Dysregulation of Dopamine Transporter Trafficking and Function after Abstinence from Cocaine Self-Administration in Rats: Evidence for Differential Regulation in Caudate Putamen and Nucleus Accumbens

Abstract

The profound alterations produced by cocaine on dopamine (DA) neurotransmission raise the possibility that dopamine transporter (DAT)-expressing neurons may modify DA transport in response to repeated cocaine exposure to maintain the appropriate efficiency of DA clearance. In this study, we determined the changes in molecular mechanisms of DAT regulation in rats with a history of repeated cocaine self-administration followed by 3 weeks of abstinence. Using ex vivo caudate putamen (CPu) and nucleus accumbens (NAcc) synaptosomal preparations, we found that DA uptake was significantly higher in the CPu and NAcc of cocaine-experienced animals compared with yoked saline animals. Surface distribution, p-Ser phosphorylation, and protein phosphatase 2A catalytic subunit (PP2Ac) interaction of DAT were all altered in the CPu. Maximal velocity (V max ) values were elevated both in the CPu and NAcc of cocaine-experienced rats compared with saline controls. Although there was no change in the apparent affinity for DA in the CPu, increased DA affinity was evident in the NAcc. Consistent with elevated DAT activity in cocaine-experienced animals, a higher level of surface DAT, DAT-PP2Ac association, and decreased serine phosphorylation of DAT were observed in the CPu, but not in the NAcc. These results, for the first time, suggest that chronic cocaine self-administration followed by abstinence leads to persisting alterations in normal DAT trafficking and catalytic regulatory cascades in the CPu and NAcc in a brain region-specific manner.Drug abuse and dependence to psychostimulants (e.g., cocaine) and other drugs of abuse constitutes a major health problem worldwide, with profound social and financial costs. Although cocaine inhibits both the serotonin transporter (SERT) and norepinephrine transporter (NET), several lines of evidence have shown that cocaine produces its primary reinforcing effects by binding to the dopamine (DA) transporter (DAT) and blocking the reuptake of DA into presynaptic terminals, thereby potentiating DA neurotransmission in mesocorticolimbic reward pathways (Wise and Rompre, 1989;Robinson and Berridge, 1993;Koob et al., 1998;McFarland and Kalivas, 2001). The relevance of DAT in DA homeostasis and in the reinforcing effects of cocaine are supported by several studies on cocaine-induced behaviors, and on neurotransmitter synthesis, storage, release, and DA receptor expression resulting from removal of DAT genes in mutant mice, and changes described in human cocaine addicts (Fumagalli et al., 1998;Jones et al., 1998;Sora et al., 2001;Jayanthi and Ramamoorthy, 2005;Jayanthi et al., 2007). Thus, long-term changes in DAT levels, kinetics, or regulation would be expected to greatly influence spontaneous and drug-induced behaviors.The search for neuroadaptations in DAT expression has focused on studies in human cocaine-dependent users and a variety of animal models of cocaine addiction. Several strategies have been adapted to quantify DAT levels, including

A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference

“…The tissue was immediately homogenized using a Teflon-glass homogenizer and centrifuged at 1000 ϫ g for 10 min at 4°C. The resulting supernatant was centrifuged at 12,000 ϫ g for 20 min, and the pellet was washed by resuspending in 0.32 M sucrose (24). Protein concentration was determined by DC protein assay (Bio-Rad) using bovine serum albumin as the standard.…”

“…Synaptosomes (300 g) treated as above or obtained from drug-administered animals were subjected to surface biotinylation and isolation of avidin-bound and -unbound fractions as described previously (1,24,25). Aliquots from total extracts (50 l) and entire eluted fractions were separated by SDS-PAGE (10%), transferred to membrane, and probed with mouse NET antibody or DAT antibody.…”

Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization

“…The tissue was immediately homogenized using a Teflon-glass homogenizer and centrifuged at 1,000 ϫ g for 10 min at 4°C. The resulting supernatant was centrifuged at 12,000 ϫ g for 20 min and the pellet was washed by resuspending in 0.32 M sucrose (24). The protein concentration was determined by DC protein assay (Bio-Rad) using bovine serum albumin as standard.…”

“…Surface Biotinylation of Synaptosomes-Rat VST synaptosomes (300 g) treated with drugs as described above (in the uptake assay) were subjected to surface biotinylation and isolation of avidin-bound and unbound fractions as described previously (24). Aliquots from total extracts (50 l) and the entire eluted fractions were separated by SDS-PAGE (10%), transferred to membrane, and probed with mouse-NET antibody.…”

“…Anti-calnexin antibody was used to validate the surface biotinylation of plasma membrane proteins. NET band densities from total and biotinylated (representing the surface pool) fractions were normalized using levels of calnexin in the total extract (24).…”