Genetic and genomic approaches to reward and addiction

Neuropharmacology 47 (2004) 101–110 www.elsevier.com/locate/neuropharm

Genetic and genomic approaches to reward and addiction Amy R. Mohn a,b, Wei-Dong Yao a,b, Marc G. Caron a,b, a b

Department of Cell Biology, HHMI Laboratories, Duke University Medical Centre, Box 3287, CARL Building, Durham NC 27710, USA Center for Models of Human Disease, Institute for Genome Sciences and Policy, Duke University Medical Center, Box 3287 or Room 487, CARL Building, Durham NC 27710, USA Received 26 May 2004; received in revised form 14 July 2004; accepted 20 July 2004

Abstract Drug addiction is recognized as a mental disease affecting the brain’s natural reward system. Drugs of abuse strongly activate reward structures in the brain and induce lasting changes in behavior that reflect changes in neuron physiology and biochemistry. With the ultimate goal of developing therapeutic interventions, it is of interest to determine the molecular and cellular components of motivation and reward, and identify those gene products that contribute to the process of drug addiction. Our laboratory has chosen three general genetic approaches to examine reward and addiction: reverse genetics to assess the role of candidate genes in drug responsiveness, forward genetics to discover novel regulators of dopamine transmission, and gene expression profiling to define gene sets in different brain structures that contribute to the molecular and neurobiological basis of reward. # 2004 Elsevier Ltd. All rights reserved. Keywords: Dopamine; Glutamate; Addiction; Mice; Psychostimulants; Reward mechanisms; Behavioral sensitization

1. Introduction In the endeavor to understand and ultimately treat drug addiction, clinicians and researchers have come to appreciate addiction as a disease affecting the brain’s natural reward system. Reward, motivation, and decision-making are fundamental to survival, and with varying degrees of sophistication these brain functions drive behaviors that are evidenced in all animals. Reward pathways are strongly activated by drugs of abuse not only in humans, but also in many animal models including rodents, insects, and fish. The primitive nature of reward learning, and the universal ability of abused drugs to target reward systems, affords us the opportunity to model drug addiction in animals. From these models we can gain insight into the molecular and cellular components of motivation and reward, and can identify those genes that contribute to the process of drug addiction.  Corresponding author. Tel.: +1-919-684-5433; fax: +1-919-6818641. E-mail address: [email protected] (M.G. Caron).

0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.07.025

Among the commonly used animal models, the mouse is well suited for the study of reward and addiction genetics. The experimental tools currently available to mouse geneticists are ample; gene function can be disrupted, attenuated, or enhanced in conditional and reversible fashion using a combination of gene targeting and transgenic approaches. Recent advances using bacterial artificial chromosomes (BACs) or RNAi add to the repertoire of methods available to manipulate gene function. Furthermore, the historical use of mice as a genetic model system has pushed it to the forefront of genomic efforts, and high-resolution physical maps and genomic sequence are available, thus making forward genetic approaches more feasible. The consequences of these genetic manipulations can then be studied in the context of addiction at the molecular, cellular, and behavioral level. There are a number of behavioral assays that are used to model individual vulnerability and different aspects of the progressive nature of addiction. These include acute response, sensitization, conditioned place preference, tolerance, withdrawal, and self-administration acquisition, extinction and relapse.

102

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

Based on recent characterization of genetically modified mice in our lab and in many others, there are several emerging themes that shape the direction of our future research. The first is that genes regulating either dopaminergic or glutamatergic neurotransmission often participate in early behavioral and cellular responses to addictive drugs. With the ever-increasing number of genetically modified animals, there is a general observation that response to drugs of abuse can be altered by mutations that target either of these pathways. The cellular points of integration of dopamine and glutamate within the reward system represent areas of future research for our laboratory. Specifically, we have focused on the integration of these two neurotransmitters within the striatum and nucleus accumbens, brain reward structures that are richly innervated by dopamine and glutamate and the primary site of action of addictive psychostimulants. Second, there is a growing appreciation of the parallels between discoveries in the fields of learning and memory and addiction (Berke and Hyman, 2000). Some of the same basic questions that have been raised in learning and memory are also of interest in addiction. Where is reward memory first made and what are the underlying molecular and cellular components? How are these memories consolidated and preserved? Can established memories, particularly in primitive, survival-oriented circuits, be forgotten or unlearned? The availability of strong pharmacological activators of the reward system should provide us with the tools to dissect the genetics, anatomy, and stages of reward learning and memory in ways that are not possible with declarative/spatial or procedural memory. Furthermore, mutant animals that have been characterized for enhanced or deficient learning and memory should be revisited for possible changes in behavioral pharmacology to drugs of abuse (Migaud et al., 1998; Tang et al., 1999; Yao et al., 2004). A multidisciplinary approach is needed to gain a fuller understanding of the mechanisms underlying dopamine and glutamate transmission in the functioning of the reward system. Our laboratory has chosen three general genetic approaches to examine reward and addiction: reverse genetics to assess the role of candidate genes in drug responsiveness, forward genetics to discover novel regulators of dopamine transmission, and gene expression profiling to define gene sets in different brain structures that contribute to the molecular and neurobiological basis of reward.

2. Candidate gene studies of dopamine transmission One of the most fruitful and exciting developments in addiction research has been the growing list of genetically modified mice that show alterations in bio-

chemical and behavioral responses to abused drugs. The complexity of addiction as a behavior is borne out by the diversity of genes that participate. In addition to those genes that encode primary targets for drug action (for example, opiate receptors), the set of candidate genes has been greatly expanded to include those that regulate intracellular signaling events well downstream of initial drug targets. This is evident in the body of research that has been performed using mice with targeted mutations in components of dopamine neurotransmission. Genes regulating dopamine transmission are of obvious interest in addiction research due to the compelling evidence that the mesolimbic dopamine system is a core component of the natural reward system, and is directly or indirectly activated by all abused drugs (Wise, 2002). Using knockout animals, those genes that regulate dopamine signal transduction pre- and postsynaptically have been shown to participate in the acute behavioral and cellular responses to a variety of drugs of abuse. Fig. 1 depicts the dopaminergic innervation of a striatal neuron and highlights some of key regulators of dopamine transmission described to date. Most of these molecules have been studied by generating null mutations in mice, and most have further been shown to play a role in the acute response to drugs of abuse and often to modulate the rewarding effects of these drugs. For example, targeted deletions have been generated for each of the five G-protein coupled receptors

