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Dopaminergic Genes and Substance Abuse
George R. Uhl,*t David J. Vandenbergh," Lawrence A. Rodriguez,* Lucinda Miner," and Nobuyuki Takahashi" *Molecular Neurobiology Branch Intramural Research Program National Institute on Drug Abuse National Institutes of Health Baltimore Maryland 2 I224 tDepartments of Neurology and Neuroscience The John Hopkins University School of Medicine Baltimore, Maryland 2 I205
Dopaminergic Genes and Substance Abuse The use of addictive substances is a complex behavioral disorder likely to represent interactions between genetic and environmental factors ( 1).Evidence from twin, adoption, and family studies indicates that drug abuse phenotypes including quantity-frequency of use and features responsible for Diagnostic and Statistical Manual (DSM) diagnoses of substance abuse-dependence are each likely to reflect significant genetic contributions (1-3). Population comorbidity studies indicate that relative risk of a drug abuse disorder is enhanced substantially in individuals with comorbid antisocial personality disorder, alcoholism, depression, or the adult residua of attention deficit-hyperactivity disorder (1).Other personality traits have also been postulated to predispose to vulnerability to specific aspects of drug abuse, ranging from initiation of use to resistance to quitting to development of the drug's control over behavior that represents a hallmark of dependence. Dopaminergic brain systems play prominent roles in drug reward (4), focusing attention on genes expressed in these circuits as candidates to contribute to substance abuse vulnerability. Psychostimulants engage these circuits with especial power, as evinced by a number of lines of evidence, including the magnitude of drug-induced dopamine spillover from nucleus accumbens doparninergic synapses following psychostimulant treatments ( 5 )and the results of lesion studies, in which dopaminergic lesions dampen brain reward systems and locomotor functions in both experimental animals and humans (6-8). A number of studies in experimental animals now suggest that manipulations of the levels of expression of several of the genes that encode proteins important for dopaminergic neurotransmission, or of genes regulated by psychostimulants, can have powerful influences on animal models of drug reward. Studies of the functional allelic variants that have been identified in several human dopaminergic genes, or of polymorphic markers that could tag heretofore underscribed I024
Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
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functional variation at other dopaminergic gene loci, have each indicated that expression of human genes with different levels of activity might contribute to differential responses to abused substances, different drug-abuse comorbidities, or different personality features of possible importance for substance abuse. In this chapter, we review several recent cases in which transgenic mouse and human data provide increasingly compelling evidence that variants in genes important for dopaminergic neurotransmission are strong candidates to contribute to interindividual differences in drug abuse vulnerabilities.
1. Transgenic Mice with Dopamine Transporter Over and Underexpressed Studies of cocaine’s primary site for reward and reinforcement in the brain have focused on the dopamine transporter (DAT) ( 9 ) .DAT is a member of the 12-transmembrane domain sodium and chloride-dependent neurotransmitter transporter family, the primary structure of which has been elucidated by cDNA cloning (10). In studying this transporter, the effect of several mutations on transporter function have been characterized. Replacement of putative transmembrane 7 serines 350 and 353 with alanines, for example, results in a transporter variant that exhibits 30% increases in dopamine transport and 30% reductions in affinity for the cocaine analog 2@-carbomethoxy-3@-(tfluorophenyl) tropane (CFT) (11).T o model the effects that a DAT gene variant might have on drug abuse phenotypes, we constructed transgenic animals that express this DAT variant in catecholaminergic neurons (12). We utilized sequences from the 5’ flanking promoter region of the rat tyrosine hydroxylase (TH) gene because of their well-characterized ability to specifically mediate gene expression in catecholaminergic neurons (13, 14). Transgenic strains THDAT2 and THDAT4 each stably pass more than 10 copies of an 8.2-kb trangene construct made up of 4.8 kb of rat TH 5’ flanking promoter sequence linked to the serine 350 and 353 alanine substituted rat DAT variant, and express the transgene mRNA and encoded protein in a region-specific manner with high levels of expression in ventral midbrain, adrenal gland, and olfactory bulb, consistent with patterns of expression previously described as mediated by the TH promoter. Heterozygous transgenic animals from each of the two lines expressing this construction display behavioral differences from wild-type littermate controls. During repeated exposure to novel testing apparatus during testing sessions spaced at 3 to 4-day intervals, both THDAT lines habituate to the testing environment more rapidly. THDAT mice are 150-230% as active following 30 mg/kg of cocaine i.p. More striking effects were observed when the reinforcing and rewarding properties of cocaine were tested using conditioned place preference to assess drug reward (15).Both THDAT2 and THDAT4 animals displayed significantly greater cocaine-induced conditioned place preferences than littermate control mice. Transgenic mice with region-specific overexpression of a DAT gene variant can manifest subtle but interesting differences in specific features related to higher order control of locomotor systems and major models of drug responsiveness.
