© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience
N E U R O B I O LO G Y O F A D D I C T I O N
REVIEW
Laboratory models of alcoholism: treatment
target identification and insight into
mechanisms
David M Lovinger & John C Crabbe
Laboratory models, including animal tissues and live animals, have proven useful for discovery of molecular targets of alcohol
action as well as for characterization of genetic and environmental factors that influence alcohol’s neural actions. Here
we consider strengths and weaknesses of laboratory models used in alcohol research and analyze the limitations of using
animals to model a complex human disease. We describe targets for the neural actions of alcohol, and we review studies in
which animal models were used to examine excessive alcohol drinking and to discover genes that may contribute to risk for
alcoholism. Despite some limitations of the laboratory models used in alcohol research, these experimental approaches are
likely to contribute to the development of new therapies for alcohol abuse and alcoholism.
The euphoria that follows the first drink at a party is familiar to many,
as is the loss of judgment and control after continuing to imbibe alcohol. Unfortunately, the escalation that leads to the desperate craving
for alcohol, destroying lives and families, is also experienced by far too
many people. Alcohol abuse and alcoholism involve interactions among
a number of neural mechanisms, including acute sensitivity to alcohol,
development of tolerance to and dependence upon the drug, and development of an intense desire to consume the drug (sometimes called
‘craving’). Alcohol has direct actions on molecules that lead to intoxication and influence responses to chronic drinking1 (Fig. 1). Other
molecules, most notably several neural proteins, interact with ethanol
less directly by influencing expression or function of molecules that are
direct ethanol targets or by altering the function of neural circuits that
participate more generally in addictive processes (Fig. 1). Molecules in
both groups contribute to the acute and chronic stages of alcohol use,
influencing the likelihood of abuse and the expression of alcoholism.
Inherited factors contribute a great deal to an individual’s susceptibility to alcohol abuse and alcoholism (Fig. 2). Genetic makeup and environmental experience interact to alter both direct alcohol actions and
molecular mechanisms that indirectly affect ethanol-related behaviors.
Such interactions influence the acute sensitivity to ethanol intoxication
and the neuroadaptive changes that take place in response to chronic
David M. Lovinger is in the Laboratory for Integrative Neuroscience, National
Institute on Alcohol Abuse and Alcoholism, National Institutes of Health,
Rockville, Maryland 20852, USA, and John C. Crabbe is in the Department of
Behavioral Neuroscience, Oregon Health & Science University, the Portland
Alcohol Research Center and the Department of Veterans Affairs Medical
Center, Portland, Oregon 97239, USA.
e-mail: [email protected]
Published online 26 October 2005; doi:10.1038/nn1581
NATURE NEUROSCIENCE VOLUME 8 | NUMBER 11 | NOVEMBER 2005
alcohol abuse. Initial sensitivity to alcohol differs among different individuals, and there is some evidence that these differences contribute to
susceptibility to later alcoholism2. Animal and human studies suggest
that differences in alcohol sensitivity, seeking and drinking behaviors
have a strong inherited component and may involve polymorphisms
in genes encoding proteins involved in intoxication3,4.
Chronic alcohol exposure causes neuroadaptations that foster
continued alcohol abuse5,6. Tolerance to ethanol (that is, decreased
sensitivity to intoxication) and dependence on it, evidenced by both
physical withdrawal and increased desire for the drug, result from
repeated exposure to alcohol. The neuroadaptations underlying these
behavioral adaptations to alcohol involve molecular mechanisms that
are affected both directly and indirectly by alcohol. The propensity for certain alcohol-related neuroadaptations is also influenced
by genetic factors and gene-environment interactions (Fig. 2). As
with other drugs of abuse, alcohol seeking in dependent individuals
may reflect its increased value as a reward, its ability to reduce the
undesirable effects of alcohol withdrawal, or both. The changes in
reward value and the consequences of withdrawal result from the
neuroadaptations brought about by chronic alcohol, and thus it is
important to understand the molecular, cellular and genetic basis of
these neuroadaptations.
Addiction to alcohol shares common neural substrates with other
addictions at the molecular, cellular and circuit levels. These commonalities include alcohol and drug actions at similar subsets of neural
proteins and neural systems that are targets for other abused substances
(such as the GABAA receptor7, brain glutamatergic systems8,9, nuclear
signaling proteins such as CREB10, stimulation of VTA dopaminergic
neurons11 and involvement of mesocorticolimbic and extended amygdala circuitry12). However, alcohol abuse and alcoholism also involve
neural processes distinct from other addictions. Attempts to understand
the common and unique aspects of alcohol addiction have spurred
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Chronic exposure
(neuroadaptation)
Functional/
biophysical
analysis
Transgenics,
knockouts,
QTL analysis,
etc.
Pharmacological,
biochemical,
molecular biological
analyses
Direct alcohol targets
© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience
Forward and
reverse genetics
Alcohol-associated proteins
Preclinical drug screening
Pharmacotherapeutic target candidates
Ann Thomson
Acute
pharmacology
Figure 1 Use of animals to identify direct and indirect alcohol targets
can lead to development of pharmacotherapies for alcohol abuse and
alcoholism. Pharmacological and genetic techniques are used alone and
in combination to uncover molecular targets that are directly affected by
alcohol (direct alcohol targets) and to identify proteins that are indirectly
altered by alcohol or that influence neural responses to alcohol (alcoholassociated proteins). These molecules can then be targeted in preclinical
screens designed to determine if pharmacotherapeutic agents or other
target-specific treatments alter responses that are predictive of alcohol
abuse or alcoholism (such as alcohol intake, alcohol reinforcement, craving
for alcohol or relapse). Molecular targets that show promise in preclinical
testing are then the focus of clinical testing for safety and efficacy.
Alcohol’s difficult pharmacology: how do we identify a ‘hit’?
Animals, animal tissues and molecules arising from experimental
animals are widely used in studies aimed at finding molecular targets
of alcohol actions. Before examining some of the latest findings in
this field, it is worthwhile to consider the limitations of experimental
searches for alcohol targets.
