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. These neuroadaptations are thought to lead to the dysregulation of behavior that characterizes addiction. Psychostimulants enhance dopamine transmission by inhibiting the dopamine transporter (DAT), a protein that clears dopamine released into the synaptic cleft. By inhibiting dopamine clearance, synaptic and extracellular neurotransmitter concentrations are increased. We previously provided evidence that synthetic k- opioid receptor agonists inhibit dopamine transmission in the nucleus accumbens and striatum by decreasing release and facilitating DAT function. At present, however, the cellular mechanism(s) mediating the interaction of kappa opioid receptors with DAT is unknown. We have used heterologous expression systems and synaptosomal preparations to address this issue. Live cell imaging of cells co-expressing the k- opioid receptor and DAT revealed that activation of k-opioid receptors by synthetic agonists produces a rapid, pertussis sensitive up-regulation of DAT function. Similar effects are observed in response to salvinorin A, a naturally occurring, high potency k- opioid receptor agonist. By examining the effects of agonists in the presence of various kinase inhibitors and TAT-peptides, we have identified a critical role of a specific kinase in mediating the k-opioid receptor upregulation of transporter function. Consistent with the presence of consensus sites for this kinase in a restricted location of the transporter and its effects on phosphorylation, we have found that truncation or single point mutation of DAT, so as to remove these sites, results in a loss of k-opioid receptor regulation of transport and decreased transporter phosphorylation. Inhibitors of this kinase microinjected into the nucleus accumbens attenuate the behavioral effects of k- opioid receptor agonists suggesting that activation of this kinase contributes to the pharmacological actions of these agents. On-going studies are examining the physiological significance of k-opioid receptor regulation of DAT regulation to mesolimbic dopamine transmission and the behavioral effects of k- ligands. K- opioid receptor agonists regulate serotonin release. However, questions exist as to whether they may also modulate serotonin transport. This question is of clinical relevance since k-opioid receptor ligands are in development for the treatment of cocaine addiction and the serotonin transporter (SERT) is a substrate of cocaine. To begin to address this issue, we developed and validated a fluorescent imaging technique which enables quantification of SERT function and trafficking in single cells and in real time. Using this technique, we have shown that k-opioid receptor agonists, down-regulate SERT function and cell surface expression. Analogous effects are observed in synaptosomal preparations of the accumbens. In contrast, to DAT regulation by k-opioid agonists, SERT regulation requires calcium and activation of CAM kinase. Using Cre-SERT and Cre-DAT mice with flox-kappa opioid receptor mice, the physiological significance of k-opioid receptor regulation of monoamine transporter regulation will be probed. Mu opioid receptors (MOR) are enriched in the ventral tegmental area (VTA). The VTA is a critical site mediating the rewarding effects of MOR agonists. MOR activation therein increases dopamine transmission in the nucleus accumbens. Morphological data suggest that MOR are located on non-dopaminergic neurons in the VTA. Intracellular recordings in slice preparations of the VTA revealed that morphine increases the firing rate of dopamine neurons but inhibits the firing rate of non-dopaminergic neurons. Although the identity of the non-dopaminergic neurons was not definitively determined, these findings led to the hypothesis that activation of MOR on GABA neurons inhibits their activity, thereby, decreasing GABA release and disinhibiting VTA dopamine neurons. However, in a slice preparation, connectivity of functional circuits is not preserved. Therefore, questions exist as to whether MOR activation affects GABA release in the awake animal. Futhermore, studies examining the functional interplay of GABA, glutamate and dopamine in the VTA are lacking. We have addressed this issue in the awake animal using microdialysis in combination with sensitive analytical techniques for the simultaneous quantification of these transmitters. Our studies show that MOR activation in the VTA increases extracellular DA concentrations. GABA concentrations were decreased whereas glutamate concentrations in the VTA were unaltered. In contrast, no change in dopamine was observed in mice lacking the gene encoding MOR. However, in these animals, basal GABA overflow was significantly increased, and glutamate overflow was decreased. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic transmission in this region. We hypothesize that increased GABAergic neurotransmission following MOR deletion is due to the elimination of a tonic inhibitory influence of MOR on GABA neurons in the VTA, whereas decreased glutamatergic transmission is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. These findings indicate a critical role of VTA MOR in maintaining a balance between excitatory and inhibitory inputs to dopaminergic neurons. Furthermore, they provide suggestive evidence that VTA MOR may modulate vulnerability to drugs of abuse by regulating GABA and glutamatergic inputs to dopaminergic neurons.Animals exposed to a cocaine-paired environment demonstrated an augmented locomotor activity and increased mPFC GABA levels in the cocaine-paired environment. Dual labeling of cFos and glutamic acid decarboxylase 67 (GAD67) immunoreactivity in mPFC neurons revealed significantly greater co-localization of these proteins following exposure to the cocaine-associated environment relative to pseudo-conditioned rats or rats exposed to the saline-associated environment indicating that the conditioned neurochemical response to the cocaine-paired environment is associated with activation of intrinsic mPFC GABA neurons. BLA inactivation prevented the increase in locomotor activity and the augmentation of mPFC GABA transmission produced by cue exposure. Intra-mPFC application of the AMPA/KA receptor antagonist, NBQX, produced similar effects. These findings indicate that exposure to a cocaine-associated environment increases mPFC GABA transmission by enhancing excitatory drive from the BLA and activation of AMPA/KA receptors on mPFC GABA neurons. Intra-VTA perfusion of the DOR agonist, DPDPE, increased somatodendritic and NAc DA overflow. These effects were associated with a decrease in VTA GABA overflow. Glutamate overflow was unaltered. VTA GABAA receptor blockade increased basal DA levels in the VTA and prevented the DA increase produced by DPDPE. Analogous to DPDPE, intra-VTA perfusion of the DOR antagonist, TIPP-psi, increased DA in the VTA and NAc. In contrast to the agonist, the antagonist perfusion enhanced GABA and glutamate overflow. Upon blockade of both GABA and NMDA receptors, DPDPE decreased VTA DA levels. The present findings provide the first demonstration of direct regulation of GABA and glutamate neurotransmission by DORs in the VTA.
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