Critical Reviews in Clinical Laboratory Sciences, 2011; 48(1): 1947

2011 Informa Healthcare USA, Inc.

ISSN 1040-8363 print/ISSN 1549-781X online
DOI: 10.3109/10408363.2011.580567

Review Article

Molecular and behavioral aspects of the actions of alcohol on

the adult and developing brain
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Silvia Alfonso-Loeches and Consuelo Guerri

Cell Pathology Laboratory, Prince Felipe Research Centre, Valencia, Spain

Abstract
The brain is one of the major target organs of alcohol actions. Alcohol abuse can lead to alterations in brain structure
and functions and, in some cases, to neurodegeneration. Cognitive deficits and alcohol dependence are highly
damaging consequences of alcohol abuse. Clinical and experimental studies have demonstrated that the developing
brain is particularly vulnerable to alcohol, and that drinking during gestation can lead to a range of physical, learning
and behavioral defects (fetal alcohol spectrum disorders), with the most dramatic presentation corresponding to
fetal alcohol syndrome. Recent findings also indicate that adolescence is a stage of brain maturation and that heavy
drinking at this stage can have a negative impact on brain structure and functions causing important short- and long-
term cognitive and behavioral consequences. The effects of alcohol on the brain are not uniform; some brain areas or
cell populations are more vulnerable than others. The prefrontal cortex, the hippocampus, the cerebellum, the white
matter and glial cells are particularly susceptible to the effects of ethanol.
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The molecular actions of alcohol on the brain are complex and involve numerous mechanisms and signaling
pathways. Some of the mechanisms involved are common for the adult brain and for the developing brain, while
others depend on the developmental stage. During brain ontogeny, alcohol causes irreversible alterations to the
brain structure. It also impairs several molecular, neurochemical and cellular events taking place during normal
brain development, including alterations in both gene expression regulation and the molecules involved in cellcell
interactions, interference with the mitogenic and growth factor response, enhancement of free radical formation and
derangements of glial cell functions. However, in both adult and adolescent brains, alcohol damages specific brain
areas through mechanisms involving excitotoxicity, free radical formation and neuroinflammatory damage resulting
from activation of the innate immune system mediated by TLR4 receptors.
Alcohol also acts on specific membrane proteins, such as neurotransmitter receptors (e.g. NMDA, GABA-A), ion channels
(e.g. L-type Ca2+ channels, GIRKs), and signaling pathways (e.g. PKA and PKC signaling). These effects might underlie the
wide variety of behavioral effects induced by ethanol drinking. The neuroadaptive changes affecting neurotransmission
systems which are more sensitive to the acute effects of alcohol occur after long-term alcohol consumption. Alcohol-
induced maladaptations in the dopaminergic mesolimbic system, abnormal plastic changes in the reward-related brain
areas and genetic and epigenetic factors may all contribute to alcohol reinforcement and alcohol addiction.
This manuscript reviews the mechanisms by which ethanol impacts the adult and the developing brain, and causes
both neural impairments and cognitive and behavioral dysfunctions. The identification and the understanding of the
cellular and molecular mechanisms involved in ethanol toxicity might contribute to the development of treatments
and/or therapeutic agents that could reduce or eliminate the deleterious effects of alcohol on the brain.
Keywords: Adolescent brain, alcohol addiction, alchohol spectrum disorders, behavioral dysfunctions,
CNS development, ethanol neurotoxicity, fetal genetic and epigenetic effects, growth factor signaling,
neuroinflammatory damage, oxidative stress
Abbreviations: 5-HT, serotonin; 5-HT3, 5-hydroxytryptamine-3; ADH, alcohol dehydrogenase; ADHD, attention
deficit hyperactivity disorder; ADNP,activity-dependent neuroprotective protein; AUD, alcohol use disorders;
BAL, blood alcohol levels; BBB, bloodbrain arrier; BOLD, brain oxygen level dependent; cAMP-PKA, cyclic AMP
dependent protein kinase A; CANX, gene encodes a member of the calnexin family of molecular chaperones;
CC, corpus callosum; CDK, cyclin-dependent kinase; CNS, central nervous system; COOH, carboxy; COX-2,
cyclooxygenase-2; CREB, cAMP-responsive element binding; CRF, corticotrophin-releasing factor; CYP2E1,
cytochrome P-450 2E1; DA, dopamine; DAMPs, damage associated molecular patterns; DARP-32, dopamine
and cAMP-regulated neuronal phosphoprotein; DRD2, dopamine receptor subunit 2; DTI, diffusion tensor

Address for Correspondence: Dr. Consuelo Guerri, Centro de Investigacin Principe Felipe, Avenida Autopista del Saler, 16, 40012-Valencia,
Spain. E-mail: guerri@cipf.es
Referee: Dr. Paula Hoffman, School of Medicine, University of Colorado Denver, Aurora, CO, USA

19
 20 S. Alfonso-Loeches and C. Guerri
imaging; ESPAD, European School Survey Project on Alcohol and other Drugs; F, fetal; FAS, fetal alcohol
syndrome; FASD, fetal alcohol spectrum disorders; G12, gestation day 12; GABA, gamma-aminobutyric acid;
GABA-A, gamma-aminobutyric acid A; GABABR1, gamma-aminobutyric acid (GABA) B receptor 1; GFAP, glial
fibrillary acidic protein; GIRK, G protein- activated potassium channel; HAS, high-alcohol-sensitive; HATs, histone
acetyltransferases; hESCs, human embryonic stem cells; HDACs, histone deacetylases; IGF, insulin-like growth
factor; iNOS, inducible nitric oxide synthase; L1, neural cell adhesion molecules L1; LAS, low-alcohol-sensitive;
LTP, long-term potentiation; MAPK/ERK, mitogen-activated protein kinases/extracellular signal-regulated kinase;
MRI, magnetic resonance imaging; mTORC1, rapamycin complex 1 signaling; NAc, nucleus accumbens; nACh,
neuronal nicotinic acetylcholine; NAP, active peptide of ADNP; NCAMs, neural cell adhesion molecules; NF-B,
nuclear factor kappa-B; NMDA, N-methyl-D-aspartate; NMDAR, N-methyl-D-aspartate receptor; NMDAR2B,
phosphorylated glutamate receptor subunit; NO/cGMP, nitric oxide/cyclic GMP; NP, neural progenitor; NSC,
neural stem cell; PEE, prenatally ethanol-exposed; PFC, prefrontal cortex; PI3K/AKT, phosphoinositide-3 kinase/
serine/threonine protein kinase; PKC, protein kinase C; PSA, polysialylated; RG, radial glia; ROS, reactive oxygen
species; Src, a non-receptor tyrosine kinase; TLRs, toll-like receptors; TM, transmembrane; VTA, ventral tegmental
area; WE, Wernickes encephalopathy; WHO, Word Health Organization.
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Introduction
Excessive alcohol consumption is the third leading
preventable cause of death in many countries1 and is
associated with multiple adverse health consequences,
including liver cirrhosis, various cancers, uninten-
tional injuries, addiction, brain damage and violence.
The World Health Organizations (WHO) Global Status
Report on Alcohol (2004 and 2009) estimates that 763.
million people worldwide have diagnosable alcohol-use
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rates over 30%. In the USA, about one third of high school
seniors reported binge drinking in the previous two
weeks6. Comparable adolescent alcohol involvement has
been noted for some Western Pacific countries, including
Australia and New Zealand4.
Research studies have identified a subtle neurochemical,
cellular, synaptic, and structural organization of the adoles-
cent brain7,8, which makes it more vulnerable to disruption
from activities such as binge drinking than the adult brain.
Alcohol-induced memory impairments, such as black-

disorders1. A recent study done in the UK that classifies
drugs by their overall harm scores, shows alcohol as the
most harmful drug to users and others2.
Despite the health consequences of alcohol abuse,
alcohol is one of the commonest drugs whose overdoses
can lead to brain damage. Even uncomplicated alcoholics,
with no specific neurological or hepatic problems, show
signs of regional brain damage and cognitive dysfunc-
tion3. Studies clearly indicate that alcohol is neurotoxic
with direct effects on nerve cells. Chronic alcohol abuse
and alcoholism are associated with multiple primary
and secondary disease states, which typically start with
experimentation and progress to addiction over a period
lasting several years. Alcohol consumption among alco-
holics is also associated with alteration in brain structure
and functions, and loss of brain mass, and these effects
could contribute to the progression of addiction.
The current trends of alcohol consumption starting
at younger age, together with the growing number of
women drinking excessively, imply increased alcohol-
related risks for the women progeny and for youths and
adolescents. Indeed, the prevalence of alcohol-related
problems and neurological deficits in adolescents is a
worldwide public health problem4. Reports from the
European School Survey Project on Alcohol and Other
Drugs (ESPAD) 20035, carried out in 35 European coun-
tries, indicate that young people today drink more and
with a clearer focus on drunkenness than earlier genera-
tions did. Binge drinking has been reported by 43% of 16
year old adolescents in the 30 days prior to questioning.
Eleven countries were noted to have rates of over 50%
(e.g. UK, 54%; Portugal, 56%) and most countries had
outs, and other cognitive effects9, are particularly common
among young drinkers and may be, at least in part, due to
disrupted neural plasticity in the prefrontal frontal cortex
and the hippocampus. In addition, adolescence is also
characterized by not only the rapid maturation of brain sys-
tems mediating reward, but by changes in the secretion of
stress-related hormones10,11, events which might enhance
anxiety and participate in the initiation pattern of alcohol
and drug consumption. Studies in human adolescents
demonstrate that drinking at early ages increases the likeli-
hood of developing alcohol-related problems.
One additional and important aspect to consider in
the actions of alcohol on the brain is the toxic effect on
the developing central nervous system (CNS). Indeed,
alcohol is one of the most common and important
substances to affect the developing fetal brain, and its
consumption during pregnancy can produce a wide
range of cognitive, behavioral and physical anomalies.
Furthermore, it is one of the leading preventable causes
of birth defects and neurodevelopmental disorders12. In
the most severe cases, these anomalies make up a pattern
of malformations termed fetal alcohol syndrome (FAS)13.
However, the effects of prenatal alcohol exposure lie in
a continuum of physical anomalies and behavioral and
cognitive deficits, and the term fetal alcohol spectrum
disorders (FASD) has been adopted14 as a non-diagnostic
umbrella term to describe this range of effects. Novel
neuroimaging techniques and experimental studies have
evidenced the vulnerability of the CNS to the effects of
alcohol, and have revealed that effects are not uniform
and that some brain areas or cell populations are more
vulnerable than others15,16.
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 21
This manuscript reviews the current knowledge of the
cellular and molecular mechanisms participating in the
effects of alcohol on the adult and developing brain, and
their correlation with the structural, cognitive, behavioral
and psychopathological deficits observed in alcoholics,
adolescents with alcohol abuse and individuals exposed
prenatally to alcohol.
Actions of alcohol on the developing brain
Fetal alcohol spectrum disorders and fetal alcohol
syndrome
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Over the last three decades, clinical and experimental
studies have provided direct evidence that the developing
brain is especially vulnerable to the toxic effects of alcohol
and that alcohol abuse during pregnancy can cause per-
manent brain damage in offspring which is associated with
life-long behavioral, social and cognitive disorders1618.
Among the most recognized consequences of prenatal
alcohol exposure is the FAS13, characterized by pre- and
postnatal growth deficiencies, craniofacial anomalies and
evidence of CNS dysfunction. FAS is now documented as
an important cause of intellectual disabilities and behavior
problems in many countries1822. However, it is now clear
that the effects of prenatal alcohol exposure lie in a contin-
uum of physical anomalies and behavioral and cognitive
deficits. This range of deleterious outcomes is now termed
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FASD14, and its prevalence (1% of all births) is higher than
that of FAS23. Notably, CNS dysfunctions, including altera-
tions in brain structure and behavior, are the most perma-
nent and devastating consequences of prenatal alcohol
exposure, and may occur in the absence of gross physical
malformations, including facial dismorphology associated
with FAS. We describe some of the most common perma-
nent changes in brain structure observed in children with
FASD, which in some cases, have been correlated with the
behavioral and cognitive dysfunctions associated with
prenatal alcohol exposure.
