Alcohol & Alcoholism Vol. 44, No. 2, pp. 128–135, 2009
Advance Access publication 20 January 2009
doi: 10.1093/alcalc/agn100
Biochemical and Neurotransmitter Changes Implicated in Alcohol-Induced Brain Damage in Chronic or
‘Binge Drinking’ Alcohol Abuse
Roberta J. Ward∗ , Frédéric Lallemand and Philippe de Witte
∗ Corresponding
Biologie du Comportement, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
author: Biologie du Comportement, Université Catholique de Louvain, Place Croix du Sud 1, 1348 Louvain-la-Neuve, Belgium.
Tel/Fax: +32-1047-4095; E-mail: [email protected]
Abstract — The brain damage, which occurs after either chronic alcoholization or binge drinking regimes, shows distinct biochemical
and neurotransmitter differences. An excessive amount of glutamate is released into specific brain regions during binge drinking (in
excess of 4- to 5-fold of the normal basal concentration) that is not evident during periods of excessive alcohol consumption in chronic
alcohol abusers. Increases in glutamate release are only observed during the initial stages of withdrawal from chronic alcoholism (∼2to 3-fold) due to alterations in the sensitivities of the NMDA receptors. Such changes in either density or sensitivity of these receptors
are reported to be unaltered by binge drinking. When such excesses of glutamate are released in these two different models of alcohol
abuse, a wide range of biochemical changes occur, mediated in part by increased fluxes of calcium ions and/or activation of various
G-protein-associated signalling pathways. Cellular studies of alveolar macrophages isolated from these two animal models of alcohol
abuse showed enhanced (binge drinking) or reduced (chronic alcoholization) lipopolysaccharide (LPS)-stimulated NO release. Such
studies could suggest that neuroadaptation occurs with the development of tolerance to alcohol’s effects in both neurotransmitter function
and cellular processes during chronic alcoholization that delay the occurrence of brain damage. In contrast, ‘binge drinking’ induces
immediate and toxic effects and there is no evidence of an increased preference for alcohol as seen after withdrawal from chronic
alcoholization.
INTRODUCTION
Chronic alcoholism is the popular term for two disorders: alcohol abuse and alcohol dependence. The key element of these
disorders is that a person’s use of alcohol has repeatedly caused
problems in his or her life. Alcohol abuse can result in a number
of problems that include memory disorders, liver disease, high
blood pressure, muscle weakness, heart problems, anaemia,
low immune function, disorders of the digestive system and
pancreatic problems. This will occur after many years of excessive alcohol intake. Alcoholism can also lead to a number of
personal problems, including depression, unemployment, family problems and child abuse [reviewed in many papers, for
example Adachi et al. (2003) and Harper (2007)]. The cost to
the economy of the use and abuse of alcohol in the UK may
well be in excess of 20 billion pounds.
However, recent media attention has been directed towards
‘binge drinking’ or heavy episodic drinkers (HED) in the
younger generation, 15–21 years, where early neurochemical
changes associated with brain damage are evident, particularly in the corticolimbic region (Obernier et al. 2002a, 2002b;
Crews et al., 2004), and occur after a relatively short time of
such alcohol abuse. This leads to differences in both mood and
cognitive performance, e.g. changes in memory (Townsend and
Duka, 2005), an increased risk of dementia (Jarvenpaa et al.,
2005) as well as susceptibility to developing chronic alcoholism
(Bates and Labouvie, 1997). It is estimated that binge drinking
costs the UK economy £20 billion a year, with 17 million working days lost to hangovers and drink-related illness each year.
This results in costs of £6.4 billion to employers and an annual
cost of alcohol harm to the National Health Service in England
of £2.7 billion. Furthermore, it has recently been mentioned
in a report from Denmark that ‘binge drinking’ three or more
times during pregnancy is associated with an increased risk
of stillbirth (Strandberg-Larsen et al., 2008). Urgent action is
therefore needed to comprehend the aetiology and pathogenesis
of the binge drinking culture, as well as to educate individuals
on the dangers of such drinking.
C
The underlying neurochemical changes in binge drinking
may be mediated by an imbalance between inhibitory and excitatory amino acids and/or changes in monoamines release,
which could drive the excessive drinking behaviour. Biochemically, alteration in phosphorylation possibly occurs, which
could lead to changes in the mediators of the inflammatory
response. In addition, there are clear and distinct differences
between binge drinking and chronic alcoholism in behavioural
characteristics, for example, in the number of visits to the
Hospital Emergency Departments for alcohol-related injuries
(Gmel et al., 2007). It is noteworthy that no alcohol-induced
preference or severe withdrawal symptoms occur in binge
drinkers after cessation of alcohol.
In this present review, data will be presented on our current
understanding of neurochemical and biochemical changes that
occur in ‘binge drinking’ individuals. Various signalling pathways will be presented, which may be responsible for the reinforcing and toxic effects of ‘binge drinking’. Finally the action
of ethanol on the immune system is discussed, with phagocytic
cells being utilized to investigate the differing action of chronic
alcoholization and binge drinking on the immune response.
Inhibitory and excitatory amino acids
Glutamate. Glutamate, when released into the synaptic cleft,
exerts its action by binding to several classes of glutamate
receptors, N-methyl-D-aspartic acid (NMDA), alpha-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and
metabotropic glutamate receptor (mGlu), which are involved
in a multitude of functions including learning and its modulation of consolidation and/or recall (Riedel et al., 2003), by
activating G-proteins leading to increased phospholipase C, diacylglycerol (IP3 DAG) and calcium-dependent protein kinases
(Fig. 1).