Investigators searching for direct targets of alcohol actions in the
CNS often use pharmacological approaches involving direct application of ethanol to neuronal preparations16. Numerous complications
arise when using these approaches. Because of the simple structure of
the ethanol molecule, only two reactive sites are present: the OH group,
which is capable of hydrogen bonding, and the short carbon backbone,
which contributes to weak hydrophobic interactions (although metal
ion interactions do occur in alcohol dehydrogenase)17. The molecule
does not form ionic or covalent bonds. These characteristics generate
poor reactivity that results in low potency of the drug, such that acute
neural effects ranging from intoxication to anesthesia18 are observed
only at blood and brain concentrations from ∼5 mM to 100 mM.
This low potency prevents the application of some pharmacological
techniques, such as radioligand binding, that can detect sites of direct
molecular interactions between proteins and compounds with high
binding affinities. Furthermore, biophysical studies of specific ethanol
interactions with molecules are often hampered by the distribution of
ethanol into many cellular compartments and the weak interactions of
the molecule with many potential target molecules. These experimental
limitations make it difficult to measure actual affinity of alcohol for
potential molecular targets, leaving only measures of potency and efficacy that are difficult to relate to occupancy of a particular molecular
site. Without measures of direct molecular interactions, it is difficult
to be certain that a particular moiety is truly an ‘alcohol binding site’.
Contrast this with the pharmacology of opiate drugs, which have high
affinity for a well-characterized G protein–coupled receptor19, and one
begins to see the problems inherent in the molecular neuropharmacology of alcohol.
Methodologies for examining direct alcohol interactions have been
applied to some proteins (such as the Drosophila melanogaster LUSH
protein; see below), but until these types of experiments can be performed on a wide variety of proteins, it is best to avoid inferring too
much about direct alcohol targets and affinity of alcohol at these targets.
The foregoing discussion provides some of the reasons why identification of direct alcohol target molecules has lagged behind the discovery
of other drug targets.
Ethanol, within the range of physiologically relevant concentrations,
nearly always produces small effects. It is not unusual to observe only a
30% change in any given measure of cellular or molecular function even
at concentrations that are near the higher limit of the sublethal range
(for example, 100 mM). This may be a blessing for the person drinking alcohol as an intoxicant, as it ensures that the desired effects occur
with a large margin of safety from outright toxicity. However, for the
neuropharmacologist, the small effect sizes are problematic. Attempts
to determine the molecular interactions that underlie relevant ethanol
effects are often confounded by signal-to-noise issues inherent in looking for changes in a small response. Small effects are often overlooked,
and changes in molecular or cellular function that result from timedependent changes in the experimental system might be mistaken for
ethanol actions. Consequently, the experimenter must have confidence
in the accuracy and range of variability of the measures that are made.
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investigators to adopt new animal models and research approaches that
have not been as widely used in investigation of other addictions.
Here we focus on two aspects of the use of animal models in alcoholism-related research that most distinguish this line of research from
investigation of other addictions. First, the basic molecular mechanisms
of alcohol action are not shared by most other drugs of abuse, and the
hunt for alcohol targets has spurred investigators to use unique experimental approaches in animal tissues, cells and molecules. Second, the
use of genetic animal models to explore mechanisms of ethanol action
has traditionally been more widespread in alcohol research than in
investigations of other addictions. Given the large role of vertebrate animal models, and more recently, invertebrate models, in alcohol research,
we feel it is important to critically analyze the strengths and weaknesses
of the animal models. We highlight findings from animal models that
indicate molecular targets and genetic underpinnings of alcoholism
and consider the limitations of using animal models as surrogates for
understanding human alcoholism.
The behavioral repertoire of laboratory animals differs from that of
humans, and many animals—most notably invertebrates—have nervous
systems that differ radically from that found in man. In typical studies,
rats are offered a choice of drinking water versus ethanol solutions, or
animals are tested in a protocol of operant self-administration, in which
a specific response yields access to alcohol. Drug state can also be repeatedly paired with specific cues, and the animal can be tested on the choice
it makes between a saline-paired or alcohol-paired cue13. Most behavioral assays of alcohol intoxication and tolerance target some aspect of
motor coordination (which turns out to be a very complex domain of
behavior genetically14) or dysregulation of body temperature, and these
methods are also used for studying a broad spectrum of drugs with
sedative-hypnotic effects. Anxiolytic effects are also studied. Laboratory
animals cannot verbally report their subjective states, which only adds to
this problem. Consequently, when using these organisms, it is difficult
to infer affective and cognitive states that are likely to be important
determinants of alcohol intoxication, abuse and alcoholism15.
REVIEW
a
Genes Proteins Pathways
Behaviors
Figure 2 Complexity of gene-environment-behavioral interactions in the
neural actions of alcohol. (a) Individual genes through their expression
lead to synthesis of specific proteins. These proteins in turn participate in
numerous cellular- and systems-level pathways that ultimately modulate
specific addiction-related behaviors. A behavioral difference can be
determined essentially by a single gene: some complex traits show purely
mendelian inheritance. More commonly, a single gene can influence
multiple behaviors (pleiotropy), or multiple genes can exert converging
influence on a single behavior, which is then variously termed multigenic,
oligogenic or polygenic. This describes what we know of addictive behaviors.
The bottom of panel a depicts the range of interest of genomics, proteomics
and metabolomics analyses. Their common feature is the concentration of
the analysis on a single behavior (such as impulsive drug-taking). Here we
also show that the relationships between proteins, pathways and behaviors
are complex. The usual case under investigation must account for both
multigenic and pleiotropic effects. (b) All of the above gene-behavior
relationships take place in environments that themselves may differ. The
only differences between the cases shown in environments 1 and 2 in the
example are that the effects of the second and third genes on the first
two behaviors differ depending on the environment—a gene-environment
interaction. The final panel shows that behaviors can themselves interact,
and that they can influence the expression of genes. The range of interest
of behavioral genomics includes all relevant genes and behaviors, including
assessment of their interactions with each other and the environments
in which they are assessed. This level of analysis will be critical to
understanding the addictions.