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The results on cocaine locomotion obtained in these mice complement those obtained by Giros et al. in DAT knockout mice (10). In these animals, although baseline locomotor activities are altered, cocaine induces virtually no excess locomotion. However, data from studies of cocaine reward in DAT knockout mice constructed independently in our laboratory suggest that cocaine-induced conditioned placed preference might also be less altered in these mice (Sora et al., in preparation). These data are also in accord with studies of the locomotor responses in dopamine D1 receptor and DAT knockout mice, in which cocaine-stimulated locomotor functions were virtually eliminated (17, 18).
II. Transgenic Mice with Vesicular Monoamine Transporter Underexpressed The brain vesicular monoamine transporter (VMAT2) pumps monoamine neurotransmitters from neuronal cytoplasm into synaptic vesicles. Vesicular monoamine stores accumulated by normal VMAT2 function may play significant roles in the locomotor stimulation and/or the behavioral reward produced by amphetamines, drugs increasingly abused in certain areas of the United States. Amphetamines dissipate proton gradients across the membranes of synaptic vesicles, disrupt VMAT2 function, enhance cytoplasmic monoamine concentrations, and cause calcium-independent, nonvesicular monoamine release into synapses (19). They also act like cocaine in blocking plasma membrane neurotransmitter transporters that use transmembrane sodium and chloride gradients to pump dopamine, norepinephrine, and serotonin from extracellular spaces into presynaptic neurons. Because little preexisting evidence documented which amphetamine action provided which contribution to amphetamineinduced locomotion or behavioral reward, we constructed transgenic VMAT2 knockout mice using a 12-kb targeting factor homologous to sequences flanking the first three exons of the murine 129 strain VMAT2 gene (20). Heterozygotes VMAT 2 knockout mice are viable into adult life and display VMAT2 levels half of wild-type values. They also display alterations in several monoaminergic markers, heart rate, and blood pressure. However, their weight gain, fertility, habituation, passive avoidance, and locomotor activities are similar to wild-type littermates (20). In these heterozygote VMAT2 knockout mice, 1- and 3-mg/kg intraperitoneal amphetamine doses enhanced locomotor activity, with increases at 1 mg/ kg 146% of those in wild-type mice. However, amphetamine-conditioned place preferences in the heterozygotes were less than those in wild-type mice. Conversely, cocaine ( 5 mg/kg), a blocker of the plasma membrane DAT devoid of significant activity on vesicular monoamine stores, induced conditioned place preferences in heterozygotes that were indistinguishable from those in wildtype mice (20). These data support the idea that plasma membrane transporter blockade by amphetamine contributes primarily to its locomotor stimulation, but that normal function of vesicular monoamine stores is necessary for full amphetamine-induced behavioral reward. They are consistent with the results
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from studies that document differences between the effects of dopamine receptor knockouts on psychostimulant-induced locomotion and reward (18) and with the differences in dose-response relationships for amphetamine-induced locomotion and reward (19).
111. Human Studies of Catechol-0-Methyltransferase Functional Alleles in Polysubstance Abusers and Nonusers Catechol-0-Methyltransferase (COMT) is expressed in dopaminergic brain regions, where its activity provides a major pathway by which extraneuronally released dopamine is inactivated (21). Three- to fourfold differences in human COMT activities are attributed to codon 158 polymorphisms that encode a valine, producing a high enzyme activity, or a methionine, yielding low activity (22-24, 38,41). To seek influences of this COMT allelic variation on substance abuse vulnerability, we compared COMT genotypes in groups of unrelated polysubstance abuser research volunteers defined on the bases of: (1) quantity-frequency of drug use or (2) Diagnostic and Statzsticai Manual of Mental Disorders Ill-Revised (DSMIII-R) criteria for substance abuse/ dependence to those of control research volunteers free of significant use of addictive substances (25, 26). Significant differences in the distributions of both COMT genotypes and allele frequencies between controls and substance abusers, defined on the basis of self-reported peak lifetime use, and between these controls and volunteers who had also been administered the Diagnostic Interview Schedule (DIS) for DSMIII-R diagnoses of substance abuse or dependence were identified (25,26). Detection of single gene effects in complex disorders can be facilitated by examining phenotypes associated with the genotypes encoding extreme values; vulnerability could be enhanced in individuals with the highest or the lowest COMT activities, for example. The proportions of high activity G/G homozygotes and low activity MA homozygotes were also examined: G/G homozygotes were nearly twice as frequent in volunteers who report high quantity-frequency drug use than in controls free of such use (25, 26). The sample defined on the basis of DSM diagnoses also shows a similar difference from control values. These data, thus, fit best with the hypothesis that high COMT activities might contribute to the genetic underpinnings of drug abuse vulnerability.