Excessive glutamate release is a major cause of neuronal cell
death, possibly involving two pathways. Firstly, excitotoxicity
that occurs through the activation of glutamatergic receptors
(Choi, 1988; Michaels & Rothman, 1990), causing Ca2+ ion
The Author 2009. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved
Biochemical and Neurotransmitter Changes Implicated in Alcohol-Induced Brain Damage
129
Dopamine D1
Adenosine A
Dopamine D2
Cannabinoid CB1
G-proteins
G-proteins
Adenylylcyclase
Ionotropic
Glutamate K+
NMDA
P
Na+
Ca++
cAMP
Nuclear Transcription
Factors (NFkappaB)
P CREB
PKA
Ca++
Ionotropic
Glutamate K+
Non-NMDA
Na+
Ca++
CBP
Nucleus
PP1
DARPP32
P
IP3R1
Metabotropic
Glutamate
mGluR1
Calcium
Binding
proteins
PLC
IP 3
Fig. 1. Signalling pathways activated by neurotransmitters binding to their respective receptors. cAMP—adenosine 3 ,5 -cyclic monophosphate; CREB—cyclic
AMP response element-binding protein; CBP—calmodulin-binding peptide; IP3 R1—inositol 1, 4, 5-trisphosphate receptors; DARPP32—dopamine and cAMP
regulated phosphoprotein (MW 32 kD); PLC—phospholipase C; IP3 —inositol trisphosphate; PP1—protein phosphatase-1.
Fig. 2. The release of glutamate into the synaptic cleft will stimulate AMPA
receptors inducing sodium (Na+ ) fluxes and NMDA receptors stimulating
calcium fluxes Ca2+ . Such calcium fluxes will bind to calcium-binding proteins
including calmodulin to activate nitric oxide synthase to enhance nitric oxide,
NO, production. Calcium fluxes will also stimulate phospholipases to generate
reactive oxygen species, ROS.
influx, with NMDA-mediated generation of nitric oxide (NO),
mitochondrial depolarization, Na+ influx leading to an unsustainable increase in ATP demand, microtubule depolymerization, mitochondrial collapse and dendritic beading (reviewed
by Greenwood et al., 2007) (Fig. 2). Secondly, oxidative glutamate toxicity, that is mediated via a series of disturbances to
the redox homeostasis of the cell (Murphy et al., 1989; Choi,
1992). Several studies have identified an increased glutamate
release in ‘binge drinking’ animals in NAC during ethanol ingestion; e.g. the scheduled high alcohol consumption murine
model (SHAC) showed enhanced glutamate release in mice
administered a 5% alcohol solution on six occasions followed
by an i.p. injection of 1.5 or 2 g/kg ethanol (Szumlinski et al.,
2007), and the rat model administered 3 g ethanol/kg two times
a week for 4 weeks followed by 5 days of abstinence, and
then a further challenge with 3 g/kg (Lallemand et al., 2008)
(Fig. 3). In a study of the anterior cingulated brain region of
13 recently abstinent young alcoholics, proton magnetic resonance spectroscopy and magnetic resonance imaging identified a significant increase in the glutamate to creatine ratio
(Lee et al., 2007). Furthermore in this latter study such alterations in glutamate correlated with altered short-term memory
function. It could be argued that such an increase in glutamate
release was related to the priming acute dose of ethanol administered immediately prior to the microdialysis experiment of
binge drinking animals. However, earlier studies showed that
acute administration of either 2 g/kg or 3 g/kg ethanol to naı̈ve
rats elicits a decrease (Moghaddam and Bolinao, 1994; Yan
et al., 1998) or no change (Dahchour et al., 1994) in glutamate release into the NAC. In contrast, in specific rat strains,
i.e. Lewis but not Fischer rats (Selim and Bradberry, 1996),
and low alcohol sensitive rats (Dahchour et al., 2000) there
were increases in glutamate release into the NAC after 1 g/kg
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Ward et al.
Glutamate
600
**
550
Water - Water (n = 9)
Percentage baseline value
500
Water - EtOH 2g/kg (n = 9)
Water - EtOH 3g/kg (n = 11)
450
400
350
300
250
200
150
100
50
0
−2
−1
0
1
2
3
4
5
Time (hour)
Fig. 3. Effect of binge drinking, of ethanol 2 or 3 g/kg, on mean glutamate levels in the nucleus accumbens compared to water, expressed as percentage of baseline
value. Rats received (a) water only, filled squares, (b) ethanol 2 g/kg, filled diamonds, or (c) ethanol 3 g/kg, filled triangles, in a ‘binge drinking’ regime. The
values before time ‘0’ represent the effect of binge drinking and the values after time ‘0’ show changes in glutamate release after the administration of either water
or the ethanol doses administered by gavage. Significant time points between ethanol doses and control are represented by ∗ P < 0.05, ∗∗ P < 0.01. Results are
presented as mean ± S.E.M.
A
Arginine
1.40E-05
Water - Water (n = 9)
1.20E-05
Water - EtOH 2g/kg (n = 9)
Water - EtOH 3g/kg (n = 11)
Concentration (M)
1.00E-05
**
8.00E-06
6.00E-06
4.00E-06
2.00E-06
0.00E+00
**
−2.00E-06
−2
−1
0
1
2
3
4
Time (hour)
Fig. 4. Effect of binge drinking, of ethanol 2 or 3 g/kg, on mean arginine levels in the nucleus accumbens compared to water expressed as concentration in NAC of
rats. (A) Non-supplemented rats that received water by gavage, filled squares, or binge drinking ethanol, 2 g/kg, filled black diamonds, or 3 g/kg, filled triangles.