Fig. 1. Key molecular components of dopamine and glutamate neurotransmission. Glutamatergic inputs from cortical and limbic brain structures (blue) and dopaminergic inputs from the ventral tegmental area (red) converge on medium spiny neurons of the nucleus accumbens. The key molecular components of neurotransmitter synthesis, vesicular packaging, and uptake are depicted within the presynaptic terminals. Postsynaptic components that participate in neurotransmission and signal transduction are highlighted, including neurotransmitter receptors, scaffolding molecules, and intracellular signaling pathways. Activation and integration of a complex array of signal transduction cascades (some of which are yet unknown) ultimately lead to changes in gene expression, synaptic structure and plasticity. For most of the signaling components depicted here, reverse genetics has been instrumental in addressing their role in the cellular and behavioral responses to drugs of abuse.

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

(GPCR) for dopamine (D1–D5). The D1 dopamine receptor knockout has significantly diminished acute psychostimulant responsiveness (Xu et al., 1994) and cocaine sensitization (Xu et al., 2000b). Targeted disruption of D3 or D4 dopamine receptors results in enhanced acute response to cocaine (Carta et al., 2000; Katz et al., 2003; Rubinstein et al., 1997; Xu et al., 1997). D2 receptor knockouts, while demonstrating normal cocaine responsiveness and self-administration (Caine et al., 2002; Chausmer et al., 2002), have alterations in their response to morphine and ethanol that indicate changes in the natural reward system. Of the dopamine receptor knockout lines, only D5 dopamine receptor knockout animals have yet to demonstrate altered acute response to drugs of abuse (Elliot et al., 2003), although only cocaine behavioral pharmacology has been examined to date. Through the neurochemical and behavioral phenotyping of each of these lines, we can begin to understand the relative contribution of each GPCR in mediating dopamine transmission and reward learning. Furthermore, there is a satisfying correlation between the behavioral phenotype of D3 knockout mice, which show enhanced cocaine responsiveness, and pharmacological studies using a D3 receptor partial agonist, BP 897, to attenuate cocaine seeking behavior in mice (Pilla et al., 1999). In this case the creation of genetically modified mice presents the opportunity to test potential therapeutic interventions for target validity and efficacy. While dopamine receptor knockouts have relatively straightforward phenotypes, those that interfere with dopamine synthesis, storage, and release have been more challenging to characterize in the context of addiction, but have also been quite informative about dopamine biology. Homozygous null alleles are lethal for tyrosine hydroxylase (TH), which is required for dopamine synthesis (Zhou et al., 1995). This is also the case for homozygous null mutations of the vesicular monoamine transporter 2 gene (VMAT2), which is required for dopamine packaging into vesicles (Fon et al., 1997; Takahashi et al., 1997; Wang et al., 1997). However, rescue of TH-deficient lethality has been accomplished by a combination of transgenic and pharmacological interventions (Zhou and Palmiter, 1995); the resulting mice are termed dopamine-deficient (DA / ) because the tyrosine hydroxylase deficiency is limited to dopamine neurons. More recently, TH function has been disrupted in the striatum by viralmediated in vivo RNAi (Hommel et al., 2003). Not surprisingly, these animal models with reduced dopamine synthesis demonstrate reduced acute responsiveness to psychostimulants (Hommel et al., 2003; Nishii et al., 1998). However, DA / mice are hypersensitive to D1 and D2 dopamine receptor agonists, illustrating how dopamine depletion leads to adaptive supersensitivity of receptors (Kim et al., 2000).

103

When dopamine transmission is reduced in VMAT2 heterozygotes, dopamine receptor supersensitivity results in enhanced acute responsiveness to psychostimulants (Wang et al., 1997). The enhanced response to psychostimulants is similar to that seen after a regimen of repeated doses of cocaine that cause behavioral sensitization, and in fact VMAT2 heterozygotes are considered ‘‘sensitized’’ from the outset and are not further sensitized by repeated doses of psychostimulants (Wang et al., 1997). Despite this dopamine receptor supersensitivity, VMAT2 heterozygotes show a reduced preference to amphetamine in a conditioned place preference paradigm (Takahashi et al., 1997). The duration and intensity of dopamine transmission are regulated to a large extent by the activity of the synaptic dopamine transporter (DAT), and genetic ablation results in profound alterations in dopamine homeostasis including persistently elevated synaptic dopamine (Giros et al., 1996; Jones et al., 1998). Among the many observations that have been made, it is notable that despite the loss of DAT as a target for cocaine action, mutant mice find cocaine rewarding, as evidenced by conditioned place preference and selfadministration studies (Rocha et al., 1998; Sora et al., 1998). Amphetamine is also strongly rewarding in DAT knockout mice, and conditioned place preference to amphetamine is still detected over 55 days after amphetamine exposure (Budygin et al., 2004). The use of DAT mutant mice in these studies has demonstrated the importance of other neurotransmitter systems, particularly serotonin, in cocaine reward (Rocha et al., 1998; Sora et al., 2001) and amphetamine reward (Budygin et al., 2004). Mateo et al. demonstrated that DAT knockout mice, as well as wildtype mice with chronic pharmacological DAT blockade, have adaptive changes in their serotonergic system within the ventral tegmental area that could lead to enhanced dopamine release in the nucleus accumbens (Mateo et al., 2004). Characterization of the neurochemistry and behavioral pharmacology of DAT mutant animals has been carried out over several years, and these animals continue to serve as a useful tool for understanding mechanisms of dopamine signal transduction in the striatum and nucleus accumbens. The aforementioned mice carry mutations in very proximal elements of dopamine transmission—synthesis, storage, release, and receptors. They provide a foundation of knowledge and a phenotypic baseline that can now be applied to our understanding of downstream events in dopamine signal transduction. For example, mutations for specific dopamine receptors can be crossed with mutations for intracellular signaling genes to determine whether a phenotype is mediated through D1 or D2 dopamine receptors. The task of tracing dopamine transmission from the plasma membrane to changes in gene transcription, synaptic plas-