Gene effects
Mendelian
Pleiotropic
Genomics
Proteomics
Metabolomics
b
Genes Proteins Pathways
Behaviors
1
1
2
2
3
3
4
4
1
1
2
2
3
3
4
4
Environments
Environment 1
Gene ҂
environment
interaction
number of cycles of exposure and withdrawal to be modeled. These
reduced systems have contributed much to our understanding of cellular
and molecular neuroadaptations to alcohol exposure.
Environment 2
Behavioral
interactions
Effects of
behavior on
gene expression
Behavioral genomics
Ann Thomson
© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience
Multigenic
Oligogenic
Polygenic
Multigenic +
Pleiotropic
Sample sizes used in experiments with ethanol often must be larger
than those used with more efficacious drugs. In addition, it is especially
important to demonstrate reversibility of the alcohol effect to ensure
that small changes are not due to ‘drift’ in the measurement over time.
The high concentrations of alcohol used in most experiments bring
into play possible artifacts related to ethanol’s solvent properties (that
is, the possibility that ethanol may redissolve a hydrophobic compound
that had previously adhered to plastic tubing or may leach plasticizing
agents out of tubing or storage vessels). Thus, great care must be taken
in designing pharmacological studies with ethanol.
An alternative approach to identifying molecules with important roles
in the neural actions of alcohol is to examine molecular changes brought
about by chronic alcohol exposure. Alcohol-induced neuroadaptations
that lead to tolerance, dependence, withdrawal signs and increased alcohol intake often involve prolonged exposure to alcohol. Many techniques
for chronic ethanol exposure in reduced neuronal preparations (such
as dispersed primary neuronal cultures or organotypic slice cultures)
allow investigators to examine molecular adaptations to ethanol in wellcontrolled experimental systems20,21. Important variables include the
concentration of alcohol applied, ethanol evaporation at physiological
temperatures, the pattern and duration of alcohol exposure, and the
NATURE NEUROSCIENCE VOLUME 8 | NUMBER 11 | NOVEMBER 2005
Modeling alcohol genetics in animals
Largely by historical accident, animal models have been used more
often in the field of alcohol genetics than in any other area of psychiatric
genetics. The wealth of alcohol response data on genetic animal models
gives us a window through which to assess the strengths and limitations
of animal models in biomedical research more generally. Some issues
may be traced to the complex genetics of alcohol dependence disorders
(Box 1; Fig. 2), and there are parallels with the pharmacological challenges just discussed. Taken together, this means that no single animal
model will convincingly capture all the features of this complex disorder, and a useful model should target certain well-defined features.
Most research with genetic animal models has therefore concentrated
on a few relatively straightforward phenotypes.
Although there are many problems in research on the cellular and
molecular neuropharmacology of ethanol and many complexities that
must be addressed by animal models, there is an abundance of candidate molecules that seem to be targets for direct and indirect actions of
ethanol in the nervous system. However, the abundance of targets also
makes it difficult to determine the most important contributors to the
neural effects of ethanol.
The direct approach: pharmacological methods
Heterologous expression of molecules allows researchers to examine
the effects of ethanol on the function of proteins expressed in a cellular
context free of many neural proteins. Heterologous expression combined with examination of the function of the same protein in neurons
has been used to characterize ethanol sensitivity of a variety of proteins,
including neurotransmitter receptors, ion channels and neurotransmitter transporters1,22. The discussion of all these molecular targets
is beyond the scope of this short review, but the following description
of alcohol actions on GABAergic synaptic transmission illustrates the
usefulness of this approach.
Interactions between ethanol and GABAergic inhibitory synaptic
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transmission have been the subject of intensive study for over
30 years7,23. Evidence from in vivo and in vitro studies supports the
idea that ethanol produces many of its intoxicating actions by enhancing GABAergic synaptic transmission23. The GABAA receptor is one
potential molecular target for ethanol, and a variety of evidence sug-
gests that alcohol potentiates GABAA receptor function. However, there
is ongoing controversy as to the prevalence and importance of ethanol
actions on this receptor. Ethanol potentiation of GABAA receptor function has been observed in some isolated neuron preparations7,24,25.
However, negative results have also been reported in numerous neuronal subtypes24. Studies in the Xenopus laevis oocyte expression system have generally found ethanol potentiation, but at relatively high
ethanol concentrations26. However, very few studies in mammalian cell
systems have produced evidence for this potentiating effect24. Clearly,
understanding the molecular basis for these discrepant results will be
a key step in resolving this controversy and determining the role of
GABAA receptors in alcohol effects.
Diversity among GABAA receptor subtypes could contribute to the
differential effects of ethanol in different cells. GABAA receptors exist
in the plasma membrane as heteropentamers of a variety of subunits
that can coassemble in various combinations. The most recent studies carried out in oocytes, isolated neurons and brain slices indicate
that ethanol can potentiate responses to GABA that are mediated by
receptors containing the α4 or α6, β2 or β3 and δ subunits25,27,28 at
ethanol concentrations as low as 1–3 mM, well within the range associated with in vivo intoxication, although there is unresolved disagreement about the effective concentrations and the shape of the ethanol
concentration-response functions observed in different laboratories.
These receptors are often found outside the synapse, where they are
thought to mediate tonic GABAergic inhibition28,29. Studies of granule
neurons in brain slices from the cerebellar cortex and dentate gyrus
demonstrate ethanol potentiation of a tonic GABAA-mediated current
that seems to involve this type of receptor28,29. What role, if any, does
this ethanol potentiation have in acute intoxication and alcohol abuse?
One clue comes from a study28 concluding that a naturally occurring
polymorphism in the rat GABAA α6 subunit may account for differences in the alcohol sensitivity of GABAA receptors. This polymorphism
is also associated with a change in acute alcohol motor impairment
observed in the rats in vivo28, and the polymorphism segregates in rat
lines bred for differential alcohol sensitivity30–32. Differences in alcohol
sensitivity have been related to differences in alcohol intake, and thus
this line of research may prove useful for understanding factors that
contribute to this link. Indeed, studies of human α6 polymorphisms
suggest some association between this subunit and alcoholism3. These
findings indicate the potential importance of direct ethanol effects on
α6-containing GABAA receptors.