The values before time ‘0’ represent the effect of chronic binge drinking and the values after time ‘0’ represent the effect of ethanol or water administration
by gavage during the microdialysis experiment. Significant time points between ethanol doses and water are represented by ∗ P < 0.05, ∗∗ P < 0.01. Results are
presented as mean ± S.E.M.
or 2 g/kg ethanol but of much smaller magnitude to that observed in these binge drinking animal models. It is noteworthy
that alcohol-preferring rats show greater region-specific brain
damage after a ‘binge-drinking’ regime than its genetic nonalcohol-preferring rat line. Such findings indicate that there
is a genetic component that possibly contributes to the brain
damage that occurs in ‘binge drinking’ individuals (Crews and
Braun, 2003). No increases in NMDA receptor density are reported in binge drinking models (Rudolph and Crews 1996).
Furthermore, glutamate antagonists, such as MK801 (NMDA
antagonist), nimodipine (voltage-gated Ca2+ channel), DNQX
(AMPA antagonist and NMDA antagonist at glycine site), do
not protect against brain damage in a ‘binge drinking’ animal
model [reviewed in Crews et al. (2004)]. Additional evidence,
which indicated that glutamate release may have an effect on
NMDA receptors, was shown in recent microdialysis experiments in the ‘binge drinking’ rat model, where significant decreases in arginine release were assayed after both 2 and 3 g/kg
ethanol (Lallemand et al., unpublished results) (Fig. 4). This
could indicate the utilization of arginine for the formation of
the signalling molecule NO mediated via the Ca2+ /calmodulin
pathway and NO synthases (Fig. 2). In one previous study
Biochemical and Neurotransmitter Changes Implicated in Alcohol-Induced Brain Damage
where chronically alcoholized rats were repeatedly withdrawn
from ethanol (Dahchour and De Witte, 2003), a significant reduction in arginine hippocampus microdialysate content was
also noted but only after the third withdrawal. It is reported
that ‘binge-drinking’ individuals, with compensated alcoholic
cirrhosis, show a transient significant increase of serum nitrite
and nitrates after an 80 g dose of ethanol (Oekonomaki et al.,
2004) that might be indirect evidence for an increase in NO
production in certain tissues, particularly the liver and brain.
No changes in the basal concentration of glutamate are apparent in various brain regions during the period of chronic alcohol
intoxication (Dahchour and De Witte, 1999, 2003) despite the
fact that blood alcohol levels are high, ∼2 g/l. However, between 3 and 5 h after the commencement of detoxification from
ethanol, there is an enormous increase in glutamate release (due
to changes in the sensitivity of NMDA receptors) (Chandler
et al., 1993), both NMDAR1 and NMDAR2B polypeptide levels being increased in numbers but decreased in sensitivity during the chronic alcoholization stage (Kumari and Anji, 2005)
in the nucleus accumbens (Dahchour and De Witte, 1999),
hippocampus (Dahchour and De Witte, 2003) and the striatum (Rossetti and Carboni, 1995). Such excesses of glutamate
release during these initial stages of detoxification cause significant behavioural disturbances as well as alcohol craving.
Elevations in the synthesis and release of the polyamines spermidine and spermine (modulators of NMDARs) contribute to
the increased activity of the receptor during ethanol withdrawal
(Gibson et al., 2003). Such adaptive changes play an important
role in the development of alcohol dependence as well as alcohol withdrawal.
Gamma-amino-butyric acid (GABA). GABA is a major inhibitory neurotransmitter, which binds to GABAA receptors,
thereby hyperpolarizing the cell membrane and inhibiting neural activity. Alcohol modulates GABA function, such that in
certain brain regions ethanol will increase GABA release (Carta
et al., 2004; Hanchar et al., 2005), possibly by inhibiting its
degradation. Ethanol will alter GABA-gated current, which is
dependent on the time course of exposure and its concentration
(Smith and Gong, 2007). Reducing GABAA activity decreases
the signs of alcohol intoxication and also alcohol’s anti-anxiety
effects by GABA. As yet, there have been no studies of the
effect of binge drinking on GABA release in different brain
regions.
Dopamine. The dopaminergic mesolimbic system plays a
significant part in the motivational and reinforcement mechanisms related to behaviour. Alcohol increases dopaminergic
transmission in the mesolimbic pathway and increases the firing
rate of dopaminergic neurons (Heidbreder and De Witte, 1993)
enhancing dopamine release, which may be mediated by an increase in the endocannabinoid tone (Cheer et al., 2007). Direct
evidence of a role for dopamine in ethanol reward comes from
the finding that rats that operantly self-administer ethanol will
stimulate its release both in the NAC (Weiss et al., 1993) and
the ventral tegmental cell body region of the meso-accumbens
dopamine reward pathway (Gatto et al., 1994). This process
can be modified by pharmacological agents that interact with
dopamine neurotransmission (McBride et al., 1990). During
chronic abuse of alcohol, larger amounts of alcohol may need
to be consumed to evoke dopamine release, in order to substantiate the pleasurable effects of alcohol intake. During alcohol
withdrawal, dopamine release will be reduced, dramatically
131
reducing the firing of related neurons leading to dysphoria,
malaise and depression. Studies are currently underway in our
laboratory to investigate changes in hippocampal dopamine release during ‘binge drinking’ in the rat model.
