Rapid regulation of the dopamine transporter: role in stimulant addiction?

Neuropharmacology 47 (2004) 80–91 www.elsevier.com/locate/neuropharm

Rapid regulation of the dopamine transporter: role in stimulant addiction? Nancy R. Zahniser , Alexander Sorkin Department of Pharmacology (N. R. Z. and A. S.) and Neuroscience Program, (N. R. Z.), University of Colorado Health Sciences Center at Fitzsimons, Aurora CO 80045–0508, USA Received 12 April 2004; received in revised form 14 June 2004; accepted 30 June 2004

Abstract Dopamine (DA) and the DA transporter (DAT) play important roles in psychomotor stimulant behavioral activation and reward. By understanding how DAT activity is regulated, we will better appreciate its contribution to normal neurotransmission and to brain diseases like drug addiction. DAT is regulated long-term by chronic drug administration. It is also regulated in a rapid, dynamic fashion by many factors—including brief exposure to DAT substrates, e.g. DA and amphetamine, and inhibitors, e.g. cocaine. We found that individual differences in the initial and sensitized locomotor responsiveness of rats to cocaine reflect differences in in vivo DAT function. Our ex vivo studies have further suggested that differences in basal and/or rapid cocaineinduced expression of functional DATs in striatum contribute to the differences in initial responsiveness. Studies in model systems have demonstrated that short-term DAT regulation occurs by altered transporter trafficking, and thereby cell surface expression. For example, a rapid, complex regulation of DAT by DA is suggested. Amphetamine causes DAT internalization into early endosomal compartments whereas cocaine appears to up-regulate surface expression of DAT. Future studies are needed to confirm these observations in neurons, as well as to elucidate the mechanisms of rapid DAT endocytic trafficking at neuronal synapses. # 2004 Elsevier Ltd. All rights reserved. Keywords: Dopamine transporter (DAT); DAT trafficking; Cocaine; Behavioral sensitization; Individual differences; Addiction

1. Introduction: focus on dopamine and the dopamine transporter Dopamine (DA) plays an important role in brain reward, both to natural reinforcers and addictive drugs (Wise and Bozarth, 1987; Carboni et al., 1989; Koob, 1992; Kelley and Berridge, 2002; Wise, 2002; Bonci et al., 2003). In fact, most addictive drugs increase extracellular concentrations of DA in nucleus accumbens (NAc) and medial prefrontal cortex (mPFC), projection areas of mesocorticolimbic DA neurons and key components of the ‘‘brain reward circuit’’. A number of lines of early evidence supported the critical role played by DA in the reinforcing effects of psychomotor  Corresponding author. Department of Pharmacology, UCHSC at Fitzsimons, Mail Stop 8303, P. O. Box 6508, Aurora, CO 800450508, USA. Tel.: +1-303-724-3661; fax: +1-303-724-3640. E-mail address: [email protected] (N.R. Zahniser).

0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.07.010

stimulant drugs like cocaine and amphetamine (Wise and Bozarth, 1987; Koob and Nestler, 1997; Wise, 1998). For example, rats will self-administer these drugs directly into NAc and/or mPFC. Antagonists of dopaminergic receptors, but not adrenergic or serotonergic receptors, reduce self-administration of cocaine and amphetamine in a dose-related manner. Likewise, lesion of mesocorticolimbic DA neurons attenuates or abolishes self-administration of these drugs. This is not to say, however, that DA is necessarily sufficient, or even required, for reward (Cannon and Palmiter, 2003). Glutamatergic, GABAergic, opioid, nicotinic cholinergic, and other brain pathways are also critical components of the brain reward circuit (Koob, 1992; Everitt and Wolf, 2002; Kalivas and McFarland, 2003; Laviolette and van der Kooy, 2004). Nonetheless, identifying factors that regulate DA neurotransmission should not only help us to gain more understanding about the molecular/cellular underpinnings of drug