However, the role of the rat α6 polymorphism in differential alcohol
sensitivity has been questioned. The specific polymorphism leading
to reduced GABA function is not observed in human or even mouse
α6, although there are interesting parallels with the human α6 P385S
polymorphism. Other polymorphisms cosegregate strongly with the
α6 difference31 and this may suggest that there is coordinated regulation of the cluster of GABAA subunit genes in this chromosomal
region. Furthermore, several acute alcohol sensitivity phenotypes do
not simply cosegregate with this polymorphism in the AT and ANT
rat lines that were selectively bred to differ in motor impairment by
ethanol32. Finally, gene-targeted mice lacking the α6 subunit do not
differ in sensitivity to ethanol motor impairment33, although this may
be due to compensations during development. Thus, it is premature
to conclude that this polymorphism is the only factor contributing to
alcohol sensitivity in rats28. Expression of the α6 subunit is restricted
to cerebellar granule neurons30, and thus this subunit would seem less
likely to participate in aspects of intoxication that do not involve motor
impairment. For this reason, it is unlikely that the α6 polymorphism
contributes to alcohol sensitivity for most aspects of intoxication. In
addition, there is little information about the direct effect of ethanol
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© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience
BOX 1 GENETICS OF COMPLEX TRAITS
The complexities involved in relating genes to addictive
behavior are becoming more amenable to analysis, thanks to
the development of new ways to measure and manipulate the
function of genes as well as large-scale applications of classical
breeding methods. Although the field is moving rapidly, addiction
will not be explained by concentrating solely on functional
genomics and its related reductions (proteomics, metabolomics;
Fig. 2a). Equal attention should be paid to the other part of the
gene-behavior nexus, the behavioral phenotypes.
Most behaviors taken to index different aspects of drug or
alcohol dependence vary continuously over a wide range in
populations, and each relevant gene influencing susceptibility
or severity usually has a small, graded (polygenic) effect on
that trait (Fig. 2a). Hence, the involvement of many genes is
required to explain the full range of genetic contribution to risk
for or severity of alcohol dependence. A gene that influences
one behavioral contributor to risk is likely to be relevant for
other behaviors or even things that may have no obvious
connection with behavior (pleiotropy). Furthermore, genes do
not act independently but typically interact with each other
(epistasis). Finally, in the same way that any statistic describes
the specific population from which it is drawn, genetic
influence reflects the environment in which a gene’s effects
on behavior are studied, and if a different environment (such
as set of families, rearing conditions, stressors) is studied,
different genes may be important, they may interact differently,
or both. (Fig. 2b).
Alcoholism and drug dependence are also complex at the
behavioral level. Although the severity of any of the various
symptoms that in the aggregate lead to diagnosis is continuous,
the diagnoses are categorical. Furthermore, unlike many
genetically influenced diseases, diagnosis does not imply a
particular pathophysiology. Thus, members of any diagnostic
category (such as ‘alcohol dependence’) are heterogeneous.
Individuals diagnosed with drug-related disorders are also
highly likely to have other (comorbid) diagnoses as well, such
as depression or anxiety disorders, each of which has its
own set of definitional and genetic complexities. Addictive
behaviors may interact as well. Anxiety may drive attempts
to alleviate it through escalation of drug ingestion, but drug
withdrawal may then engender more anxiety, not less. In
addition, there are developmental shifts in the characteristics
of the disorder across the life span. For example, any amount of
drinking in an 8-year-old may be considered excessive, whereas
high consumption levels in 20-year-olds are seen as less
diagnostic than a similar level would be in a 65-year-old.
Confronted with this daunting complexity, the field often
progresses in small steps. A study may identify one or two
relevant genes and assess their interactions with other factors.
Gradually, genetic knowledge from many studies then can be
assembled into a larger system of interactants that enables
us to understand a set of related behaviors. We term this
perspective behavioral genomics (Fig. 2b).
© 2005 Nature Publishing Group http://www.nature.com/natureneuroscience
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in mammalian heterologous expression systems or on isolated neurons
containing α6-δ receptors. It is always reassuring to observe drug effects
in a single neuron under tightly controlled physiological and pharmacological conditions, because this rules out many indirect effects that
could contribute to ethanol effects in more intact tissues. Clearly, further work is needed to determine the factors that contribute to ethanol
effects on GABAA receptors and the scope of the involvement of this
receptor in alcohol effects on the brain and behavior.
The long-held belief that ethanol potentiation of GABAergic transmission arises solely from increased GABAA receptor function is being
challenged by studies indicating that ethanol potentiates GABA release
in several brain regions. This potentiation is observed at reasonable
ethanol concentrations and thus could certainly contribute to intoxication and other alcohol-related behaviors. As yet, there is little information about the molecular mechanisms underlying this potentiation.
A recent review34 nicely describes this growing area of investigation.
Here we simply note that ethanol increases GABAergic synaptic input
onto neurons, including those in the central amygdala, cerebellum and
hippocampus35–37. At many of these synapses, ethanol alters several
electrophysiological measurements indicating an increase in presynaptic GABA release. These include increased paired-pulse facilitation and
increases in the frequency of miniature inhibitory postsynaptic currents
(mIPSCs). In cerebellum, an increase in firing rate of GABAergic interneurons also contributes to the increased GABAergic input36. Because
transmission at most GABAergic CNS synapses is mediated predominantly by GABAA receptors, presynaptic ethanol potentiation must
be considered when formulating hypotheses about effects of GABAA
receptor–targeted drugs on alcohol-related behaviors and changes in
alcohol responsiveness or alcohol drinking in gene-targeted mice lacking GABAA receptor subunits.