Serotonin. Serotonin plays a role in the regulation of mood,
eating arousal, sleep pain and many other behaviours (Carlson,
1998). Alcohol increases serotonin release in the CNS affecting emotion, mood and thinking. There are several types of
serotonin receptors, 5-HT1A , 5-HT1B , 5-HT2 and 5-HT3 , each
of which has its own specific influence on behaviour related
to the consumption of alcohol (Lovinger, 1999). The 5-HT3
receptor, a ligand-gated ion channel, has been localized to several regions of the brain and appears to be involved in many
neuronal functions including responses to alcohol and other
drugs of abuse. There is an extensive and growing literature
indicating that 5-HT3 receptors are involved in several facets
of alcohol-seeking behaviour, alcohol intoxication and addiction. In addition, there is strong evidence that ethanol alters
the function of the 5-HT3 receptor, possibly through actions
on its receptor protein (Rodd et al., 2007). In a recent study
(Szumlinski et al., 2007), it was shown that repeated alcohol
consumption in mice (i.e. presentation of 5% alcohol every
third day for 18 days for increasing time periods) deregulates
serotonin function within the nucleus accumbens, by reducing
the extracellular concentration of serotonin.
Opioid system. The opioid system includes endorphin,
enkephalin and other endogenous substances that modulate
pain, mood reinforcement and response to stress. Endorphin
and enkephalin are related to reinforcement from alcohol. Alcohol administration enhances endogenous opioid activity in
both rats and man, by increasing opioid release, after in vitro
brain slices and of blood levels in humans in vivo. In occasional
alcohol users, alcohol-induced dopamine release may produce
reinforcement as this is regulated by opioids (Herz, 1997). In
contrast, chronic alcohol abuse leads to reduced brain levels of
endorphin, which contribute to the negative emotional states
that accompany alcoholic withdrawal, as well as increasing
the CNS expression of inhibitory G-proteins, thereby reducing
adenylate cyclase (Wand et al., 1993). There are few reports of
the effects of ‘binge drinking’ on opioid function in adolescent
or mature rodents, although binge-like exposure in newborn
rats increases the number of apoptotic β-endorphin neurons in
the arcuate nucleus of the hypothalamus (Sarkar et al., 2007).
Taurine. Some investigators have suggested that binge drinking intoxicated rats showed significant cortical oedema (Crews
et al., 2004), which could be reduced by the administration
of diuretics such as furosemide and acetazolamide. No significant change in the basal levels of taurine was observed in
binge drinking rats, which might have been apparent if oedema
was occurring since taurine functions as an osmoregulator
(Lallemand et al., 2008). Small transient changes in the taurine
microdialysis content were evident after ethanol administration
to ‘binge drinking’ rats. These were comparable to the pattern
of responses seen after acute intraperitoneal ethanol injections
(Dahchour et al., 1994), which are attributed to ethanol-induced
osmolarity changes.
Biochemical changes
Chronic alcohol abuse and ‘binge drinking’ induce a wide range
of biochemical changes in the brain that are related to changes
132
Ward et al.
on the intricate signalling pathways (Hoek and Kholodenko,
1998; Pandey, 1998), such as NO synthesis, protein phosphorylation and the action and release of trophic factors, MAPK
and NFkappaB pathways. The generation of free radicals in
the brain, generated by the inducible MEOS system (Sohda
et al., 1993) or acetaldehyde toxicity, (Heap et al., 1995) may
contribute to the alcohol-induced brain damage (Pratt et al.,
1990), which will be minimized if adequate cytoprotection can
be upregulated, e.g. reduced glutathione, in the specific brain
regions affected.
Changes in phosphorylation. Cellular signalling events rely
heavily on protein phosphorylation (with protein kinases initiating phorphorylation while protein phosphatases will dephosphorylate), thereby activating and inactivating signal transduction cascades.
Neurotransmitters interact with specific cell surface receptors that are coupled to the stimulatory G protein Gs , thereby
activating cAMP, which results in phosphorylation of the transcription factor to the cyclic AMP responsive element-binding
protein, CREB (Fig. 1). This latter protein serves as both an
up- and down-stream molecular target for the action of brainderived neurotrophic factors by inducing key genes that improve neuronal vitality, growth and resistance to insults (Walton
and Dragunow, 2000; Mantamadiotis et al., 2002). CREB requires phosphorylation to initiate transcription of pro-survival
neuronal factors (Fig. 1). Both ‘binge drinking’ and chronic alcohol intoxication reduce the amount of phosphorylated CREB
immunoreactivity in the hippocampal dentate gyrus during intoxication (Yang et al., 1998; Bison and Crews, 2003). In contrast, an acute ethanol challenge, 3 g/kg, induced an increase in
the phosphorylated form of CREB in the rat cerebellum.
Changes in phosphorylation of the protein phosphatase inhibitor DARPP-32 may play an important role in reducing
ethanol inhibition of NMDA receptors. In a recent study of cultured neurons and brain slices, Maldve et al. (2002) showed that
activation of dopamine D1 receptors may over-ride the alcoholinduced inhibition of NMDA receptors in the NAC. This was
dependent on a series of intracellular signalling events involving the activation of adenylate cyclase, the subsequent activation of the cyclic AMP-dependent protein kinase A (PKA) and
phosphorylation of the protein phosphatase inhibitor DARPP32 (Fig. 5). Activation of D1 receptors also increased phosphorylation of the NR1 subunit of the NMDA receptor, presumably because phosphorylation of DARPP-32 inhibits the
protein phosphatase PP-1 that normally removes the phosphate
group at serine 897 within the NR1 protein. Such interactions
between alcohol, dopamine and glutamate may be important
in the development of ‘binge drinking’, with D1 receptors and
DARPP-32 mediating such effects.
