs

spatial memory but not stimulus/response memory in the rat. Alcohol

Clin Exp Res 19, 902–909.

Matthews, D. B., Devaud, L. L., Fritschy, J. -M., Sieghart, W., & Morrow,

A. L. (1998a). Differential regulation of GABAA receptor gene expres-

sion by ethanol in the rat hippocampus vs. cerebral cortex. J Neurochem

70, 1160–1166.

Matthews, D. B., Devaud, L. L., Kralic, J. E., Fritschy, J. -M., Sieghart, W.,

& Morrow, A. L. (1998b). Chronic MK-801 administration alters

GABAA receptor subunit peptide expression and function in the rat.

Soc Neurosci Abstr 24, 1831.

McDonald, B. J., & Moss, S. J. (1997). Conserved phosphorylation of the

intracellular domains of GABAA receptor h2 and h3 subunits by

cAMP-dependent protein kinase, cGMP-dependent protein kinase, pro-

tein kinase C and Ca2 + /calmodulin type II-dependent protein kinase.

Neuropharmacology 36, 1377–1385.

McDonald, B. J., Amato, A., Connolly, C. N., Benke, D., Moss, S. J., &

Smart, T. G. (1998). Adjacent phosphorylation sites on GABAA recep-

tor h subunits determine regulation by cAMP- dependent protein ki-

nase. Nature Neurosci 1, 23–28.

Mehta, A. K., & Ticku, M. K. (1989). Chronic ethanol treatment alters the

behavioral effects of Ro 15-4513, a partially negative ligand for ben-

zodiazepine binding sites. Brain Res 489, 93–100.

Messing, R. O., Peterson, P. J., & Henrich, C. J. (1991). Chronic ethanol

exposure increases levels of protein kinase Cy and q and protein kinase

C-mediated phosphorylation in cultured neural cells. J Biol Chem 266,

23428–23432.

Mhatre, M. C., & Ticku, M. K. (1992). Chronic ethanol administration

alters g-aminobutyric acid A receptor gene expression. Mol Pharmacol

42, 415–422.

Mhatre, M. C., & Ticku, M. K. (1993). Chronic ethanol treatment down-

regulates GABAA receptor a subunit polypeptide expression in rat

cerebral cortex and cerebellum. Alcohol Clin Exp Res 17, 442.

Mihic, S. J., Ye, Q., Wick, M. J., Koltchine, V. V., Krasowski, M. A.,

Finn, S. E., Mascia, M. P., Valenzuela, C. F., Hanson, K. K., Green-

blatt, E. P., Harris, R. A., & Harrison, N. L. (1997). Sites of alcohol

and volatile anaesthetic action on GABAA and glycine receptors.

Nature 389, 385–389.

Mody, I. (2001). Distinguishing between GABA(A) receptors responsible

for tonic and phasic conductances. Neurochem Res 26, 907–913.

Mohler, H., Fritschy, J. -M., Luscher, B., Rudolph, U., Benson, J., &

Benke, D. (1996). The GABAA receptors: from subunits to diverse

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226 225

functions. In T. Narahashi (Ed.), Ion Channels, vol. 4 (pp. 89–111).

New York: Plenum.

Moran, M. H., & Smith, S. S. (1998). Progesterone withdrawal I: pro-

convulsant effects. Brain Res 807, 84–90.

Moran, M. H., Goldberg, N., & Smith, S. S. (1998). Progesterone with-

drawal: II. insensitivity to the sedative effects of a benzodiazepine.

Brain Res 807, 91–100.

Morrow, A. L., Suzdak, P. D., Karanian, J. W., & Paul, S. M. (1988).

Chronic ethanol administration alters g-aminobutyric acid, pentobarbi-

tal and ethanol-mediated 36Cl� uptake in cerebral cortical synaptoneur-

osomes. J Pharmacol Exp Ther 246, 158–164.

Morrow, A. L., Herbert, J. S., & Montpied, P. (1992). Differential effects of

chronic ethanol administration on GABAA receptor a1 and a6 subunit

mRNA levels in rat cerebellum. Mol Cell Neurosci 3, 251–258.