104

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

ticity, synapse structure and neuron function is one that will remain relevant to drug abuse research for many years to come. The application of reverse genetics to this effort has generated several mutant mouse lines that highlight critical elements in dopamine signaling and the natural reward system. A few examples of these efforts are described here in illustration. An early molecular event in postsynaptic dopamine neurotransmission involves the attenuation of the signal through desensitization of the activated receptor. Postsynaptic dopamine receptor activation leads to coupling with either Ga/s (in the case of D1 receptors) or Ga/i (in the case of D2 receptors) to stimulate or inhibit adenylate cyclase respectively and modulate intracellular cAMP levels (Fig. 1). GPCR signaling is terminated through desensitization, in which the receptor is phosphorylated by one of seven known GRKs (G protein-coupled receptor kinases). This phosphorylation leads to the recruitment of barrestin, which prevents further signal transduction through G proteins even in the continued presence of agonist. Desensitization could potentially contribute to reward and addiction mechanisms by regulating the intensity and duration of the dopamine signal; this hypothesis has been tested through the characterization of transgenic mice that lack various components of the GRK/ barrestin machinery. Mice lacking each of the genes for GRK1–7 have been generated and, through the pharmacological characterization of these mice, it seems evident that some level of GRK specificity exists for a given GPCR receptor. For example, GRK5-deficient mice display a loss of muscarinic receptor desensitization, but dopamine receptor desensitization appears normal. Knockout of GRK2 is embryonically lethal, but dopamine behavioral pharmacology has been performed on GRK2 heterozygous mice. These mice do show significant enhancement of locomotor response to a 20 mg/kg dose of cocaine. However, other doses of cocaine do not induce an enhanced response, nor does amphetamine or apomorphine (Gainetdinov et al., 2004). From the comparison of knockout lines for GRK2–6, it is suggested that dopamine receptor desensitization is most affected by the loss of GRK6 (Gainetdinov et al., 2004). Both homozygous and heterozygous GRK6 mutant mice display enhanced coupling of D2 receptors to G proteins and enhanced behavioral responsiveness to cocaine and amphetamine (Gainetdinov et al., 2003). Dopamine receptor desensitization presumably requires barrestin recruitment and receptor internalization. However, it has been difficult to test this hypothesis in vivo using the current genetic models of barrestin deficiency. There are two non-visual barrestins encoded by two genes, barr1 and barr2. Knockout mice for each of these genes have been gen-

erated, and mice carrying either null mutation are viable (Bohn et al., 1999; Conner et al., 1997). Initial studies of barr1 or barr2 knockout mice did not reveal major alterations in dopamine receptor desensitization based on their acute locomotor responses to cocaine (Bohn et al., 2003; Gainetdinov et al., 2004). It is likely that either barr1 or barr2 is able to interact with dopamine receptors, and that one isoform can compensate for the other’s absence in vivo. This hypothesis is not easily tested since compound homozygous null animals are embryonic lethals (Kohout et al., 2001). Future studies, perhaps using RNAi to disrupt normal function of both barrestins in striatum, may be necessary to elucidate the role of barrestins in dopamine receptor desensitization and in the rewarding effects of psychostimulants. Another mechanism to negatively modulate postsynaptic dopamine response occurs through the action of RGS9-2 (regulators of G protein signaling), a striatum-enriched splice variant of RGS9 (Rahman et al., 2003). Through the use of mice deficient in RGS9, and through viral-mediated overexpression of RGS9-2 in rats, Rahman et al. have demonstrated that RGS9-2 negatively regulates D2 (but not D1) dopamine receptor signaling. Accordingly, cocaine responsiveness is enhanced in RGS9 knockout mice and inhibited in RGS9-2 overexpressing rats. RGS9 knockout mice display enhanced acute responses to amphetamine and apomorphine as well, show an enhanced level of cocaine sensitization, and display increased conditioned place preference to cocaine.

3. Candidate gene studies of glutamate transmission Many of the molecules governing postsynaptic dopamine transmission directly or indirectly influence glutamatergic transmission. In addition to mesolimbic dopaminergic projections, glutamate projections from the frontal cortex represent the other major input pathway into the striatum and nucleus accumbens. These two neurotransmitter systems converge and are integrated by medium spiny neurons of the striatum and nucleus accumbens; glutamate provides the major excitatory input and dopamine serves as a modulator of excitatory tone. It should not be surprising then that disruption of glutamate transmission could influence the cellular and behavioral responses to drugs of abuse. Some of the molecular components of glutamate signal transduction are depicted in Fig. 1, including elements of the post-synaptic density scaffold. For the majority of these molecules, knockout animals have been created to examine their function in vivo. Though not all of the knockout animals have been assessed for neurochemical, cellular, or behavioral responses to drugs of

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

abuse, there are several mutant lines that exhibit addiction-relevant phenotypes. The most notable of these is the knockout of mGluR5, a group I metabotropic glutamate receptor that couples to Gq to generate diacylglycerol and inositol (1,4,5)-triphosphate through phospholipase C activation. mGluR5 is expressed in several areas of the reward system, including the ventral tegmental area, nucleus accumbens, amygdala, and hippocampus (Kenny and Markou, 2004). Mice lacking mGluR5 show disruptions in acute cocaine responsiveness and more remarkably fail to self-administer cocaine (Chiamulera et al., 2001). Pharmacological inhibition of mGluR5 using MPEP produced similar disruptions in cocaine’s stimulant and rewarding properties (Chiamulera et al., 2001), and also attenuates amphetamine stimulant effects (McGeehan et al., 2004). These observations have fueled research to determine whether mGluR5 signaling is most relevant to psychostimulant addiction, or plays a role in the rewarding properties of other drugs of abuse. Towards this end, MPEP has been shown to reduce ethanol seeking and relapse behavior (Backstrom et al., 2004) and to decrease nicotine self-administration in rats and mice (Paterson et al., 2003). However, it has also been reported that MPEP does not impair conditioned place preference to ethanol or nicotine (McGeehan and Olive, 2003). mGluR5, as well as group I receptor mGluR1, interacts with the scaffolding protein Homer (Tu et al., 1998; Xiao et al., 1998). In turn, Homer1 links these receptors to AMPA and NMDA ionotropic glutamate receptors at the postsynaptic density (PSD) through proteins of the PSD scaffold (Tu et al., 1999). It has been suggested that Homer influences glutamatergic signaling through the localization and trafficking of mGluRs in the scaffold, as well as through the modulation of intracellular calcium signaling pathways (Kammermeier et al., 2000). Based on the association of Homer with group I mGluRs and the striking phenotype of mGluR5-deficient mice, the role of Homer1 in cocaine action has recently been investigated. It has been shown that the Homer1b/c isoform is decreased in the nucleus accumbens following withdrawal from repeated cocaine administration (Swanson et al., 2001). Also, there are indications that mice deficient in Homer2, another scaffolding protein that interacts with group I mGluR’s, display increased sensitivity to the rewarding effects of cocaine and ethanol (Szumlinski et al., 2003). The characterization of mice deficient in AMPA and NMDA ionotropic receptors, as well as a large body of pharmacological literature, suggests that glutamate transmission in general is involved in the acute response and rewarding effects of abused drugs. AMPA receptors, which mediate the major component of the glutamatergic excitatory postsynaptic potential, are