Transcription factors were the first signalling proteins to be
identified as redox sensitive, which are regulated through sensitive cysteine residues, that need to be reduced for activity.
The transcription factor NFκappaB (NFκB) is sequestered in
the cytoplasm where it is associated with a member of the
IκB family of inhibitory proteins (Fig. 6). For the activation
of NFκB, rapid phosphorylation of its inhibitory subunit, IκB,
by specific IκB kinases is necessary. IκB is then released from
NFκB to be ubiquinated and degraded by proteosomes. NFκB
is then translocated to the nucleus where it mediates transcriptional initiation of a number of cytokines. Originally it was
thought that NFκB activation was due to the production of
Fig. 5. Intracellular molecular events linking dopamine receptor activation
to increased NMDA phosphorylation and decreased inhibition by alcohol
[adapted from Lovinger (2002)]. N-methyl-D-aspartate receptors (NMDARs)
are ligand-gated ion channels. Functional NMDARs are heterotetrameric assemblies of NR1 subunits with at least one type of NR2 subunits. PKA—protein
kinase A; DARPP—dopamine and cAMP regulated phosphoprotein (MW
32 kD).
Fig. 6. NFkappaB activation.
reactive oxygen species (Schreck et al., 1992), but later studies revealed that, in vitro, such NFκB activation by exogenous
H2 O2 was cell specific (Bowie and O’Neill, 2000). Although
one study did show some benefit of specific anti-oxidants, i.e.
furosemide and butylated hydroxytoluene, in preventing neurodegeneration in binge drinking animal models (Crews et al.,
2004), no results for NFκB activation were presented. In our
previous studies of NFκB activation in the cortex of chronically
alcoholized rats (Ward et al., 1996), it was evident that this transcription factor was severely down regulated by comparison to
the high activation of NFκB after an acute ethanol dose. Such
neuroadaptations may not occur in the binge drinking model,
since it was noted that there was excessive generation of ROS,
particularly hydroxyl radicals, during the binge drinking regime
as well as after further acute ethanol challenges (Lallemand
et al., 2008).
Glial and macrophages
Glial cells play an important function in the brain, nurturing
neurons and facilitating neuronal activity. Four different types
Biochemical and Neurotransmitter Changes Implicated in Alcohol-Induced Brain Damage
133
**
35
**
30
Nitrite release (µM)
200,00 cells Incubated 24h
25
20
**
15
10
5
0
CON
CON + Stim
EtOH
EtOH + Stim
80
Fig. 7. Schematic diagram of microglial signal transduction following the
exposure to lipopolysaccharide (LPS), interferon gamma (IFNγ ) and other
inflammatory stress signals. After treatment of microglia with LPS, TLR4
signalling is initiated with NFkappaB activation and IFNβ production. Ethanol
may also act on the co-activator p300 to inhibit TLR4 signalling. The effect
of ethanol on MAPK signalling has not been investigated in microglia [from
Suk (2007)]. TLR4—toll-like receptor 4; MAPK—mitogen-activated protein
kinases.
of glial cells exist, e.g. oligodendrocytes, ependymal cells, astrocytes and microglia. Microglial cells are closely related to
monocytes and macrophages (Stoll and Jander, 1999) and play
a pivotal role in CNS immunity. Glial cells, including astrocytes, are essential for the regulation of released glutamate
and its conversion to glutamine through the enzyme glutamine
synthetase. Activated microglia secretes neurotoxic inflammatory cytokines and mediators such as tumour necrosis factor
(TNFα), and NO, which may initiate or amplify the neuroinflammatory responses. The activation of microglial cells may
be aimed at initially protecting the neurons but latterly could
induce neuronal destruction. Deregulation by ethanol of the inflammatory activation signalling of microglia may contribute to
the derangement of CNS immune and inflammatory responses.
Chronic ethanol consumption did not alter the density of microglial cells that reside in the molecular layer of the cerebellar
cortex (Dlugos and Pentney, 2001) while intermittent ethanol
exposure increased the number of cerebellar microglia, indicating microgliosis (Riikonen et al., 2002). Therefore, understanding the regulatory processes of such neuro-inflammation
is pivotal to understanding the mode of neuropathology in
alcohol-induced brain damage in both chronic alcohol abusers
and ‘binge drinkers’.
Many intracellular signalling pathways are mobilized by microglial activation and include NFκB, MAPK, PKA, PKC,
JAK-STAT, NOS and TLRs (Fig. 7). Many of the glial signalling pathways are affected by ethanol, e.g. NFκB, MAPKs
and JAK-STAT, although there is no consensus as to whether
ethanol suppresses, enhances or has no effect [reviewed by Suk
(2007)]. The cell type, ethanol concentration dose and timing
of exposure may be important factors to consider.