Morrow, A. L., VanDoren, M. J., & Devaud, L. L. (1998). Effects of

progesterone or neuroactive steroid? Nature 395, 652–653.

Morrow, A. L., Janis, G. C., VanDoren, M. J., Matthews, D. B., Samson, H.

H., Janak, P. H., & Grant, K. A. (1999). Neurosteroids mediate phar-

macological effects of ethanol: a new mechanism of ethanol action?

Alcohol Clin Exp Res 23, 1933–1940.

Morrow, A. L., Kumar, S., & Wehner, J. M. (2001a). Chronic ethanol

consumption increases endocytosis of a1 subunit containing GABAA

receptors in rat cortex by a PKC-dependent mechanism. ACNP 40th

Annual Meeting (p. 215). Waikoloa, HI: ACNP Secretariat.

Morrow, A. L., VanDoren, M. J., Penland, S. N., & Matthews, D. B.

(2001b). The role of GABAergic neuroactive steroids in ethanol

action, tolerance and dependence. Brain Res Brain Res Rev 37,

98–109.

Morrow, A. L., Khisti, R. T., Tokunaga, S., McDaniel, J. R., & Matthews,

D. B. (2003). GABAergic neuroactive steroids modulate selective eth-

anol actions: mechanisms and significance. In S. H. Smith (Ed.), Neuro-

steroid Effects in the Central Nervous System: The Role of the GABAA

Receptor ( pp. 219–245). Miami, FL: CRC Press.

Moss, S. J., Doherty, C. A., & Huganir, R. L. (1992a). Identification of the

cAMP-dependent protein kinase and protein kinase C phosphorylation

sites within the major intracellular domains of the h1, g2S and g2L

subunits of the g-aminobutyric acid type A receptor. J Biol Chem 267,

14470–14476.

Moss, S. J., Smart, T. G., Blackstone, C. D., & Huganir, R. L. (1992b).

Functional modulation of GABAA receptors by cAMP-dependent pro-

tein phosphorylation. Science 257, 661–665.

Narita, M., Tamaki, H., Kobayashi, M., Soma, M., & Suzuki, T. (2001).

Changes in Ca2+-dependent protein kinase C isoforms induced by

chronic ethanol treatment in mice. Neurosci Lett 307, 85–88.

Nusser, Z., Sieghart, W., & Mody, I. (1999). Differential regulation of

synaptic GABAA receptors by cAMP-dependent protein kinase in

mouse cerebellar and olfactory bulb neurones. J Physiol 521 (Pt 2),

421–435.

Nymann-Andersen, J.,Wang, H., Chen, L., Kittler, J. T.,Moss, S. J., &Olsen,

R. W. (2002). Subunit specificity and interaction domain between

GABA(A) receptor-associated protein (GABARAP) and GABA(A)

receptors. J Neurochem 80, 815–823.

Obrietan, K., Gao, X. B., & Van Den Pol, A. N. (2002). Excitatory actions

of GABA increase BDNF expression via a MAPK-CREB-dependent

mechanism—a positive feedback circuit in developing neurons. J Neu-

rophysiol 88, 1005–1015.

Oh, S., Jang, C. G., Ma, T., & Ho, I. K. (1999). Activation of protein kinase

C by phorbol dibutyrate modulates GABAA receptor binding in rat

brain slices. Brain Res 850, 158–165.

Olive, M. F., & Hodge, C. W. (2000). Co-localization of PKCq with variousGABAA receptor subunits in the mouse limbic system. NeuroReport 11,

683–687.

Papadeas, S., Grobin, A. C., & Morrow, A. L. (2001). Chronic ethanol

consumption differentially alters GABAA receptor a1 and a4 subunit

peptide expressions and GABAA receptor-mediated 36Cl� uptake in

mesocorticolimbic regions of rat brain. Alcohol Clin Exp Res 25,

1270–1275.

Peng, Z., Hauer, B., Mihalek, R. M., Homanics, G. E., Sieghart, W., Olsen,

R. W., & Houser, C. R. (2002). GABA(A) receptor changes in delta

subunit-deficient mice: altered expression of alpha4 and gamma2 sub-

units in the forebrain. J Comp Neurol 446, 179–197.

Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., & Sperk, G.

(2000). GABAA receptors: immunocytochemical distribution of 13 sub-

units in the adult rat brain. Neuroscience 101, 815–850.

Poisbeau, P., Cheney, M. C., Browning, M. D., & Mody, I. (1999). Mod-

ulation of synaptic GABAA receptor function by PKA and PKC in adult

hippocampal neurons. J Neurosci 19, 674–683.

Porter, N. M., Twyman, R. E., Uhler, M. D., & Macdonald, R. L. (1990).

Cyclic AMP-dependent protein kinase decreases GABAA receptor cur-

rent in mouse spinal neurons. Neuron 5, 789–796.

Proctor, W. R., Soldo, B. L., Allan, A. M., & Dunwiddie, T. V. (1992).

Ethanol enhances synaptically evoked GABAA receptor-mediated

responses in cerebral cortical neurons in rat brain slices. Brain Res

595, 220–227.

Proctor, W. R., Poelchen, W., Bowers, B. J., Wehner, J. M., Messing, R. O.,

& Dunwiddie, T. V. (2003). Ethanol differentially enhances hippocam-

pal GABAA receptor-mediated responses in protein kinase C gamma

(PKC gamma) and PKC epsilon null mice. J Pharmacol Exp Ther 305,

264–270.

Puia, G., Santi, M., Vincini, S., Pritchett, D. B., Purdy, R. H., Paul, S. M.,

Seeburg, P. H., & Costa, E. (1990). Neurosteroids act on recombinant

human GABAA receptors. Neuron 4, 759–765.

Puia, G., Vincini, S., Seeburg, P. H., & Costa, E. (1991). Influence of

recombinant GABA-A receptor subunit composition on the action of

allosteric modulators of GABA-gated Cl � currents. Mol Pharmacol

39, 691–696.

Reynolds, J. N., Prasad, A., & MacDonald, J. F. (1992). Ethanol modula-

tion of GABA receptor-activated Cl� currents in neurons of the chick,

rat and mouse central nervous system. Eur J Pharmacol 224, 173–181.

Robello, M., Amico, C., & Cupello, A. (1993). Regulation of GABAA

receptor in cerebellar granule cells in culture: differential involvement

of kinase activities. Neuroscience 53, 131–138.

Robello, M., Amico, C., & Cupello, A. (1997). A dual mechanism for

impairment of GABAA receptor activity by NMDA receptor activation

in rat cerebellum granule cells. Eur Biophys J 25, 181–187.

Roberto, M., Madamba, S. G., Moore, S. D., Tallent, M. K., & Siggins, G.

R. (2003). Ethanol increases GABAergic transmission at both pre- and

postsynaptic sites in rat central amygdala neurons. Proc Natl Acad Sci

USA 100, 2053–2058.

Romeo, E., Brancati, A., De Lorenzo, A., Fucci, P., Furnari, C., Pompili,

G. F., Sasso, G. F., Spalletta, G., Troisi, A., & Pasini, A. (1996).

Marked decrease of plasma neuroactive steroids during alcohol with-

drawal. J Pharmacol Exp Ther 247, 309–322.

Sapp, D. W., & Yeh, H. H. (1998). Ethanol-GABAA receptor interactions: a

comparison between cell lines and cerebellar Purkinje cells. J Pharma-

col Exp Ther 284, 768–776.

Sassoe-Pognetto, M., Panzanelli, P., Sieghart, W., & Fritschy, J. M. (2000).

Colocalization of multiple GABA(A) receptor subtypes with gephyrin

at postsynaptic sites. J Comp Neurol 420, 481–498.

Seeburg, P. H., Wisden, W., Verdoorn, T. A., Pritchett, D. B., Werner, P.,

Herb, A., Luddens, H., Sprengel, R., & Sakmann, B. (1990). The

GABAA receptor family: molecular and functional diversity. Cold

Spring Harbor Symp Quant Biol 55, 29–38.

