Laboratory models of alcoholism: treatment target identification and insight into mechanisms

NEUROBIOLOGY OF ADDICTION 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 drinking 1 (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: lovindav@mail.nih.gov Published online 26 October 2005; doi:10.1038/nn1581 alcohol abuse. Initial sensitivity to alcohol differs among different individuals, and there is some evidence that these differences contribute to susceptibility to later alcoholism 2. 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 intoxication 3,4. Chronic alcohol exposure causes neuroadaptations that foster continued alcohol abuse 5,6. 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 GABA A receptor 7, brain glutamatergic systems 8,9, nuclear signaling proteins such as CREB 10, stimulation of VTA dopaminergic neurons 11 and involvement of mesocorticolimbic and extended amygdala circuitry 12 ). 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 NATURE NEUROSCIENCE VOLUME 8 NUMBER 11 NOVEMBER 2005 1471

Acute pharmacology Functional/ biophysical analysis Direct alcohol targets Chronic exposure (neuroadaptation) Pharmacological, biochemical, molecular biological analyses Preclinical drug screening Pharmacotherapeutic target candidates Forward and reverse genetics Transgenics, knockouts, QTL analysis, etc. Alcohol-associated proteins 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 cue 13. 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 genetically 14 ) 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 alcoholism 15. 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 preparations 16. 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, Ann Thomson 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. 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 anesthesia 18 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 receptor 19, 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. 1472 VOLUME 8 NUMBER 11 NOVEMBER 2005 NATURE NEUROSCIENCE

a b Genes Proteins Pathways Behaviors Genomics Proteomics Metabolomics Genes Proteins Pathways Behaviors 1 2 3 4 1 2 3 4 Behavioral genomics Gene effects Mendelian Pleiotropic Multigenic Oligogenic Polygenic Multigenic + Pleiotropic Environments Environment 1 Gene environment interaction Environment 2 Behavioral interactions Effects of behavior on gene expression 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 systems 20,21. Important variables include the concentration of alcohol applied, ethanol evaporation at physiological temperatures, the pattern and duration of alcohol exposure, and the 1 2 3 4 1 2 3 4 Ann Thomson 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. 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. 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 transporters 1,22. 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 NATURE NEUROSCIENCE VOLUME 8 NUMBER 11 NOVEMBER 2005 1473

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). transmission have been the subject of intensive study for over 30 years 7,23. Evidence from in vivo and in vitro studies supports the idea that ethanol produces many of its intoxicating actions by enhancing GABAergic synaptic transmission 23. The GABA A receptor is one potential molecular target for ethanol, and a variety of evidence suggests that alcohol potentiates GABA A receptor function. However, there is ongoing controversy as to the prevalence and importance of ethanol actions on this receptor. Ethanol potentiation of GABA A receptor function has been observed in some isolated neuron preparations 7,24,25. However, negative results have also been reported in numerous neuronal subtypes 24. Studies in the Xenopus laevis oocyte expression system have generally found ethanol potentiation, but at relatively high ethanol concentrations 26. However, very few studies in mammalian cell systems have produced evidence for this potentiating effect 24. Clearly, understanding the molecular basis for these discrepant results will be a key step in resolving this controversy and determining the role of GABA A receptors in alcohol effects. Diversity among GABA A receptor subtypes could contribute to the differential effects of ethanol in different cells. GABA A 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 subunits 25,27,28 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 inhibition 28,29. Studies of granule neurons in brain slices from the cerebellar cortex and dentate gyrus demonstrate ethanol potentiation of a tonic GABA A -mediated current that seems to involve this type of receptor 28,29. What role, if any, does this ethanol potentiation have in acute intoxication and alcohol abuse? One clue comes from a study 28 concluding that a naturally occurring polymorphism in the rat GABA A α6 subunit may account for differences in the alcohol sensitivity of GABA A receptors. This polymorphism is also associated with a change in acute alcohol motor impairment observed in the rats in vivo 28, and the polymorphism segregates in rat lines bred for differential alcohol sensitivity 30 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 alcoholism 3. These findings indicate the potential importance of direct ethanol effects on α6-containing GABA A 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 difference 31 and this may suggest that there is coordinated regulation of the cluster of GABA A 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 ethanol 32. Finally, gene-targeted mice lacking the α6 subunit do not differ in sensitivity to ethanol motor impairment 33, 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 rats 28. Expression of the α6 subunit is restricted to cerebellar granule neurons 30, 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 1474 VOLUME 8 NUMBER 11 NOVEMBER 2005 NATURE NEUROSCIENCE