Neurobehavioral disorders associated with
FASD
Heavy prenatal alcohol exposure has been associated
with widespread neuropsychological deficits across sev-
eral domains, including general intelligence, memory,
language, attention, learning, visuospatial abilities,
executive functioning, fine and gross motor skills, and
social and adaptive functioning24. The mean IQ of chil-
dren with FAS is estimated to be in the low 70s24, and FAS
is the leading preventable cause of mental retardation25.
However, the majority of children with FAS are not men-
tally retarded and their IQ scores range widely (e.g. from
20 to 120)24. Individuals prenatally exposed to high lev-
els of alcohol, but without all the characteristics of FAS,
have average IQs in the low 80s24. Children exposed to
alcohol prenatally also display poorer academic achieve-
ment and higher learning disabilities rates than non-
exposed children26, which may relate to impairments in
verbal and non-verbal learning and memory2729. Motor
dysfunctions, such as tremors, weak grasp, poor hand/
eye coordination, gait and balance difficulties, motor
response programming and movement time have also
been described in FASD children24,30,31, and persist into
adulthood in heavily exposed populations32.
Some studies suggest that the long-term retention of
verbal information is intact in alcohol-exposed individu-
als, but that initial encoding processes may be impaired28,33.
In addition to non-verbal memory difficulties, individuals
with FASD show visuospatial processing deficits27, sug-
gesting abnormality in the frontal-subcortical pathway
and greater damage to the left hemisphere. Attention def-
icits are also frequently cited in the FASD population34,35,
while attention deficit hyperactivity disorder (ADHD) is
the most frequent comorbid psychiatric disorder diag-
nosed in alcohol-exposed children36. A recent collabora-
tive study also suggests that executive function and spatial
processing are especially sensitive to prenatal alcohol
exposure37.
FASD individuals have social and adaptive deficits, since
they are more likely to be rated as hyperactive, disruptive,
impulsive or delinquent than non-exposed children38,39.
Furthermore, and based on parents ratings of their childs
behavior, children with histories of prenatal alcohol expo-
sure displayed significant and profound impairment,
and showed particular difficulties in social, attention and
aggressive domains40,41. One interesting finding is the high
share of secondary disabilities and mental illness in adults
with FASD, as well as other psychopathological condi-
tions, such as depression, anxiety disorders, suicidal ide-
ation and attempts throughout their life42,43. These results
highlight not only the importance in identifying FASD at
early ages, but also the provision of specific treatments to
address this developmental disability since early identifi-
cation and treatment have been demonstrated to be pro-
tective against more serious secondary disabilities44.
Influence of prenatal alcohol exposure on
brain structure
The effects of alcohol on brain structure originate from
the first neuropathological findings of children with FAS.
These studies have revealed widespread damage through-
out the brain, including microcephaly, errors in migra-
tion, agenesis of the corpus callosum (CC) and anterior
commissure, as well as cerebellar and brainstem anoma-
lies. Likewise, abnormalities of the basal ganglia, dien-
cephalon, cerebellum, brainstem, optic nerve, olfactory
bulb, hippocampus, pituitary and neural tube have been
reported13,4549, The variability of brain malformations can
explain the lack of specific brain abnormalities in FAS46,48.
However, in recent years, the appearance of novel neu-
roimaging techniques (e.g. magnetic resonance imaging
(MRI), functional MRI, diffusion tensor imaging (DTI),
magnetic resonance spectroscopy, positron emission
tomography, and single phton emission computed tomog-
raphy) has enabled the study of living individuals with
 22 S. Alfonso-Loeches and C. Guerri
FASD, and has provided new insights into the structural and
functional alterations caused by prenatal alcohol exposure.
Consistent with autopsy findings, MRI studies have reported
overall volume reductions in the cranial, cerebral, and cer-
ebellar vaults in FASD20,5056. Abnormal cortical thickness
and brain-behavior correlation have also been observed in
individuals with heavy prenatal alcohol exposure57. These
studies have also demonstrated that the effects of alcohol
are not uniform since some areas of the same region are
more vulnerable to the effects of alcohol than others. For
example, the parietal lobe50,55,58,59, portions of the frontal
lobe55 and specific areas of the cerebellum20,60,61, appear to
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be especially sensitive to alcohol insult.
Abnormalities of the CC, including complete and
partial agenesis56,62,63, hypoplasia20,51,56,62, displacement in
posterior regions54, regional surface area reductions in
anterior and posterior areas54,63, and increased variability
in shape64,65, have also been reported. More recently, (DTI)
has been used to examine the white matter integrity of the
CC66,67. Among young adults, individuals with FAS have
presented diminished white matter integrity in the genu
and splenium of the CC when compared to controls66,
while only diffusion differences in the isthmus of the CC
have been noted for individuals with more moderate lev-
els of exposure67. One important association found is that
of CC abnormalities with the neuropsychological deficits
commonly observed in alcohol-exposed populations,
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such as bimanual coordination29, attention34, verbal learn-
ing ability54 and executive functioning65,68.
The cerebellum has also been identified as being partic-
ularly vulnerable to prenatal alcohol exposure. Reductions
in both the surface area20 and the cerebellar volume50,51,
have been described in children with FAS and FASD. In
addition, some studies have confirmed that the volume
of the anterior cerebellar vermis, an early developing part
of the cerebellum, is reduced in FASD if compared with
controls, even after controlling for overall brain size20,61.
FAS subjects also show more severe displacement in the
superior and anterior edges of the anterior vermis than
non-dysmorphic FASD individuals60. Given that the cer-
ebellum is responsible for the execution of motor behavior,
such as posture, balance and coordination, the structural
abnormalities observed may help explain the motor defi-
cits often seen in FASD individuals29,30. The cerebellum is
also involved in other functions, such as attention regula-
tion69 and classical conditioning70. Accordingly, deficits in
classically conditioned eye blink responses are noted in
children with FAS71 while cerebellar vermis displacement
is negatively correlated with verbal learning and memory
in FASD60.
Risk factors influencing the effects of
prenatal alcohol exposure on the developing
brain: critical periods
Clinical data indicate that the range and magnitude of
prenatal alcohol-induced effects on brain abnormalities
and behavioral outcomes considerably vary. Studies into
humans and experimental FASD models have provided
a number of biological and environmental factors that
could influence the effects of alcohol on the developing
brain. The most important factors include dose of alcohol
and exposure pattern, developmental timing of exposure,
mothers and fetuss genetic background, maternal age and
nutrition, socio-economic status, and synergistic reactions
with other drugs. Among these, the levels of alcohol reach-
ing the fetal brain and duration of exposure markedly influ-
ence the type and extent of damage. In this context, both
human and animal studies have found that binge drink-
ing (drinking a large amount of alcohol in a short period
of time), which produces high blood alcohol levels (BAL
200mg/dL), is more damaging to the fetus than chronic
alcohol exposure that produces lower BALs ( 150mg/dL)72.
In addition, the mothers and fetuss genetic background,
such as variations in alcohol metabolism, also influences
the risk of alcohol-induced malformations in the fetus,
which is another risk factor in alcohol-induced fetal brain
anomalies. For example, the more efficient alcohol dehy-
drogenase (ADH) allele, ADH 1B*3, affords protection for
FASD outcomes73, while the maternal and fetal ADH1B*2
allele lowers the risk of FAS in a South African population
(comparing to ADH1B*1)74. Experimental animal studies
have also demonstrated the differential vulnerability to
motor deficits in rat lines selectively bred for the extremes
in alcohol-induced sleep time (high-alcohol-sensitive
(HAS) and low-alcohol-sensitive (LAS)), when exposed to
alcohol during the neonatal period75.
Furthermore, the specific affected brain structure
and the magnitude of damage are also strongly influ-
enced by the developmental timing of alcohol exposure
(Figure 1). For example, facial dysmorphology, a salient
feature of FAS13, appears to arise only when high-peak
BALs occur during the embryonic stage of gastrulation76.
Mice exposed to alcohol on embryonic day 7 (E7) or E876
at BALs of ~250mg/dL (reached after binge-drinking),
and macaques exposed to alcohol (BAL=223mg/dL) on
E19 or E2077 exhibit FAS-associated facial dismorphia.
Chronic exposure to alcohol during gastrulation (at BAL
~150mg/dL) also has a negative impact on the develop-
ing brain as it reduces the neural progenitors pool78 and
causes long-term effects on both the forebrain79,80, and
mature brainstem nuclei structures81. Human studies
have demonstrated that both binge drinking and chronic
alcohol abuse during the early human embryogenesis
stage (corresponding to the third week of human gesta-
tion) are associated with a greater incidence of cranio-
facial defects and mental disabilities82. By using human
neural progenitors (NPs) to assess early stages of brain
development, a recent study has revealed how 200mg/
dL alcohol exposure impairs human NPs survival and
affects their differentiation to mature neural cells83.
Alcohol also affects other ontogenetic stages of brain
development. For instance, a second critical period is the
neuroepithelial cell proliferation and migration stage,
which occurs from weeks 7 to 20 of human gestation84, and
from gestation day 12 (G12) to G2021 in rats. At this stage,
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 23
most of the brain areas (except the cerebellum) begin to
differentiate. Experimental studies have reported that alco-
hol exposure (BAL= ~125150mg/dL) at this stage alters
neuronal migration, impairs cell generation timing and
reduces the number of neurons and glial cells in the neo-
cortex, the hippocampus and the sensory nucleus78,8588. As
with other neurodevelopmental disorders84, perturbation
of neuroglial proliferation and migration by alcohol can
cause long-term abnormalities in the cerebral cortex and
brain size, as noted in FAS and FASD individuals50,55,58, and
likely contributes to the cognitive defects observed in adults
with FASD. Additionally, since the formation of the CC and
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the glial midline starts at around 7 weeks in humans89,90,
exposure to alcohol at this stage might disrupt early events
in callosal formation, leading to agenesis, hypoplasia, or
abnormalities in the CC56,62,63. Accordingly, a recent study
using a combined transcriptome analysis of human and
mouse fetal cerebral cortices exposed to alcohol in vitro
and in vivo, respectively, has demonstrated that in vitro
human cortical fetal tissue (between 1518 gestational
weeks) exposed to 235mg/dL alcohol for 24h leads to the
alteration of many genes, including the down-regulation of
the expression of those genes participating in the specifica-
tion of callosal projection neurons91. These findings suggest
the sensitivity of the CC to the effects of alcohol during the
mid gestational stage.
Finally, alcohol also interferes with the brain growth
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spurt which, in the rat occurs, from gestational day 20
to postnatal day 19 (roughly the equivalent to the third
trimester of human gestation), although brain growth
does continues into the first two years of life in humans
and extends until postnatal day 25 in rats. This period is
Figure 1. Developmental effects of alcohol during central nervous system ontogeny in humans and rats. NSCs, neural stem cells; F, fetal; P,
postnatal. Adapted from Moore and Persaud 1973424.
characterized by a remarkable development of glial cells
and dendritic arborization, and the cerebellum under-
goes its most rapid period of development. Several stud-
ies have revealed how this period is especially vulnerable
to alcohol-induced microcephaly, neuronal cell loss72,92,93,
synaptogenesis and glial derangements94, and can cause
learning/memory deficits95,96. A single administration of a
high dose of alcohol that produces a BAL above 200mg/
dL for 8h to neonatal mice can trigger significant neu-
roapoptosis in several brain regions93,97,98. However, the
caudate nucleus, the frontal and parietal cortices are the
most vulnerable regions to the neuroapoptotic effects of
alcohol99. In line with these data, alterations in both the
caudate nucleus and the frontal cortices have also been
noted in individuals with FASD100,101. Animal studies have
also revealed that alcohol effects are permanent since
neonatal binge-like alcohol exposure (BAL=330.03mg/
dL) results in long-term deficits of the hippocampal neu-
rogenesis in young adult rats102 (Figure 1).