Sibille, E., Pavlides, C., Benke, D., & Toth, M. (2000). Genetic inactivation

of the serotonin1A receptor in mice results in downregulation of major

GABAA receptor a subunits, reduction of GABAA receptor binding,

and benzodiazepine-resistant anxiety. J Neurosci 20, 2758–2765.

Sieghart, W. (1989). Multiplicity of GABAA receptors. Trends Pharmacol

Sci 10, 407–409.

Sieghart, W. (2000). Unraveling the function of GABAA receptor subtypes.

Trends Pharmacol Sci 21, 411–413.

Sieghart, W., & Sperk, G. (2002). Subunit composition, distribution and

function of GABA(A) receptor subtypes. Curr Top Med Chem 2,

795–816.

S. Kumar et al. / Pharmacology & Therapeutics 101 (2004) 211–226226

Sieghart, W., Fuchs, K., Tretter, V., Ebert, V., Jechlinger, M., Hoger, H., &

Adamiker, D. (1999). Structure and subunit composition of GABAA

receptors. Neurochem Int 34, 379–385.

Sik, A., Gulacsi, A., Lai, Y., Doyle, W. K., Pacia, S., Mody, I., & Freund, T.

F. (2000). Localization of the A kinase anchoring protein AKAP79 in

the human hippocampus. Eur J Neurosci 12, 1155–1164.

Smith, S. S., Gong, Q. H., Hsu, F. -C., Markowitz, R. S., Ffrench-Mullen, J.

M. H., & Li, X. (1998a). GABAA receptor a4 subunit suppression

prevents withdrawal properties of an endogenous steroid. Nature 392,

926–930.

Smith, S. S., Gong, Q. H., Li, X., Moran, M. H., Bitran, D., Frye, C. A.,

Hsu, F. (1998b). Withdrawal from 3a-OH-5a-pregnan-20-one using a

pseudopregnancy model alters the kinetics of hippocampal GABAA-

gated current and increases the GABAA receptor a4 subunit in associ-

ation with increased anxiety. J Neurosci 18, 5275–5284.

Soldo, B. L., Proctor, W. R., & Dunwiddie, T. V. (1994). Ethanol differ-

entially modulates GABAA receptor-mediated chloride currents in hip-

pocampal, cortical, and septal neurons in rat brain slices. Synapse 18,

94–103.

Sundstrom-Poromaa, I., Smith, D. H., Gong, Q. H., Sabado, T. N., Li, X.,

Light, A., Wiedmann, M., Williams, K., & Smith, S. (2002). Hormon-

ally regulated a4h2y GABAA receptors are a target for alcohol. Nat

Neurosci 5, 721–722.

Takebayashi, M., Kagaya, A., Uchitomi, Y., Yokota, N., Horiguchi, J., &

Yamawaki, S. (1998). Differential regulation by pregnenolone sulfate of

intracellular Ca2 + increase by amino acids in primary cultured rat

cortical neurons. Neurochem Int 32, 205–211.

Tapia-Arancibia, L., Rage, F., Givalois, L., Dingeon, P., Arancibia, S., &

Beauge, F. (2001). Effects of alcohol on brain-derived neurotrophic

factor mRNA expression in discrete regions of the rat hippocampus

and hypothalamus. J Neurosci Res 63, 200–208.

Thiele, T. E., Willis, B., Stadler, J., Reynolds, J. G., Bernstein, I. L., &

McKnight, G. S. (2000). High ethanol consumption and low sensitivity

to ethanol-induced sedation in protein kinase A-mutant mice. J Neuro-

sci 20, RC75.

Ticku, M. K., & Mehta, A. K. (1990). Gamma-aminobutyric acidAreceptor desensitization in mice spinal cord cultured neurons: lack

of involvement of protein kinases A and C. Mol Pharmacol 38,

719–724.

Tretter, V., Ehya, N., Fuchs, K., & Sieghart, W. (1997). Stoichiometry and

assembly of a recombinant GABAA receptor subtype. J Neurosci 17,

2728–2737.

Ulfig, N., & Setzer, M. (1999). Expression of a kinase anchoring protein 79

in the human fetal amygdala. Microsc Res Tech 46, 48–52.

