Author(s): Eric J. Nestler 
Social and psychological factors contribute to addiction, but it is clear that genetic factors also weigh in. Epidemiological studies have long established that alcoholism, for example, is familial, with estimates that genetic factors account for 40-60% of risk . More recent studies indicate similar rates of heritability for other drug addictions, including addiction to opiates and cocaine [2, 3]. Numerous linkage and association studies are now underway, with a view to identifying specific genetic variants that confer risk [4, 5]. Several relatively large chromosomal regions have been implicated in addiction vulnerability--although specific genetic polymorphisms have yet to be identified by this approach. It has been established, however, that some East Asian populations carry variations in enzymes (for example, the alcohol and aldehyde dehydrogenases) that metabolize alcohol. Such variants increase sensitivity to alcohol, dramatically ramping up the side effects of acute alcohol intake [6, 7]. Consequently, alcoholism is exceedingly rare in individuals, for example, that are homozygous for the ALDH2* 2 allele, which encodes a less active variant of aldehyde dehydrogenase type2.[illus. 1]
It is well established that inbred strains of mice and rats show robust differences in behavioural and biochemical responses to drugs of abuse [8, 9, 10, 11]. Lines of rodents have been selectively bred for increased or decreased responsiveness to alcohol or other drugs [12, 13]. They have also been bred for altered rates of alcohol drinking. Whereas genetic variations found in these models may be different from those of humans, their identification should shed light on mechanisms underlying the addiction process. Accordingly, several groups are attempting to identify the genetic basis of these behavioural differences among rodent strains and lines by use of quantitative trait locus (QTL) analysis. In one early study, a predeliction for drinking water laced with morphine was mapped to three large chromosomal regions . Several other groups have identified chromosomal regions that may contain QTLs related to particular responses to alcohol, cocaine or opiates. But as is the case with humans, no genetic polymorphism has yet been identified with certainty .
The difficulty in finding genes that contribute to risk for addiction parallels the difficulty in finding genes for other psychiatric disorders and, in fact, for most common diseases. There are many reasons for this difficulty [14, 15], including the fact that addiction is a complex trait. And so any single gene might produce a relatively small effect and would therefore be difficult to detect experimentally. It is also possible that variants in different genes may contribute to addiction in different lineages. And, as epidemiological studies have shown, non-genetic factors (for example, poverty, crime and delinquency) also affect risk, although they remain vaguely defined. Animal studies also show that environmental factors such as stress can interact with an animal's genotype to determine its ultimate response to a drug of abuse . As a result, delineating the mechanisms by which specific genetic variations and environmental factors interact (see Fig. 1) is an important focus of investigation.
Another obstacle to identifying genes that determine vulnerability to addiction is the difficulty in quantifying behavioural endpoints, which have a greater degree of variance and are perhaps more susceptible to environmental influence than many non-behavioural phenotypes. A major focus of behavioural research is to establish behavioural endpoints with the same degree of sophistication and interassay reliability as is the case for non-behavioural traits. (See the Correspondence  on page 263 for discussion of this issue.)
A gene might contribute to addiction vulnerability in several ways. A mutant protein (or altered levels of a normal protein) could change the structure or functioning of specific brain circuits during development or in adulthood. These altered brain circuits could change the responsiveness of the individual to initial drug exposure or the adaptations that occur in the brain after repeated drug exposure (see Fig. 1). In a similar manner, environmental stimuli could affect addiction vulnerability by influencing these same neural circuits. Perhaps combining genetic approaches with one of these specific (and more narrowly defined) phenotypes would facilitate the identification of addiction vulnerability genes in both humans and animal models.
In contrast with the slow progress in identifying genes that affect risk for addiction in humans, great strides have been made in demonstrating the role of specific gene products as assessed in animal models. The general strategy is to modify the amount of a particular gene product or the product itself, and to characterize the consequences of such modifications in behavioural tests. Mice with constitutive mutations continue to deliver insights into drug mechanisms, and mice with inducible and tissue-specific mutations (Table 1) are increasingly used to overcome limitations of constitutive mutants . Other genetic approaches include viral-mediated gene transfer, intra-cerebral infusions of antisense oligonucleotides and mutations in non-mammalian model organisms.
Animals with altered levels of a particular gene product in the brain are subjected to a variety of behavioural tests to assess their responses to drugs of abuse (Table 2). These include measures of locomotor activity (most drugs of abuse increase activity when given acutely), and the progressive increase in locomotor activity (locomotor sensitization) that occurs with repeated drug exposure [19, 20, 21]. The rationale behind these tests rests on the finding that locomotor responses are mediated by the mesolimbic dopamine system, which is also implicated in reward and addiction [22, 23, 24, 25, 26], but it should be noted that the relationship between locomotor responses, drug reward and addiction is a matter of some debate.