105

heteromers of GluR subunits 1–4. Mice deficient in GluR1 display altered behavioral responses to morphine and amphetamine. Sensitization to morphine is enhanced in GluR1-deficient mice, and amphetamine induces an enhanced sensitized response but only when delivered within a context-specific environment (Vekovischeva et al., 2001). GluR1-deficient mice also exhibit reduced tolerance to morphine antinociception and reduced withdrawal symptoms following cessation of chronic morphine. The role of AMPA receptors in addiction has been underscored by a recent study demonstrating that AMPA receptor number is increased in the nucleus accumbens shell during the extinction phase of cocaine self-administration (Sutton et al., 2003). Viral-mediated overexpression of GluR1 and GluR2 in the nucleus accumbens was shown to increase the extinction of cocaine self-administration. Several studies have also reported increased GluR1 subunit expression in the ventral tegmental area following repeated exposure to cocaine, morphine, and ethanol (Churchill et al., 1999; Fitzgerald et al., 1996; Ortiz et al., 1995) but see Lu et al. (2002). Continued study of mutant mice with altered AMPA receptor function is warranted, particularly since regulation of AMPA receptor trafficking appears to be a common mechanism to change synaptic efficacy (Bredt and Nicoll, 2003), and since exposure to drugs of abuse causes increases in synaptic strength at dopaminergic neurons (Saal et al., 2003; Ungless et al., 2001) and decreases in synaptic efficacy in the nucleus accumbens (Thomas et al., 2001). The correlations between drug exposure and changes in synaptic efficacy lend support to the view of addiction as an example of pathological reward learning and memory. In other brain structures where memory is studied, such as the hippocampus, NMDA receptors play a critical role in the establishment of long-term memories. Functional NMDA receptors are composed of heteromers of the requisite NR1 subunit, which is expressed throughout the CNS, NR2 subunits A–D, which have temporally and spatially restricted expression patterns, and/or NR3 subunits A–B. Pharmacological evidence suggests that behavioral responses to psychostimulants can be attenuated by administration of non-competitive NMDA receptor antagonists such as MK-801 (Vanderschuren and Kalivas, 2000; Wolf, 1998) but see Tzschentke and Schmidt (1998). Additionally, NMDA receptor antagonists have been proposed as a potential therapy in relapse and withdrawal prevention (Bisaga and Popik, 2000). Initial studies using mice with altered NMDA receptor function have been performed to assess their role in reward and addiction. While NR1 null mice die perinatally (Forrest et al., 1994; Li et al., 1994), other genetic models of decreased NMDA receptor function are viable as adults. Mice lacking NR2A subunit of NMDA recep-

106

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

tors have been studied for behavioral responses to morphine, methamphetamine, and phencyclidine (Miyamoto et al., 2004). Most notable was the observation that NR2A mutant mice have an attenuated locomotor response to methamphetamine and a slower development of sensitization only with lower doses of methamphetamine. However, conditioned place preference to methamphetamine appears to be intact. Our laboratory has focused on the neurochemistry and behavioral pharmacology of NR1-deficient mice, which are viable with only 5–10% of normal NMDA receptor level (Laakso et al., 2002; Mohn et al., 1999). Our initial characterization of these mice in addiction paradigms suggests a similar phenomenon to that described by Miyamoto et al., such that NR1-deficient mice have clear alterations in their acute response to drugs of abuse, but are still able to experience addictive drugs as rewarding (unpublished observations). 4. Forward genetic approaches to identify novel mutations relevant to addiction Reverse genetics has provided the means to test the role of a gene in specific aspects of addiction biology, particularly when pharmacological inhibitors are unavailable. For those genes that merit the extra effort and energy involved, conditional mutagenesis may provide some of the most valuable information about reward circuitry and memory (Cui et al., 2004). A limitation of reverse genetics, apart from the time and expense of generating transgenic mice, is that it is by definition limited to the study of those genes that have already been implicated in addiction through prior studies. Recognizing that the cellular mechanisms of reward learning and memory are far from understood, we and others are also taking an unbiased approach to addiction through the application of forward genetic approaches. Forward genetics is useful to find central regulators of a biological pathway and has been used for decades to unravel key genetic determinants for cell cycle regulation, development and organogenesis, and growth and metabolism. In addition, it has been used (primarily in fruit flies) to identify genes that regulate complex behaviors such as mating, circadian rhythm, and learning and memory (Benzer, 1973). There is a long history of forward genetics in the mouse for the genetics of behavior, but technical challenges have made gene cloning more difficult than that for Drosophila or C. elegans. However, a mouse mutagenesis screen led to the identification and cloning of clock, a central regulator of the mammalian circadian rhythm that had eluded investigators for decades (King et al., 1997; Vitaterna et al., 1994). This effort used the chemical supermutagen ethylnitrosourea (ENU) to induce germline random base pair mutations at high frequency (on the order of 1 sequence change per 105