Collectively, the experimental data indicate that although
alcohol can interfere with important ontogenetic stages of
the mammalian brain, the levels of alcohol reaching the
fetal brain and the developmental timing of alcohol expo-
sure are important determining factors of specific affected
brain structures and the resulting degree of damage.
Mechanisms of the actions of alcohol on the
developing brain
There is experimental evidence to demonstrate that alco-
hol interferes with several of the molecular, biochemical
and cellular events participating in the correct formation
 24 S. Alfonso-Loeches and C. Guerri
of the developing CNS, and that these effects appear
to be temporally specific and involve stage-specific
changes in cell function or gene regulation. Therefore,
depending on the time and levels of alcohol exposure,
alcohol could interfere with cell proliferation, migra-
tion, growth and differentiation, and may even cause cell
death. For example, chronic exposure to alcohol (BAL=
137157mg/dL) during early embryonic development,
when cells undergo rapid division, inhibits proliferation
and reduces the number of cells generated87, including
neural progenitor cells (e.g. radial glia (RG))88,103,104, and
neurons, and may cause abnormal cell migration105,106.
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However, if alcohol exposure occurs later when cells dif-
ferentiate and synapses are established, it can lead to a
reduced number of formed synapses and cell death97,107.
Among the molecular mechanisms proposed as the
potential candidates responsible for the FASD range, we
find the following mechanisms15,108,109.
Alterations in gene expression regulation
and homeobox genes
A large body of evidence demonstrates the role of retinoic
acid in embryonic development, which is an important
regulator of the Hox gene expression110. Deficiencies in
retinoic acid or in its receptors cause neural crest death
and nervous system defects111. Some earlier studies sug-
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gested that alcohol-induced embryo-fetal abnormalities
can be caused by reduced retinoic acid due to the alco-
hol-induced inhibition of retinol oxidation112. Indeed,
alcohol has been shown to compete with retinol for the
binding to ADH (Class IV form), leading to a deficient
conversion of retinol into retinoic acid113. This hypothesis
is supported by findings showing that high alcohol levels
(BAL=400mg/dL) lower the amount of retinoic acid in
neural cells113. One study114 has revealed how the binge
pattern of alcohol exposure suppresses the expression of
hemeobox gene, msx2, in the developing mouse embryo.
Conversely, another study which used chick embryos
encountered no alterations in either the msx2 expression
or the related growth factor, BMP4, after alcohol expo-
sure106. Further research is warranted to understand the
molecular mechanisms which control embryonic devel-
opment and the involvement of Hox gene expression
alterations in pathological conditions, such as alcohol
exposure or alcohol embryopathy.
More recent studies have suggested the relevance of
epigenetic changes in the FASD etiology115,116. Epigenetics
involves modifications in the activation of certain genes,
but not the basic DNA structure. During brain develop-
ment, epigenetic modifications, such as histone acetyla-
tion and DNA methylation, play an important role in both
the stability and plasticity of developing neuronal circuits,
and new data indicate the impact of epigenetics on the
pathogenesis of brain diseases117. Recent findings have
revealed that maternal alcohol consumption alters the
epigenotype and the phenotype of offspring in a fetal alco-
hol mouse model116 and induces epigenetic modifications
in critical genes in mediating dysmorphology in neurode-
velopment115. Further research is necessary to understand
the molecular mechanisms participating in the alcohol-
induced epigenetic modification, and to elucidate
whether these events occur in specific genes with a func-
tional spectrum relating to development. Accordingly, an
earlier study established that prenatal alcohol exposure
impairs GFAP demethylation, thus delaying the GFAP
expression during rat brain development88.
Interference with the mitogenic and growth
factor responses involved in neural stem cell
proliferation, migration and differentiation
Neurotrophic factors, particularly neurotrophins, play
a crucial role during development in neuronal survival
and maturation, and they are important regulators of
naturally occurring cell death118. Several studies have
demonstrated that alcohol exposure interferes with the
production and/or the response of several trophic fac-
tors by impairing cell growth, division and survival and
by affecting nervous cells proliferation, survival and dif-
ferentiation (See reviews119121). Trophic factors are able
to protect some neuronal populations against alcohol-
induced neurotoxicity122124. In vivo studies have shown
that exposure to alcohol (maternal BAL=125130mg/
dL) during brain development lowers the levels of the
brain-derived neurotrophic factor and alters its recep-
tor, TrkB, by impairing the function of the intracellular
signaling pathways involved in cell survival (MAPK/ERK
and PI3K/AKT pathways). These effects are associated
with enhanced cell death by apoptosis and necrosis in
the postnatal rat cerebral cortex125. These data suggest
that any alteration in growth factor functions may under-
lie some alcohol-induced alterations in the developing
CNS.
Alcohol can also interfere with the growth factors
relating to cell-cycle events and factors associated with
cell differentiation and survival. For example, a number
of studies have shown that alcohol increases the cell
cycle (particularly G1) and reduces the number of prolif-
erating cells both in vivo and in vitro122,126,127. Regulation
of cell-cycle events occurs through the induction of those
genes encoding for cyclins, which, in turn, interact with
cyclin-dependent kinases (CDKs), to further modulate
cell-cycle regulatory proteins and cell-cycle timing.
Alcohol, and specifically ethanol, has been also shown to
inhibit cyclins and CDKs in cerebellar tissue128,129, but the
specific role of ethanol-induced changes in the expres-
sion of cyclins and CDKs for teratogenic deficits in cell
acquisition and cell loss remains unclear. Finally, inhibi-
tion of the intracellular signaling of insulin-like growth
factor (IGF)-mediated cell proliferation and survival has
also been shown in not only cerebellar granule neurons
exposed to ethanol (150160mg/dL), but also in animals
FASD models130,131. If we consider that growth and neu-
rotrophic factors play critical roles during CNS formation,
disruption of their support might underlie the short- and
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 25
long-term neurodevelopmental dysfunctions associated
with FASD.
Disturbances in the molecules that mediate
cell-cell interactions
Cell adhesion molecules belong to the immunoglobulin
superfamily and play a crucial role in cellcell interac-
tions, which form the basis for brain organizations by
participating in activity-dependent plasticity during
development, and synaptic plasticity in adults132. Among
these molecules, neural cell adhesion molecules, NCAM
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
and L1 have received particular attention in alcohol-
induced developmental brain abnormalities because
they participate in several aspects of neural development
including cell migration, morphogenesis, synaptic estab-
lishment and the synaptic plasticity of the nervous system.
Knockout NCAM mice display overall brain size reduc-
tion and persistent behavioral deficits133, while defects
in L1 can lead to abnormalities in brain development
and mental deficiencies134. Ethanol has been found to
interact with L1135 to disrupt L1-mediated cell-cell adhe-
sion in cultured cells136, and to inhibit L1 cell adhesion
molecule tyrosine phosphorylation and dephosphory-
lation137. Octanol antagonizes L1-mediated cell adhe-
sion and prevents ethanol-induced apoptosis in mouse
culture embryos138. Chronic alcohol exposure (maternal
For personal use only.
BAL=125140mg/dL) also alters the expression pattern
of NCAM during rat postnatal cerebral cortex develop-
ment by increasing the embryonic highly polysialylated
NCAM (PSA-NCAM) form, and by reducing the expres-
sion of adult, less sialylated NCAM isoforms with more
adhesive properties, during the synaptogenesis period139.
Alterations in both the NCAM levels and the NCAM sialy-
lation state have been observed in developmental defects
and degenerative diseases, and after exposure to some
neurotoxins during development139.
Finally, if we consider that both L1 and NCAM are
involved in the morphogenesis and plasticity of the CNS
by regulating cellcell interactions, alcohol-induced
disruptions in these neural cells adhesion molecules
might contribute to the defects in morphogenesis, neu-
ral migration, glial development, synaptogenesis and in
the plasticity observed in animals exposed in utero to
alcohol.
Oxidative stress and free radical damage
The developing brain is particularly vulnerable to reactive
oxygen and reactive nitrogen species-mediated damage.
This vulnerability is partly due to its high concentrations
of unsaturated fatty acids, high oxygen consumption
rate, low anti-oxidant concentrations and high content
of metals catalysing free radical formation. Alcohol can
increase the reactive oxygen species (ROS) generated
directly via mitochondrial respiration forming superox-
ide, hydroxyl radicals or nitrogen radicals or via the oxi-
dation of ethanol by enzymes such as cytochrome P-450
2E1 (CYP2E1) to generate hydroxyethyl radicals. Indeed,
alcohol increases the CYP2E1 expression, the formation
of ROS and lipid peroxidation in the brain of both chronic
alcohol-fed rats140 and fetuses exposed prenatally to
alcohol141,142. Likewise, a relationship between ROS for-
mation and free radicals has been clearly established,
while alterations in the cytoskeleton and cell functions
have been demonstrated in astrocytes143, neural crest
cells144, cultured cortical neurons145, cerebellar granule
cells, and cerebellar tissue146,147.
Alcohol can also reduce endogenous antioxidant lev-
els in vivo and in vitro143,148,149, thus favouring an increased
in lipid peroxidation. Indeed, there is growing evidence
demonstrating that antioxidant supplementation can
prevent some alcohol-induced damage123,142, or patho-
genesis in certain experimental models of fetal alcohol
exposure, and it has been suggested as a potential treat-
ment to prevent alcohol toxicity during development.
Alcohol-induced teratogenesis in mouse embryos can be
diminished by adding superoxide dismutase150. Vitamin
E supplementation has been seen to prevent alcohol-
induced cell loss in hippocampal cultures151, cerebellar
granule cell cultures152, and the cerebellar Purkinje cells of
alcohol-exposed postnatal rats123. Collectively, although
antioxidants can be protective in some in vitro and in
vivo models, it is uncertain whether antioxidant supple-
ments may serve as effective potential interventions.
Dysregulation of the molecular signaling
involved in cell survival
A number of studies have indicated that alcohol promotes
the signaling pathways leading to cell death, but at the
same time interferes with those pathways involved in cell
survival such as those associated with neurotrophic fac-
tors action125, (see above). Recent reports have suggested
that alcohol interferes with cyclic AMP (cAMP) produc-
tion and transcription factor CREB153. Conversely, alcohol
facilitates those signaling pathways classically associated
with cell death, such as the esphigomyelinase-ceramide
pathway, which leads to apoptosis154. Likewise, by acti-
vating RhoA/ROCK-I/MLC signaling in proliferating
cultured astrocytes155, alcohol induces changes in both
focal adhesions and the actin-myosin system, leading to
cell contraction, membrane blobbing, and cell death by
anokis155.
Alcohol exposure during brain developmental
also promotes caspase-3 activation, an executioner
protease that is activated during apoptotic cell death156.
For instance, human neuroprogenitors exposed to physi-
ologically relevant concentrations of ethanol (25mmol/L
or ~115mg/dL) trigger apoptosis and caspase-3 acti-
vation83. Furthermore, binge-like alcohol exposure
(BAL=250mg/dL) exposure during the neonatal period
in rodents (equivalent to the third trimester of human
gestation) has induced the death of the post-mitotic neu-
rons in the cerebral cortex99,157, and the cerebellum158,159.
In both cases, cell death appears to be associated with
 26 S. Alfonso-Loeches and C. Guerri
caspase-3 activation99,159. In the cerebellum, caspase-3
activation has been found to be dose-dependent and
occurs during a period of enhanced vulnerability to
alcohol-induced cell death (postnatal day 4). However,
the up-regulation of caspase-3 in the cerebral cortex has
been associated with the inhibition of NMDA-glutamate
receptors NMDAR and the activation of GABA receptors,
which can lead to apoptotic-neurodegeneration157.
Other signaling pathways involved in the developmen-
tal mechanisms of the nervous system and in cell survival
are those associated with neurotransmitter receptors,
such as glutamate and GABA receptors. Nevertheless,
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if we consider the critical roles of glutamate receptors,
particularly NMDAR -mediated Ca2+ influx during brain
ontogeny, most studies have focused on these receptors.
