Drug addiction: Knockout mice and dirty drugs George R. Uhl, David J. Vandenbergh and Lucinda L. Miner
Recent studies with knockout mice implicate the dopamine transporter as the target of the locomotor effects of the addictive psychomotor drugs cocaine and amphetamine; studies of reward in these animals are eagerly awaited. Address: Department of Health and Human Services, National Institutes of Health, National Institute on Drug Abuse, Addiction Research Center, PO Box 5180, Baltimore, Maryland 21224, USA. Current Biology 1996, Vol 6 No 8:935–936 © Current Biology Ltd ISSN 0960-9822
The addictive drugs cocaine and amphetamine stimulate locomotion and provide behavioral reward. They have multiple molecular sites of action, of which those on sodium- and chloride-dependent neurotransmitter transporters are thought to be particularly important [1,2]. Both drugs bind to the plasma-membrane transporters for dopamine, norepinephrine and serotonin, inhibiting reuptake into neurons of each of these monoamines. Amphetamine can also disrupt the hydrogen ion gradient across the membranes of the synaptic vesicles that store monoamines, releasing them into the cytoplasm from where they can be exported out of the cell by mechanisms suggested to include ‘reverse’ transport by the plasma-membrane transporters . Cocaine also blocks voltage-gated sodium channels at higher concentrations . These wide-ranging effects make cocaine and amphetamine prototypical ‘dirty drugs’, and have led to persistent uncertainties about which sites contribute to their locomotor effects and which to their rewarding behavioral actions. The cloning of genes encoding the plasma membrane monoamine transporters has made it possible to test, by genetic manipulation, the effects of altering transporter expression on animal responses to psychostimulant drugs. The elimination of a response to a drug when one of several potential sites of action is removed suggests that that site is necessary for the response. Now Giros et al.  have reported studies using dopamine transporter (DAT) gene ‘knockout’ mice, which provide evidence that the dopamine transporter is involved in locomotor responses to psychomotor stimulant drugs. Homozygous DAT mutant mice exhibited no locomotor response to amphetamine or cocaine at doses that led to significant responses in wild-type and heterozygous mice. This result strongly supports the view that much of the locomotor activation caused by cocaine and amphetamine can be attributed to DAT blockade and effects on brain dopamine systems. It should be noted, however that the mutant mice also
showed behavioural abnormalities in the absence of any drug — up-to four-fold increases in their nocturnal locomotor activity levels — so the observed effects of DAT gene inactivation on drug responses are superimposed on differences in baseline levels of locomotion. The brains of the DAT mutant mice showed expected alterations in dopaminergic function. Dopamine released from striatal slices under depolarizing conditions persisted in the extracellular fluid far longer in mutant than wild-type slices, consistent with a failure of dopamine reuptake in the mutants. Interestingly, amphetamine failed to release dopamine into extracellular fluid perfusing mutant slices, supporting roles for the membrane transporter in ‘reverse’ transport and/or in amphetamine accumulation. These important results need to be interpreted with caution, however, as the mutant mice show a number of developmental adaptations to the loss of the transporter. Thus, their dopamine D1 and D2 receptor densities are lower than normal. Neurotransmitter peptides in locomotor circuits are altered in the mutant mice, preproenkephalin expression is substantially downregulated and preprodynorphin expression is somewhat increased . There may also have been adaptive changes in systems that were not studied. These adaptive changes may have contributed to the altered druginduced behaviours of the mutant mice, tempering conclusions about possible direct roles of the transporter. There are also pharmacological and genetical considerations in interpreting these data. In a preliminary study on the C57BL/6J mouse strain that was one of the progenitors of the DAT mutant mice, amphetamine was found to induce locomotion with a striking, inverted-U-shaped dose–effect relationship (our unpublished data). The dose used to test the DAT mutant mice falls on the descending limb of this curve, so single-dose assessments cannot distinguish between shifts in drug potency (rightward shift in dose-response curves) and reduced drug responsiveness (downward shift in dose-response curves). The use of different animal strains with different baseline activity levels could also complicate matters. The C57BL/6J and the 129/Sv parental lines of the DAT mutants differ 3–4-fold in their locomotor activity levels in a novel environment, while the stress induced by saline injections elicited much more locomotor activity in 129/Sv than in C57BL/6J mice . These sorts of data mandate careful attention to the effects of background genotype, especially in interpreting important but subtle differences in locomotor responses such as those reported by Giros et al. .
