Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7699–7704, June 1998 Neurobiology Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice ICHIRO SORA*, CHRISTINE WICHEMS†,NOBUYUKI TAKAHASHI*, XIAO-FEI LI*, ZHIZHEN ZENG*, RANDAL REVAY*, KLAUS-PETER LESCH‡,DENNIS L. MURPHY†, AND GEORGE R. UHL*§¶ *Molecular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224; §Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21224; †Laboratory of Clinical Science, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-1264; and ‡Department of Psychiatry, University of Wuerzburg, Wuerzburg 97080, Germany Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 16, 1998 (received for review December 12, 1997) ABSTRACT Cocaine and methylphenidate block uptake norepinephrine transport, but only weakly inhibits serotonin by neuronal plasma membrane transporters for dopamine, transport, this difference from cocaine could conceivably serotonin, and norepinephrine. Cocaine also blocks voltage- contribute to a distinct profile on tests of reward (14–16). gated sodium channels, a property not shared by methylpheni- These and other more indirect lines of evidence support the date. Several lines of evidence have suggested that cocaine idea that cocaine’s inhibition of serotonin uptake could also blockade of the dopamine transporter (DAT), perhaps with provide an alternative and plausible molecular site for contri- additional contributions from serotonin transporter (5-HTT) butions to cocaine reward (17–19). recognition, was key to its rewarding actions. We now report To test the dopamine- or serotonin-transporter dependence that knockout mice without DAT and mice without 5-HTT of cocaine reward, we have constructed DAT knockout mice establish cocaine-conditioned place preferences. Each strain and assessed cocaine-conditioned place preferences in these displays cocaine-conditioned place preference in this major DAT knockout mice and in previously described serotonin mouse model for assessing drug reward, while methylpheni- transporter (5-HTT) knockout mice (20). We have focused on date-conditioned place preference is also maintained in DAT conditioned place preference because it provides a technically knockout mice. These results have substantial implications for tractable and robust measure of drug reward in mice, although understanding cocaine actions and for strategies to produce other drug features occasionally contaminate this test (21, 22). anticocaine medications. This assay is able to detect the rewarding properties of virtually every class of abused substance. Mice express their drug Cocaine use is a principal drug abuse problem in the United preference 24 hr after the last drug administration, when they States and other countries, contributing to substantial mor- are likely to be free from acute cocaine effects on motor bidity and mortality among the millions of individuals who use performance (5–7). it each year (1). No current medication provides effective treatment for cocaine dependence (2). These facts give par- ticular importance to defining the sites for cocaine reward in MATERIALS AND METHODS the brain so that they can be more accurately targeted by Targeted Disruption of the Murine DAT Gene. DAT knock- potential therapeutic agents. out mice were produced by standard techniques (23). Thirteen- Several lines of evidence have provided support for a role of and 15-kb DAT genomic fragments were isolated from a l-FIX the dopamine transporter (DAT) as a primary site for cocaine II genomic library (Stratagene) prepared from the 129SvEv reward. Structure–activity studies document good correlations mouse strain (24), and a targeting vector constructed by using between psychostimulant properties in tests of reward and a 5.0-kb BamHI–SmaI59 fragment and a 5.5-kb SpeI–BamHI their abilities to block DAT; poorer correlations are noted with 39 fragment subcloned into pPGKneo. The final construct, their potencies in blocking other transporters (3, 4). Dopami- designated pDATKO, contains the herpes simplex virus thy- nergic lesions blunt cocaine influences in model systems that midine kinase (TK) gene driven by the MC1 polyoma enhancer test reward (5–7). Psychostimulants enhance dopamine release at the 39 end of a SpeI–BamHI 39 fragment. The first and from dopaminergic circuits (8). Transgenic mice that overex- second exons of the murine DAT gene are flanked by 5.0 kb press DAT display enhanced cocaine-conditioned place pref- of 59 and 5.5 kb of 39 sequence. Twenty-five micrograms of erence (G.R.U., et