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 GABA A 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 GABA A 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 review 34 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 hippocampus 35 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 input 36. Because transmission at most GABAergic CNS synapses is mediated predominantly by GABA A receptors, presynaptic ethanol potentiation must be considered when formulating hypotheses about effects of GABA A receptor targeted drugs on alcohol-related behaviors and changes in alcohol responsiveness or alcohol drinking in gene-targeted mice lacking GABA A 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 proteins 38,39, 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 CNS 40. 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 ethanol 41. Furthermore, alcohol intake can be regulated by treatments that affect the camp-pka signaling system in the nucleus accumbens 42. 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 flies 43. The 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 crystallography 44. However, other researchers disagree that LUSH is an alcohol binding protein and have challenged the idea that flies avoid high ethanol concentrations 45. 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 neurons 46, 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 terminals 47, 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 intoxication 48. 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 behavior 49 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 NATURE NEUROSCIENCE VOLUME 8 NUMBER 11 NOVEMBER 2005 1475

systems. For example, octopamine is a monoamine neurotransmitter implicated in ethanol actions in D. melanogaster 38. 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 alcohol 7 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 elsewhere 52,53. 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 responses 53 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 GABA A receptors. Blockade of opioid or 5-HT 3 receptors also reduces intake, and these classes of agents have some efficacy in the clinic with alcoholics 56,57. 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. 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 alcohol 58. 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 gene 59. 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 withdrawal 60. 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 response 59. 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 elsewhere 61. 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 informative 62. 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 GABA A receptor subunit genes (α1, α6, β2, γ2), depends upon the presence of one or more genes within the chromosome 1 QTL 63. 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 pursued 55. 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 traits 65 and are now being used on alcohol-related QTL 66 70. For example, a locus very near the dopamine D2 receptor gene strongly affects its expression 68. 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. 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 GABA A receptor has been fairly thoroughly studied using genetic engineering strategies, as this receptor system has long 1476 VOLUME 8 NUMBER 11 NOVEMBER 2005 NATURE NEUROSCIENCE

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-administration 71. 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 GABA A 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 response 71. 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 circuits 72, 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 alcohol 73. Some rhesus monkeys given voluntary access to alcohol daily for long periods will develop patterns of repeated drinking that are obviously excessive 74. 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 laboratory 75 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 dark 78,80, limiting access to fluids for a brief period 80 82 or establishing chronic levels of alcohol before offering it for self-administration 83,84 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 seems 15. 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 rodents 85, 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-HT 3 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 success 86. However, low serotonin transporter activity is also seen in rhesus monkeys with an analogous short variant promoter polymorphism in the transporter gene 87. 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 NATURE NEUROSCIENCE VOLUME 8 NUMBER 11 NOVEMBER 2005 1477

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 depression 88. 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 reared 89. 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 diagnoses 90. 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 phobia 91. 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 relapse 56. 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 alcoholics 92. 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 patchy 93. Some promising new targets are emerging. For example, noncompetitive antagonists of the NMDA receptor, a neurotransmitter that is directly inhibited by ethanol 94, are being tested for efficacy in reducing alcoholic relapse 95. 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 models 96,97. 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 addictions 98,99. Indeed, early clinical indicators suggest that drugs targeted at the CB1 receptor will be useful in the treatment of obesity and cessation of cigarette smoking 100. These findings suggest that CB1- targeted 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 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 pharmacotherapy 93. Two metabolic genes (alcohol dehydrogenase and acetaldehyde dehydrogenase) have specific polymorphic variants whose unpleasant effects in their bearers significantly protect against alcoholism 3. 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/ reprintsandpermissions/ 1. Diamond, I. & Gordon, A.S. Cellular and molecular neuroscience of alcoholism. Physiol. Rev. 77, 1 20 (1997). 2. Schuckit, M.A., Smith, T.L. & Kalmijn, J. The search for genes contributing to the low level of response to alcohol: patterns of findings across studies. Alcohol. Clin. Exp. Res. 28, 1449 1458 (2004). 3. Oroszi, G. & Goldman, D. 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