base pairs) (Beier, 2000). Although base pair mutations are more difficult to clone than mutations induced by transgene insertions, recent advances in physical mapping and sequencing of the mouse genome have made it a realistic endeavor and several large-scale ENU mutagenesis efforts are underway (Bucan and Abel, 2002; Hrabe de Angelis and Balling, 1998; Justice et al., 1999). The principal advantage of forward genetics is the lack of bias in the approach, and hence the ability to identify novel and sometimes unexpected players in a pathway. Our laboratory is participating in two ENU mutagenesis screens that are designed to identify novel genes regulating psychostimulant behavioral responses or dopamine neurotransmission. A large-scale recessive screen conducted at the Neurogenomics Center at Northwestern University (http://genome.northwestern. edu) is identifying mutant mice with alterations in acute locomotor responses to cocaine. In collaboration with Joseph Takahashi, we are characterizing the dopamine neurochemistry and behavioral pharmacology of novel mutants identified in this screen. It is notable that several of the mouse knockouts displaying alterations in acute response to cocaine also show alterations in more sophisticated measures of addiction behaviors, such as conditioned place preference and self-administration (Laakso et al., 2002). This would suggest that initial screening for alterations in acute response will be useful to identify genes that also regulate later stages of drug responsiveness and addiction. It should be noted that the Neurogenomics Center is one of three NIH-funded neuroscience mutagenesis centers that is generating publicly available collections of ENU-induced mutant mouse lines (Bucan and Abel, 2002; Bult et al., 2004). Partnered with the Neurogenomics Center are the Neuroscience Mutagenesis Facility at the Jackson Laboratory (http://www.jax. org/nmf) and the Neuromutagenesis Project of the Tennessee Mouse Genome Consortium (http://www. tnmouse.org). Mutants generated from these centers are identified first through a number of behavioral screens and heritability is confirmed before they are listed on a common website at http://www.neuromice. org. Publicly available ENU-induced mouse mutants can also be found from the ENU Mutagenesis Program at the MRC Mammalian Genetics Unit at Harwell (http://www.mgu.har.mrc.ac.uk) and from the Mouse Mutagenesis Screen in the German Human Genome Project (http://www.gsf.de/ieg/groups/enu-mouse. html). The second mutagenesis screen that we have undertaken within our laboratory is smaller in scale and is focused more specifically on identifying novel genes that regulate dopamine transmission. To achieve this, we are screening for new dominant-acting mutations that modify the dopamine transporter knockout

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

phenotype. The DAT knockout animal serves as a sensitized genetic background, having chronic elevations in extracellular dopamine and displaying marked hyperactivity in novel environments. Random point mutations are added onto this sensitized background by breeding DAT mutant mice with ENU-mutagenized mice. The resulting mice are then screened by evaluating their locomotor activity in a novel environment. DAT knockout mice with significant attenuation or exacerbation of their normal hyperactivity are identified from this screen; these mice represent potential carriers of DAT modifier mutations. After testing for trait heritability, the mutation can be cloned by a combination of interstrain crosses for positional cloning and candidate gene analysis once a chromosomal region is defined. Due to its focus and scale (in comparison to that of mutagenesis centers), this effort is quite feasible for a single lab to undertake. To date we have identified four lines that demonstrate trait heritability. Two lines in particular show a DAT enhancer phenotype and are now committed to positional cloning efforts. Based on our initial results, we anticipate that the screen will generate two to five mutants each year that warrant positional cloning, and that 1X genome coverage can be accomplished over the next two years. 5. Identification of molecular alterations underlying behavioral sensitization in the reward circuitry The basic reward circuit has been identified in part by observing which brain regions are commonly activated by addictive drugs having very distinct pharmacological profiles. We have employed a similar line of investigation to study the phenomenon of sensitization, where an animal has an enhanced locomotor response after repeated exposure to an addictive drug. Within our laboratory we have a number of mutant mouse lines that have a common phenotype of being presensitized to psychostimulants or dopamine agonists, such that their first locomotor response is elevated to the level of a wildtype mouse subjected to a sensitization regimen. We have used these models as a tool to detect common changes in transcription that occur in a sensitized state. As mentioned previously, DAT knockout mice display disrupted dopamine clearance and elevated extracellular dopamine concentration, leading to increased postsynaptic responsiveness (Gainetdinov et al., 1999). Norepinephrine transporter (NET) knockout mice demonstrate prolonged synaptic norepinephrine clearance, elevated extracellular norepinephrine, but diminished dopamine concentration, presumably underlying the postsynaptic D2/ D3 dopamine receptor supersensitivity observed in these mice (Xu et al., 2000a). While mice lacking vesicular monoamine transporters (VMAT2 / ) die

107

shortly after birth, VMAT2+/ mice show diminished extracellular dopamine levels and release, and display enhanced sensitivity to direct and indirect dopamine receptor agonists as well as ethanol (Wang et al., 1997). These mice, along with normal mice treated with repeated intermittent psychostimulants (Fig. 1A), offer the basis for a phenotype-driven, microarray-based genomic screen for common genes affected in the brain reward circuitry of these models, which may serve general mechanisms underlying core features of behavioral plasticity elicited by psychostimulants. Our first study focused on the striatum with its ventral extension, the nucleus accumbens, a nucleus that plays an indispensable role in the development and maintenance of behavioral sensitization. We identified six genes that are consistently altered in the striatum of the four genetically or pharmacologically sensitized mice. Interestingly, most of the small set of co-affected genes are implicated in synaptic function, plasticity, learning, and most remarkably, drug-dependent behavioral plasticity, such as sensitization, conditioned place preference, and self-administration (Yao et al., 2004). One gene identified in this screen encodes the synaptic scaffolding protein PSD-95 abundant in the excitatory glutamatergic synapses. Both the mRNA and protein levels of PSD-95 are reduced by chronic, but not acute administration of cocaine in normal animals, and in the three genetically sensitized mice. Moreover, the reduction of striatal PSD-95 is specific to this member of the large family of MAGUK proteins and to the striatum and lasts for at least two months. At the synaptic level, electrophysiological experiments reveal enhanced long-term potentiation (LTP) at the nucleus accumbens in all three genetically as well as the pharmacologically sensitized animals, providing a common synaptic mechanism for the phenomenon. Furthermore, targeted deletion of PSD95 in an independent line of mice enhances LTP, augments the acute locomotor-stimulating effects of cocaine, but essentially eliminates further behavioral plasticity to chronic cocaine. These results uncover a previously unappreciated role of PSD-95 in psychostimulant action and identify a molecular and cellular mechanism shared between drug-related plasticity and learning, thus demonstrating the power of the unbiased forward genetic approach. There is now a greater appreciation that the reward circuit consists not only of the mesolimbic dopamine pathway, but also includes inter-connected cortical and limbic structures. Emerging evidence suggests addiction may result from a hijacked brain reward pathway involving essentially all of the structures in the reward circuitry. From a molecular and cellular perspective, it is postulated that repeated drug exposure causes stable changes in gene expression, post-transcriptional modifications, intracellular signaling, and synaptic plasticity

108

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

in each structure of the reward circuitry. Moreover, the complex behavioral manifestations of addiction, e.g. sensitization, tolerance, dependence, craving, compulsive drug taking, and relapses may be due to distinct molecular and cellular mechanisms in different structures. Thus, we are currently extending the phenotypedriven genomic approach described above to the entire reward circuitry to identify common changes in gene expression in each nucleus using the cohort of genetic and pharmacological sensitization models.