The spineless approach: invertebrate models
Studies in invertebrate models have unearthed molecules with potential
roles in alcohol intoxication and alcoholism, along with unexpected
concordance with previous findings from studies of vertebrates. For
example, several genes that influence acute alcohol sensitivity and tolerance have been identified in mutant Drosophila melanogaster fruit flies,
and intracellular signaling pathways involving cAMP and protein kinase
A (PKA), as well as transcription-associated proteins38,39, are beginning
to be implicated. Powerful molecular genetic methods can be applied in
D. melanogaster to alter protein expression in only a subset of neurons
within the nervous system much more rapidly than is possible in mammals. Selective overexpression of a PKA inhibitor leads to localization
of the cAMP-mediated signaling important for regulation of ethanol
sensitivity in a subset of putative neurosecretory neurons in the CNS40.
This finding rules out a host of more trivial interpretations of previous
findings (such as alterations in muscle function or metabolism), and
helps validate D. melanogaster as a model for study of ethanol effects
on the CNS. Studies using cell lines derived from rodents had already
implicated cAMP-associated signaling proteins in effects of acute and
chronic ethanol41. Furthermore, alcohol intake can be regulated by
treatments that affect the cAMP-PKA signaling system in the nucleus
accumbens42. Thus, symmetry is emerging between the findings in D.
melanogaster and rodent models, but it is not yet clear whether any of
these proteins are direct targets of alcohol.
Another D. melanogaster mutant may provide one of the best models
for direct ethanol binding to a protein. The lush mutant lacks an olfactory protein involved in detecting ethanol (from rotting fruit)43. Flies
lacking the LUSH protein do not avoid alcohol, in contrast to wild-type
flies. The alcohol avoidance behavior typical of wild-type flies can be
reinstated by reexpression of the LUSH protein in mutant flies43. The
NATURE NEUROSCIENCE VOLUME 8 | NUMBER 11 | NOVEMBER 2005
lush gene product is putatively involved in olfactory detection of ethanol and other short carbon-chain alcohols such as butanol, and it has
an alcohol-binding site identified by NMR and X-ray crystallography44.
However, other researchers disagree that LUSH is an alcohol binding
protein and have challenged the idea that flies avoid high ethanol concentrations45. These investigators attribute the avoidance behavior to
plasticizing contaminants in ethanol solutions, again raising the specter
of problems brought about by alcohol’s solvent properties. This issue
has yet to be resolved, but the crystal structure of LUSH certainly indicates how an alcohol-binding pocket in a protein might look.
The nematode Caenorhabditis elegans has also been used to probe
the genetic and molecular basis of acute ethanol sensitivity. Mutant
C. elegans lines with decreased sensitivity to ethanol-induced disruption of acute movement and egg-laying behavior have a loss-of-function mutation in the gene that codes for the pore-forming subunit
of the large-conductance calcium-activated potassium channel (BK
channel)46. Acute ethanol exposure also enhances BK channel function in C. elegans neurons46, which could alter motor and egg-laying
function. This elegant work demonstrates the power of invertebrate
models for analysis of susceptibility genes and molecular targets of
ethanol action. Similarly, in rodents, ethanol potentiates BK channels
in neurohypophyseal peptidergic terminals47, which likely contributes
to alcohol-induced changes in vasopressin secretion and diuresis within
this neuroendocrine system. The role of BK channels in mammalian
intoxication remains to be determined.
The use of invertebrate animal models has also revealed potential
molecular targets of ethanol action that had not been previously examined. For example, ethanol sensitivity in D. melanogaster is regulated by
insulin, the insulin receptor and insulin-producing neurons. Mutation
of the insulin receptor or its substrate in a subset of neurons within the
D. melanogaster CNS increases sensitivity to intoxication48. It will be
interesting to determine to what extent the interactions between insulin
and the cAMP signaling system are involved in fruit fly intoxication.
Behavioral screening methods for zebra fish (a vertebrate model,
of course) have also been developed to study effects of ethanol on
behavior49–51. Thus, the rapidly emerging use of invertebrate models for alcohol research may be complemented by the use of simpler
vertebrate models that are amenable to genetic analysis and genetic
manipulation.
The early promise of invertebrate and non-mammalian vertebrate
model systems has spurred intense interest in the alcohol research field.
The tractability of these organisms for genetic studies such as mutagenesis screens suggests that further interesting developments may be on
the way. However, their utility for understanding human alcohol abuse
and alcoholism is still in question. It remains to be seen if the nervous
system circuitry of invertebrates can be generalized meaningfully to the
mammalian brain, as it is possible that neural circuits contributing to
behavioral effects of alcohol differ in invertebrates and mammals even
if the behavioral outcomes are similar in D. melanogaster and C. elegans.
For example, the effects of ethanol on basic motor control circuits seem
to underlie incoordination in invertebrates, whereas more sophisticated
circuits such as the basal ganglia and cerebellum contribute to motor
impairment in rodents. Future studies must focus on determining the
comparability of molecular and cellular effects of ethanol in the different circuits found in vertebrate and invertebrate organisms.
Invertebrate and vertebrate model organisms, however, do share considerable commonalities in neurochemistry. Neurotransmitters such
acetylcholine, dopamine and glutamate are present in the nervous systems of all these organisms, and many neuropeptides are also commonly
expressed. However, there are notable differences in neurotransmitter
types and receptors that could alter the effect of ethanol in the different
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systems. For example, octopamine is a monoamine neurotransmitter
implicated in ethanol actions in D. melanogaster38. This neurotransmitter is not found in mammals, but may have analogous cellular functions to norepinephrine in the mammalian CNS. Significant questions
remain as to whether neuronal systems implicated in alcohol actions in
mammals will have a role in invertebrate responses to the drug.
Thus far, we have stressed similarities between molecular substrates
of alcohol responsiveness in D. melanogaster, C. elegans and rodents.