al., unpublished observations). Finally, pDATKO DNA was linearized with NotI and transfected by ‘‘indifference’’ to cocaine has been inferred from the reduced electroporation into 107 J1 embryonic stem (ES) cells derived cocaine-stimulated locomotion recently described in mice that from 129ySv mice [a generous gift from R. Jaenisch (25)]. J1 lack DAT (9, 10). cells in which homologous recombination had occurred were There are also limitations to postulated direct relationships selected by 8 days of growth in Dulbecco’s modified Eagle’s between DAT blockade and psychostimulant-induced reward. medium (DMEM) containing 15% fetal bovine serum (Hy- Among these are the failure of several compounds that po- Clone), 0.1 mM 2-mercaptoethanol, 500 mgyml G418, and 2 tently inhibit dopamine uptake, including mazindol, to display mM ganciclovir (a generous gift from Syntex). EcoRI digests substantial abuse liability in humans or animal model studies of DNA prepared from 400 G418- and ganciclovir-resistant (11–13). Because mazindol potently inhibits dopamine and clones were screened by Southern blot analyses using a 371-bp The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: ES cells, embryonic stem cells; DAT, dopamine trans- accordance with 18 U.S.C. §1734 solely to indicate this fact. porter; 5-HTT, serotonin transporter; TH, tyrosine hydroxylase. © 1998 by The National Academy of Sciences 0027-8424y98y957699-6$2.00y0 ¶To whom reprint requests should be sent at: Molecular Neurobiology, PNAS is available online at http:yywww.pnas.org. Box 5180, Baltimore, MD 21224. e-mail: [email protected]. 7699 Downloaded by guest on September 26, 2021 7700 Neurobiology: Sora et al. Proc. Natl. Acad. Sci. USA 95 (1998) EcoRI–BamHI fragment. Four positive cell lines from the mined by the Bradford method (Bio-Rad) and results analyzed doubly resistant colonies displayed the 5-kb EcoRI fragment by using MACLIGAND. anticipated of homologous recombinants, which was readily Immunohistochemical Analyses. DAT and tyrosine hydrox- distinguishable from the 12-kb fragment obtained from wild- ylase (TH) immunohistochemistry were performed as previ- type DNA. Two ES clones from the positive cell lines were ously described (27). Specificity of primary antisera was evi- used to establish mutant mice. Chimeric mice were generated dent in ELISAs, preadsorption tests, and the anatomic distri- by injecting 15–20 homologous recombinant ES cells into bution of immunoreactivity (data not shown). blastocysts harvested from C57BLy6J mice (The Jackson Behavioral Tests. Mice were housed at 24°C in 50% relative y y Laboratory) and implanting the blastocysts into uteri of pseu- humidity with a 12 12 hr light dark cycle with lights on at 7:00 dopregnant CD-1 mice (Charles River) 2.5 days after coitus. a.m. and off at 7:00 p.m. and ad libitum access to food and Chimeric mice were mated with wild-type C57BLy6J mice to water under American Association for Laboratory Animal Care guidelines, as described (28). Mice tested for each produce F1 offspring. Southern analyses of DNA extracted from tail-tip specimens of F mice revealed that germ-line experiment were compared with littermate controls to main- 1 tain near-identity of average genetic background. Locomotor transmission was achieved from crosses between 4 chimeric activity was assessed as total distance traveled, which was animals. F homozygous, heterozygous, and wild-type off- 2 calculated from measurement of the number of beam breaks spring of F 3 F intercrosses were used for further biochem- 1 1 when mice were placed individually in 46 3 25 3 19 cm clear ical and behavioral testing. plastic cages inside Optovarimex activity monitors (Columbus Radioligand Binding Studies. Washed membranes prepared Instruments, Columbus, OH), to which the mice had not been from rapidly dissected striatal specimens were incubated with previously exposed, under dim-light sound-attenuated condi- 3 2 b b [ H]CFT [( )-2- -carbomethoxy-3- -(4-fluorophenyl)tro- tions (29). Distance traveled was monitored for 3 hr. Imme- y pane-1,5-naphthalenedisulfonate] (WIN35,428) (83.5 Ci diately after baseline activity testing, the mice were removed y 5 mmol; NEN DuPont; 1 Ci 37 GBq) in ice-cold 0.32 M from the monitors, injected with 10 mgykg cocaine hydrochlo- sucrose containing 10 mM sodium phosphate, pH 7.4, as ride, and returned to the same chamber, where distance described (3). Results from parallel incubations with 0.1 mM traveled was monitored for an additional 1 hr (28). Reward was cocaine hydrochloride