A more direct measure of drug reward is conditioned place preference, where an animal learns to prefer an environment that is paired with drug exposure . It, too, is mediated partly by the mesolimbic dopamine system and is thought to model some of the powerful conditioning effects of drugs of abuse that are seen in humans. As with measures of locomotor activity, place-conditioning assays are amenable to relatively high-throughput design, which explains their popularity. But neither test directly measures the behavioural abnormalities (compulsive drug-seeking and -taking) that are the core features of human addiction.
To get closer to such abnormalities, operant tests must be applied, including self-administration, intracranial self-stimulation and conditioned reinforcement paradigms [23, 24, 25, 26] (Table 2). These tests, however, are quite complicated. Indeed, because of their intricacy, especially in mice, they have been used on a comparatively small number of genetically altered animals [27, 28]. A major challenge is to devise schemes that make application of these behavioural tests more widely available. One approach is to use oral self-administration procedures, which are much easier to implement than intravenous methods [11, 28].
Other aspects of drug exposure have been studied in genetic models. For example, withdrawal symptoms have been used as a measure of opiate dependence on many occasions . The aversive (negative emotional) effects of drug withdrawal can be measured by conditioned place-avoidance assays, in which animals learn to avoid an environment associated with withdrawal. Moreover, investigators have measured the rate and degree of tolerance (or insensitivity) to the analgesic effects of opiates [30, 31]. This is important because the molecular basis of tolerance, which limits the use of opiates in the treatment of chronic pain, is poorly understood.
Confirming initial drug targets
A straightforward use of genetic tools in the dissection of addiction is in the confirmation of targets of drugs. Pharmacological approaches can be used to identify drug targets, but they frequently fail to identify which of several subtypes is most relevant. In contrast are studies involving `knockout' mice, which have led to the identification of the [mu]-opioid receptor, the dopamine transporter, the CB1 cannabinoid receptor and the [beta]2 nicotinic acetylcholine receptor as targets that mediate rewarding and other effects of opiates, stimulants, cannabinoids and nicotine, respectively [32, 33, 34]. Such success inspires hope that knockouts will also lead to targets (of drugs of abuse) where none are presently known--for example, inhalants. In some cases, knockout of the primary drug target has revealed the existence of secondary targets that partially compensate the loss of the primary target .
In addition to secondary targets, there are numerous neurotransmitters, their receptors and post-receptor signalling pathways that modify responses to acute and chronic drug exposure. Genetic tools have not only confirmed pharmacological studies that implicate neurotransmitter pathways, they have provided fundamentally new insights into their mechanism.
For example, several behavioural aspects of mice lacking the serotonin 5HT 1B receptor indicate enhanced responsiveness to cocaine and alcohol; notably, they self-administer both drugs at higher levels than wild-type controls [27, 28]. The mice also express higher levels of [DELTA]FosB (a Fos-like transcription factor implicated in addiction) under basal conditions. These observations point to the involvement of serotonergic mechanisms in addiction. There are many other such examples. Mice deficient in the dopamine D2 receptor or the cannabinoid CB1 receptor have a diminished rewarding response to morphine, implicating dopaminergic systems and endogenous cannabinoid-like systems in opiate action [34, 36]. Mice lacking the [beta]2 subunit of nicotinic cholinergic receptors show reduced rewarding responses to cocaine .
Several neuropeptides are implicated in drug responses. Mice with low levels of neuropeptide Y drink more alcohol--and those with high levels are more likely to abstain [38, 39]. Mice deficient in certain neurotrophic factors have abnormal behavioural responses to opiates and to cocaine--BDNF knockouts show reduced responsiveness, whereas GDNF knockouts are more responsive [40, 41]. These findings support the view that trophic mechanisms (for example, alterations in neural structure) may mediate some of the effects of drug administration on brain function.
Even though they may not develop the more complex aspects of addiction seen in mammals, non-mammalian model organisms can be used to identify biochemical pathways through which drugs act [42, 43, 44]. A recent study determined that the Drosophila melanogaster mutant inactive , which is deficient in tyrosine decarboxylase and therefore cannot synthesize tyramine, remains sedate despite repeated doses of cocaine . This abnormality is reversed by administration of tyramine and raises the possibility that related biochemical pathways in mammals may modulate long-term adaptation to chronic drug exposure. In addition, the locomotor responses of flies to cocaine (acting through dopamine pathways) are remarkably similar to those of mammals, indicating that dopaminergic pathways were obligated in neuronal circuits controlling movement over one billion years ago.