However, there are no findings to date implicating GABAergic or
glutamatergic transmission in invertebrate alcohol effects, despite
the known importance of these neurotransmitters in mammalian
responses to alcohol7–9,28. It may well be that mammalian brains have
evolved alcohol-sensitive circuitry and neurotransmitter systems that
are not prominent in the invertebrate brain. It will also be important
to determine which invertebrate proteins are direct targets of ethanol
action (such as the BK channel) and which indirectly influence alcohol sensitivity. Examination of the effects of alcohol in invertebrate
models is a relatively new research area, and thus it is premature to
speculate too strongly on the similarities and differences in the systems.
Ultimately, more complete characterization of the molecular actions of
ethanol in multiple nervous systems will be needed to determine which
mechanisms are most common. Comparison of the effects of alcohol
on neural circuits and molecules across organisms should yield some
consensus on the similarity of intoxication in the different models.
Invertebrate models show great promise for alcohol research at present,
and it is likely that information gained from such models will complement, but certainly not replace, that gained from the continued use of
mammalian laboratory animals.
Genetic animal models and alcohol responses
Evaluation of the validity and utility of rodent animal models is also
important for developing better tools to analyze the genetics of alcohol-related behaviors. A wealth of historical data has been reviewed
elsewhere52,53. Selective breeding has been used since the late 1940s to
develop lines of rats and mice that differ markedly in voluntary alcohol
drinking. Inbred strains of mice that preferred to drink alcohol, or
shunned it, were first identified in 1959. At least seven different pairs
of lines of rats have been bred for high versus low alcohol preference.
These genetically high and low drinkers have some fairly consistent differences in the neurobiology of alcohol responses53–55. High drinkers
tend to have low synaptic levels of serotonin and dopamine, and activation of serotonin 5-HT1A or 5-HT2 receptors reduces alcohol intake, as
does stimulation of dopamine D2 or D3 or GABAA receptors. Blockade
of opioid or 5-HT3 receptors also reduces intake, and these classes of
agents have some efficacy in the clinic with alcoholics56,57. Rather than
review the older material, we concentrate on a few recent examples
of genetic animal model research drawn from quantitative trait locus
(QTL) gene mapping approaches and studies of null mutants, including
some gene expression studies.
alcohol58. The primitive tools then available and the sparsity of the
mouse map of genomic markers made progress initially slow, but as the
technology has improved, the rate of progress has greatly accelerated in
the past few years. To date in addiction research, a single QTL has been
definitively traced to the underlying gene59. In multiple genetic animal
models (standard and recombinant inbred mouse strains, segregating populations, short-term selectively bred lines, congenic strains),
a QTL on mouse chromosome 4 has been linked to the severity of
acute alcohol and pentobarbital withdrawal60. The QTL region initially contained several hundred genes, and polymorphisms and gene
expression data allowed the strong inference that the gene Mpdz, which
codes for a multiple PDZ domain protein, was the only remaining gene
in the region that affected the withdrawal response59. MPDZ protein
facilitates coupling of ligands and receptors and interacts with 5-HT2A,
5-HT2B and 5-HT2C receptors as well as cKIT (a membrane tyrosine
kinase receptor) and p75 (a Trk-associated neurotrophin receptor).
A more detailed description of this project, which is also pursuing
withdrawal QTL on chromosomes 1 and 11, and of the strengths and
weaknesses of QTL mapping approaches, is given elsewhere61. Because
of the close similarity (>85%) between the mouse and human genomes
owing to their shared ancestor, gene findings in mice and humans are
increasingly reciprocally informative62. Not surprisingly, alcoholrelated QTL do not act in isolation. The influence on acute alcohol
and pentobarbital withdrawal of a chromosome 11 QTL, whose interval contains multiple GABAA receptor subunit genes (α1, α6, β2, γ2),
depends upon the presence of one or more genes within the chromosome 1 QTL63. Similarly, some QTL affecting chronic ethanol withdrawal severity are apparent only when considered epistatically with
another genome region. The chromosome 4 QTL containing the Mpdz
gene also affects chronic alcohol withdrawal severity in combination
with a QTL on chromosome 8 (ref. 64). Numerous QTL affecting the
tendency of mice to seek to drink or to avoid alcohol solutions are also
being pursued55.
One of the methods recently brought into play to accelerate the progress of gene mapping efforts is to synthesize information about both
gene expression- and gene sequence–based sources of genetic influence. Until recently, QTL mapping efforts simply identified associations between the occurrence of the mapped trait and markers that
varied in DNA base pair sequence. Positional cloning of a candidate
gene near the markers was followed by functional studies showing
that the gene’s different protein variants had different neurobiological functions affecting the trait. We now know that QTL ‘signals’ may
also derive ultimately from differential expression of the underlying
gene. For example, a specific genetic variation in a promoter region
leading to greater expression of a gene could be found preferentially in
high alcohol responders. Methods exploring both gene sequence and
gene expression variation are proving to be powerful tools to identify
genes for complex traits65 and are now being used on alcohol-related
QTL66–70. For example, a locus very near the dopamine D2 receptor
gene strongly affects its expression68. Levels of DRD2 protein are correlated with ethanol’s motor stimulant effects, as well as with its efficacy
to produce a conditioned taste aversion. Both these traits, which are
thought to model different aspects of ethanol’s reinforcing effects, show
a strong QTL signal in this region as well.
Finding new genes through gene mapping
Mapping approaches to identify genes affecting alcohol responses were
initiated in the early 1990s. For reasons discussed earlier (Box 1), individual differences in responses to alcohol are not generally all-or-none
but rather are quantitatively distributed in populations (that is, they are
quantitative traits). In gene mapping, individuals’ behavioral responses
are first compared with their genotype at many polymorphic markers
scattered across the chromosomes. A pattern of association between a
marker and degree of response provisionally identifies a quantitative
trait locus (QTL). Of well over 100 QTL for many responses to seven
different drugs of abuse cited in an early review, over half were for
Genetically engineered candidate genes
Mouse stocks genetically engineered to have a null mutation for one
of about 50 genes or to overexpress them have been studied for one or
more alcohol responses. We know of no recent, systematic review of the
mutant studies. The GABAA receptor has been fairly thoroughly studied
using genetic engineering strategies, as this receptor system has long
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been a target of mechanistic studies (see above). Null mutants and/or
transgenic overexpression mutants have been generated for the α1, α2,
α5, α6, β2, β3, γ2L, γ2S and δ receptor subunit genes, as well as for the
GABA transporter gene.