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addiction, but also may offer insights as to new strategies for drug abuse prevention and treatment. Removal of DA from the extracellular space and its transport back into DA neurons is an important mechanism controlling DA neurotransmission. This removal occurs via Na+-/Cl -dependent uptake by the DA transporter (DAT). Thus, DAT-mediated uptake is the primary mechanism that terminates DA neurotransmission. The importance of DAT in shaping the temporal and spatial dynamics of DA signaling in the brain has been appreciated for some time because of the intense psychomotor stimulation produced by drugs that block DAT activity. However, its importance was perhaps most dramatically demonstrated in mice with genetic deletions of DAT (Giros et al., 1996; Gainetdinov et al., 1999). Despite marked compensatory changes to downregulate DA neurotransmission, these DAT knock-out mice still have higher than normal extracellular concentrations of DA, which is manifested by hyperactivity and other behavioral changes. DAT is clearly an important site of action for psychomotor stimulants. Stimulants like cocaine and methylphenidate competitively inhibit DAT, resulting in increased synaptic concentrations of DA released from axon varicosities and dendrites, prolonged interactions of DA with both its postsynaptic and presynaptic receptors and behavioral activation. Amphetamine-based stimulants also increase extracellular concentrations of DA, but by a different mechanism. Amphetamines are substrates not only for the plasmalemma DAT, but also for the vesicular monoamine transporter 2 (VMAT2) localized on DA storage vesicles. Uptake of amphetamines inhibits transport of DA by both DAT and VMAT2. More importantly, however, once taken up, amphetamines induce reverse transport of DA, thereby causing release of DA and greatly increasing both its intra- and extracellular concentrations. Thus, cocaine’s ability to increase extracellular DA concentrations is strictly dependent on DA released by neuronal activity, whereas amphetamine’s ability is not. Cocaine and amphetamine also have relatively high affinities for the other plasmalemma monoamine transporters, viz. norepinephrine and serotonin transporters (NETs and SERTs, respectively). In terms of stimulant abuse, however, drug interactions with DAT have been thought to be more important than with either NET or SERT. The receptor antagonist studies mentioned above support this idea. Likewise, the potencies of cocaine-like drugs to inhibit binding to DAT, as compared with binding to NET and SERT, are best correlated with their potencies for self-administration (Ritz et al., 1987). However, more recent investigations using mice with null mutations of one or two of the monoamine transporters suggest that although DAT contributes to the reinforcing/motivational rewarding

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effects of psychomotor stimulants, it is unlikely to be the sole mediator. These studies have demonstrated that DAT knockout mice still self-administer cocaine and exhibit conditioned place preference for cocaine and have highlighted the importance of SERT (Uhl et al., 2002; Rocha, 2003). It is also interesting to note that cocaine and amphetamine, but not the selective DAT inhibitor GBR 12909, increase extracellular DA concentrations in NAc of DAT knockout mice (Carboni et al., 2001). Of course, widespread compensatory changes occur in DAT knockout mice; and extrapolation of results from these animals to normal, intact animals may, or may not, be appropriate. In summary, DAT is critical for terminating DA neurotransmission and contributes to the abuse potential of psychomotor stimulants. Thus, we need to understand more about its functional regulation. In particular, by knowing more about how DAT activity is regulated, we will better appreciate its role in normal neurotransmission, as well as its role in brain diseases like drug addiction.

2. Long-term regulation of DAT and cocaineinduced behavioral sensitization In the past, neurotransmitter transporters were considered to be relatively stable constituents of the plasma membrane—essentially they were thought to function like ever-present synaptic vacuum cleaners that were either ‘‘on’’ or ‘‘off’’. However, within the last 10–15 years, it has become apparent that this picture is incorrect. Many labs, including our own, have shown that the activity of DAT is regulated not only long-term by drug administration, but also short-term in a rapid, dynamic fashion by many factors (Zahniser and Doolen, 2001; Gulley and Zahniser, 2003; Kahlig and Galli, 2003; Mortensen and Amara, 2003). This is also the case for other members of the Na+/Cl dependent transporter gene family—notably the GABA transporter GAT1, as well as NET and SERT (Beckman and Quick, 1998; Zahniser and Doolen, 2001; Robinson, 2002; Torres et al., 2003b). Chronic interactions of ligands with their receptors often result in either up- or down-regulation of the number of functional receptors at the cell surface. If transporters were regulated in a similar manner, then prolonged exposure to transporter substrates and inhibitors would also be expected to regulate the number of active transporters expressed at the cell surface. This type of drug-induced regulation has been observed in some cases (Zahniser and Doolen, 2001). Regulation of DAT by chronic cocaine administration provides a well-studied example of long-term inhibitor-induced regulation of transporter expression, and thereby activity, which has been observed in both rodent and human brain (Zahniser et al., 1995; Kuhar and Pilotte,