Indeed, NMDAR are involved in neuronal differentiation
and survival, neuronal migration, synaptogenesis, struc-
tural remodeling, long-lasting forms of synaptic plastic-
ity and higher cognitive functions160. Prenatal and early
postnatal alcohol exposure have been shown to impair
NMDAR function and expression161, to reduce both
NMDA-stimulated Ca2+ entry into neonatal neurons
and [3H]MK-801 binding to NMDA, and to affect the cell
surface expression of NMDARs NR1 splice variants and
NR2 subunits161164. Conversely, chronic prenatal alcohol
exposure (maternal BAL=328mg/dL) up-regulates the
GABA type A (GABA-A) receptor expression in both the
For personal use only.
cerebral cortex and the hippocampus of adult animals165.
Alterations in the NMDA and GABA-A receptors during
brain ontogeny may underlie some learning and memory
deficits observed following fetal alcohol exposure.
Derangements in glial proliferation,
differentiation and functioning
Alterations in glial development are suspected of contrib-
uting to adverse alcohol effects on the developing brain
because neuroglial heterotopias, alterations in neuronal
migration, agenesis of the CC and anterior commissure
(areas originally formed by glial cells) have been observed
in post-mortem studies of children with FAS46.
Glial cells are present throughout CNS development
and play critical roles in multiple developmental events.
During embryogenesis, RG provide physical and chemi-
cal guidance for the migration of young neurons, and
more recent data indicate that RG serve as a multipoten-
tial neural precursor cell with the potential to self-renew
and to generate neurons166 and oligodendrocytes167.
During synaptogenesis, glial cells and glial-derived
factors promote the formation of mature functional
synapses168, regulate neurotransmitters and energy in
the brain169, and play key roles in synaptic function and
behavior170172. Disturbance of either glia or neuronal-
glial communication during the well-established criti-
cal periods of brain development can cause irreversible
deficits in the CNS function173175. Alteration of astrocytes
and neuronal-glial interactions and signaling could also
impair cortical plasticity176, along with the generation of
oligodendrocytes and myelin177,178, thus causing abnor-
malities in white matter.
Clinical and experimental studies have provided
compelling evidence that fetal and/or neonatal expo-
sure to alcohol profoundly affects glial functions94,179,180.
Experimental studies have demonstrated that alcohol
exposure during embryogenesis reduces the telenceph-
alic RG progenitor pool, as well as its transformation into
neurons and astrocytes78, and that it impairs RG morphol-
ogy. Alcohol also impairs astroglial proliferation, survival
and functions179,180, which can affect many developmental
processes, such as the availability of trophic support mol-
ecules involved in neuronal survival, modulation of the
formation of mature synapses, and regulation of synaptic
transmission and plasticity176,181. Indeed, alcohol reduces
the glial factor, the activity-dependent neuroprotective
protein (ADNP), in both the cerebral cortex and astrocytes
from prenatally alcohol-exposed (PAE) fetuses. Co-cultures
of PAE astrocytes with control neurons markedly decrease
neuronal growth, differentiation and synaptic connec-
tions, and these effects can be reverted with the addition
of NAP, the active peptide of ADNP, which suggest the role
of astroglial factors in neuronal plasticity181. NAP admin-
istration is able to restore brain weight, brain volume and
regional brain size in an experimental FAS model182.
Neuroimaging studies of individuals with FASD have
shown more important losses in white matter structures
(mainly composed of astrocytes, oligodendrocytes and
myelin) than in gray matter structures50,58,59,183, which
supports the experimental data. Changes in brain
metabolism in adolescents and young adults with FASD
have also been reported101 using magnetic resonance
spectroscopy to assess neuronal and glial markers. The
metabolic anomalies observed in different brain struc-
tures (parietal and frontal cortices, frontal white matter,
CC, thalamus and cerebellar dentate nucleus) of FASD
individuals are consistent with abnormalities in the glial
cell pool rather than in neurons. As astroglial cells play
critical roles in the metabolic processes linked to neu-
ronal activity such as blood flow, energy and glucose
utilization169, these results suggest that ethanol-induced
glial impairment during brain development could affect
neuronal activity, leading to permanent metabolic and
structural brain alterations.
In short, the evidence deriving from clinical and
experimental data highlights the vulnerability of human
glial cells to the teratogenic effects of alcohol and their
permanent consequences. Recent findings also suggest
that white matter is a target of alcohol teratogenesis and
that the neurobehavioral sequelae associated with ges-
tational alcohol exposure, especially in areas of execu-
tive dysfunction and visual processing deficits, may be
related to white matter anomalies184.
Summary and potential FASD treatments
Our current understanding of pathogenesis mechanisms
suggests that alcohol acts through multiple targets on
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 27
different cell types and developmental stages. Therefore,
prevention programme and social and political efforts
to mitigate or eliminate drinking during pregnancy, are
presently the best ways to limit the consequences of
maternal alcohol consumption and the appearance of
FASD. One particular approach is to find specific phar-
macotherapeutic or nutritional interventions during
pregnancy that could be applied to the at-risk popula-
tions that are most likely to give birth to children with
FASD. For example, the aforementioned experimental
studies demonstrate that nutritional supplementation
with antioxidants142, choline185 or D-NAP peptide182 dur-
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
ing gestation can ameliorate some deficits induced by
prenatal or neonatal alcohol exposure.
At the same time, public health and economic concerns
warrant continued research to develop systematic identi-
fication, effective interventions and improved outcomes
for individuals with FASD. Research on biomarkers of
fetal alcohol damage186 and studies into neuroimaging
techniques may advance diagnostic possibilities, which
will improve the future of those individuals affected by
prenatal alcohol exposure. Along these lines, experimen-
tal studies have demonstrated that experience-dependent
complex motor learning187 and environmental enrich-
ment can ameliorate some behavioral deficits associated
with prenatal alcohol-induced brain damage. Until risky
alcohol abuse patterns during pregnancy are detected and
For personal use only.
eliminated, prevention policies will be an important step to
abolish alcohol-induced developmental brain disorders.
Impact of alcohol abuse on the adolescent
brain
Alcohol is one of the first drugs of choice among young
people and adolescents, and heavy binge drinking is
becoming increasingly frequent among high school
students in different countries. Recent epidemiologi-
cal data have drawn attention to the high rates of risky
drinking patterns, such as binge drinking, in adolescents
and young adults, and to a surge in the prevalence of
alcohol dependence in these populations. Indeed, the
number of high school students consuming alcohol and
the widespread occurrence of episodes of drunkenness
and binge drinking in the USA have considerably risen
in the last 10 years6,188. Reports from the ESPAD5, indicate
that young people today drink more and with a clearer
focus on drunkenness than earlier generations did. In
Mediterranean countries, concerns about youth drink-
ing have substantially grown in recent years. One clear
trend is a shift to binge drinking as a natural habit which
is associated with drunkenness, especially among teen-
agers, in all the wine cultures with moderate alcohol
consumption189,190. This alcohol consumption tendency
is also observed in eastern countries such as Hong Kong
and Singapore, whose adolescents have recently reported
increases in binge-pattern191,192.
Concerns about heavy drinking during adolescence
have emerged after new evidence demonstrating that
adolescence is a brain maturation stage vulnerable to
alcohol toxicity, alcohol and substance use problems and
psychiatric disorders193. Indeed, findings in the last decade
have clearly shown that adolescence is a critical devel-
opmental period in which certain brain areas undergo
important structural and functional changes in both syn-
aptic plasticity and neural connectivity8,194. These struc-
tural changes concomitantly occur with modifications in
some neurotransmitter systems and hormone secretion,
which markedly influence the refinement of neural cir-
cuits and certain brain areas such as the prefrontal cortex
(PFC), subcortical areas and the hippocampus.
Alterations in brain connectivity along with hormonal
changes occurring during puberty (e.g. secretion of
gonadal steroids and stress-related hormones)10,195, are
associated with adolescents specific behavioral charac-
teristics, such as increased sensation-seeking, risk-taking
behaviors, low levels of harm avoidance, impulsivity and
anxiety196, and might well explain the initiation pattern of
alcohol and drug consumption. Likewise, the relatively
late development of the PFC circuits involved in judge-
ment and inhibitory control may underlie the propensity
of adolescents to impulsivity, and to ignore the negative
consequences of their behavior, both of which could
increase the risk of substance abuse.
By considering the enormous plastic changes occur-
ring during teenage/adolescent brain maturation stages
and the vulnerability of the developing brain to the dam-
aging effects of alcohol, one could expect that alcohol
consumption in juvenile and adolescent stages would
have a significant impact on cognitive and behavioral
functions in adulthood197. Certainly in recent years, a
number of clinical and animal studies have shown the
impact of alcohol on the adolescent brain, and progress
has been made in linking behavioral effects of adolescent
drinking to underlying cellular and molecular mecha-
nisms. Furthermore, excellent reviews have been recently
published on this topic11,198200.
Neurodevelopmental processes during
adolescence and the impact of alcohol
drinking
As previously mentioned, the brain undergoes significant
changes during adolescence, particularly the PFC and the
hippocampus. The two major cellular processes that con-
tinue during adolescence are myelination of axons, and
overproduction and elimination of synapses, or pruning.
Although myelination is completed in the sensory and
motor cortices within the first few years of life, the forma-
tion of myelin sheaths around axons continues in the fron-
tal cortex during adolescence201. Progressive myelination of
axons results in developmental increases in cortical white
matter throughout adolescence and into adulthood, which
might serve to accelerate the information flow58,202. The sec-
ond process to occur in the prefrontal cortex during ado-
lescence is increased synaptic density, followed by a period
of synaptic pruning. Histological evidence has shown that
 28 S. Alfonso-Loeches and C. Guerri
the overproduction and elimination of synapses in the PFC
cortex in humans and monkeys extends throughout ado-
lescence and into young adulthood203. What is important to
note is that this brain area is involved in executive functions
such as cognitive flexibility, self-regulation and evaluation
of risk and reward (See reviews11,204).
MRI in humans has confirmed that the volume of PFC
lowers from adolescence to young adulthood205207, and
that this reduction is associated with refinement in the
neural circuits and connectivity. For instance, loss of syn-
apses, especially the excitatory glutamatergic inputs to
the PFC, occurs during adolescence208, while dopamine
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(DA) and serotonin (5-HT) input to the PFC increase dur-
ing adolescence but decrease later on in life209,210. These
remodeling changes in neural circuits are associated
with both cognitive functional modifications and the
acquisition of executive functions (e.g. response inhibi-
tion, attention, working memory)211,212. For example, level
of intelligence is associated with the trajectory of cortical
development, primarily in the frontal regions (e.g. the
PFC) implicated in the maturation of intelligent activ-
ity. More intelligent children demonstrate a particularly
plastic cortex with an initial accelerated and prolonged
phase of cortical increase, which leads to equally vigor-
ous cortical thinning by adolescence213.
Given the above neurochemical, cellular, synaptic, and
structural organization of the teenage/adolescent brain
For personal use only.
areas and the vulnerability of the developing brain to the
damaging effects of alcohol, one might expect that alcohol
consumption in the juvenile and adolescent stages would
have a greater impact than in mature adult brain func-
tions197. Indeed, clinical and experimental studies have
revealed that alcohol affects adolescent and adult brain
functions and behaviors differently, and that adolescents
are more vulnerable to the deleterious effects that alcohol
has on brain functions and behavior. Adolescent binge
drinking is associated with cognitive deficits, poor aca-
demic achievements214,215, and attentional and visual work-
ing memory processing9. In fact, memory problems are
among the most common dysfunctions in teenagers with
alcohol use disorders (AUD), and these effects have been
associated with brain structural abnormalities and the
brains response to a spatial working memory task. Studies
using MRI and DTI have shown PFC abnormalities in ado-
lescent binge drinkers, including reduced integrity of the
white matter of adolescents with AUD216,217, and those not
meeting the diagnostic criteria for AUD218. Notable gender
differences have been observed in alcohol-induced brain
damage. Thus, although PFC abnormalities have been
noted in both young men and women with AUD, female
subjects exhibit smaller volumes than their male counter-
parts who, in turn, present larger volumes compared with
same-gender controls217. These findings correlate with the
functional neuroimaging studies which report that male
subjects with AUD present increased frontal activation,
while female drinkers display limited frontal activation in
response to spatial working memory. Alcohol also induces
greater impairments in both brain functioning and gray
matter volume for female adolescents than male. This sug-
gests that female adolescents are more vulnerable to adverse
alcohol effects than their male counterparts, and may even
be at an increased risk of behavioral deficits219, thus con-
firming the well-established notion that women are more
vulnerable than men to alcohol-related organ damage220.