References Backstrom, P., Bachteler, D., Koch, S., Hyytia, P., Spanagel, R., 2004. mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior. Neuropsychopharmacology 29, 921–928. Beier, D.R., 2000. Sequence-based analysis of mutagenized mice. Mamm. Genome 11, 594–597. Benzer, S., 1973. Genetic dissection of behavior. Sci. Am. 229, 24–37. Berke, J.D., Hyman, S.E., 2000. Addiction, dopamine, and the molecular mechanisms of memory. Neuron 25, 515–532. Bisaga, A., Popik, P., 2000. In search of a new pharmacological treatment for drug and alcohol addiction: N-methyl-d-aspartate (NMDA) antagonists. Drug Alcohol Depend. 59, 1–15. Bohn, L.M., Lefkowitz, R.J., Gainetdinov, R.R., Peppel, K., Caron, M.G., Lin, F.T., 1999. Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 286, 2495–2498. Bohn, L.M., Gainetdinov, R.R., Sotnikova, T.D., Medvedev, I.O., Lefkowitz, R.J., Dykstra, L.A., Caron, M.G., 2003. Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice. J. Neurosci. 23, 10265–10273. Bredt, D.S., Nicoll, R.A., 2003. AMPA receptor trafficking at excitatory synapses. Neuron 40, 361–379. Bucan, M., Abel, T., 2002. The mouse: genetics meets behaviour. Nat. Rev. Genet. 3, 114–123. Budygin, E.A., Brodie, M.S., Sotnikova, T.D., Mateo, Y., John, C.E., Cyr, M., Gainetdinov, R.R., Jones, S.R., 2004. Dissociation of rewarding and dopamine transporter-mediated properties of amphetamine. Proc. Natl. Acad. Sci. USA. Bult, C., Kibbe, W.A., Snoddy, J., Vitaterna, M., Swanson, D., Pretel, S., Li, Y., Hohman, M.M., Rinchik, E., Takahashi, J.S., et al., 2004. A genome end-game: understanding gene function in the nervous system. Nat. Neurosci. 7, 484–485. Caine, S.B., Negus, S.S., Mello, N.K., Patel, S., Bristow, L., Kulagowski, J., Vallone, D., Saiardi, A., Borrelli, E., 2002. Role of dopamine D2-like receptors in cocaine self-administration: studies with D2 receptor mutant mice and novel D2 receptor antagonists. J. Neurosci. 22, 2977–2988. Carta, A.R., Gerfen, C.R., Steiner, H., 2000. Cocaine effects on gene regulation in the striatum and behavior: increased sensitivity in D3 dopamine receptor-deficient mice. Neuroreport 11, 2395–2399. Chausmer, A.L., Elmer, G.I., Rubinstein, M., Low, M.J., Grandy, D.K., Katz, J.L., 2002. Cocaine-induced locomotor activity and cocaine discrimination in dopamine D2 receptor mutant mice. Psychopharmacology (Berlin) 163, 54–61. Chiamulera, C., Epping-Jordan, M.P., Zocchi, A., Marcon, C., Cottiny, C., Tacconi, S., Corsi, M., Orzi, F., Conquet, F., 2001. Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat. Neurosci. 4, 873–874. Churchill, L., Swanson, C.J., Urbina, M., Kalivas, P.W., 1999. Repeated cocaine alters glutamate receptor subunit levels in the nucleus accumbens and ventral tegmental area of rats that develop behavioral sensitization. J. Neurochem. 72, 2397–2403.

Conner, D.A., Mathier, M.A., Mortensen, R.M., Christe, M., Vatner, S.F., Seidman, C.E., Seidman, J.G., 1997. beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation. Circ. Res. 81, 1021–1026. Cui, Z., Wang, H., Tan, Y., Zaia, K.A., Zhang, S., Tsien, J.Z., 2004. Inducible and reversible NR1 knockout reveals crucial role of the NMDA receptor in preserving remote memories in the brain. Neuron 41, 781–793. Elliot, E.E., Sibley, D.R., Katz, J.L., 2003. Locomotor and discriminative-stimulus effects of cocaine in dopamine D5 receptor knockout mice. Psychopharmacology (Berlin) 169, 161–168. Fitzgerald, L.W., Ortiz, J., Hamedani, A.G., Nestler, E.J., 1996. Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents. J. Neurosci. 16, 274–282. Fon, E.A., Pothos, E.N., Sun, B.C., Killeen, N., Sulzer, D., Edwards, R.H., 1997. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19, 1271–1283. Forrest, D., Yuzaki, M., Soares, H.D., Ng, L., Luk, D.C., Sheng, M., Stewart, C.L., Morgan, J.I., Connor, J.A., Curran, T., 1994. Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death. Neuron 13, 325–338. Gainetdinov, R.R., Jones, S.R., Caron, M.G., 1999. Functional hyperdopaminergia in dopamine transporter knock-out mice. Biol. Psychiatry 46, 303–311. Gainetdinov, R.R., Bohn, L.M., Sotnikova, T.D., Cyr, M., Laakso, A., Macrae, A.D., Torres, G.E., Kim, K.M., Lefkowitz, R.J., Caron, M.G., Premont, R.T., 2003. Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice. Neuron 38, 291–303. Gainetdinov, R.R., Premont, R.T., Bohn, L.M., Lefkowitz, R.J., Caron, M.G., 2004. Desensitization of G protein-coupled receptors and neuronal functions. Annu. Rev. Neurosci. 27, 107–144. Giros, B., Jaber, M., Jones, S.R., Wightman, R.M., Caron, M.G., 1996. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606– 612. Hommel, J.D., Sears, R.M., Georgescu, D., Simmons, D.L., DiLeone, R.J., 2003. Local gene knockdown in the brain using viral-mediated RNA interference. Nat. Med. 9, 1539–1544. Hrabe de Angelis, M., Balling, R., 1998. Large scale ENU screens in the mouse: genetics meets genomics. Mutat. Res. 400, 25–32. Jones, S.R., Gainetdinov, R.R., Jaber, M., Giros, B., Wightman, R.M., Caron, M.G., 1998. Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc. Natl. Acad. Sci. USA 95, 4029–4034. Justice, M.J., Noveroske, J.K., Weber, J.S., Zheng, B., Bradley, A., 1999. Mouse ENU mutagenesis. Hum. Mol. Genet. 8, 1955–1963. Kammermeier, P.J., Xiao, B., Tu, J.C., Worley, P.F., Ikeda, S.R., 2000. Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels. J. Neurosci. 20, 7238–7245. Katz, J.L., Chausmer, A.L., Elmer, G.I., Rubinstein, M., Low, M.J., Grandy, D.K., 2003. Cocaine-induced locomotor activity and cocaine discrimination in dopamine D4 receptor mutant mice. Psychopharmacology (Berlin) 170, 108–114. Kenny, P.J., Markou, A., 2004. The ups and downs of addiction: role of metabotropic glutamate receptors. Trends Pharmacol. Sci. 25, 265–272. Kim, D.S., Szczypka, M.S., Palmiter, R.D., 2000. Dopamine-deficient mice are hypersensitive to dopamine receptor agonists. J. Neurosci. 20, 4405–4413. King, D.P., Zhao, Y., Sangoram, A.M., Wilsbacher, L.D., Tanaka, M., Antoch, M.P., Steeves, T.D., Vitaterna, M.H., Kornhauser,