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Elevations in spontaneous locomotor activity have been noted in animals with genetic manipulations affecting several other genes encoding components of the dopaminergic system. D1 and D3 receptor knockout mice showed hyperactivity, or enhanced spontaneous locomotion [7–10]. Conversely, transgenic mice overexpressing the DAT in catecholaminergic neurons showed unchanged spontaneous locomotor activities (our unpublished observations). Different behavioral systems might be engaged to produce enhanced locomotor stimulation or reduced locomotor inhibition through actions in the several distinct circuits in which neurons express the D1 receptor, D3 receptor or DAT. The fact that DAT inactivation eliminated the effects of both amphetamine and cocaine on locomotion could be viewed as supporting the view that those effects predominantly reflect drug actions directly on the transporter itself. But in the case of amphetamine, at least, the effect could be less direct: loss of the DAT prevents extracellular release by ‘reverse transport’ of the cytoplasmic dopamine whose concentrations are enhanced following amphetamine-induced vesicular release. One way of testing this idea could come from studies of the tissue contents of dopamine and its metabolites. When we have examined dopamine content in animals with modest alterations in DAT expression, we have found significant changes in tissue dopamine levels (our unpublished observations). Conceivably, amphetamine effects may be altered as a result of adaptive changes in vesicular dopamine stores due to loss of the DAT. Despite the use of the term ‘indifference’ to cocaine and amphetamine in the title of their paper, Giros et al.  did not report any direct tests of the motivational effects of these drugs in the DAT mutant mice. Several studies in rodents have suggested some parallels between intensities of drug effects on locomotion and reward systems [11,12], but recent work has shown that effects on locomotion and reward are readily separable. Thus, cocaine administration to D1 receptor knockout mice gave a blunted locomotion response but a strikingly intact reward response . Several other lines of evidence, however, have suggested that cocaine reward is largely due to its inhibition of DAT: lesions of DAT-expressing neurons result in striking reductions in psychostimulant reward [13,14]; psychostimulants enhance synaptic concentrations of the dopamine released by DAT-expressing neurons ; the relative potencies of cocaine analogs in tests of behavioral reward and DAT inhibition correlate well ; and transgenic mice overexpressing DAT in catecholaminergic neurons display enhanced cocaine-induced reward (our unpublished observations). The direct examination of cocaine reward and reinforcement in DAT knockout animals will allow another test of the idea that DAT is a necessary component for cocaine and amphetamine behavioral reward.
The work of Giros et al.  serves as a major additional piece of evidence in support of the view that the DAT has a central role in the locomotor actions of the ‘dirty drugs’ cocaine and amphetamine. Additional data will be eagerly sought to test the DAT’s role in the induction of reward and reinforcement by psychomotor stimulants, and to assess whether DAT knockout mice really are indifferent to cocaine and amphetamine. References 1. Amara SG, Kuhar MJ: Neurotransmitter transporters: recent progress. Annu Rev Neurosci 1993, 16:73–93. 2. Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ: Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 1987, 237:1219–1223. 3. Seiden LS, Sabol KE, Ricaurte GA: Amphetamine: effects on catecholamine systems and behavior. Annu Rev Pharmacol Toxicol 1993, 32:639–677. 4. Weidman S: Effects of calcium ions and local anesthetics on the electrical properties of Purkinje fibers. J Physiol 1955, 129:568–582. 5. Giros B, Jaber M, Jones SR, Wightman RM, Caron MG: Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 1996, 379:606–612 6. Miner LL: Cocaine reward and locomotor activity in C57BL/6J and 129/SvJ inbred mice and their F1 cross. Pharmacol Biochem Behav 1996, in press. 7. Accili D, Fishburn CS, Drago J, Steiner H, Lachowicz JE, Park B-H, Gauda EB, Lee EJ, Cool MH, Sibley DR, et al.: A targeted mutation of the D3 dopamine receptor gene is associated with hyperactivity in mice. Proc Natl Acad Sci USA 1996, 93:1945–1949. 8. Xu M, Moratalla R, Gold LH, Hiroi N, Koob GF, Graybiel AM, Tonegawa S: Dopamine D1 receptor mutant mice are deficient in striatal expression of dynorphin and in dopamine-mediated behavioral responses. Cell 1994, 79:729–742. 9. Xu M, Hu X-T, Cooper DC, Moratall R, Graybiel AM, White FJ, Tonegawa S: Elimination of cocaine-induced hyperactivity and dopamine-mediated neurophysiological effects in dopamine D1 receptor mutant mice. Cell 1994, 79:945–955. 10. Miner LL, Drago J, Chamberlain PM, Donovan D, Uhl GR: Retained cocaine conditioned place preference in D1 receptor deficient mice. Neuroreports 1995, 6:2314–2316. 11. Wise RA, Bozarth MA: A psychomotor stimulant theory of addiction. Psychol Rev 1987, 94:469–492. 12. Piazza PV, Deminiere J-M, LeMoal M, Simon H: Factors that predict individual vulnerability to amphetamine self-administration. Science 1989, 245:1511–1513. 13. Roberts DCS, Koob GF, Klonoff R, Fibiger HC: Extinction and recovery of cocaine self-administration following 6hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 1980, 12:781–787. 14. Roberts DCS, Koob GF: Disruption of cocaine self-administration following 6-hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol Biochem Behav 1982, 17:901–904. 15. Petit HO, Justice JB: Effect of dose on cocaine self-administration behavior and dopamine levels in the nucleus accumbens. Brain Res 1991, 539:94–102.