Indeed, female alcoholics also appear to be more suscep-
tible to frontal lobe gray matter reduction than their male
counterparts221,222. Although the mechanisms involved in
gender differences remain elusive, several factors could
influence brain-related alcohol effects in female adoles-
cents, including gender-specific brain development223,224,
differential gene expression linked to enhanced alcohol-
related neurotoxicity in women225, increased blood alcohol
concentration among women despite similar drinking pat-
terns, and differences in alcohol metabolism226. Hormonal
and receptor differences in response to alcohol may also
play a critical role in these gender differences. Hormonal
receptor levels are associated with not only gender differ-
ences in brain functioning during spatial tasks227, but also
alcohol-induced changes in hormone distributions191,228,
which could partially account for the gender differences
noted in alcohol-related neural abnormalities.
Another brain area that undergoes important remod-
eling changes is the hippocampus, a brain region known
to participate in learning and memory functions. MRI
reveals that the hippocampus volume is significantly
larger in older male adolescents than in younger male
adolescents. As in PFC, less cerebral gray matter volume
and significantly greater cerebral white matter volume are
found in older adolescents compared with younger ado-
lescents229, and these changes might reflect the maturation
of memory functions. Adolescents who begin drinking at
earlier ages have proportionally smaller hippocampal vol-
umes compared with those who start this habit later230,231,
suggesting that differences in size are induced by alcohol.
Likewise, reductions of the brain oxygen level dependent
(BOLD) response in the left precentral gyrus, the bilateral
cerebellar areas and in bilateral parietal cortices, have
also been encountered in binge-drinking adolescents232.
Neuropsychological studies have revealed alterations in
visual-spatial functioning and in both verbal and non-
verbal information in adolescents who report alcohol epi-
sodes233, or in adolescents with AUD during abstinence234.
These results indicate that drinking at early ages could
modify brain maturation processes in specific brain areas
that are critical for cognitive processes. In addition, subtle
but important brain changes occur among adolescents
with AUD and result in a worsened ability in problem
solving, verbal and non-verbal retrieval, visuospatial skills
and working memory.
Mechanisms underlying the pathological
consequences of adolescent drinking:
experimental animal studies
Studies using experimental animal models have demon-
strated that both the ontogenetic transitions pattern and
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 29
the physiological and behavioral characteristics in ado-
lescents are common among mammalian species (see
Spear204,235). For instance, absolute PFC volume declines
in adolescent rats205, while synapse elimination also
occurs during adolescence in the PFC of non-human
primates210. Maturation changes are also evident in the
hippocampus of adolescent rodents236. Although the
complexity of the human brain and behavior cannot be
extrapolated to experimental animals, there are numer-
ous similarities between human adolescents and adoles-
cents of other species.
Regarding alcohol exposure effects during adoles-
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cence, a number of studies on experimental animal
models have also provided evidence of the vulnerability
of the adolescent brain to the toxic effects of alcohol237241
particularly the cortex and hippocampus, which are
key regions for mediating memory processes216,230,231,242.
Compared with adults, alcohol-exposed adolescent
animals are more likely to show cognitive deficits,
including learning and memory dysfunctions243249.
Some cognitive effects, such as learning impairments,
induced by repeat alcohol treatment in adolescent rats
notably continue into adulthood241,250.Conversely, ado-
lescent rats are less vulnerable than adults to a number
of acute effects of ethanol, including ethanol-induced
sedation244, motor impairments248,251, and acute with-
drawal/hangover effects of alcohol. The insensitivity to
For personal use only.
the acute effects of alcohol might enable adolescents to
drink larger amounts of alcohol without being aware of
its intoxicating effects, which logically places them at
risk for AUD.
Although the behavioral and neurobiological mecha-
nisms for the ontogenetic differences in alcohol sen-
sitivity remain unclear, adolescents are more sensitive
than adults are to both the memory impairing effects of
alcohol and the impact of alcohol on the brain function
underlying memory formation. Thus, the use of hip-
pocampal slices taken from adolescent (30-day-old) and
adult (90-day-old) rats243249 show how that alcohol more
potently inhibits the induction of long-term potentiation
(LTP) in immature versus mature animals. According to
these in vitro data, adolescent rats present greater impair-
ment in accomplishing a spatial memory task after acute
alcohol exposure than adults243. These results suggest
that alcohol disrupts memory and memory-related brain
functions (e.g. LTP) more strongly in adolescent animals
than in adults: these effects could contribute to adoles-
cents greater vulnerability to alcohol-induced memory
impairments and reduced synaptic plasticity.
Regarding the toxic effects of alcohol on the adolescent
brain, some studies have shown that young rats exposed
to heavy binge-like episodes of alcohol display greater
damage in the frontal-anterior cortical regions than
adults, including the olfactory frontal cortex, the anterior
perirhinal and the piriform cortex237. Intermittent alcohol
administration to juvenile/adolescent rats (from postna-
tal days 30 to 45) also increases neural death in both the
hippocampus and the PFC and these animals present
behavioral and cognitive deficits at the end of alcohol
treatments and at the adult stage241. These results sug-
gest that binge-like alcohol drinking during adolescence
induces brain damage, thus impairing normal brain
maturation and plasticity processes, and causing long-
lasting behavioral consequences.
The underlying mechanisms of alcohol-induced brain
damage during adolescence are poorly understood,
although two mechanisms have been proposed. The first
suggests that intermittent alcohol drinking might induce
brief episodes of withdrawal from alcohol and excitotoxic
neuronal damage. Alcohol withdrawal has been shown
to increase the aberrant synaptic activation of NMDA-
glutamate receptors252 and to produce a marked cellular
injury, as the CA1 pyramidal neurons from hippocam-
pal explants have demonstrated253,254. This process can
result in discrete excitotoxic neuronal damage or a more
severe and complicated alcohol withdrawal syndrome,
including withdrawal seizures255. However, DeBellis
et al. reported that only two of the 14 adolescents with
AUD who presented PFC reduction displayed withdrawal
symptoms216. In addition, excitotoxicity appears to result
in enhanced glutamatergic transmission by up-regulat-
ing the NMDA-glutamate receptors after chronic alcohol
consumption. Moreover, reduced levels and function
of the NMDA receptors has been observed in animals
exposed to alcohol during adolescence256258.
The second mechanism proposes that alcohol induces
adolescent brain damage by neuroinflammation, which
occurs by activation of innate immune system and toll-like
receptors (TLR4) in glial cells259,260 (see below). Indeed, a
number of studies of recent years have reported that alco-
hol intake induces inflammatory mediators in the brain
by activating intracellular signaling pathways which in
turn trigger the induction of pro-inflammatory cytokines
(IL-1, TNF-), cyclooxygenase-2 (COX-2), inducible
nitric oxide synthase (iNOS) and neural cell death261,262.
Recent findings have demonstrated that intermittent
alcohol administration to adolescent rats up-regulates
the COX-2 and iNOS levels, and increases cell death in
the neocortex, hippocampus and the cerebellum241. It is
noteworthy that an increase in brain inflammatory medi-
ators concomitantly occurs with short- and long-term
cognitive deficits in animals treated with alcohol during
the adolescent stage241, thus suggesting an association
between brain damage and long-term cognitive effects.
Likewise, alcohol administration during middle adoles-
cence has been shown to reduce hippocampal neurogen-
esis by impairing neural survival in young adult rats and
non-human primates263,264. These results indicate that the
hippocampal neurogenic niche during adolescence is
highly vulnerable to alcohol and that alcohol alters the
ongoing process of neuronal development, which might
underlie the deficits in hippocampus-associated cogni-
tive deficits observed in adolescent drinkers264.
Concerning potential treatments for the damag-
ing effects of alcohol on the adolescent brain, we
have demonstrated241 that the administration of a
 30 S. Alfonso-Loeches and C. Guerri
non-steroidal anti-inflammatory compound, indo-
methacin (an inhibitor of COX-2 activity)265,266, before
administering alcohol to adolescent rats, not only
abolishes the induction of COX-2 and iNOS expres-
sions and cell death, but prevents alcohol-induced
behavioral deficits241. Remarkably, elevated levels
of COX-2 and iNOS have also been observed during
excitotoxicity, ischemic and neural injury267269. while
COX-2 inhibitors have been shown to prevent neu-
ronal loss and to ameliorate brain injury caused by
excitotoxicity270, ischemic brain injury, brain inflam-
mation and neurodegeneration271,272. COX-2 inhibitors
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
also improve the behavioral and cognitive functions
associated with neurodegeneration272.
In summary, human and animal studies have evi-
denced the vulnerability of the adolescent brain to
alcohols harmful effects. The results suggest that binge
episodes of heavy drinking, which likely take place during
adolescence, could interfere with neural developmental
and plasticity and brain maturation, particularly in the
PFC and the hippocampus, which could lead to lifelong
behavioral impairments247. Furthermore, dysfunctions in
the PFC also occur in alcoholics and have been associ-
ated with impaired cognitive functions and compulsive
behavior, events that predispose to alcohol abuse273,274.
Therefore, alcohol-induced PFC impairments in ado-
lescents might not only underlie cognitive dysfunctions,
For personal use only.
but also predispose to alcohol abuse, impulsivity and
dependence11.
Adolescence, a period of increased risk for
alcohol abuse
Another important long-lasting consequence of alco-
hol use during adolescence is the higher risk of devel-
oping alcohol abuse and dependence in adulthood.
Prospective and retrospective human studies suggest
that early onset of alcohol use typically emerges as a
reliable predictor of both later problematic use, and
dependence on alcohol and other drugs275278. People
who begin drinking before the age of 15 are four times
more likely to develop alcohol dependence at some time
in their lives compared with those who start drinking at
the age of 20, or beyond25. It is not clear whether start-
ing to drink at an early age actually causes alcoholism
or whether it simply indicates an existing vulnerability
to AUD. Some studies suggest the existence of certain
predispositions, such as personality characteristics
with strong tendencies to impulsiveness, or a genetic
background in certain adolescents for substance abuse
disorders (e.g. adolescents with AUD)279. Nevertheless,
experimental animal studies have suggested that
despite genetic factors possibly playing a role in early
alcohol consumption, exposure to alcohol during the
juvenile/adolescent stage can also sensitize the brain
regions and/or developmental processes involved in
drug addiction, which may also play a role in increasing
propensity to later alcohol abuse.
There is substantial evidence to demonstrate that the
mesocorticolimbic DA system also undergoes impor-
tant remodeling changes during adolescence, and that
this system participates in the rewarding and reinforced
effects of drugs of abuse, including alcohol280282. For
example, D1 and D2 receptors show greater overproduc-
tion and pruning at puberty in the striatum than in the
nucleus accumbens283,284. Basal DA synthesis and turnover
increase in the nucleus accumbens during adolescence,
but decrease in the PFC285. In addition, connectivity
continues to develop between the frontal cortex and the
subcortical structures throughout adolescence and into
adulthood, as evidenced by the increased density of pre-
frontal-nucleus accumbens projections286 and by the den-
sity of dopaminergic and glutamatergic innervation from
the ventral tegmental area209 and amygdala287,288. These
remodeling changes in the neurocircuitry involving the
DA projections to mesolimbic brain regions and the PFC
could acquire special functional significance for adoles-
cence since this circuitry forms part of the reward system
that modulates typical adolescent behavior204,235,238, and
motivation to natural (food) and unnatural (alcohol and
other drug of abuse) rewards289. Therefore, neurochemi-
cal immaturity of the mesocorticolimbic system might
contribute to initiation of alcohol intake initiation and to
adolescent-specific vulnerability to drug addiction.