Among the many inflammatory mediators produced by activated phagocytic cells, i.e. macrophages and microglia, NO
production has been widely regarded as representative of inflammatory activation. Microglia-derived NO will exert direct
Nitrite release (µM)
70
60
**
Controls
Chronic ethanol
*
50
40
*
30
20
10
0
Con
LPS
IFN
LPS+ IFN
Fig. 8. Release of nitrite into the extracellular fluid after incubation of alveolar macrophages isolated from binge drinking rats (EtOH) or chronically
alcoholized rats, after incubation with stimulants, either lipopolysaccharide
(LPS) alone or together with interferon gamma (INFγ ).
toxicity towards neurons (Liu et al., 2002). Exposure of the
immortalized cell line BV-2 microglia cells to ethanol, 10 or
100 mM, significantly reduced the LPS-induced NO production. Ethanol also inhibited NFκB signalling (Lee et al., 2004),
although the mechanism did not involve changes either in the
ability of NFκB to bind to its cognate DNA sequences or IκB
degradation. This would indicate that the site of ethanol’s action
is between DNA binding of NFκB and the transactivation of
target genes. One of the co-activators involved in this process,
p300, may be the site of ethanol’s action. Importantly IFNγ signalling in microglia is not affected by ethanol; neither STAT1
nor IRF-1 (constituting the main components of the IFN-γ signalling pathway) is altered after ethanol administration. In our
previous studies, alveolar macrophages (which are the functional equivalent of brain microglia) isolated from various animal models of alcohol toxicity, i.e. chronic alcohol intoxication
(Zhang et al., 1998) and ‘binge drinking’ model (Ward et al.,
2008), showed variable responses in LPS-induced NO release
(Fig. 8). As can be clearly observed, the LPS-stimulated NO
release from the chronically alcoholized macrophages was significantly reduced by comparison to controls. In contrast, binge
drinking significantly increased LPS-stimulated NO release
both at the end of the binge-drinking regime and after 28 days
of no further treatment. Further studies are currently underway
to identify whether various signalling pathways, such as p38,
MAPK, JAK/STAT1 and IRF-1, are altered by these different
drinking regimes in these phagocytic cells.
Similar results were also reported for astrocytes. The induction of iNOS activity by stimulants in A172 astroglia cell
line and C6 glioma cells was suppressed by high ethanol
134
Ward et al.
concentrations, >100 mM (Militante et al., 1997; Davis and
Syapin 2004, respectively). However, COX-2 expression and
the resulting PGE2 production was increased by ethanol in rat
astrocyte cultures (Luo et al., 2001). Further studies are needed
to identify the specific effects of ethanol, when administered
acutely, chronically or intermittently, on the intracellular signal transduction that may be different among the glial cell
population.
Considerable research efforts are necessary to ascertain how
the various signalling pathways are altered by alcohol when
administered either chronically or intermittently. The results
of such studies will enable pharmacological intervention to be
made to prevent the onset and development of alcohol-induced
brain damage. There is little doubt that the problem of binge
drinking, particularly by adolescents, needs to be addressed
urgently, to prevent cognitive impairment, which could lead to
irreversible brain damage.
Acknowledgements — R.J.W. gratefully acknowledges grants from IREB and ERAB and
COST D34.
REFERENCES
Adachi J, Asano M, Uen Y et al. (2003) Alcoholic muscle disease and
biomembrane perturbations (review). J Nutr Biochem 14:616–25.
Bates M, Labouvie EW. (1997) Adolescent risk factors and the prediction of persistent alcohol and drug use into adulthood. Alcohol
Clin Exp Res 21:944–95.
Bison S, Crews F. (2003) Alcohol withdrawal increases neuropeptide
Y immunoreacivity in rat brain. Alcohol Clin Exp Res 27:1173–83.
Bowie A, O’Neill LA. (2000) Oxidative stress and nuclear factorkappaB activation: a reassessment of the evidence in the light of
recent discoveries. Biochem Pharmacol 59:13–23.
Carlson NR. (1998) Physiology of Behaviour. Boston, MA: Allyn &
Bacon.
Carta M, Mameli M, Valenzuela CF. (2004) Alcohol enhances
GABAergic transmission to cerebellar granule cells via an increase
in Golgi cell excitability. J Neurosci 24:3746–51.
Chandler LJ, Sumners C, Crews FT. (1993) Ethanol inhibits NMDA
receptor-mediated excitotoxicity in rat primary neuronal cultures.
Alcohol Clin Exp Res 17:54–60.
Cheer JF, Wassum KM, Sombers LA et al. (2007) Phasic dopamine
release evoked by abused substances requires cannabinoid receptor
activity. J Neurosci 27:791–5.
Choi DW. (1988) Glutamate neurotoxicity and disease of the nervous
system. Neuron 1:623–34.
Choi DW. (1992) Excitotoxic cell death. J Neurobiol 23:1261–76.
Crews FT, Braun CJ. (2003) Binge ethanol treatment causes greater
brain damage in alcohol-preferring P rats than in alcohol-nonpreferring rats. Alcohol Clin Exp Res 27:1075–82.
Crews FT, Collins MA, Dlugos C et al. (2004) Alcohol-induced
neurodegeneration: when, where and why. Alcohol Clin Exp Res
28:350–64.
Dahchour A, De Witte P. (1999) Excitatory and inhibitory amino acid
changes during repeated episodes of ethanol withdrawal: an in vivo
microdialysis study. Eur J Pharmacol 17:171–8.
Dahchour A, De Witte P. (2003) Effects of acamprosate on excitatory
amino acids during multiple ethanol withdrawal periods. Alcohol
Clin Exp Res 27:465–70.
Dahchour A, Hoffman A, Deit Rich R et al. (2000) Effects of ethanol
on extracellular amino acid levels in high- and low-alcohol sensitive
rats: a microdialysis study. Alcohol Alcohol 35:548–53.