IV. Human Studies of DRD4 Functional Alleles in “Novelty Seeking” and Related Personality Substance Abuse Predispositions Two recent studies have suggested an association between D4 dopamine receptor polymorphisms and measures designed to assess novelty-seeking behaviors, characterized by “exhilaration or excitement in response to novel stimuli” (27, 28). Such an association was plausible for several reasons. This
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personality dimension has been postulated to be substantially influenced by dopaminergic neurotransmission in one major formulation of personality typology and neurochemical correlates. The dopaminergic D4 receptor is expressed in human brain regions implicated in reward and mood. This receptor displays functional allelic variants that alter its efficiency in G-protein coupling and, thus, change features of dopaminergic neurotransmission in these circuits. However, the data from Benjamin et al., suggest that only some individual item scores on the NEO-PI-R correlate with D4 genotype. These results were at least as consistent with the idea that happy, positive feelings or a ratio between positive and conscientious feelings could represent the underlying DRD4associated phenotype. We approached this question by assessing DRD4 genotypes in a sample taken from the Baltimore Longitudinal Study of Aging (29),a well-characterized population of research volunteers whose characterized personality types allowed us to select the individuals with the highest and with the lowest values on novelty-seeking scales and measures of “happiness,” defined as ratios between measurements of excitement seeking to neuroticism (Em). We found only limited generalization of DRD4 genotype associations with specific personality features contributing to measures of novelty seeking; only the E/N ratio correlated with genotype (25). While our failure to identify the association to the original novelty-seeking item scores in this population strongly suggest that the findings of Benjamin et al. and Ebstein et al. cannot be generalized to all populations, the items positively correlated with DRD4 genotype (warmth, excitement seeking, and positive emotions) and the item that provided a negative correlate, deliberation, provide a ratio whose value did correlate weakly with DRD4 genotype. These results support the idea that a more modest association with an assessment of a ratio of positive and negative feelings might represent a result of differential expression of D4 receptor genotypes. Indeed, given the relatively low levels of expression of the dopamine DRD4 gene, its limited brain distribution, and the substantially greater number of dopamine D2 receptors, even in areas of which the D4 receptors expressed, biologicai fit with this modest receptor expression and a more subtle personality influence on personality make some sense.
V. Other Dopamine Receptors The status of dopamine D2-receptor polymorphisms in drug abuse has been reviewed (1, 42). More recent studies of a polymorphic marker at the dopamine D3 receptor have shown no association with substance abuse vulnerabilities (Rodriguez et al., in preparation).
VI. Dopamine Transporter Examination of association between variable number tandem repeat (VNTR) markers at the human DAT locus revealed no assocation (30). How-
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ever, more recent observations may indicate association between polymorphic markers at this locus and two drug-associated conditions. Individuals who report paranoid experiences during cocaine administration display the DAT allele marked by a nine-copy VNTR marker more frequently than control subjects (31). Attention deficit-hyperactivity disorder (ADHD) risk is also increased in individuals with the DAT allele marked by the 10-copy VNTR (32). Because individuals with ADHD can display psychostimulant responses that differ from those of normal individuals, this evidence also plausibly links DAT allelic variants with alterations in human drug responses.
VII. Future Directions This current review supports potentially prominent roles for dopaminergic gene variants in contributing to individual differences in vulnerability to drug abuse. However, other genes are quite likely also to be involved. Seeking drug-regulated genes provides one avenue to find other classes of drug-abuse vulnerability genes in humans. New approaches, such as subtracted differential display, will allow us to identify hundreds of genes regulated by drug administration in experimental animals (33). Polymorphic markers at these gene loci can provide new tools to identify possible gene substrates for human individual-to-individual differences in drug abuse vulnerability. Positional cloning of drug abuse vulnerability genes may also now become possible. Recent initial attempts to model this process (34) have been based on genetic heritability hZ estimates of 0.45 and assumptions that environmental influences on drug abuse vulnerability are largely nonshared, both based on twin study data (1).An initial estimate of the relative risk for developing a substantial drug abuse disorder in the sibling of an abuser (As) is three to fourfold, based on a group of family studies (1).The two to fourfold risk in excess of that in the general population can then be modelled as due to both some oligogenic and some polygenic influences. In quantitative trait locus studies in mice, the genetics of drug responsiveness can often be modelled as based on the detectable contributions of a limited number of genes, with a residual genetic variance assumed to be polygenic, due to effects of genes whose influences may be too small to determine individually (3.5-37). If these indications from mouse genetics are applicable to humans, a case in which five loci equally contribute to a 75% oligogenic influence could represent a plausible starting point for beginning to model the human condition. This would provide a locus-specific As value of about 1.4 for the human condition. Power calculations based on the work of Reisch (1990) and others indicate that evaluating 300 polymorphic markers spaced at 10 centimorgan intervals across the human genome in 400 nuclear affected sib pair pedigrees would provide an 80% opportunity to detect a locus of such an effect size (34). Although many of the assumptions used to make these power calculations require better and better estimates over time, these values also suggest that an affected sib pair approach to identification of drug abuse vulnerability genes is possible, with multicenter collaboration and powerful genotyping assistance.