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110 P.L., Lowrey, P.L., et al., 1997. Positional cloning of the mouse circadian clock gene. Cell 89, 641–653. Kohout, T.A., Lin, F.S., Perry, S.J., Conner, D.A., Lefkowitz, R.J., 2001. beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking. Proc. Natl. Acad. Sci. USA 98, 1601–1606. Laakso, A., Mohn, A.R., Gainetdinov, R.R., Caron, M.G., 2002. Experimental genetic approaches to addiction. Neuron 36, 213–228. Li, Y., Erzurumlu, R.S., Chen, C., Jhaveri, S., Tonegawa, S., 1994. Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice. Cell 76, 427–437. Lu, W., Monteggia, L.M., Wolf, M.E., 2002. Repeated administration of amphetamine or cocaine does not alter AMPA receptor subunit expression in the rat midbrain. Neuropsychopharmacology 26, 1–13. Mateo, Y., Budygin, E.A., John, C.E., Jones, S.R., 2004. Role of serotonin in cocaine effects in mice with reduced dopamine transporter function. Proc. Natl. Acad. Sci. USA 101, 372–377. McGeehan, A.J., Olive, M.F., 2003. The mGluR5 antagonist MPEP reduces the conditioned rewarding effects of cocaine but not other drugs of abuse. Synapse 47, 240–242. McGeehan, A.J., Janak, P.H., Olive, M.F., 2004. Effect of the mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) on the acute locomotor stimulant properties of cocaine, d-amphetamine, and the dopamine reuptake inhibitor GBR12909 in mice. Psychopharmacology (Berlin). Migaud, M., Charlesworth, P., Dempster, M., Webster, L.C., Watabe, A.M., Makhinson, M., He, Y., Ramsay, M.F., Morris, R.G., Morrison, J.H., et al., 1998. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439. Miyamoto, Y., Yamada, K., Nagai, T., Mori, H., Mishina, M., Furukawa, H., Noda, Y., Nabeshima, T., 2004. Behavioural adaptations to addictive drugs in mice lacking the NMDA receptor epsilon1 subunit. Eur. J. Neurosci. 19, 151–158. Mohn, A.R., Gainetdinov, R.R., Caron, M.G., Koller, B.H., 1999. Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell 98, 427–436. Nishii, K., Matsushita, N., Sawada, H., Sano, H., Noda, Y., Mamiya, T., Nabeshima, T., Nagatsu, I., Hata, T., Kiuchi, K., 1998. Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission. J. Neurosci. Res. 54, 450–464. Ortiz, J., Fitzgerald, L.W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R.S., Nestler, E.J., 1995. Biochemical actions of chronic ethanol exposure in the mesolimbic dopamine system. Synapse 21, 289–298. Paterson, N.E., Semenova, S., Gasparini, F., Markou, A., 2003. The mGluR5 antagonist MPEP decreased nicotine self-administration in rats and mice. Psychopharmacology (Berlin) 167, 257–264. Pilla, M., Perachon, S., Sautel, F., Garrido, F., Mann, A., Wermuth, C.G., Schwartz, J.C., Everitt, B.J., Sokoloff, P., 1999. Selective inhibition of cocaine-seeking behaviour by a partial dopamine D3 receptor agonist. Nature 400, 371–375. Rahman, Z., Schwarz, J., Gold, S.J., Zachariou, V., Wein, M.N., Choi, K.H., Kovoor, A., Chen, C.K., DiLeone, R.J., Schwarz, S.C., et al., 2003. RGS9 modulates dopamine signaling in the basal ganglia. Neuron 38, 941–952. Rocha, B.A., Fumagalli, F., Gainetdinov, R.R., Jones, S.R., Ator, R., Giros, B., Miller, G.W., Caron, M.G., 1998. Cocaine selfadministration in dopamine-transporter knockout mice. Nat. Neurosci. 1, 132–137. Rubinstein, M., Phillips, T.J., Bunzow, J.R., Falzone, T.L., Dziewczapolski, G., Zhang, G., Fang, Y., Larson, J.L., McDougall, J.A., Chester, J.A., et al., 1997. Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell 90, 991–1001.