Dahchour A, Quertemont E, De Witte P. (1994) Acute ethanol increases taurine but neither glutamate nor GABA in the nucleus
accumbens of male rats: a microdialysis study. Alcohol Alcohol
29:485–7.
Davis RL, Syapin PJ. (2004) Acute ethanol exposure modulates
the expression of inducible nitric oxide synthase in human mi-
croglia: evidence for a transcriptional mechanism. Alcohol 32:195–
202.
Dlugos CA, Pentney RJ. (2001) Quantitative immunocytochemistry
of glia in the cerebellar cortex of old ethanol-fed rats. Alcohol 23:
63–9.
Gatto GJ, McBride WJ, Murphy JM et al. (1994) Ethanol self-infusion
into the ventral tegmental area by alcohol preferring P rats Alcohol
11:557–64.
Gibson DA, Harris BR, Prendergast MA et al. (2003) Polyamines
contribute to ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through interactions with the NMDA receptor. Alcohol Clin Exp Res 27:1099–106.
Gmel G, Givel J-C, Yersin B et al. (2007) Injury and repeated injurywhat is the link with acute consumption, binge drinking and chronic
heavy alcohol use. Swiss Med Wkly 137:642–8.
Greenwood SM, Mizielinska SM, Frenguelli BG et al. (2007) Mitochondrial dysfunction and dendritic beading during neuronal toxicity. J Biol Chem 282:26235–44.
Hanchar HJ, Dobson PD, Olsen RW et al. (2005) Alcohol-induced
motor impairment caused by increased extrasynaptic GABA(A)
receptor activity. Nat Neurosci 8:339–45.
Harper C. (2007) The neurotoxicity of alcohol. Hum Exp Toxicol
26:251–7.
Heap L, Ward RJ, Abiaka C et al. (1995) The influence of brain
acetaldehyde on oxidative status, dopamine metabolism and visual
discrimination task. Biochem Pharmacol 17:263–70.
Heidbreder C, De Witte P. (1993) Ethanol differentially affects extracellular monoamines and GABA in the nucleus accumbens. Pharmacol Biochem Behav 46:477–81.
Herz A. (1997) Endogenous opioid systems and alcohol addiction.
Psychopharmacology 99, 99–111.
Hoek JB, Kholodenko BN. (1998) The intracellular signalling network
as a target for ethanol. Alcohol Clin Exp Res 22:224S–30S.
Hopf FW, Martin M, Chen BT et al. (2007) Withdrawal from intermittent ethanol exposure increases probability of burst firing in VTA
neurons in vitro. J Neurophysiol 98:2297–310.
Jarvenpaa T, Rinne JO, Koskenvuo M et al. (2005) Binge drinking in
midlife and dementia risk. Epidemiology 16:766–71.
Kumari M, Anji A. (2005) An old story with a new twist: do NMDAR1
mRNA binding proteins regulate expression of the NMDAR1 receptor in the presence of alcohol. Ann N Y Acad Sci 1053:311–
8.
Lallemand F, Schol F, Javaux C et al. (2007) Heavy episodic drinking
induces significant alterations in amino acid content of the nucleus accumbens but not in alveolar macrophages. Alcohol Alcohol
42:i30.
Lee E, Jang DP, Kim JJ et al. (2007) Alteration of brain metabolites in
young alcoholics without structural changes. Neuroreport 18:1511–
4.
Lee H, Jeong J, Son E et al. (2004) Ethanol selectively modulates
inflammatory activation signalling of brain microglia. J Neuroimmunol 156:88–95.
Liu B, Gao HM, Wang JY et al. (2002) Role of nitric oxide
in inflammation-mediated neurodegeneration. Ann N Y Acad Sci
962:318–31.
Lovinger DM. (1999) The role of serotonin in alcohol’s effects on the
brain. Curr Separations 18:23–8.
Lovinger DM. (2002) NMDA receptor lose their inhibitions. Nat Neurosci 5:614–6.
Luo J, Linstrom CL, Donahue A et al. (2001) Differential effects of
ethanol on the expression of cyclo-oxygenase in cultures cortical
astrocytes and neurons. J Neurochem 76:1354–63.
Maldve RE, Zhang TA, Ferrani-Kile K et al. (2002) DARPP-32 and
regulation of the ethanol sensitivity of NMDA receptors in the
nucleus accumbens. Nat Neurosci 5:641–8.
Mantamadiotis T, Lemberger T, Bleckmann SC et al. (2002) Disruption of CREB function in brain leads to neurodegeneration Nat
Genet 31:47–54.
McBride WJ, Murphy JM, Lumeng L et al. (1990) Serotonin dopamine
and GABA involvement in alcohol drinking. Alcohol 7:199–
203.
Michaels RL, Rothman SM. (1990) Glutamate neurotoxicity in vitro;
antagonist pharmacology and intracellular calcium concentrations
J Neurosci 10:283–92.
Biochemical and Neurotransmitter Changes Implicated in Alcohol-Induced Brain Damage
Militante JD, Feinstein DL, Syapin PL. (1997) Suppression by ethanol
of inducible nitric oxide synthase expression in C6 glioma cells.
J Pharmacol Exp Ther 281:558–65.
Moghaddam B, Bolinao ML. (1994) Biphasic effect of ethanol on
extracellular accumulation of glutamate in the hippocampus and
the nucleus accumbens. Neurosci Lett 178:99–102.
Murphy TH, Miyamoto M, Sastre A et al. (1989) Glutamate toxicity in
a neuronal cell line involves inhibition of cystine transport leading
to oxidative stress. Neuron 2:1547–58.