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As genome scaning-positional cloning approaches to detection of genes contributing to human drug abuse vulnerability become available, however, the study of candidate loci such as those expressed in the dopaminergic systems important for reward processes appears to provide a valuable initial route to identification of genes that contribute in both animal models and humans to interindividual differences in drug abuse vulnerability.
Acknowledgment This work was largely supported by the Intraumural Research Program, National Institute on Drug Abuse, National Institutes of Health.
References 1. Uhl, G. R., Elmer, G. I., Labuda, M. C., Pickens, R. W. (1995). Genetic influences in drug abuse. Psychopharmacology: The Fourth Generation of Progress (F. E. Bloom and D. J. Kupfer, eds.). Raven Press, New York. 2. Goldberg, J., Lyons, M. J., Eisen, S. A., True, W. R., and Twang, M. (1993). Genetic influence on drug use: A preliminary analysis of 2674 Vietnam era veteran twins. Behav. Genet. 23, 552. 3. Johnson, E. O., van den Bree, M. B. M., Uhl, G. R., and Pickens, R. W. (1996). Indicators of genetic and environmental influences in drug abusing individuals. Drug Alcohol Depend. 41,17-23. 4. Kuhar, M. J., Ritz, M. C., and Boja, J. W. (1991). The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299-302. 5. Di Chiara, G., and Imperato, A. (1988). Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl. Acad. Sci. U.S.A. 85, 5274-5278. 6. Roberts, D. C. S., and Koob, G. F. (1982). Description of cocaine self-administration following 6-hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol. Biochem. Behav. 17, 901-904. 7. Pettit, H. O., Ettenberg, A., and Bloom, F. E. (1984). Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats. Psychopharmacology 84, 167-173. 8. Roberts, D. C. S., Corcoran, M. E., and Fibiger, H. C. (1977). On the role of ascending catecholamine systems in intravenous self-administration of cocaine. Pharmacol. Bzochem. Behav. 6 , 615-620. 9. Ritz, M. C., Lamb, R. J., Goldberg, S. R., and Kuhar, M. J. (1987). Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237,12191223. 10. Shimada, S., Kitayama, S., Lin, C.-L., Patel, A,, Nanthakumar, E., Gregor, P., and Uhl, G. R. (1991). Cloning and expression of a cocaine sensitive dopamine transporter cDNA. Science 254, 576-578. 11. Kitayama, S., Wang, J.-B., and Uhl, G. R. (1993).Dopamine transporter mutants selectively enhance MPP+ transport. Synapase 15, 58-62. 12. Miner, L. L., Donovan, D. M., Perry, M., Revay, R., Sharpe, L. G., Przedborski, S., Isenwasser, S., Rothman, R. R., Schindler, C., and Uhl, G. R. Cocaine reward: Alteration by regional variant dopamine transporter overexpression. 1. Neurosci. (submitted).
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13. Harrington, C. A,, Lewis, E. J., Krzemien, D., and Chikaraishi, D. M. (1987).Identification and cell type specifically of the tyrosine hydroxylase gene promoter. Nucleic Acids Res. 15, 2363-2384. 14. Banerjee, S. A,, Hoppe, P., Brilliant, M., and Chikaraishi, D. M. (1992). 5’ Flanking sequences of the rat tyrosine hydroxylase gene target accurate tissue-specific, developmental, and transsynaptic expression in transgenic mice. ]. Netrrosci. 12, 4460-4467. 15. Carr, G. D., Fibiger, H. C., and Phillips, A. G. (1984). Conditioned place preference as a measure of drug reward. In Neuropharmacological Basis of Reward, pp. 264-319. Oxford New York. 16. Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., and Caron, M. G. (1996).Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606-612. 17. Drago, J., Gerfen, C. R., Lachowicz, J. E., Steiner, H., and Hollon, T. R. (1994). Altered striatal function in a mutant mouse lacking D1A dopamine receptors. Proc. Natl. Acad. Sci. 91,12564-12568. 18. Miner, L. L., Drago, J., Chamberlain, P. M., Donovan, D., andUhl, G. R. (1995).Retained cocaine conditioned place preference in D1 receptor deficient mice. Neuroreport 6,23142316. 19. Cho, A. K., and Segal, D. S., eds. (1994).Amphetamine and its Analogs. Academic Press, San Diego. 20. Takahashi, N., Miner, L., Sora, I., Ujike, H., Revay, R., Kostic, V., Przedborski, S., and Uhl, G. R. VMATZ knockout mice: Heterozygotes display reduced amphetamineconditioned reward, enhanced amphetamine locomotion and enhanced MPTP toxicity. Proc. Nut. Acad. Sci. USA (in press). 