The stability of the behavioural abnormalities that characterize addiction indicates that drug-induced changes in gene expression may be involved . As classic pharmacological agonists and antagonists to most proteins are not available, genetic tools are all the more attractive.
One demonstration of this approach is in the exploration of [DELTA]FosB, a Fos-like transcription factor. [DELTA]FosB accumulates in the nucleus accumbens (a target of the mesolimbic dopamine system) after chronic, but not acute, exposure to any of several drugs of abuse [46, 47], including opiates, cocaine, amphetamine, alcohol, nicotine and phencyclidine (also known as PCP or `angel dust'). This is in contrast with other Fos-like proteins, which are much less stable than [DELTA]FosB and induced only transiently after acute drug administration. Consequently, [DELTA]FosB persists in the nucleus accumbens long after drug-taking ceases. Adult mice in which [DELTA]FosB can be induced selectively within the same subset of neurons in the nucleus accumbens as those in which it is induced by drug administration show a marked predilection for cocaine [46, 47, 48]. It would seem that [DELTA]FosB is a relatively sustained molecular `switch' that contributes to a state of addiction.
The expression of another transcription factor, CREB, is also important. CREB knockout mice are less likely to develop opiate dependence --observing the effects of injecting antisense oligonucleotides to CREB into different parts of the brain points to the locus coeruleus (a brain region important for such dependence) as one relevant site of action . Overexpression of CREB in the nucleus accumbens counters the rewarding properties of opiates and cocaine; overexpression of a dominant-negative CREB mutant has the opposite effect [50, 51]. These findings suggest that CREB promotes certain aspects of addiction (for example, physical dependence), while opposing others (for example, reward), and highlight that the same biochemical adaptation can have very different behavioural effects depending on the type of neuron involved.
A challenge of current research is to identify target genes through which [DELTA]FosB, CREB and other transcription factors act. Two strategies are used. One considers candidate genes: for example, genes that contain putative response elements for the transcription factor in question or whose products are implicated in relevant mechanisms in a relevant region of the brain. This approach has led to the identification of the AMPA glutamate receptor subunit GluR2 as a mediator of [DELTA]FosB action . In a similar manner, the opioid peptide dynorphin was identified as a target for CREB, and shown to partly mediate CREB-induced repression of drug reward .
New molecular substrates
Candidate gene approaches are limited by our rudimentary knowledge of the gene products and the complex mechanisms underlying addiction. As a result, more open-ended strategies are needed, such as those based on analysis of differential gene expression in certain brain regions under control and drug-treated conditions. Differential display, for example, enabled the identification of NAC-1, a transcription factor-like protein, which is induced in nucleus accumbens by chronic cocaine and is now known to modulate the locomotor effects induced by cocaine . The neuropeptide CART was also first identified by differential display of mRNA in the nucleus accumbens before and after drug exposure. Whereas it may have a role in addiction, its current claim to fame is as a potent anorexigenic (anti-appetite) factor  that acts in the hypothalamus.
Various types of microarray analysis have led to the identification of large numbers of drug-regulated genes; it is typical for 1-5% of the genes on an array to show consistent changes in response to drug regulation . But without a better means of evaluating this vast amount of information (other than exploring the function of single genes using traditional approaches), it is impossible to identify those genes that truly contribute to addiction. Strategies under current evaluation include the analysis of detailed time courses of drug action and expression changes unique to carefully characterized behavioural states.
And, in the end. . .
Animal models have proved to be pivotal to our understanding of neurobiological mechanisms involved in the addiction process. One drawback (and one that is not limited to the field of addiction) is that sometimes a genetic mutation is found to result in a phenotype without any plausible scheme as to how the mutation actually causes that phenotype. Fortunately, the increasing sophistication of genetic tools, together with the increasing predictive value of animal models of addiction, makes it increasingly feasible to fill in the missing pieces--to understand the cellular mechanisms and neural circuitry that ultimately connect molecular events with complex behaviour.
Figure 1: Scheme showing genetic and environmental factors combining to influence the process by which repeated exposure to a drug of abuse causes addiction. [see PDF for image]
Genetic and environmental factors, by establishing all aspects of normal synaptic structure and function, determine an individual's inherent sensitivity to initial drug exposure. These factors also establish how individual nerve cells, and the circuits in which they operate, adapt over time to chronic drug exposure, which in turn determines the development of addiction.
Credit: Bob Crimi
Table: Genetic tools to study addiction [see PDF for image]
Table: Some behavioural tests commonly used to study addiction [see PDF for image]
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 Department of Psychiatry and Center for Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.