Although relatively few studies have linked the behav-
ioral effects of adolescent drinking to underlying neural
mechanisms, studies with animal models have begun to
explore the impact of the mesocorticolimbic DA path-
way response on adolescent and adult animals. Indeed,
some recent studies have shown258 that alcohol exposure
during adolescence increases adult alcohol preferences
and intake. These behavioral effects have been associ-
ated with the reduced protein levels of both the DA
receptor subunit D2 (DRD2) and the phosphorylated
glutamate receptor subunit 2B (NMDAR2B), and with
changes in both chromatin remodeling and the acety-
lation of histones H3 and H4 in several brain regions
(prefrontal cortex, nucleus accumbens, and striatum).
Alcohol also tends to elicit a higher DA response in the
nucleus accumbens (NAc) core of adolescent rats than
in adult rats pre-treated with multiple doses of alcohol,
although the basal DA levels were higher in alcohol-
treated adolescents290,291. Similarly, alcohol drinking
during peri-adolescence by alcohol-preferring rats,
which present high extracellular accumbal DA levels292,
enhances alcohol self-administration in adulthood293.
Studies conducted with C57BL/6J and BAL mice294,295,
have also confirmed that post-weaning two-bottle choice
exposure slightly, but significantly, increases alcohol
consumption and preference in adulthood. Conversely,
forced alcohol exposure during adolescence in rats does
not enhance the reinforcing properties of alcohol in adult
animals205,296,297. Although several factors (e.g. inter-spe-
cies differences, genetic factors, alcohol administration
paradigm, age of initiation or housing conditions) may
explain the variations in findings across different studies,
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 31
changes in extracellular DA during adolescence could
sensitize the dopaminergic system by causing maladap-
tive brain developmental processes11,239, and mediating
the increased likelihood of engaging in drug use initia-
tion during adolescence.
To summarize, the data available suggest that neuro-
chemical immaturity and heightened neuroplasticity in
the limbic brain regions might confer greater sensitivity
to adolescents addictive drug actions. Sensitization of the
mesocorticolimbic DA pathway, along with changes in
glutamatergic and dopaminergic neurotransmission and
chromatin remodeling might mediate adolescents vul-
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
nerability to long-term alcohol addiction consequences.
Despite the progress made in adolescent binge drinking
consequences and in finding links between behavioral
effects and neurochemical mechanisms, there are still
significant gaps in our understanding of the cellular
and molecular bases of cognitive deficits (e.g. attention,
memory, executive functioning) caused by heavy alcohol
consumption during adolescence. Studies that integrate
multiple approaches such as genetic, hormonal, neural
and behavioral features are needed to identify biomarkers
of at-risk adolescents and treatment targets. Longitudinal
behavioral studies are also important to assess the poten-
tial reversible effects of alcohol use during adolescence.
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Behavioral and neurobiological actions of
alcohol on the adult brain
Mechanisms of the behavioral effects of alcohol
Alcohol is the most widely used drug in our society. Alcohol
abuse and alcoholism not only contribute to a wide range
of medical complications, but also lead to alcohol addic-
tion characterized by compulsive drug use, craving, and
chronic relapses during abstinence. It is remarkable to
note that, despite alcohol having been used and misused
for hundreds of years it is still uncertain how alcohol
actually acts to induce its effects on the brain. Recent
molecular and pharmacological studies have demon-
strated, however, that alcohol and particularly ethanol,
acts on specific membrane proteins, such as receptors,
ion channels, and signaling pathways298301. These pro-
teins include NMDA-glutamate, -aminobutyric acid
A (GABA-A), glycine, 5-hydroxytryptamine-3 (5-HT3),
and neuronal nicotinic acetylcholine (nACh) receptors,
as well as L-type Ca2+ channels and G protein-activated
inwardly rectifying K+ channels302. Following the first
hit of alcohol on specific targets in the brain, a second
wave of indirect effects on a variety of neurotransmitter/
neuropeptide systems is initiated302, leading to the typical
acute behavioral effects of alcohol that range from disin-
hibition to sedation, and even hypnosis, with increasing
concentrations of alcohol.
In addition, alcohol activates some specific signaling
pathways, leading to alterations in both gene expression
and the neuroadaptations underlying alcohol-related
disorders303305. As a result of repeated alcohol intake, the
long-lasting cellular and neurophysiological changes
that trigger alcohol-seeking behavior become appar-
ent in the brain reinforcement system303,305. Whether or
not this behavioral response transforms into addictive
behavior eventually depends on genetic and environ-
mental factors301.
Alcohol-sensitive sites in receptors and ion
channels
Alcohol produces a wide variety of behavioral effects, but
exactly how it acts to produce these effects is still poorly
understood. Although alcohol has long been believed to
act non-specifically by disordering lipids in cell mem-
branes, proteins are at the core of most current theories of
its mechanisms of action. Studies undertaken in the last
few decades have demonstrated that ethanol (the main
toxic component of all of the alcoholic beverages), at
physiologically relevant concentrations (520 mmol/L),
directly interferes with the function of specific recep-
tors and ion channels. Among these receptors, NMDA
glutamate receptors and GABA-A receptors in alcohol
actions on the brain have drawn special attention299. As
mentioned earlier, these receptors are also a target of the
effects of alcohol during brain ontogeny, including fetal
and postnatal development and adolescence.
In the adult brain, several studies have demonstrated
that NMDARs are an important target of the actions of
alcohol in the brain, as they participate in alcohol depen-
dence, tolerance, and withdrawal299,306308. NMDARs are
heteromeric ligand-gated ion channels composed of an
obligatory NR1 subunit and combinations of NR2 and
sometimes NR3 subunits309, including NR2A-D which
confers specific functional properties to NMDARs309.
Ethanol inhibits NMDARs over the 550 mmol/L range
in a concentration-dependent manner in hipocam-
pal neurons310. Although the molecular mechanisms
of ethanol-induced NMDAR inhibition are uncertain,
recent experimental studies using site-directed muta-
genesis have revealed putative binding sites of ethanol
with transmembrane (TM) domains TM3 and TM4 of
the NR1 and NR2A subunits, respectively311. Inhibition
of these receptors by ethanol is compensated in chronic
ethanol treatment, resulting in an increased NMDAR-
mediated function after ethanol removal in cultures of
cortical neurons312. This increase in NMDARs, especially
the NR2B subunit, may contribute to the alcohol with-
drawal syndrome308,312,313, which is characterized by both
behavioral and electrophysiological parameters in alco-
hol-dependent rats314. These results suggest the potential
role of NMDARs in alcohol tolerance and withdrawal315.
Substantial evidence also shows that GABAergic neu-
rotransmission and GABA-A receptors are important
targets for many behavioral actions of alcohol. There are
also reports spanning over 30 years in the literature show-
ing how low to moderate (330 mmol/L) concentrations
of ethanol enhance GABAergic neurotransmission. The
GABA-A receptor is not only a pentameric ligand-gated Cl
ion channel, but the major inhibitory neurotransmitter
 32 S. Alfonso-Loeches and C. Guerri
receptor in the mammalian brain. Several subunits have
been identified, where the majority of GABA-A receptors
are composed of -, -, -, and -subunits316. A region
in the TM domains of the / subunits of the GABA-A
receptor has been identified through the use of different
receptor constructs. GABA-A is involved in the action of
alcohol317, and can potentially bind to a water-filled pro-
tein cavity between the second and third TM segments of
these receptor subunits299,318. Ethanol also affects glycine
receptors, and there is evidence that ethanol acts on spe-
cific residues in the TM domains317, on the extracellular
domain of glycine receptors, and that the net effect on
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the receptor function is the summation of the positive
and negative modulatory effects of ethanol at different
ethanol-sensitive binding sites319.
However, the effects of ethanol on GABA-A receptors
strongly depend on the subunit composition and con-
centration of ethanol. Thus, while most subunit compo-
sitions of GABA-A receptors display responses to ethanol
only at high concentrations (> 60 mmol/L), very low
concentrations (13 mmol/L) of ethanol are capable of
altering GABA-A receptors containing the delta-subunit,
which is located outside of the synapses. These GABA
receptors are exclusively associated with 46 subunits
and the 3 subunit in vivo. Moreover, in 4 subunit
combinations, those receptors containing the 3 subunit
have been found to be almost 10 times more sensitive
For personal use only.
than those receptors containing the 2 subunit, suggest-
ing that the 3 subunit is also an ethanol-sensitive site320.
Nevertheless, mouse models, in which the 3 subunit
was genetically deleted or knock-in mice carried a single
point mutation in the subunit, do not differ in their
acute response to ethanol if compared with wild-type
animals321. These findings suggest that the extrasynap-
tic subunits containing GABA-A receptors, but not 3,
are primary targets for alcohol.
Other alcohol targets include members of the super-
family of GABA-A receptors such as neuronal nACh322
and the 5-HT3 receptor function323. The 5-HT3 receptor
mediates fast synaptic transmission at postsynaptic sites,
regulates neurotransmitter release presynaptically, and
its ethanol sensitivity has been consistently shown in dif-
ferent in vitro preparations324. Non-ligand ion channels,
such as dihydropyridinesensitive L-type Ca2+ channels
and K+ channels known as G protein-activated potas-
sium (GIRK) channels), are also ethanol targets325. Thus,
whereas ethanol inhibits L-type channels, it opens GIRK
channels326. The selective enhancement of the GIRK2
function by intoxicating concentrations of ethanol has
been proved for homomeric and heteromeric channels,
and a 43 amino-acid region in the carboxy COOH) ter-
minus, which is critical for the action of ethanol in these
channels, has been identified326,327.
Finally, neuroadaptive changes in neurotransmission
systems, which are more sensitive to acute effects of alcohol,
occur after long-term alcohol consumption. Both studies
into experimental animal models and findings obtained
in humans have shed light on the adaptive changes in
not only the signaling systems involving neurotransmit-
ters glutamate, gamma-aminobutyric acid (GABA), DA,
and 5-HT, but also other signaling molecules, including
endogenous opioids and the corticotrophin-releasing fac-
tor (CRF). These systems adaptations to chronic alcohol
exposure have been associated with behavioral effects,
such as changes in reinforcement, enhanced anxiety, and
increased sensitivity to stress, all of which may contrib-
ute to a relapse in drinking among abstinent alcoholics.
Moreover, some of these systems are targets of currently
available therapeutic agents for alcohol dependence315.
Neurobiological basis involved in alcohol
addiction
As previously mentioned, alcohol acts on specific targets
in the brain and triggers a variety of neurotransmit-
ter/neuropeptide systems that lead to the typical acute
behavioral effects of alcohol, ranging from disinhibition
to sedation, and even hypnosis, with increasing concen-
trations of alcohol301,302. However, apart from these acute
pharmacodynamic aspects of alcohol, other mechanisms
and signaling pathways are involved in the initiation and
maintenance phase of alcohol drinking behavior. Activity
of the mesolimbic dopaminergic system, opioids and
endocannabinoids is crucial for the initiation of alcohol
reinforcement and reward.
The mesocorticolimbic DA pathway compromises the
ventral tegmental area (VTA), the NAc and the associ-
ated limbic structures. This pathway is not only involved
in modulating behavioral responses to stimuli that acti-
vate feelings of reward (motivation) and reinforcement
through the neurotransmitter DA, but also participates
in the reward and reinforced effects of drugs of abuse,
including alcohol280,281,328. Numerous in vivo pharmaco-
logical studies have revealed the alcohol-induced activa-
tion of mesolimbic A10 neurons, and that these effects
are apparently associated with the reinforcing properties
of alcohol since rats will directly self-administer alco-
hol into the VTA329, and specifically into the posterior,
but not the anterior VTA330. These results indicate that
alcohol acts within the posterior VTA and suggest that
VTA DA neuron activation is involved in this process.
The role of DA in mediating alcohol reinforcement in
the human brain has also been confirmed331, suggesting
that enhanced DA release occurs in response to alcohol
drinking. The degree of psychostimulation is mediated,
at least in part, by augmented extracellular DA level: D1,
D2 and D3 receptor agonists and antagonists are capable
of modulating alcohol consumption in common stock
rats332 and in alcohol-preferring rats333,334.