21. Cooper, J. R., Bloom, F. E., and Roth, R. H. (1991).The Biochemical Basis of Neuropharmacology. Oxford Press, New York. 22. Aksoy, S., Klener, J., and Weinshilboum, R. M. (1993). Catechol-0-methyltransferase pharmacogenetics: Photoaffinity labelling and Western blot analysis of human liver samples. Pharmacogenetics 3, 116-122. 23. Bertocci, B., Miggiano, V., Da Prada, M., Dembric, Z., Lahm, H.-W., and Malherbe, P. (1991). Human catechol-0-methyltransferase: Cloning and expression of the membrane associated form. Proc. Natl. Acad. Sci. 88, 1416-1420. 24. Tenhunen, J., Salminen, M., Lundstrom, K., Kiviluoto, T., Savolainen, R., and Ulmanen, I. (1994). Genomic organization of the human catechol-0-methyltransferase gene and its expression from two distinct promoters. Eur. J. Btochem. 223, 1049-1054. 25. Vandenbergh, D. A., Zonderman, A., Wang, J., Uhl, G. R., and Costa, P. A. Failure to replicate the association between long-repeat alleles of the novelty seeking and dopamine D4 receptor polymorphisms: Evidence for limited generalization. Mol. Psychia. (in press). 26. Vandenbergh, D. J., Rodriguez, L. A,, Miller, I., Uhl, G. R., and Lachman, H. M. A Highactivity catechol-0-methyltransferase allele is more prevalent in polysubstance abusers. Psych. Genetics (in press). 27. Benjamin, J., Li, L., Patterson, C., Greenberg, B. D., Murphy, D. L., and Hamer, D. H. (1996). Population and familial association between the D4 dopamine receptor gene and measures of novelty seeking. Nut. Genet. 12, 81-84. 28. Ebstein, E. P., Novick, O., Umansky, R., Priel, B., Osher, Y., Blaine, D., Bennett, E. R., Nemanov, L., Katz, M., Belmaker, R. H. (1996). Dopamine D4 receptor (D4DR) exon 111 polymorphism associated with the human personality trait of novelty seeking. Nut. Genet. 12, 78-80. 29. Shock, N. W., et al. Normal Human Aging: The Baltimore Longitudinal Study of Aging (1984).NIH Publication No. 84-2450, U.S. Government Printing Office, Washington, DC. 30. Persico, A. M., Vandenbergh, D. J., Smith, S. S., and Uhl, G. R. (1993). Dopamine transporter gene polymorphisms are not associated with polysubstance abuse. Biol. Psychiatry 34, 265-267.
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31. Gelernter, J., Kranzler, H. R., Satel, S . L., and Rao, P. A. (1994). Genetic association between dopamine transporter protein alleles and cocaine-induced paranoia. Neuropsychopharmogy 11,195-200. 32. Cook, E. H., Stein, M. A., Krasowski, M. D., Cox, N. J., Olken, D. M., Kieffer, J. E., and Leventhal, B. L. (1995). Association of attention deficit disorder and the dopamine transporter gene. Am. J. Hum. Genet. 56, 993-998. 33. Wang, X.-B., Funada, M., Imai, Y., Revay, R., Ujike, H., and Uhl, G. R. rGP1: A psychostimulant-regulated gene essential for establishing cocaine sensitization J. Neurosci. (submitted) 34. Uhl, G. R., Gold, L. H., Risch, N., and Wilson, A. Analyses of complex behavioral disorders. Proc. Natl. Acad. Sci. USA, 94,(in press). 35. Crabbe, J. C., Belknap, J, K., and Buck, K. J. (1994). Genetic animal models of alcohol and drug abuse. Science 264, 1715-1723. 36. Miner, L. L., and Marley, R. J. (1995).Chromosomalmapping of loci influencing sensitivity to cocaine-induced seizures in BXD recombinant inbred strains of mice. PsychopharmacolOgy 117, 62-66. 37. Miner, L. L., and Marley, R. J. (1995). Chromosomal mapping of the psychomotor stimulant effects of cocaine in BXD recombinant mice. Psychopharmacology 122, 209-214. 38. Lachman, H., Papolos, D. F., Saito, T., Yu, Y. M., Szurnlanski, C. L., and Weinshilboum, R. M. (1996). Human catechol-0-rnethyltransferase pharmacogenetics: Description of a functional polymorphism and its potential application to neuropsychiatric disorders. Pharmacogenetics 6, 243-250. 39. Spielman, R. S., and Weinshilboum, R. M. (1981). Genetics of red cell COMT activity: Analysis of thermal stability and family data. Am. J. Med. Genet. 10, 279-290. 40. Weinshilboum, R. M., and Raymond, F. A. (1977).Inheritance of low erythrocyte catechol0-methyl transferase activity in man. Am. J. Hum. Genet 29, 125-135. 41. Uhl, G., Blum, K., Noble, E., and Smith, S. (1994). Substance abuse vulnerability and D2 receptor gene. Trends Neurosci. 16, 83-88.