109

Saal, D., Dong, Y., Bonci, A., Malenka, R.C., 2003. Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37, 577–582. Sora, I., Wichems, C., Takahashi, N., Li, X.F., Zeng, Z., Revay, R., Lesch, K.P., Murphy, D.L., Uhl, G.R., 1998. Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice. Proc. Natl. Acad. Sci. USA 95, 7699–7704. Sora, I., Hall, F.S., Andrews, A.M., Itokawa, M., Li, X.F., Wei, H.B., Wichems, C., Lesch, K.P., Murphy, D.L., Uhl, G.R., 2001. Molecular mechanisms of cocaine reward: combined dopamine and serotonin transporter knockouts eliminate cocaine place preference. Proc. Natl. Acad. Sci. USA 98, 5300–5305. Sutton, M.A., Schmidt, E.F., Choi, K.H., Schad, C.A., Whisler, K., Simmons, D., Karanian, D.A., Monteggia, L.M., Neve, R.L., Self, D.W., 2003. Extinction-induced upregulation in AMPA receptors reduces cocaine-seeking behaviour. Nature 421, 70–75. Swanson, C.J., Baker, D.A., Carson, D., Worley, P.F., Kalivas, P.W., 2001. Repeated cocaine administration attenuates group I metabotropic glutamate receptor-mediated glutamate release and behavioral activation: a potential role for Homer. J. Neurosci. 21, 9043–9052. Szumlinski, K.K., Toda, S., Middaugh, L.D., Worley, P.F., Kalivas, P.W., 2003. Evidence for a relationship between Group 1 mGluR hypofunction and increased cocaine and ethanol sensitivity in Homer2 null mutant mice. Ann. NY Acad. Sci. 1003, 468–471. Takahashi, N., Miner, L.L., Sora, I., Ujike, H., Revay, R.S., Kostic, V., Jackson-Lewis, V., Przedborski, S., Uhl, G.R., 1997. VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity. Proc. Natl. Acad. Sci. USA 94, 9938– 9943. Tang, Y.P., Shimizu, E., Dube, G.R., Rampon, C., Kerchner, G.A., Zhuo, M., Liu, G., Tsien, J.Z., 1999. Genetic enhancement of learning and memory in mice. Nature 401, 63–69. Thomas, M.J., Beurrier, C., Bonci, A., Malenka, R.C., 2001. Longterm depression in the nucleus accumbens: a neural correlate of behavioral sensitization to cocaine. Nat. Neurosci. 4, 1217–1223. Tu, J.C., Xiao, B., Yuan, J.P., Lanahan, A.A., Leoffert, K., Li, M., Linden, D.J., Worley, P.F., 1998. Homer binds a novel prolinerich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21, 717–726. Tu, J.C., Xiao, B., Naisbitt, S., Yuan, J.P., Petralia, R.S., Brakeman, P., Doan, A., Aakalu, V.K., Lanahan, A.A., Sheng, M., Worley, P.F., 1999. Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23, 583–592. Tzschentke, T.M., Schmidt, W.J., 1998. Does the noncompetitive NMDA receptor antagonist dizocilpine (MK801) really block behavioural sensitization associated with repeated drug administration? Trends Pharmacol. Sci. 19, 447–451. Ungless, M.A., Whistler, J.L., Malenka, R.C., Bonci, A., 2001. Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons. Nature 411, 583–587. Vanderschuren, L.J., Kalivas, P.W., 2000. Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacology (Berlin) 151, 99–120. Vekovischeva, O.Y., Zamanillo, D., Echenko, O., Seppala, T., UusiOukari, M., Honkanen, A., Seeburg, P.H., Sprengel, R., Korpi, E.R., 2001. Morphine-induced dependence and sensitization are altered in mice deficient in AMPA-type glutamate receptor-A subunits. J. Neurosci. 21, 4451–4459. Vitaterna, M.H., King, D.P., Chang, A.M., Kornhauser, J.M., Lowrey, P.L., McDonald, J.D., Dove, W.F., Pinto, L.H., Turek, F.W., Takahashi, J.S., 1994. Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science 264, 719–725.

110

A.R. Mohn et al. / Neuropharmacology 47 Supplement No. 1 (2004) 101–110

Wang, Y.M., Gainetdinov, R.R., Fumagalli, F., Xu, F., Jones, S.R., Bock, C.B., Miller, G.W., Wightman, R.M., Caron, M.G., 1997. Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 19, 1285–1296. Wise, R.A., 2002. Brain reward circuitry: insights from unsensed incentives. Neuron 36, 229–240. Wolf, M.E., 1998. The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog. Neurobiol. 54, 679–720. Xiao, B., Tu, J.C., Petralia, R.S., Yuan, J.P., Doan, A., Breder, C.D., Ruggiero, A., Lanahan, A.A., Wenthold, R.J., Worley, P.F., 1998. Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins. Neuron 21, 707–716. Xu, M., Hu, X.T., Cooper, D.C., Moratalla, R., Graybiel, A.M., White, F.J., Tonegawa, S., 1994. Elimination of cocaine-induced hyperactivity and dopamine-mediated neurophysiological effects in dopamine D1 receptor mutant mice. Cell 79, 945–955. Xu, M., Koeltzow, T.E., Santiago, G.T., Moratalla, R., Cooper, D.C., Hu, X.T., White, N.M., Graybiel, A.M., White, F.J.,

Tonegawa, S., 1997. Dopamine D3 receptor mutant mice exhibit increased behavioral sensitivity to concurrent stimulation of D1 and D2 receptors. Neuron 19, 837–848. Xu, F., Gainetdinov, R.R., Wetsel, W.C., Jones, S.R., Bohn, L.M., Miller, G.W., Wang, Y.M., Caron, M.G., 2000a. Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat. Neurosci. 3, 465–471. Xu, M., Guo, Y., Vorhees, C.V., Zhang, J., 2000b. Behavioral responses to cocaine and amphetamine administration in mice lacking the dopamine D1 receptor. Brain Res. 852, 198–207. Yao, W.D., Gainetdinov, R.R., Arbuckle, M.I., Sotnikova, T.D., Cyr, M., Beaulieu, J.M., Torres, G.E., Grant, S.G., Caron, M.G., 2004. Identification of PSD-95 as a regulator of dopamine-mediated synaptic and behavioral plasticity. Neuron 41, 625–638. Zhou, Q.Y., Palmiter, R.D., 1995. Dopamine-deficient mice are severely hypoactive, adipsic, and aphagic. Cell 83, 1197–1209. Zhou, Q.Y., Quaife, C.J., Palmiter, R.D., 1995. Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development. Nature 374, 640–643.