VanDoren, M. J., Matthews, D. B., Janis, G. C., Grobin, A. C., Devaud, L.

L., & Morrow, A. L. (2000). Neuroactive steroid 3a-hydroxy-5a-preg-

nan-20-one modulates electrophysiological and behavioral actions of

ethanol. J Neurosci 20, 1982–1989.

Wafford, K. A., Thompson, S. A., Thomas, D., Sikela, J., Wilcox, A. S., &

Whiting, P. J. (1996). Functional characterization of human gamma-

aminobutyric acidA receptors containing the alpha4 subunit. Mol Phar-

macol 50, 670–678.

Wallner, M., Hanchar, H. J., & Olsen, R. W. (2003). Ethanol enhances

a4h3y and a6h3yg-aminobutyric acid type A receptors at low concen-

trations known to affect humans. Proc Natl Acad Sci USA 100 (25),

15218–15223.

Wan, F. -J., Berton, F., Madamba, S. G., Francesconi, W., & Siggins, G. R.

(1996). Low ethanol concentrations enhance GABAergic inhibitory

postsynaptic potentials in hippocampal pyramidal neurons only after

block of GABAB receptors. Proc Natl Acad Sci USA 93, 5049–5054.

Wang, H. B., & Olsen, R. W. (2000). Binding of the GABAA receptor-

associated protein (GABARAP) to microtubules and microfilaments

suggests involvement of the cytoskeleton in GABARAP-GABAA re-

ceptor interaction. J Neurochem 75, 644–655.

Wang, Y., Freund, R. K., & Palmer, M. R. (1999). Potentiation of ethanol

effects in cerebellum by activation of endogenous noradrenergic inputs.

J Pharmacol Exp Ther 288, 211–220.

Weiner, J. L., Zhang, L., & Carlen, P. L. (1994). Potentiation of GABAA-

mediated synaptic current by ethanol in hippocampal CA1 neurons:

possible role of protein kinase C. J Pharmacol Exp Ther 268,

1388–1395.

Weiner, J. L., Gu, C., & Dunwiddie, T. V. (1997a). Differential ethanol

sensitivity of subpopulations of GABAA synapses onto rat hippocampal

CA1 pyramidal neurons. J Neurophysiol 77, 1306–1312.

Weiner, J. L., Valenzuela, C. F., Watson, P. L., Frazier, C. J., & Dunwiddie,

T. V. (1997b). Elevation of basal protein kinase C activity increases

ethanol sensitivity of GABAA receptors in rat hippocampal CA1 pyra-

midal neurons. J Neurochem 68, 1949–1959.

Wetsel, W. C., Khan, W. A., Merchenthaler, I., Rivera, H., Halpern, A. E.,

Phung, H. M., Negro-Vilar, A., & Hannun, Y. A. (1992). Tissue and

cellular distribution of the extended family of protein kinase C isoen-

zymes. J Cell Biol 117, 121–133.

Whittemore, E. R., Yang, W., Drewe, J. A., & Woodward, R. M. (1996).

Pharmacology of the human gamma-aminobutyric acidA alpha4 subunit

expressed in Xenopus laevis oocytes. Mol Pharmacol 50, 1365–1375.

Wisden, W., Herb, A., Wieland, H., Keinanen, K., Luddens, H., & Seeburg,

P. H. (1991). Cloning, pharmacological characteristics and expression

pattern of the rat GABAA receptor4 subunit. FEBS Lett 289, 227–230.

Yoshida, Y., Huang, F. L., Nakabayashi, H., & Huang, K. P. (1988). Tissue

distribution and developmental expression of protein kinase C iso-

zymes. J Biol Chem 263, 9868–9873.

Zafra, F., Castren, E., Thoenen, H., & Lindholm, D. (1991). Interplay

between glutamate and gamma-aminobutyric acid transmitter systems

in the physiological regulation of brain-derived neurotrophic factor and

nerve growth factor synthesis in hippocampal neurons. Proc Natl Acad

Sci USA 88, 10037–10041.