A recent review of this work compiled behavioral data for several
ethanol responses, including high-dose sensitivity (loss of righting
reflex), withdrawal severity and tendency toward self-administration71.
This review compared the transgenic behavioral data with those for
expression of the various subunits in brain. Because the chromosomal
location of these genes is known, it was also possible to compare behavioral sensitivity in the mutants and gene expression patterns taken from
a publicly available set of informatics tools and data sets, WebQTL
(http://www.genenetwork.org) with the location of QTL that had been
mapped for the same behavioral responses.
For example, by QTL mapping efforts, the GABAA receptor subunit–
rich chromosome 11 region was found to harbor a gene or genes affecting sensitivity to alcohol-induced loss of righting reflex in some mouse
genotypes. The α1 subunit gene was differentially expressed in mouse
strains that differed in sensitivity, and gene deletion of the α1 subunit
gene affected sensitivity in one of two such null mutant models. This,
and similar data for the α2 subunit gene on chromosome 5, strongly
suggests a role for the α1 and α2 subunit genes in this response71.
Because many different ethanol-related responses have been studied
and because many different genotypes were used, the data matrix available for analysis is frustratingly incomplete. However, such syntheses
of available data will serve to identify the lacunae in our knowledge,
allowing the complexities of GABA-ethanol interactions to be reduced
to a more tractable set of possibilities.
Genetic models: challenges and future directions
Studies of tolerance and dependence seem to have reasonable face
validity across the biomedical science community. Dependent animals,
including those from which alcohol is subsequently withdrawn, display
apparent dysregulation of multiple neural systems that normally are
held within bounds by homeostatic feedback loops. Alcohol dependence
in humans obviously must disrupt in some way the overall calculation
of an individual of the risks and benefits of drinking a lot, but how these
miscalculations play out in the brain’s reward and stress axis circuitry is
not yet clear. Functional and structural brain imaging studies suggest
that chronic alcohol abuse has localized effects on brain circuits72, and
functional MRI signals measured during a behavioral inhibition task
may prove to be useful to predict individuals’ expectancies about the
positive or negative effects of drinking alcohol73.
Some rhesus monkeys given voluntary access to alcohol daily for long
periods will develop patterns of repeated drinking that are obviously
excessive74. However, all the rodent genetic animal models of alcohol
drinking mentioned above, even those animals intensely selectively
bred for preference, show patterns of drinking that do not look exactly
like human alcoholism. Mice and rats will drink until they begin to
approach brain alcohol levels that produce clear signs of intoxication,
but they will rarely continue to drink thereafter until their blood levels
subside somewhat. In other words, rodents seem to have some internal
controls limiting intake that are not shared by susceptible humans.
Several behavioral manipulations can overcome these controls in the
laboratory75–79, but these generally require fairly long-duration exposure and are labor intensive. Thus, it would be useful to have more highthroughput rodent models in which animals voluntarily self-administer
ethanol to excess. Attempts to create such models are supported by the
Integrative Neuroscience Initiative on Alcoholism of the US National
Institute on Alcohol Abuse and Alcoholism. Results have supported the
idea that scheduling access to alcohol during the circadian dark78,80,
NATURE NEUROSCIENCE VOLUME 8 | NUMBER 11 | NOVEMBER 2005
limiting access to fluids for a brief period80–82 or establishing chronic
levels of alcohol before offering it for self-administration83,84 can convince some rodents to drink to intoxication on a daily basis. To date,
most of these studies have focused on the C57BL/6J mouse, known to
drink large quantities of alcohol. Although it is encouraging that we
seem to be able to overcome the (unknown) factors that tend to selflimit ingestion in these creatures, the ultimate utility of these newer
models will be achieved if it proves possible to selectively breed for
very high intakes.
Other challenges facing animal model research are more generic.
Some of the principal defining characteristics of the alcohol dependence disorders are behavioral (for example, interference with work
or social life). Because animals cannot self-report, we must infer their
subjective state from their behavior, which is not as straightforward as
it seems15. There are many rodent behavioral assays designed to reflect
an anxious state, and anxiety is reported by alcoholics when they are
withdrawing from a period of chronic drinking. However, a recent
review of rat and mouse studies of anxiety during alcohol withdrawal
found that nearly all such studies (at least with mice) showed a marked
reduction in general activity of the animals during withdrawal. Because
reduced activity in an apparatus such as the elevated plus maze makes
it difficult to infer anxiety specifically, it is methodologically tricky to
study withdrawal anxiety in rodents85, although some protocols seem
to work reproducibly.
Invertebrate models are not exempt from these challenges. To date,
they have been able to model simple sensitivity to the sedative effects of
alcohol, and the reduction in that sensitivity (tolerance) with chronic
exposure. Given the behavioral repertoire of flies and worms, it will
be difficult to devise assays tapping such internal states as anxiety,
response inhibition or its opposite (impulsivity), craving and reduced
or increased sensitivity to reward. We are fairly confident that the
rodent models in place are a reasonable reflection of these psychological
aspects of alcohol dependence. However, some features (for example,
the intense social pressures loosely called ‘peer pressures’ experienced by
adolescents) will never be modeled in non-human species. In general,
the feelings experienced by alcoholics at different stages of their disease
have been less convincingly modeled than the biological sequelae of
chronic administration.
Serotonin and alcohol: translating animal research to humans
The neurotransmitter serotonin (5-HT) is important for normal and
dysregulated emotional behavior, including depression and alcoholism.
