Rapid substrate-induced down-regulation in function and surface localization of dopamine transporters: rat dorsal striatum versus nucleus accumbens.

The neurotransmitter dopamine (DA) plays an essential role in normal CNS functions such as cognition, locomotion, and reward (Iversen and Iversen 2007). The duration and extent of DA neurotransmission is largely limited by DA transporters (DATs) (Jaber et al. 1997). The DAT is a member of the neurotransmitter:sodium symporter family, which also includes GABA, norepinephrine, and serotonin transporters (Chen et al. 2004). DATs are localized to dopaminergic neurons (Nirenberg et al. 1996), where they are functional when expressed on the plasma membrane. In particular, DATs located within striatal brain reward circuitry help to mediate the psychostimulant actions of d-amphetamine (AMPH) and methamphetamine (METH) (Koob and Nestler 1997; Wise and Bozarth 1987). DAT function can be rapidly regulated by exposure to DAT substrates (e.g. DA, AMPH, and METH), DAT inhibitors (e.g. cocaine), ligands for various presynaptic receptors (e.g. DA D2 receptors), and several signaling systems (e.g. protein kinase C (PKC)) (for reviews see: Gulley and Zahniser 2003; Mortensen and Amara 2003). Here, we focused on further understanding the relatively rapid and transient down-regulation of DAT activity and cell surface expression induced by its substrates. This has been shown to occur following their initial uptake by DAT, inhibition/reversal of DAT, and ultra rapid up-regulation of DATs in dorsal striatum (dSTR) and cells expressing cloned DATs (Fleckenstein et al. 1997; Saunders et al. 2000; Chi and Reith 2003; Johnson et al. 2005; Kahlig et al. 2006; Boudanova et al. 2008). Although it may be counterintuitive why exposure to DAT substrates results in not only increased DA in the synapse, but also down-regulation of DAT uptake activity, this latter effect may be a protective mechanism since cytoplasmic DA can be toxic to neurons (Ziv et al. 1994). Interestingly, two published studies have suggested that DAT activity may be differentially regulated by substrates within subregions of striatum (Kokoshka et al. 1998; Gulley et al. 2002;). Specifically, systemic administration of METH decreased DAT activity measured ex vivo in well-washed synaptosomes prepared from rat dSTR but not from nucleus accumbens (NAc) (Kokoshka et al. 1998). Additionally, brief, rapidly repeated local application of DA decreased in vivo DA clearance, consistent with loss of DATs, in rat dSTR but not in NAc (Gulley et al. 2002). Since NAc has 40-60% fewer DATs than dSTR (Marshall et al. 1990), it could be important for DATs in NAc to be more resistant to substrate-induced down-regulation. However, whether this is the case and the explanation for this putative brain regional difference are not known. The DAT protein is a single gene product, and to date only one regional difference has been identified (i.e. glycosylation of DATs differs in rat dSTR and NAc) (Lew et al. 1992). Although lack of N-glycosylation reduces DAT activity and surface expression, it is not essential for DAT surface expression (Li et al. 2004). Post-translational modifications and accessory proteins involved in DAT trafficking could also differ between dSTR and NAc, but these have not been reported. It is important to understand if DATs in dSTR and NAc differ in substrate-induced regulation because this could contribute to the differential dopaminergic neuronal function reported for these two brain regions. For instance, AMPH- and cocaine-induced increases in extracellular DA are greater in NAc than in dSTR (Carboni et al. 1989; Cass et al. 1992; Kuczenski and Segal 1992), but the vulnerability to the neurotoxic effects of METH is greater in dSTR than in NAc (Haughey et al. 1999; Wallace et al. 1999). Furthermore, for psychostimulants (e.g. AMPH, METH, and cocaine), dSTR and NAc are suggested to play differing roles in the transition from initial drug use to drug addiction, with NAc being particularly important during the early stages and dSTR being more important in the later stages (Everitt and Robbins 2005). Therefore, the purpose of the present study was to compare directly the effect of relatively brief in vitro pre-exposure to DAT substrates, with an emphasis on AMPH, on DAT uptake activity, kinetics, and cell surface expression in synaptosomes prepared from rat dSTR versus NAc. Since PKC has been previously shown to play a role in substrate-mediated regulation of DATs in dSTR and cells expressing cloned DATs (Giambalvo 1992a,b; Vaughan et al. 1997; Zhang et al. 1997; Zhu et al. 1997; Daniels and Amara 1999; Blakely and Bauman 2000; Chi and Reith 2003), follow-up experiments were performed to assess if this was also the case in NAc synaptosomes. Lastly, the effects of systemic administration of AMPH were compared on ex vivo DAT kinetics and surface expression to explore further regionally selective substrate-induced DAT regulation.