Psychostimulants increase transmission of the neurotransmitter, dopamine, in the nucleus accumbens and prefrontal cortex. This action contributes to the rewarding effects of these agents and the initiation of drug abuse. Following continued drug use, enduring changes in brain chemistry are observed within the nucleus accumbens, prefrontal cortex and other regions of the prefrontal-cortico-striatal loop, a circuit that controls incentive motivation, learning and impulsivity. Increasing evidence suggests that these neuroadaptations lead to the dysregulation of behavior that characterizes addiction. Psychostimulants enhance dopamine transmission by inhibiting the dopamine transporter, a protein that clears dopamine released into the synaptic cleft. By inhibiting dopamine clearance, synaptic and extracellular neurotransmitter concentrations are increased. We and others have shown that D2 and D3 dopamine receptors receptors regulate extracellular dopamine concentrations in the nucleus accumbens and striatum. Extracellular dopamine concentrations are decreased in response to D2/D3 receptor agonists and increased in response to antagonists. Our in-vivo and in-vitro studies provided evidence that D2/D3 receptors regulate extracellular dopamine concentrations by affecting both transporter mediated uptake and release. Due to the lack of ligands selective for these receptor sub-types and the presence of both D2 and D3 receptors in the brain regions examined, the role of each in the regulation of dopamine dynamics could not be delineated.? ? Using live-cell imaging techniques, our studies have provided the first direct demonstration that D2 receptors regulate dopamine transporter activity in heterologous expression systems. We monitored dopamine transporter function in real time using the high affinity, fluorescent dopamine transporter substrate, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). Addition of D2/D3 agonists (e.g., quinpirole, PD128907) to cells co-expressing these proteins induced a rapid and concentration-dependent increase in ASP+ accumulation. The increase in uptake was pertussis toxin-sensitive indicating a Gi/Go dependent mechanism. D2 receptor activation induced phosphorylation of extracellular related kinase (ERK1/2) and Akt, a major target of phosphoinositide 3-kinase (PI3K), confirming that the D2 receptor signals through these kinases. ERK 1/2 inhibition prevented the effects of D2/D3 receptor agonists on ASP+ accumulation. PI3K inhibition was without effect indicating that D2 receptor stimulation upregulates DAT function via an ERK1/2-dependent and PI3K-independent mechanism. Biochemical and imaging studies revealed that the dopamine transporter and D2 receptor are in close physical proximity providing a cellular basis for the interaction of these two proteins. Mutation studies revealed that the N-terminus of the dopamine transporter is not required for D2/DAT interactions.? ? We have completed studies assessing the role of D3 receptors in DAT regulation. In cells co-expressing D3 receptors and the dopamine transporter, D3 receptor activation rapidly increased ASP+ uptake. D3 receptor stimulation, like D2 receptor stimulation, activated ERK 1/2 and PI3K. In contrast, however, to D2 receptor stimulation, inhibition of either kinase prevented the increase in uptake evoked by D3 receptor activation. Inhibition of other kinases (e.g. p38 MAPK) was without effect. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression as well as increased dopamine transporter exocytosis. In contrast, following prolonged D3 receptor activation, dopamine transporter function is down-regulated and transporter cell surface expression is reduced. ? ? K-opioid receptor (KOPr) agonists decrease extracellular dopamine concentrations in the nucleus accumbens and dorsal striatum. Acute administration of the potent KOPr agonist, salvinorin A, an alkaloid found in the mint leaf and used by Indian tribes for its psychotomimetic actions, produces similar effects. We previously showed that synthetic KOPr agonists decrease dopamine concentrations in the nucleus accumbens by increasing dopamine transporter function and inhibiting release. Using the technique of no-net flux microdialysis under transient conditions we examined whether local perfusion of salvinorin A produces similar effects in the striatum. In contrast to what we have observed in the nucleus accumbens, we found no effect of salvinorin A on dopamine uptake. However, dopamine release was markedly depressed. Although these findings suggest that salvinorin A may not modulate dopamine uptake, our ex-vivo studies demonstrate marked changes in striatal uptake following brief incubation (e.g., 5 min) of striatal tissue with this KOPragonist or synthetic agonists. Therefore, we hypothesize that the failure to observe regulation of uptake in-vivo may be due to the longer duration of receptor activation used in in-vivo studies. On-going studies are examining this issue since identification of the mechanisms by which salvinorin modulates dopamine transmission is important for understanding the increase in recreational use of this opiate as well as its psychotomimetic and dysphoric effects. ? ? Cocaine binds with high affinity to the dopamine transporter as well as to other monoamine transporters. Using heterologous systems we have found that KOPr agonists also modulate the serotonin transporter. In contrast, however to the dopamine transporter, KOPr agonist regulation of the serotonin transporter varies as a function of the duration of receptor activation. Whereas transient receptor activation increases serotonin transporter function, more prolonged receptor activation decreases function. These effects are observed in response to synthetic agonists and in response to salvinorin A. Our previous studies have shown that KOPr agonists attenuate several behavioral and neurochemical effects of cocaine. We hypothesize that the cocaine-antagonist-like effects of these agents result at least in part from their ability to regulate dopamine and serotonin transport. On-going studies seek to elucidate the cellular mechanisms by which KOPr systems regulate monoamine transporters and whether blockade of this regulation attenuates the cocaine-antagonist like actions of KOPr agonists. ? ? The repeated use of morphine and other mu opioid receptor (MOPr)agonists leads to tolerance and dependence. These effects limit the clinical use of opiates in the treatment of pain and are also thought to play an important role in the maintenance of opiate addiction, once developed. The repeated intermittent administration of morphine can result in an enhancement of its behavioral effects (e.g., sensitization), a phenomenon implicated in drug-craving. Studies in the spinal cord have suggested that delta opioid receptors (DOPr) may contribute to the development of analgesic tolerance and to opiate dependence. Whether DOPr play a role in mediating the rewarding effects of opiates or the development of sensitization is unknown. We addressed this issue using mice which lack the gene encoding the DOPr. Our studies revealed that mice lacking DOPr exhibit an augmentation of context-dependent sensitization and that tolerance is reduced relative to wildtype mices. The conditioned rewarding effects of morphine were also reduced in these mice reduced and similar findings were obtained in response to pharmacological blockade of DOPr in wildtype mice. These findings indicate that the endogenous DOPr system is recruited in response to both repeated and chronic morphine administration and that this recruitment serves an essential function in the development of tolerance, behavioral sensitization, and the conditioning of opiate reward.