Compounds targeted at brain serotonergic systems, including SSRIs
and 5-HT3 antagonists, were proposed as potential treatments for alcoholism on the basis of animal studies. Brain levels of 5-HT are regulated
by several proteins, including the serotonin transporter (5-HTT) that
removes serotonin from the synapse. The specific serotonin reuptake
inhibitors (SSRIs), most frequently used to treat depression, act by
inhibiting 5-HTT. Activity in the 5-HTT gene is affected by a polymorphism (5-HTTLPR) in its promoter region, with three common
genotypes termed s/s, l/l, or s/l for short (s) or long (l) variants. One or
more short alleles leads to reduced 5-HTT function, but attempts to
relate the 5-HTTLPR polymorphism to depression or other psychiatric
diagnoses have met with varied success86.
However, low serotonin transporter activity is also seen in rhesus
monkeys with an analogous short variant promoter polymorphism in
the transporter gene87. The s/l heterozygotes show greater alcohol selfadministration than l/l homozygotes. Experimental studies seek to constrain complexity by using uniform environmental conditions, but this
approach may obscure genetic effects. The effect was seen only in monkeys separated from their mothers at birth and reared with peers after
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five weeks of nursery rearing. Peer rearing is demonstrably stressful, so
there is an intriguing parallel with a study showing a similar gene-byenvironment interaction between the transporter polymorphism and
life stress on human depression88. When subjected to isolation stress,
monkeys of both sexes showed elevated adrenocorticotropic hormone
(ACTH) if they were l/s heterozygotes, but l/s females showed an even
greater increase in ACTH if they were peer reared and no increase if
they were mother reared89. In a human genetics study, one variant
of the l allele of 5-HTTLPR significantly predicted both alcohol use
disorders and a low response to alcohol, which itself is predictive of
later alcohol-related diagnoses90. This study awaits replication. The
5-HTTLPR polymorphism has pleiotropic effects. A different
(missense) mutation in the 5-HTT gene itself has been identified in two
generations of a family with a high incidence of obsessive-compulsive
disorder. Individuals with the missense mutation who were also
l/l homozygotes have extremely high levels of 5-HT transporter activity,
possibly owing to their ‘double hit’ at the two mutant genes, and suffer
from multiple disorders, including alcohol and drug abuse, Asperger’s
syndrome and social phobia91.
Basic-clinical translation: developing pharmacotherapies
As the ultimate goal of much alcohol research is development of effective therapeutics, there is great interest in translating basic findings on
the neural actions of alcohol into potential clinical applications. Two
medications currently used in the treatment of alcoholism—acamprosate (a taurine analog with no well-established molecular target
in CNS) and naltrexone (an opiate receptor antagonist)—target neural circuitry involved in alcohol ‘craving’ and relapse56. Acamprosate
was not developed on the basis of data from animal models, whereas
laboratory animal studies indicated that opiate receptor antagonists
reduce alcohol intake before the first tests of naltrexone in alcoholics92.
Researchers have focused on developing and testing new pharmacotherapies aimed at molecular targets identified by their sensitivity to
alcohol or the apparent involvement of the target in control of alcohol
drinking in rodent models. A parallel effort is examining the efficacy of
potential therapeutics in the broader array of behavioral assays mentioned above. Analogous to the situation with null mutants, and with
the exception of naltrexone, acamprosate and the SSRIs, the current
matrix of compounds and behavioral tests is frustratingly patchy93.
Some promising new targets are emerging. For example, noncompetitive antagonists of the NMDA receptor, a neurotransmitter that is directly
inhibited by ethanol94, are being tested for efficacy in reducing alcoholic
relapse95. Antagonists of the CB1 cannabinoid receptor and the mGluR5
metabotropic receptors reduce alcohol intake in several animal models,
and gene-targeted animals lacking CB1 show reduced intake in some
behavioral models96,97. Plans are underway to evaluate the therapeutic
potential of CB1 and mGluR5 antagonists in human alcoholics.
The roles of CB1 and mGlu receptors in brain reinforcement and
control of addictive behaviors are not confined to interactions with
ethanol. There is a substantial and growing literature implicating the
brain cannabinoid system in opiate, nicotine and even food-related
addictions98,99. Indeed, early clinical indicators suggest that drugs targeted at the CB1 receptor will be useful in the treatment of obesity and
cessation of cigarette smoking100. These findings suggest that CB1targeted therapeutics are successful because they tap into generalized
brain reward systems. The finding that alcohol abuse is likely to be
closely related to other addictions that involve these systems comes as
no surprise. However, the finding that the glutamate and cannabinoid
systems may have generalized roles suggests that caution should be
exercised in touting these neurochemicals as targets for treatment of any
one addiction disorder. Long-term studies may yet reveal that general
1478
interference with brain reinforcement systems might have untoward
consequences, such as anhedonia or disruption of normal neurophysiological drives. The CB1 and mGlu receptors are just two among many
molecular targets being considered for alcoholism pharmacotherapy93.
Two metabolic genes (alcohol dehydrogenase and acetaldehyde dehydrogenase) have specific polymorphic variants whose unpleasant effects
in their bearers significantly protect against alcoholism3. The known
therapeutic agent disulfiram (Antabuse) is directed at this metabolic
susceptibility. The wide array of potential therapeutic targets raises
the hope that effective treatments will be found in the not-too-distant
future.
Summary
Much recent progress has been achieved on both fronts highlighted in
this review. Several interesting targets for potential pharmacotherapies have emerged from continuing pharmacological and physiological
studies. New genetic animal models are emerging in both vertebrate
and invertebrate organisms that will lead to greater levels of behavioral
dysregulation of alcohol self-administration, as well as identification
of new genes and their proteins that are involved in alcohol’s neural
actions. Significant barriers still must be surmounted before we can
successfully use information gained from animal models for translation of basic findings into clinical treatments, but that end point seems
closer than ever.
ACKNOWLEDGMENTS
The authors are supported by the US Department of Veterans Affairs (J.C.C.), and
the US National Institute on Alcohol Abuse and Alcoholism (AA10760, AA12714
and AA13519 to J.C.C) and the Division of Intramural Clinical and Basic Research
(D.M.L.). We thank M. Rutledge-Gorman for help in preparing the manuscript,
and G. McClearn for many previous versions of Figure 2.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/
Reprints and permissions information is available online at http://npg.nature.com/
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