Experimental studies have also shown that although
mesolimbic DA activation may play a crucial role in the
reinforcing effects of alcohol, this process is modulated by
several neurochemical signals, including GABA-A, 5-HT3,
nACh, and glycine receptors, plus the endocannabinoid
and endogenous opioid system301. Likewise, different sig-
nal transduction pathways within the NAc and other areas
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 33
receiving input from A10 neurons have been suggested
as participating in alcohol reinforcement. Among these
pathways, cAMP-dependent protein kinase A (cAMP-
PKA) signaling is suspected of mediating the effects of
alcohol and of influencing CREB-mediated processes. In
addition, cAMP-PKA signaling in medium spiny neurons
affects the DARPP-32 function, which in turn is an impor-
tant regulator of the NMDA receptor function within the
reinforcement system, and may play an important role in
neuroadaptations in response to alcohol exposure. NMDA
receptors are closely associated with NO/cGMP signaling,
and this pathway plays a critical role in mediating not only
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alcohol reinforcement, but also other alcohol-induced
behavioral responses. Finally, PKC signaling is also affected
by ethanol, which in turn, influences the GABA-A receptor
function. Hence, ethanol affects the functioning of those
relevant receptors (NMDA and GABA-A) to synaptic plas-
ticity301. A recent report has also implicated the mammalian
target of rapamycin complex 1 (mTORC1) signaling within
NAc as a molecular mechanism underlying alcohol drink-
ing behavior303. Administration of rapamycin, a mTORC1
inhibitor, decreases the expression of alcohol-induced
locomotor sensitization and place preference, as well as
excessive alcohol intake in preclinical rodent models of
alcohol abuse303.
Drug-induced adaptations in the mesolimbic system
reveal that glutamatergic synapses on DA neurons in the
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VTA, and in other brain regions such as the amygdala,
undergo plastic changes following the administration of
drugs of abuse, including alcohol335. These changes have
been suggested as the neural adaptation that underlies
alcohol reinforcement, alcohol seeking or alcohol-
induced habit formation. However, direct experimental
evidence for the behavioral significance of these drug-
induced synaptic changes involving glutamate receptors
is still lacking.
Finally, neuroimaging research in humans confirms
the experimental data and contributes greatly to our
knowledge of the neuroanatomical and neurochemical
substrates of addictive behavior. This line of research
indicates the involvement of the extended amygdala,
including the NAC, the orbitofrontal cortex, and the dor-
sal striatum; these brain areas are responsible for rein-
forcement, decision making and impulse control. The
hypofunction of the dopaminergic system and alterations
within endogenous opioid systems appear to correlate
with craving and relapse behavior336,337. Recent advances
in glutamate spectroscopy and the development of the
NMDA receptor and metabotropic glutamate receptor
PET ligands338 will assist in transferring this knowledge to
alcohol-dependent patients.
Genetic factors influencing alcohol
dependence
The importance of genetic factors in alcohol dependence
has been supported for many years. Indeed, studies
since 1974339 have demonstrated that adopted children of
alcoholics are at a four-fold risk of alcohol dependence.
This trend is also noted in relatives of alcoholics and in
identical twins of alcohol-dependent subjects, who are
at greater risk of this disorder than fraternal twins or full
siblings340. More recent studies have supported the notion
that, although it is possible for a small number of genes
to directly influence alcohol dependence, it is more likely
that a relevant group of genes could influence a range
of genetic personality characteristics that predispose
to alcoholism341. For example, several studies report an
association of phenotypes of impulsivity, disinhibition
and related characteristics with a polymorphism of the
GABA-A receptor, the alpha 2 gene in chromosome 4342344.
At the behavioral level, this gene variation also relates to
conduct disorder and the antisocial personality disorder,
and is associated with predisposition to dependence on
illicit substances and alcohol342. Several other polymor-
phisms that are potentially associated with disinhibition
or related cognitive-based mechanisms include a varia-
tion of the cholinergic receptor (e.g. muscarinic 2) and
the ADH 4 genes, where the latter potentially carries its
impact via changes in the DA reward system. Conversely,
other groups of polymorphisms appear to lower the risk of
repeated heavy drinking and associated problems, such
as the mutation in the ALDH*2 gene which results in an
enzyme incapable of destroying acetaldehyde. About 10%
of Asian (Japanese, Chinese and Korean) individuals are
ALDH2*2 homozygotes with a resulting highly intense
aversive response to drinking that contributes to a near-
zero rate of alcoholism, and does not appear to affect the
risk of dependence on other drugs (see revision344).
Regarding the more specific gene expression profiling
in alcoholics, several studies have identified differentially
expressed genes in cortical brain regions in response to
long-term alcohol consumption by using human post-
mortem brains and microarrays analyses341,345350. Several
gene expression studies have evaluated the prefrontal
cortex because of its susceptibility to damage by alcohol
abuse. For example, loss of white matter volume351,352,
and neuronal loss in gray matter352 have been reported
in post-mortem brains of long-term alcohol abusers.
Major changes have been observed in the genes gener-
ally involved in myelination, ubiquitination, apoptosis,
cell adhesion, neurogenesis and neural disease, showing
altered expression levels. It is interesting to note that the
genes involved in neurodegenerative diseases, such as
Alzheimers, are also significantly altered (presenilin 1
and transferrin), suggesting a link between alcoholism
and other neurodegenerative conditions. Many stud-
ies have reported down-regulated myelination-related
genes including transferring347, UDP glycosyltransferase
8347, peripheral myelin protein 22345,347, and proteolipid
protein 1347. Other differentially expressed genes include
lysosomal-associated membrane protein 2345,349,350, pro-
teasome subunit b type 2,345 CANX,350 GABBR1345, solute
carrier family 12, member 2341 and transketolase353. The
alcohol-responsive transcripts identified in the frontal
cortex by Flatscher-Bader et al. include those encoding
 34 S. Alfonso-Loeches and C. Guerri
transcription factors, DNA-binding proteins, mitochon-
drial proteins and neuroprotection-/apoptosis-related
proteins354. Conversely, in the nucleus accumbens, the
differentially expressed genes associated with synaptic
vesicles and cytoarchitecture are significantly down-reg-
ulated. The authors suggest that these changes in the NAc
gene expression in response to long-term alcohol abuse
might result in deficits in normal synaptic transmission
and altered plasticity.
Essentially, these data suggest that the transcriptional
response of the brain to chronic alcohol abuse affects
numerous genes in multiple functional systems in dif-
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
ferent regions of the human brain. These changes at the
transcriptional level likely reflect both pre-existing dif-
ferences in gene expression and those altered by alcohol
consumption. An important goal for addiction biologists
is to understand the role that these widespread changes
in cellular regulation play in alcohol dependence.
Epigenetic effects induced by alcohol
Changes in gene expression in brain reward regions are
believed to contribute to the pathogenesis and persis-
tence of drug addiction. Recent studies in experimental
animals have suggested that drugs of abuse and related
environmental stimuli, such as drug-associated cues
or stress, converge on the genome to alter certain gene
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programs. Specifically, the epigenetic mechanisms alter-
ing the chromatin structure in specific gene promoters
can lead to potent and often long-lasting changes in
gene expression which contribute to neurobehavioral
alterations or addictive-like behavior in experimental
animals355.
A common form of epigenetic modification involves
the addition of molecules to DNA structure. Thus, post-
translational modifications in the chromatin, such as
acetylation, phosphorylation and methylation of histones
on DNA, positively or negatively modulate the transcrip-
tional activity of the underlying genes without causing
major alterations to the genetic structure356. Enzymes
which induce histone acetylation, such as histone acetyl-
transferases (HATs), cause nucleosome relaxation and
promote gene expression, whereas those enzymes which
remove acetyl groups from the histone, such as histone
deacetylases (HDACs), pack DNA into a more condensed
chromatin, block access to transcriptional activators and
lower the gene expression357. These associations exist in
brain in vivo studies in response to drugs of abuse in ani-
mal models.
Alcohol exposure during adolescence induces changes
in the acetylation of histones H3 and H4 in the rat frontal
cortex, NAc and the striatum258, and these changes are
associated with subsequent drinking behavior. Likewise,
studies in experimental animals have also revealed that
decreased HDAC activity along with an up-regulation in
histone acetylation (H3 and H4), levels of CREB (cAMP-
responsive element binding) binding protein (CBP) and
neuropeptide Y (NPY) expression are associated with
the anxiolytic effects produced by acute alcohol treat-
ment. During withdrawal, however, increased HDAC
activity and decreased acetylation of both H3 and H4 are
noted in the amygdala. Blocking increased HDAC activ-
ity during alcohol withdrawal with the HDAC inhibitor
trichostatin A rescues the deficits in H3 and H4 in the
amygdala and prevents alcohol withdrawal-related
anxiety in rats358.
Alterations in DNA methylation in the promoter regions
of -synuclein might prove to be an important example
of maladaptive molecular responses to chronic alcohol
exposure. Alpha-synuclein belongs to a quantitative trait
locus for increased alcohol consumption. Its expression
is elevated in different brain areas of rats with inbred
alcohol preference359. It is further involved in the regula-
tion of DA biosynthesis and dopaminergic neurotrans-
mission360. Therefore, alterations in the -synuclein gene
may have profound effects on DA-dependent alcohol
seeking. In fact, an increased expression of -synuclein
in alcohol-dependent patients has been correlated with
obsessive craving361. In these alcoholics, a significant
increase in -synuclein promoter DNA methylation has
been observed, which is significantly associated with
their elevated homocysteine levels. However, no signifi-
cant differences have been found for the promoter DNA
methylation within a control gene (presenilin-1) in alco-
holics and controls362. These results suggest gene-specific
DNA promoter hypermethylation within the -synuclein
gene after chronic alcohol consumption.
In summary, alcohol-induced alterations in methy-
lation and acetylation patterns may have an impact on
long-lasting alterations in gene expression. Nevertheless,
these findings suggest that epigenetic effects induced by
chronic alcohol exposure are likely to identify the molec-
ular mechanisms underlying addictive behavior.
Mechanisms of alcohol-induced neurological
deficits and brain damage
Alcohol abuse and chronic alcohol consumption can
result in significant alterations to the brain structure,
physiology and function. Even uncomplicated alcoholics,
with no specific neurological or hepatic problems, show
signs of regional brain damage and cognitive dysfunction3.
Post-mortem studies in human alcoholic brains have
proved that alcoholics have reduced brain weight com-
pared with non-drinking controls363, and that the degree
of brain atrophy correlates with the rate and amount
of alcohol consumed over a persons lifetime. Reduced
brain weight and volume have been attributed to loss of
white matter352,364, which occurs primarily in the frontal
lobe that is specifically susceptible to alcohol-related
brain damage365367, although neuronal loss has also been
documented in specific regions of the cerebral cortex, the
hypothalamus and the cerebellum of alcoholic brains368370.
The reduction mechanisms for white matter loss remain
unclear, although glial impairments in conjunction with
astrocytic loss have been reported in the prefrontal cortex
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 35
and the hippocampus of human alcoholic brains352,371373.
Changes in myelinization might also occur during chronic
alcoholism since the gene expression of both glial fibrillary
acidic protein (GFAP, astrocyte marker) and myelin-asso-
ciated genes are down-regulated in alcoholics brains349,370.
Indeed, both myelin disruptions and astrocyte death play
an important role in alcohol-associated disorders374, and
that glutamate excitotoxicity375,376, has been suggested as
playing a role in myelin alterations.
One inherent problem in examined adult post-mortem
brain tissue, when adequate information about the indi-
viduals history of alcohol exposure is lacking, is whether
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the brain structural changes observed in alcoholics may
have occurred as a result of alcohol abuse or of the effects
of alcohol during early development. It is known that
prenatal exposure to alcohol can lead to structural and
functional changes in the brain, and to problems with
alcohol and drug abuse later on in life42,43. Likewise, pre-
frontal cortex abnormalities, along with reduced integrity
of white matter, have been demonstrated in adolescents
with alcohol-use disorders216,217. These results suggest
that some brain changes observed in alcoholics may
originate during an early development stage, although
alcohol abuse can aggravate brain injury.