Dopaminergic Genes and Substance Abuse The use of addictive substances is a complex behavioral disorder likely to represent interactions between genetic and environmental factors ( 1).Evidence from twin, adoption, and family studies indicates that drug abuse phenotypes including quantity-frequency of use and features responsible for Diagnostic and Statistical Manual (DSM) diagnoses of substance abuse-dependence are each likely to reflect significant genetic contributions (1-3). Population comorbidity studies indicate that relative risk of a drug abuse disorder is enhanced substantially in individuals with comorbid antisocial personality disorder, alcoholism, depression, or the adult residua of attention deficit-hyperactivity disorder (1).Other personality traits have also been postulated to predispose to vulnerability to specific aspects of drug abuse, ranging from initiation of use to resistance to quitting to development of the drug's control over behavior that represents a hallmark of dependence. Dopaminergic brain systems play prominent roles in drug reward (4), focusing attention on genes expressed in these circuits as candidates to contribute to substance abuse vulnerability. Psychostimulants engage these circuits with especial power, as evinced by a number of lines of evidence, including the magnitude of drug-induced dopamine spillover from nucleus accumbens doparninergic synapses following psychostimulant treatments ( 5 )and the results of lesion studies, in which dopaminergic lesions dampen brain reward systems and locomotor functions in both experimental animals and humans (6-8). A number of studies in experimental animals now suggest that manipulations of the levels of expression of several of the genes that encode proteins important for dopaminergic neurotransmission, or of genes regulated by psychostimulants, can have powerful influences on animal models of drug reward. Studies of the functional allelic variants that have been identified in several human dopaminergic genes, or of polymorphic markers that could tag heretofore underscribed I024
Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
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functional variation at other dopaminergic gene loci, have each indicated that expression of human genes with different levels of activity might contribute to differential responses to abused substances, different drug-abuse comorbidities, or different personality features of possible importance for substance abuse. In this chapter, we review several recent cases in which transgenic mouse and human data provide increasingly compelling evidence that variants in genes important for dopaminergic neurotransmission are strong candidates to contribute to interindividual differences in drug abuse vulnerabilities.
1. Transgenic Mice with Dopamine Transporter Over and Underexpressed Studies of cocaine’s primary site for reward and reinforcement in the brain have focused on the dopamine transporter (DAT) ( 9 ) .DAT is a member of the 12-transmembrane domain sodium and chloride-dependent neurotransmitter transporter family, the primary structure of which has been elucidated by cDNA cloning (10). In studying this transporter, the effect of several mutations on transporter function have been characterized. Replacement of putative transmembrane 7 serines 350 and 353 with alanines, for example, results in a transporter variant that exhibits 30% increases in dopamine transport and 30% reductions in affinity for the cocaine analog 2@-carbomethoxy-3@-(tfluorophenyl) tropane (CFT) (11).T o model the effects that a DAT gene variant might have on drug abuse phenotypes, we constructed transgenic animals that express this DAT variant in catecholaminergic neurons (12). We utilized sequences from the 5’ flanking promoter region of the rat tyrosine hydroxylase (TH) gene because of their well-characterized ability to specifically mediate gene expression in catecholaminergic neurons (13, 14). Transgenic strains THDAT2 and THDAT4 each stably pass more than 10 copies of an 8.2-kb trangene construct made up of 4.8 kb of rat TH 5’ flanking promoter sequence linked to the serine 350 and 353 alanine substituted rat DAT variant, and express the transgene mRNA and encoded protein in a region-specific manner with high levels of expression in ventral midbrain, adrenal gland, and olfactory bulb, consistent with patterns of expression previously described as mediated by the TH promoter. Heterozygous transgenic animals from each of the two lines expressing this construction display behavioral differences from wild-type littermate controls. During repeated exposure to novel testing apparatus during testing sessions spaced at 3 to 4-day intervals, both THDAT lines habituate to the testing environment more rapidly. THDAT mice are 150-230% as active following 30 mg/kg of cocaine i.p. More striking effects were observed when the reinforcing and rewarding properties of cocaine were tested using conditioned place preference to assess drug reward (15).Both THDAT2 and THDAT4 animals displayed significantly greater cocaine-induced conditioned place preferences than littermate control mice. Transgenic mice with region-specific overexpression of a DAT gene variant can manifest subtle but interesting differences in specific features related to higher order control of locomotor systems and major models of drug responsiveness.
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The results on cocaine locomotion obtained in these mice complement those obtained by Giros et al. in DAT knockout mice (10). In these animals, although baseline locomotor activities are altered, cocaine induces virtually no excess locomotion. However, data from studies of cocaine reward in DAT knockout mice constructed independently in our laboratory suggest that cocaine-induced conditioned placed preference might also be less altered in these mice (Sora et al., in preparation). These data are also in accord with studies of the locomotor responses in dopamine D1 receptor and DAT knockout mice, in which cocaine-stimulated locomotor functions were virtually eliminated (17, 18).
