Repeated Exposure to Methamphetamine, Cocaine or Morphine Induces Augmentation of Dopamine Release in Rat Mesocorticolimbic Slice Co-Cultures

Repeated intermittent exposure to psychostimulants and morphine leads to progressive augmentation of its locomotor activating effects in rodents. Accumulating evidence suggests the critical involvement of the mesocorticolimbic dopaminergic neurons, which project from the ventral tegmental area to the nucleus accumbens and the medial prefrontal cortex, in the behavioral sensitization. Here, we examined the acute and chronic effects of psychostimulants and morphine on dopamine release in a reconstructed mesocorticolimbic system comprised of a rat triple organotypic slice co-culture of the ventral tegmental area, nucleus accumbens and medial prefrontal cortex regions. Tyrosine hydroxylase-positive cell bodies were localized in the ventral tegmental area, and their neurites projected to the nucleus accumbens and medial prefrontal cortex regions. Acute treatment with methamphetamine (0.1–1000 µM), cocaine (0.1–300 µM) or morphine (0.1–100 µM) for 30 min increased extracellular dopamine levels in a concentration-dependent manner, while 3,4-methylenedioxyamphetamine (0.1–1000 µM) had little effect. Following repeated exposure to methamphetamine (10 µM) for 30 min every day for 6 days, the dopamine release gradually increased during the 30-min treatment. The augmentation of dopamine release was maintained even after the withdrawal of methamphetamine for 7 days. Similar augmentation was observed by repeated exposure to cocaine (1–300 µM) or morphine (10 and 100 µM). Furthermore, methamphetamine-induced augmentation of dopamine release was prevented by an NMDA receptor antagonist, MK-801 (10 µM), and was not observed in double slice co-cultures that excluded the medial prefrontal cortex slice. These results suggest that repeated psychostimulant- or morphine-induced augmentation of dopamine release, i.e. dopaminergic sensitization, was reproduced in a rat triple organotypic slice co-cultures. In addition, the slice co-culture system revealed that the NMDA receptors and the medial prefrontal cortex play an essential role in the dopaminergic sensitization. This in vitro sensitization model provides a unique approach for studying mechanisms underlying behavioral sensitization to drugs of abuse

Structural and Functional Characterization of the Interaction of Snapin with the Dopamine Transporter: Differential Modulation of Psychostimulant Actions

International audienceThe importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity

Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter

The dopamine transporter (DAT) plays a key role in the regulation of dopaminergic signaling wherein it controls both the spatial and temporal actions of dopamine. Here we evaluated the behavioral and neurochemical consequences of increased DAT function by generating DAT transgenic mice (DAT-tg) that overexpress the transporter. These mice were generated by pronuclear injection of a bacterial artificial chromosome containing the mouse DAT locus, yielding an anatomical expression pattern of DAT-tg identical to WT. In DAT-tg mice there is a 3-fold increase in the levels of total and membrane-expressed DAT, but synaptic plasma membrane fractions of DAT-tg mice show only a 30% increase in transporter levels. Functional studies reveal that in the DAT-tg animals there is a 50% increase in the rate of dopamine (DA) uptake resulting in extracellular levels of DA that are decreased by ≈40%. Behaviorally, DAT-tg animals display similar locomotor stimulation when treated with DAT blockers such as GBR12909, methylphenidate, and cocaine. However, these mice demonstrate markedly increased locomotor responses to amphetamine compared with WT animals. Furthermore, compared with controls, there is a 3-fold greater increase in the amount of DA released by amphetamine in DAT-tg mice that correlates with the 3-fold increase in protein expression. Finally, DAT-tg animals show reduced operant responding for natural reward while displaying preference for amphetamine at much lower doses (0.2 and 0.5 mg/kg) than WT mice (2 mg/kg). These results suggest that overexpression of DAT leads to a marked increase in sensitivity to psychomotor and rewarding properties of amphetamine

Three Ubiquitin Conjugation Sites in the Amino Terminus of the Dopamine Transporter Mediate Protein Kinase C–dependent Endocytosis of the Transporter

Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT amino-terminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery

Dopamine transporter cell surface localization facilitated by a direct interaction with the dopamine D2 receptor

Altered synaptic dopamine levels have been implicated in several neurological/neuropsychiatric disorders, including drug addiction and schizophrenia. However, it is unclear what precipitates these changes in synaptic dopamine levels. One of the key presynaptic components involved in regulating dopaminergic tone is the dopamine transporter (DAT). Here, we report that the DAT is also regulated by the dopamine D2 receptor through a direct protein–protein interaction involving the DAT amino-terminus and the third intracellular loop of the D2 receptor. This physical coupling facilitates the recruitment of intracellular DAT to the plasma membrane and leads to enhanced dopamine reuptake. Moreover, mice injected with peptides that disrupt D2–DAT interaction exhibit decreased synaptosomal dopamine uptake and significantly increased locomotor activity, reminiscent of DAT knockout mice. Our data highlight a novel mechanism through which neurotransmitter receptors can functionally modulate neurotransmitter transporters, an interaction that can affect the synaptic neurotransmitter levels in the brain