Presynaptic D 2 dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D 2 DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D 2 ϩ/ϩ ), one (D 2 ϩ/Ϫ ), or no (D 2 Ϫ/Ϫ ) functional copies of the gene coding for the D 2 DA receptor. In vivo microdialysis studies demonstrated that basal and K ϩ -evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D 2 Ϫ/Ϫ mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D 2 ϩ/ϩ mice. In D 2 ϩ/ϩ mice, but not D 2 Ϫ/Ϫ mice, local application of the D 2 -like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [ 3 H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D 2 DA receptor subtype modulates activity of the striatal DAT. Key Words: D 2 dopamine autoreceptorGene knockout mice-Dopamine uptake-In vivo microdialysis-In vivo electrochemistry-Striatum. J. Neurochem. 72, 148 -156 (1999).Dopaminergic neurotransmission in the CNS plays a key role in the control of motor, cognitive, and reward processes. Dopaminergic neurotransmission is a complex and tightly controlled process that involves synthesis, storage, release, receptor binding, subsequent activation of signal transduction systems, and termination of the action of the neurotransmitter. Extracellular levels of dopamine (DA) within the striatum are thought to largely depend on a balance between vesicular release of DA and reuptake of the released DA through the DA transporter (DAT). Multiple mechanisms have been identified for the short-and long-term regulation of DA release, whereas relatively little is known about mechanisms by which DAT may be acutely regulated.One well-characterized mechanism by which DA release can be modulated is via DA autoreceptors. DA receptors are divided into two general classes, the D 1 -like family, composed of the D 1 and D 5 receptor subtypes, and the D 2 -like family, composed of the D 2 , D 3 , and D 4 subtypes (see Sokoloff and Schwartz, 1995). Release-modulating DA autoreceptors belong to the D 2 -like family (see Langer, 1997). Studies using in vitro slice techniques, synaptosomes, and in vivo microdialysis have provided strong evidence that activation of these terminal autoreceptors with D 2 -like receptor agonists inhibits DA release within the rodent striatum (Dwoskin and Zahniser, 1986;Altar et al., 1987;Westerink and de Vries, 1989). D 2 DA receptors have been localized on dopaminergic axon terminals (Sesack et al., 1994), and at least a subpopul...

Behavioral responses of rodents to cocaine are characterized by marked individual variability. Here, outbred male SpragueDawley rats were profiled based on concomitant recording of behavioral and electrochemical responses. Rats were categorized as either low or high cocaine responders (LCRs or HCRs, respectively) based on their differential locomotor responsiveness to an acute, low-dose injection of cocaine (10 mg/kg i.p.). LCRs and HCRs also differed in other cocaine-induced behaviors. The role of the dopamine transporter (DAT) in mediating the behavioral differences in cocaine responsiveness in LCRs and HCRs was investigated by high-speed chronoamperometric recording of exogenous dopamine (DA) clearance signals in nucleus accumbens (NAc) and dorsal striatum (dSTR). Higher volumes of DA were required in NAc of HCRs, than of LCRs, to produce equivalent peak DA signal amplitude (A max ) responses.In HCRs, systemic cocaine administration evoked an immediate and prolonged 2-fold augmentation in A max in both brain regions, coincident with locomotor activation. The cocaineinduced decrease in the efficiency of DA clearance (k) in NAc of HCRs was more immediate and prolonged than in dSTR, where the transient decrease coincided with maximal stereotypic behavior. In contrast, in LCRs, A max was not altered by cocaine, and decay rate constant (k) was transiently attenuated only in dSTR. Correlation analyses of individual responses revealed that cocaine-induced changes in DA clearance signal parameters accounted for 20 to 40% of the variation in behavioral responsiveness to cocaine. Overall, our findings emphasize the importance of characterizing individual responses to understand more fully the range of functional consequences resulting from DAT inhibition.Psychomotor stimulants such as cocaine bind to the dopamine (DA) transporter (DAT), thereby inhibiting the machinery primarily responsible for the clearance of DA from the extracellular space and termination of DA neurotransmission (Ritz et al., 1987). The consequences of DAT inhibition in vivo have been investigated using either microdialysis or voltammetry. These studies have demonstrated that both local and systemic administrations of psychomotor stimulants elicit an increase in extracellular DA concentration and prolongs the time course of DA clearance within rat nucleus accumbens (NAc) and dorsal striatum (dSTR) (Nomikos et al., 1990;Kuczenski and Segal, 1992;Cass et al., 1993b;Zahniser et al., 1999). It has been postulated that this spatial and temporal augmentation in DA signaling following reuptake blockade mediates the behavioral activating and addictive properties of stimulants that target DAT (Kuhar et al., 1991).A common spectrum of behavioral effects, including locomotor activation, stereotyped behaviors, and self-administration, is produced by DAT inhibitors. Nonetheless, individual differences have been observed in the behavioral responsiveness to acute or repeated administration of either cocaine (Hooks et al., 1991a;Cass et al., 1993a; Djano an...

Behavioral sensitization to cocaine reflects neuroadaptive changes that intensify drug effects. However, repeated cocaine administration does not induce behavioral sensitization in all male Sprague-Dawley rats. Because cocaine inhibits the dopamine (DA) transporter (DAT), we investigated whether altered DAT function contributes to these individual differences. Freely moving rats had electrochemical microelectrode/microcannulae assemblies chronically implanted in the nucleus accumbens so that exogenous DA clearance signals were recorded simultaneous with behavior. The peak DA signal amplitude (A max ) and efficiency of clearance (k) were used as indices of in vivo DAT function. Low and high cocaine responders (LCRs and HCRs, respectively) were identified based on their locomotor responsiveness to an initial injection of cocaine (10 mg/kg i.p.). Consistent with DAT inhibition, cocaine elevated A max and reduced k in HCRs, but not in LCRs. The same dose of cocaine was administered for six additional days and after a 7-day withdrawal. Baseline behavioral and dopamine clearance indices were unaltered by repeated cocaine or after withdrawal. Only LCRs expressed cocaine-induced sensitized locomotor activation, and this was accompanied by cocaine-induced elevations in A max and reductions in k. These sensitized responses to cocaine persisted in LCRs after withdrawal. In contrast, neither locomotor nor electrochemical responses were altered by repeated saline administration or a saline challenge after repeated cocaine administration, suggesting that conditioning did not significantly contribute. Our results suggest that increased DAT inhibition by cocaine is associated with locomotor sensitization and that DAT serves as a common substrate for mediating both the initial and sensitized locomotor responsiveness to cocaine.

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The galactose--glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains. The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees. Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins. Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain. Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations. When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s-1. Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold. Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales. The functional implications of such motions are discussed.