II. Transgenic Mice with Vesicular Monoamine Transporter Underexpressed The brain vesicular monoamine transporter (VMAT2) pumps monoamine neurotransmitters from neuronal cytoplasm into synaptic vesicles. Vesicular monoamine stores accumulated by normal VMAT2 function may play significant roles in the locomotor stimulation and/or the behavioral reward produced by amphetamines, drugs increasingly abused in certain areas of the United States. Amphetamines dissipate proton gradients across the membranes of synaptic vesicles, disrupt VMAT2 function, enhance cytoplasmic monoamine concentrations, and cause calcium-independent, nonvesicular monoamine release into synapses (19). They also act like cocaine in blocking plasma membrane neurotransmitter transporters that use transmembrane sodium and chloride gradients to pump dopamine, norepinephrine, and serotonin from extracellular spaces into presynaptic neurons. Because little preexisting evidence documented which amphetamine action provided which contribution to amphetamineinduced locomotion or behavioral reward, we constructed transgenic VMAT2 knockout mice using a 12-kb targeting factor homologous to sequences flanking the first three exons of the murine 129 strain VMAT2 gene (20). Heterozygotes VMAT 2 knockout mice are viable into adult life and display VMAT2 levels half of wild-type values. They also display alterations in several monoaminergic markers, heart rate, and blood pressure. However, their weight gain, fertility, habituation, passive avoidance, and locomotor activities are similar to wild-type littermates (20). In these heterozygote VMAT2 knockout mice, 1- and 3-mg/kg intraperitoneal amphetamine doses enhanced locomotor activity, with increases at 1 mg/ kg 146% of those in wild-type mice. However, amphetamine-conditioned place preferences in the heterozygotes were less than those in wild-type mice. Conversely, cocaine ( 5 mg/kg), a blocker of the plasma membrane DAT devoid of significant activity on vesicular monoamine stores, induced conditioned place preferences in heterozygotes that were indistinguishable from those in wildtype mice (20). These data support the idea that plasma membrane transporter blockade by amphetamine contributes primarily to its locomotor stimulation, but that normal function of vesicular monoamine stores is necessary for full amphetamine-induced behavioral reward. They are consistent with the results
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from studies that document differences between the effects of dopamine receptor knockouts on psychostimulant-induced locomotion and reward (18) and with the differences in dose-response relationships for amphetamine-induced locomotion and reward (19).
111. Human Studies of Catechol-0-Methyltransferase Functional Alleles in Polysubstance Abusers and Nonusers Catechol-0-Methyltransferase (COMT) is expressed in dopaminergic brain regions, where its activity provides a major pathway by which extraneuronally released dopamine is inactivated (21). Three- to fourfold differences in human COMT activities are attributed to codon 158 polymorphisms that encode a valine, producing a high enzyme activity, or a methionine, yielding low activity (22-24, 38,41). To seek influences of this COMT allelic variation on substance abuse vulnerability, we compared COMT genotypes in groups of unrelated polysubstance abuser research volunteers defined on the bases of: (1) quantity-frequency of drug use or (2) Diagnostic and Statzsticai Manual of Mental Disorders Ill-Revised (DSMIII-R) criteria for substance abuse/ dependence to those of control research volunteers free of significant use of addictive substances (25, 26). Significant differences in the distributions of both COMT genotypes and allele frequencies between controls and substance abusers, defined on the basis of self-reported peak lifetime use, and between these controls and volunteers who had also been administered the Diagnostic Interview Schedule (DIS) for DSMIII-R diagnoses of substance abuse or dependence were identified (25,26). Detection of single gene effects in complex disorders can be facilitated by examining phenotypes associated with the genotypes encoding extreme values; vulnerability could be enhanced in individuals with the highest or the lowest COMT activities, for example. The proportions of high activity G/G homozygotes and low activity MA homozygotes were also examined: G/G homozygotes were nearly twice as frequent in volunteers who report high quantity-frequency drug use than in controls free of such use (25, 26). The sample defined on the basis of DSM diagnoses also shows a similar difference from control values. These data, thus, fit best with the hypothesis that high COMT activities might contribute to the genetic underpinnings of drug abuse vulnerability.