Neuroimaging studies have confirmed that chronic
alcohol abuse induces global changes in brain morphol-
ogy, such as cortical and subcortical atrophy. These studies
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have verified that the frontal lobes, the thalamus, the CC
and the cerebellum, among others, are especially vulner-
able to the effects of alcohol, and that the contraction of
these brain areas is mainly due to loss of white matter377,378.
Alcohol abuse displays memory and concentration defi-
cits, which are consistent with hippocampal dysfunction
and damage, as demonstrated by the reduction in the hip-
pocampal volumes observed in alcoholics379,380. Notably,
reduction in the prefrontal cortex, the cerebelllum and
the hippocampus have also been observed in individuals
with FASD50,56,381, and in adolescents with heavy alcohol
consumption216,217,230, suggesting the vulnerability of these
brain areas to the actions of alcohol.
Clinical studies have also shown how heavy alcohol
intake can cause severe brain injury367,382, and in some
cases can actually correlate with the wide spectrum of
neural damage observed in neurodegenerative disorders.
Indeed, alcoholism is associated with important cognitive
function impairments and social deep drinkers who have
non-specific hepatic problems or thiamine deficiencies
show regional brain damage and cognitive dysfunctions.
The neurological dysfunctions commonly observed in
alcohol reliance include deficits in abstract reasoning,
visuospatial abilities, postural stability and verbal learn-
ing, declarative and procedural memory, perceptual
motor skills and increased risk for dementia383387. These
abnormalities are more severe and present new areas of
damage in alcoholics with a deficit in thiamine388, such
as Wernickes encephalopathy (WE), Wernicke-Korsakoff
syndrome and Korsakoffs psychosis (alcohol amnesic
disorder)389.
Experimental studies have also shown that alcohol is
toxic for neural cells in culture390,391, and that both acute
and chronic alcohol intake can cause brain injury, and
can even lead to neurodegeneration259,262,392. Although
the neuropathological processes underlying alcohol-in-
duced neural damage are largely unknown, some studies
in experimental animals have shown that binge alcohol
intoxication and withdrawal can induce brain edema,
oxidative stress, and can even cause neurodegeneration
associated with neuroinflammatory processes393. MRI
studies in humans have revealed an association between
brain choline metabolites (an indicator of neurodegen-
eration) and brain damage associated with long-term
alcohol consumption394,395. Although neurodegeneration
markers can diminish during abstinence, both glial acti-
vation and proinflammatory gene expression produced
by a prolonged blood alcohol concentration may persist
for long periods in the brain, and even worsen to neuro-
degeneration396. Other postulated mechanisms include
the participation of excitotoxic events and nitric oxide
generation397, involvement of glial swelling and brain
edema398,399, production of free radicals causing oxidative
stress, activation of nuclear factor kappa-B (NF-B)400
and an increase in free radical species and lipid peroxida-
tion induced by CYP2E1 activation140,143. Studies in post-
mortem human alcoholic brains have also suggested that
deficits in acetylcholine homeostasis, insulin/insulin-like
growth factor (IGF) signaling and oxidative stress might
be involved in alcohol-induced neurodegeneration.
Insulin and IGF types I and II are involved in maintaining
cognitive and motor functions in the CNS such as neu-
ronal survival energy metabolism and plasticity, and rep-
resent potentially important targets in alcohol-induced
neurotoxicity401.
Recently, a number of studies have indicated that alco-
hol can induce brain injury and neurodegeneration by
mechanisms involving inflammatory damage259,261,393,402.
These studies indicate that both acute and chronic alco-
hol treatments increase pro-inflammatory cytokines
in the rat brain and in cultured astrocytes by activating
the microglia402 and the signaling pathways classically
associated with inflammation (MAPKs, NF-B, AP-1)262.
Moreover, they suggest that these events concomitantly
occur with increased cell death262,403. Likewise, other
studies using human brain microvascular endothelial
cells in culture have proposed that alcohol induces neu-
roinflammatory processes by promoting bloodbrain
barrier (BBB) disruption leading to leukocyte migration
across the BBB404 (Figure 2), although the underlying
mechanism of BBB dysfunction caused by alcohol abuse
remains elusive.
Inflammation plays a crucial role in the pathogenesis
of several CNS disorders, including chronic neurodegen-
erative diseases such as Parkinsons disease405,406, and
Alzheimers disease407. The hallmark of brain inflamma-
tion is the activation of glial cells, primarily microglia
and astrocytes, which release free radicals, cytokines and
inflammatory mediators that can induce brain damage408.
 36 S. Alfonso-Loeches and C. Guerri
The inflammatory response in the brain is usually associ-
ated with innate immunity activation, specifically with
toll-like receptors (TLRs), the key host molecules in
immune response regulation during infections and CNS
damage. Activation of TLRs triggers the downstream
stimulation of nuclear factor-B (NF-B) and the induc-
tion of genes that encode inflammation-associated mol-
ecules and cytokines409. Recent evidence has indicated
that TLRs respond to both pathogens and to host tissue
injury410,411, which participate not only in controlling the
hostdefence response, but also in neuroinflammation
and neurodegeneration412,413.
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
The down-regulation of TLR4 protects against oxidative
stress in Alzheimers disease416, focal cerebral ischemia417
and ischemic brain injury418, thus supporting the role of
TLR4 in brain injury.
In summary, a number of human and experimental
studies have shown that alcohol is toxic for the brain and
that both alcohol abuse and chronic alcohol consump-
tion can induce severe brain damage, and can even cause
neurodegeneration in some cases. Current studies sup-
port that the activation of the innate immune system,
TLR receptors and glial cells259,419, may contribute to
alcohol-induced neuroinflammation and neurodegen-

Current reports reveal that alcohol is capable of act-
ing as a ligand of TLR4 to cause not only the activation of
astrocytes in culture261 and in cultured microglia cells260,
but also the production of cytokines and inflammatory
mediators in rat brains259,262. Indeed, a recent report has
clearly shown the critical role of TLR4 in alcohol-induced
brain injury since elimination of TLR4 receptors protects
against both the activation of microglia and astroglial
cells and the production of cytokines and inflammatory
mediators, and also against apoptosis induced by long-
term alcohol consumption in mice259 (Figure 2). Other
studies have suggested that alcohol regulates the produc-
tion of pro-inflammatory mediators in human astrocytes
in culture by interacting with Src kinase and TLR4414,
while others have used brain slice cultures to propose
For personal use only.
eration (Figure 2). The activation of the innate immune
system and TLR4 may also participate in alcohol-
induced neuroinflammation in adolescents who binge
drink. Nonetheless, it is unknown whether the activation
of TLRs can mediate some of the detrimental effects on
fetal brain development.
Summary and perspectives
According to the WHO420 alcohol is the most prevalent
psychoactive substance, responsible for 5.4% of total
disease burden. Although alcohol can affect almost all
body tissues and organs, the brain is considered the most
significant target of alcohol use and/or abuse. Alcohol
can alter the function and the structure of both the devel-

that alcohol induces the production of cytokines by acti- oping and the adult brain, causing neurological, neu-
vating the innate immune gene expression cascade415. ropsychological and cognitive dysfunctions. Although

Figure 2. Neuroinflammatory components in ethanol-induced brain damage. Ethanol, by activating toll-like receptor 4 (TLR4) and
interleukin-1 receptor (IL-1RI), stimulates microglial and astroglial cells, which trigger signaling pathways (MAPKs, NF-kB, AP-1) and
the production of proinflammatory mediators (iNOS and COX-2), cytokines (IL-6, IL-1 and TNF-), reactive oxygen species (ROS) and
nitric oxide (NO), leading to neuroinflammatory damage. Products derived from microglia and astrocytes act in combination to promote
neurotoxicity and finally cell death. These inflammatory events may also increase following cell injury via the activation of TLR4 receptors
by endogenous TLR ligands (e.g. DAMPs, damage-associated molecular patterns, including cellular debris) allowing an amplification of
the ethanol-induced inflammatory response in a cyclic manner. BBB, bloodbrain barrier.
 Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain 37
alcohol exposure is particularly damaging for the brain
during prenatal development, the adolescent, and even
the adult brain is also affected by this neurotoxin. There
is evidence indicating that alcohol consumption pat-
tern influences the neurotoxic effects of alcohol on the
brain, while binge drinking is particularly detrimental
for fetal development and during adolescence. The brain
regions/structures that are apparently the most sensitive
to the effects of alcohol include the prefrontal cortex, the
CC, the hippocampus and the cerebellum. The alcohol-
triggered alterations in these areas may underlie some
of the behavioral and cognitive dysfunctions in exposed
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by McMaster University on 05/11/13
individuals in all age groups, including children with
FASD.
The molecular actions of alcohol on the brain are
complex, and involve numerous mechanisms and signal-
ing pathways. Some of these actions, such as increased
oxidative stress, interference with mitogenic and growth
factor responses, altered gene expression, and epigenetic
effects, as well as alterations in neurotransmitter receptors
such as glutamate and GABA, occur in both the adult and
the developing brain. Nonetheless, the behavioral and
cognitive consequences may differ in accordance with
the brain ontogeny period, when the effects occurring
during brain development are the more important and
permanent ones. For example, alcohol abuse and binge
drinking can result in prefrontal cortex damage, reduced
For personal use only.
executive functions and can contribute to compulsive
behavior, all of which are events that can predispose to
alcohol abuse421. However, while later effects in adults
seem to be reversible during abstinence421, these effects
in adolescents can predispose to alcohol consumption421,
whereas they may induce irreversible deficits in executive
function and spatial processing in children with FASD68.
The impact of alcohol on brain functioning also plays
a central role in addictive and permanent adaptations,
such as the development of alcohol dependence with its
features of tolerance or withdrawal upon discontinuation
of alcohol use. However, despite the huge progress made
towards understanding those mechanisms underlying
the effects of alcohol on the brain, researchers continue
to explore the processes involved in addiction develop-
ment with a view to identifying new targets for treatment
and to developing new medications that address differ-
ent aspects of the dependence syndrome.
Further studies are required to understand the mech-
anisms by which alcohol activates the innate immune
system and TLR4 receptors, although it is clear that these
receptors are mediators of alcohol-induced inflam-
matory damage in the adolescent and adult brain259.
Nevertheless, it is unknown whether TLR4 participates in
the effects of alcohol on fetal development. In the search
for answers, it is necessary to use a multidisciplinary
approach capable of linking the neuroinflammation
with the specific behavioral and neuropsychological
effects of alcohol abuse, and is able to provide potential
new treatments and/or intervention strategies to restore
alcohol-induced brain damage and to correct behavioral
dysfunctions. In line with this, new findings demonstrate
the role of TLR4 in the cognitive and anxiety-related
behavioral alterations induced by alcohol422, and the
expression of TLR4 in the central amygdala seems to also
contribute binge drinking, and may play a key role in
early neuroadaptation in excessive drinking423.
There are still substantial gaps in our understanding
of the cellular and molecular bases of cognitive deficits
(e.g. attention, memory, executive functioning) caused
by heavy alcohol consumption during adolescence. We
also need to improve our knowledge about the mecha-
nism participating in adolescents greater sensitivity to
the addictive drug actions. Longitudinal behavioral stud-
ies are also important to assess the potentially reversible
effects of alcohol use during adolescence.
Finally, research into biomarkers of fetal alcohol dam-
age186 and studies on neuroimaging techniques may
help us advance in diagnoses, and possibly improve the
future of those individuals affected by prenatal alcohol
exposure.
Acknowledgment
We thank M. March for the excellent professional assis-
tance in this manuscript.
Declaration of interest
The authors experimental work has been supported by
grants from the Spanish Ministry of Science and Innovation
(SAF 2009-07503), the Spanish Ministry of Health, the
Carlos III Institute (RTA Network, RD06/0001/0019),
PNSD (2010I037), PROMETEO/ 2009/072 and General
Direction of Drug Dependence, GV. The authors alone
are responsible for the content and writing of the paper.
All authors declare they have no actual or potential com-
peting financial interest.
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