Obernier JA, Bouldin TE, Crews FT. (2002a) Binge ethanol exposure in adult rats causes necrotic cell death. Alcohol Clin Exp Res
26:547–57.
Obernier JA, White AM, Swartzwelder HS et al. (2002b) Cognitive
deficits and CNS damage after a 4-day binge ethanol exposure in
rats. Pharmacol Biochem Behav 72:521–32.
Oekonomaki E, Notas G, Mouzas IA et al. (2004) Binge drinking and
nitric oxide metabolites in chronic liver disease. Alcohol Alcohol
39:106–9.
Pandey SC. (1998) Neuronal signalling systems and ethanol dependence. Mol Neurobiol 17:1–15.
Pratt OE, Rooprai HK, Shaw GK et al. (1990) The genesis of alcoholic
brain tissue injury. Alcohol Alcohol 25:217–30.
Riedel G, Platt B, Micheau J. (2003) Glutamate receptor function in
learning and memory. Behav Brain Res 140:1–4.
Riikonen J, Jaatinen P, Rintala J et al. (2002) Intermittent ethanol
exposure increases the number of cerebellar microglia. Alcohol Alcohol 37:421–6.
Rodd ZA, Gryszowka VE, Toalston JE et al. (2007) The reinforcing
actions of a serotonin-3 receptor agonist within the ventral tegmental area: evidence for subregional and genetic differences and involvement of dopamine neurons. J Pharmacol Exp Ther 321:1003–
12.
Rossetti ZL, and Carboni S. (1995) Ethanol withdrawal is associated
with increased extracellular glutamate in the rat striatum. Eur J
Pharmacol 283:177–83.
Rudolph JG, Crews FT (1996) No change in NMDA receptor binding
in rat brain following chronic ethanol treatment. Alcohol Clin Exp
Res 20:8A.
Sarkar DK, Kuhn P, Marano J et al. (2007) Alcohol exposure during the developmental period indices β-endorphin neuronal death
and causes alteration in the opioid control of stress axis function.
Endocrinology 148:2828–34.
Schreck R, Albermann K, Baeuerle PA. (1992) Nuclear factor
kappa B: an oxidative stress-responsive transcription factor of
eukaryotic cells (a review). Free Radic Res Commun 17:221–
37.
Selim M, Bradberry CW. (1996) Effect of ethanol on extracellular 5HT and glutamate in the nucleus accumbens and prefrontal cortex:
135
comparison between the Lewis and Fischer 344 rat strains. Brain
Res 716:157–64.
Smith SS, Gong QH. (2007) Ethanol effects on GABA-gated current in
a model of increased alpha4betadelta GABAA receptor expression
depend on time course and preexposure to low concentrations of
the drug. Alcohol 41:223–31.
Sohda T, Shimizu M, Kamimura S et al. (1993) Immunohistochemical
demonstration of ethanol-inducible P450 2E1 in rat brain. Alcohol
Alcohol (Suppl 1B):69–75.
Stoll G, Jander S. (1999). The role of microglia and macrophages in
the pathophysiology of the CNS. Prog Neurobiol 58:233–47.
Strandberg-Larsen K, Nielsen NR, Gronbaek M et al. (2008) Binge
drinking in pregnancy and risk of fetal death. Obstet Gynecologists
111:602–9.
Suk K. (2007) Microglial signal transduction as a target of alcohol
action in the brain. Curr Neurovasc Res 4:131–42.
Szumlinski KK, Diab ME, Friedman R et al. (2007) Accumbens neurochemical adaptations produced by binge-like alcohol consumption.
Psychopharmacology (Berlin) 190:415–31.
Townsend JM, Duka T. (2005) Binge drinking, cognitive performance
and mood in a population of young social drinkers. Alcohol Clin
Exp Res 29:317–25.
Walton MR, Dragunow M. (2000) Is CREB a key to neuronal survival?
Trends Neurosci 23:48–53.
Wand GS, Diehl AM, Levine MA et al. (1993) Chronic ethanol treatment increases expression of inhibitory G-proteins and reduces
adenylcyclase activity in the CNS of two lines of ethanol-sensitive
mice. J Biol Chem 268:2595–601.
Ward RJ, Lallemand F, De Witte P et al. (2008) Anti-inflammatory
actions of taurine analogues in phagocytic cells. BrJ Pharmacol
(submitted)
Ward RJ, Zhang Y, Crichton RR et al. (1996) Expression of NFKappaB in rat brain after administration of ethanol in vivo. FEBS Lett
389:119–22.
Weiss F, Lorang MT, Bloom FE et al. (1993) Oral alcohol selfadministration stimulates dopamine release in the rat nucleus accumbens: genetic and motivational determinants. J Pharmacol Exp
Ther 267:250–8.
Yan OS, Reith ME, Yan SG et al. (1998) Effect of systemic ethanol on
basal and stimulated glutamate releases in the nucleus accumbens of
freely moving Sprague-Dawley rats: a microdialysis study. Neurosci
Lett 258:29–32.
Yang X, Horn K, Wand GS. (1998) Chronic ethanol exposure impairs
phosphorylation of CREB and CRE-binding activity in rat striatum.
Alcohol Clin Exp Res 22:382–90.
Zhang Y, Crichton RR, Jorens PG et al. (1998) Decreased release of
nitric oxide by alveolar macrophages after in vivo loading with iron
or ethanol. Biochem Pharmacol 55:21–5.