IV. Human Studies of DRD4 Functional Alleles in “Novelty Seeking” and Related Personality Substance Abuse Predispositions Two recent studies have suggested an association between D4 dopamine receptor polymorphisms and measures designed to assess novelty-seeking behaviors, characterized by “exhilaration or excitement in response to novel stimuli” (27, 28). Such an association was plausible for several reasons. This
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personality dimension has been postulated to be substantially influenced by dopaminergic neurotransmission in one major formulation of personality typology and neurochemical correlates. The dopaminergic D4 receptor is expressed in human brain regions implicated in reward and mood. This receptor displays functional allelic variants that alter its efficiency in G-protein coupling and, thus, change features of dopaminergic neurotransmission in these circuits. However, the data from Benjamin et al., suggest that only some individual item scores on the NEO-PI-R correlate with D4 genotype. These results were at least as consistent with the idea that happy, positive feelings or a ratio between positive and conscientious feelings could represent the underlying DRD4associated phenotype. We approached this question by assessing DRD4 genotypes in a sample taken from the Baltimore Longitudinal Study of Aging (29),a well-characterized population of research volunteers whose characterized personality types allowed us to select the individuals with the highest and with the lowest values on novelty-seeking scales and measures of “happiness,” defined as ratios between measurements of excitement seeking to neuroticism (Em). We found only limited generalization of DRD4 genotype associations with specific personality features contributing to measures of novelty seeking; only the E/N ratio correlated with genotype (25). While our failure to identify the association to the original novelty-seeking item scores in this population strongly suggest that the findings of Benjamin et al. and Ebstein et al. cannot be generalized to all populations, the items positively correlated with DRD4 genotype (warmth, excitement seeking, and positive emotions) and the item that provided a negative correlate, deliberation, provide a ratio whose value did correlate weakly with DRD4 genotype. These results support the idea that a more modest association with an assessment of a ratio of positive and negative feelings might represent a result of differential expression of D4 receptor genotypes. Indeed, given the relatively low levels of expression of the dopamine DRD4 gene, its limited brain distribution, and the substantially greater number of dopamine D2 receptors, even in areas of which the D4 receptors expressed, biologicai fit with this modest receptor expression and a more subtle personality influence on personality make some sense.
V. Other Dopamine Receptors The status of dopamine D2-receptor polymorphisms in drug abuse has been reviewed (1, 42). More recent studies of a polymorphic marker at the dopamine D3 receptor have shown no association with substance abuse vulnerabilities (Rodriguez et al., in preparation).
VI. Dopamine Transporter Examination of association between variable number tandem repeat (VNTR) markers at the human DAT locus revealed no assocation (30). How-
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ever, more recent observations may indicate association between polymorphic markers at this locus and two drug-associated conditions. Individuals who report paranoid experiences during cocaine administration display the DAT allele marked by a nine-copy VNTR marker more frequently than control subjects (31). Attention deficit-hyperactivity disorder (ADHD) risk is also increased in individuals with the DAT allele marked by the 10-copy VNTR (32). Because individuals with ADHD can display psychostimulant responses that differ from those of normal individuals, this evidence also plausibly links DAT allelic variants with alterations in human drug responses.
VII. Future Directions This current review supports potentially prominent roles for dopaminergic gene variants in contributing to individual differences in vulnerability to drug abuse. However, other genes are quite likely also to be involved. Seeking drug-regulated genes provides one avenue to find other classes of drug-abuse vulnerability genes in humans. New approaches, such as subtracted differential display, will allow us to identify hundreds of genes regulated by drug administration in experimental animals (33). Polymorphic markers at these gene loci can provide new tools to identify possible gene substrates for human individual-to-individual differences in drug abuse vulnerability. Positional cloning of drug abuse vulnerability genes may also now become possible. Recent initial attempts to model this process (34) have been based on genetic heritability hZ estimates of 0.45 and assumptions that environmental influences on drug abuse vulnerability are largely nonshared, both based on twin study data (1).An initial estimate of the relative risk for developing a substantial drug abuse disorder in the sibling of an abuser (As) is three to fourfold, based on a group of family studies (1).The two to fourfold risk in excess of that in the general population can then be modelled as due to both some oligogenic and some polygenic influences. In quantitative trait locus studies in mice, the genetics of drug responsiveness can often be modelled as based on the detectable contributions of a limited number of genes, with a residual genetic variance assumed to be polygenic, due to effects of genes whose influences may be too small to determine individually (3.5-37). If these indications from mouse genetics are applicable to humans, a case in which five loci equally contribute to a 75% oligogenic influence could represent a plausible starting point for beginning to model the human condition. This would provide a locus-specific As value of about 1.4 for the human condition. Power calculations based on the work of Reisch (1990) and others indicate that evaluating 300 polymorphic markers spaced at 10 centimorgan intervals across the human genome in 400 nuclear affected sib pair pedigrees would provide an 80% opportunity to detect a locus of such an effect size (34). Although many of the assumptions used to make these power calculations require better and better estimates over time, these values also suggest that an affected sib pair approach to identification of drug abuse vulnerability genes is possible, with multicenter collaboration and powerful genotyping assistance.
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As genome scaning-positional cloning approaches to detection of genes contributing to human drug abuse vulnerability become available, however, the study of candidate loci such as those expressed in the dopaminergic systems important for reward processes appears to provide a valuable initial route to identification of genes that contribute in both animal models and humans to interindividual differences in drug abuse vulnerability.
Acknowledgment This work was largely supported by the Intraumural Research Program, National Institute on Drug Abuse, National Institutes of Health.
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