Release of the neurotransmitter dopamine in the mesolimbic system of the brain mediates the reinforcing properties of several drugs of abuse, including nicotine. Here we investigate the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the beta2 subunit of this receptor. We found that nicotine stimulates dopamine release in the ventral striatum of wild-type mice but not in the ventral striatum of beta2-mutant mice. Using patch-clamp recording, we show that mesencephalic dopaminergic neurons from mice without the beta2 subunit no longer respond to nicotine, and that self-administration of nicotine is attenuated in these mutant mice. Our results strongly support the idea that the beta2-containing neuronal nicotinic acetylcholine receptor is involved in mediating the reinforcing properties of nicotine.

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Adenosine and dopamine signaling exert opposite effects in the basal ganglia, a brain region involved in sensory-motor integration. Thus, adenosine agonists induce motor depression and adenosine antagonists, such as caffeine, produce motor activation (1). These opposite effects result from specific antagonistic interactions between subtypes of adenosine and dopamine receptors in the striatum, the main input structure of the basal ganglia. In fact, striatal dopamine receptors and, to some extent, adenosine receptors are segregated in the two main populations of ␥-aminobutyric acid (GABA) efferent neurons. EXPERIMENTAL PROCEDURESCell Cultures-Maintenance of SH-SY5Y cells (parental and D 2 Rtransfected cells) as well as the pharmacological characterization and maintenance of D 2 R-and D 1 R-transfected mouse fibroblast Ltk Ϫ cells are described in detail elsewhere (7-9). For primary cultures, striata were removed from 16-day-old Sprague-Dawley rat embryos (B&K Universal) in Ca 2ϩ /Mg 2ϩ -free PBS supplemented with 20 units/ml penicillin and 20 g/ml streptomycin (Invitrogen). The tissue fragments were pooled and mechanically dissociated in SFM Neurobasal serum-free medium (Invitrogen), supplemented with B27 (Invitrogen), glutamine (2 mM; Invitrogen), penicillin/streptomycin (20 units/ml/20 g/ml; Invitrogen), and ␤-mercaptoethanol (25 M) (Invitrogen). Cells were collected by centrifugation at 100 ϫ g for 5 min and resuspended in fresh medium. The resulting single-cell suspension was seeded on 24-well plates coated with gelatin (Sigma) and poly-L-lysine (Sigma), and cells were grown at 37°C in saturation humidity with 5% CO 2 .Immunolabeling Experiments-Neuroblastoma cells were grown on glass coverslips coated with poly-L-lysine (Sigma) and exposed to vari-* This work was

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Since high-affinity adenosine A2 receptors (A2u) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2 receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-p-(2-carboxyethyl)phen- ethylaminoJ-5'-N-ethylcarboxamidoadenosine (CGS 21680) Adenosine has been shown to function as a neuromodulator in many areas of the mammalian central nervous system (1-3). These actions of adenosine are mediated by receptors that can be subdivided into Al and A2 subtypes based on relative agonist and antagonist potencies (4, 5). Al activation inhibits and A2 activation stimulates adenylate cyclase (4, 6, 7). The A2 receptors have been further subclassified into high-affinity (A2a) and low-affinity (A2b) receptors, based on agonist potencies with regard to adenylate cyclase activation (4) and receptor binding (5). The A2b receptors are widely distributed in the brain and mediate the stimulatory action of high concentrations of adenosine agonists on cAMP formation, which could affect dopamine release and synthesis (8,9). In contrast, A2a receptors are exclusively localized to dopamine-innervated areas ofthe central nervous system (10, 11), with a postsynaptic distribution (12) similar to that of postsynaptic D1 and D2 receptors (13). By using in situ hybridization the recently cloned A2a receptors (14) have been found to be localized to striatal medium-sized neurons (15).Adenosine agonists inhibit, whereas adenosine antagonists, including caffeine, enhance spontaneous (16, 17) and dopamine-induced locomotor activity (16,(18)(19)(20). The potencies of adenosine agonists in producing hypomotility correlate with their affinities for Au adenosine receptors (21,22), suggesting that A2. receptors mediate most of the behavioral effects of adenosine agonists. The hypomotility induced by adenosine agonists resembles that induced by classical neuroleptics (22), which act by blocking postsynaptic D2 receptors (23). In fact, behavioral evidence for a negative interaction between postsynaptic A2. and D2 receptors has recently been obtained using acutely reserpinized mice (24,25). Since activation of postsynaptic D2 receptors seems to be a necessary step for locomotor behavior, a negative interaction between postsynaptic A2. and D2 receptors could explain the hypomotility induced by adenosine agonists and the enhancement of locomotor activity induced by adenosine antagonists, including caffeine (24,25).In contrast to the A2a receptor, the D2 receptor mediates an inhibition of adenylate cyclase (26