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1996; Mash et al., 2002). In contrast, relatively few studies have focused on chronic cocaine-induced regulation of SERT or NET. Nonetheless, chronic cocaine use has been fond to up-regulate both of these transporters in specific brain regions of non-human primates and humans (Jacobsen et al., 2000; Mash et al., 2000; Macey et al., 2003). Interestingly, however, chronic treatment with selective SERT or NET inhibitors produces the opposite effect in rat brain: down-regulation of SERT or NET, respectively (Benmansour et al., 1999 and 2004). In general, the studies that have investigated chronic cocaine-induced regulation of DAT have shown that during repeated cocaine administration and at short times after cessation of drug treatment, a relatively short-lived increase in the number of DAT binding sites and the velocity of DA uptake is observed. This up-regulation is consistent with more rapid clearance of synaptic DA and behavioral tolerance to the effects of cocaine. In contrast, however, at longer times after cessation of repeated cocaine treatment, DAT mRNA, binding sites and activity are reduced, particularly in limbic brain regions. This persistent, but not permanent, DAT down-regulation is consistent with the ability of a cocaine challenge—given to rodents days to weeks, and even months, after withdrawal from repeated cocaine—to increase extracellular DA concentrations and DA-mediated behaviors to a greater extent than the same dose of cocaine did initially. Expression of persistent, drug-induced ‘‘behavioral sensitization’’ has been suggested to contribute to craving and the high relapse rate of cocaine addicts. Specifically Robinson and Berridge (2003) have suggested that sensitization causes the CNS to attribute greater incentive salience to the drug, resulting in compulsive motivation to take additive drugs. Certainly, a number of labs have recently found many drug-induced molecular and cellular changes suggestive that drug addiction is an aberrant form of learning and memory (Nestler, 2001; Wolf, 2002; Gerdeman et al., 2003). Since cocaine’s direct action is to inhibit the DAT, we focused our investigations on how DAT regulation might contribute to cocaine-induced behavioral sensitization. Throughout our studies we consistently found that only 50%–60% of outbred male Sprague–Dawley rats were behaviorally responsive to the initial, relatively low dose of cocaine that we administered— 10 mg/kg, i.p. Furthermore, the less responsive rats became behaviorally sensitized with repeated, oncedaily treatment with cocaine. In contrast, the rats that were more responsive initially did not. To assess in vivo DAT activity, we used high-speed chronoamperometry to measure clearance of exogenous DA applied locally into the rats’ dorsal striatum (dSTR) or core subregion of the NAc. Our early studies showed that following withdrawal from repeated cocaine

administration, a cocaine challenge reduced clearance of DA in NAc, but not in dSTR (Cass et al., 1993). These results were consistent with cocaine-induced sensitization of locomotor activity because reduced DA clearance in NAc core would prolong the interaction of DA with its receptors in this brain region known to be crucial for locomotor activity. However, since not all of the rats were behaviorally sensitized and the electrochemical recordings of DA clearance were done while the rats were anesthetized with urethane, the relationship between the altered DA clearance and behavior needed to be clarified. Thus, we modified our electrochemical recording technique so that behavior and DA clearance could be measured simultaneously in individual unanesthetized, freely moving rats (Sabeti et al., 2002, 2003). Using this approach, we again found that the initial dose of cocaine (10 mg/kg, i.p.) induced a wide range of magnitudes of open-field locomotor activity in outbred male Sprague–Dawley rats. Furthermore, categorizing each rat as either a low or high cocaine responder (LCR or HCR, respectively), based on the median split of the cocaine-induced activation, we found that basal DAT activity in NAc core and cocaine-induced inhibition of DA clearance were greater in both NAc and dSTR of HCRs, than in LCRs. Also, regardless of the LCR/HCR classification, the higher the initial cocaineinduced locomotor activation was, the greater the cocaine inhibition of DA clearance was, particularly in NAc (Sabeti et al., 2002). With once-daily, repeated administration of 10 mg/kg cocaine, LCRs began to show both significant drug-induced behavioral activation and DA clearance inhibition after 5 days of treatment, whereas the responsiveness of HCRs remained unchanged from their initial high level (Sabeti et al., 2003). Thus, LCRs, but not HCRs, exhibited sensitized cocaine-induced locomotor activation and DAT inhibition; and the heightened responsiveness of the LCRs persisted when they were given a cocaine challenge 1 week after withdrawal from the repeated treatment. Overall, our results suggest that increased cocaine inhibition of DAT activity contributes to locomotor sensitization and that DAT serves as a common substrate for mediating both the initial and sensitized locomotor responsiveness of male Sprague–Dawley rats to cocaine. Our findings also emphasize the importance of taking into account differential individual responsiveness to stimulants like cocaine and understanding the basis/bases for these differences.

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3. Differences in DAT and individual responsiveness to cocaine Differences in initial responsiveness of individuals to drugs of abuse reflect both genetic and environmental influences and are certainly one factor that determines whether or not an individual will become addicted to that drug (e.g. cocaine; Haertzen et al., 1983; Davidson et al., 1993). Thus, we next investigated what might differ about cocaine and its actions in LCRs versus HCRs. We first showed that brain levels of cocaine and the affinity of DAT for cocaine in NAc are similar in LCRs and HCRs (Gulley et al., 2003). Also, LCRs and HCRs do not differ in the total number of DAT binding sites, as measured by saturation analysis with the cocaine analog [3H]WIN 35,428 in crude membranes from NAc. However, this approach measures the total number of cellular transporters—i.e. functional transporters in the plasma membrane, as well as nonfunctional transporters in intracellular membranes—so that the number of functional DATs at the cell surface could still differ between LCRs and HCRs. Furthermore, brief exposure to cocaine can rapidly up-regulate cell surface expression of DATs (Daws et al., 2002; Little et al., 2002); but this has not been observed in all cases (Fleckenstein et al., 1999; Chi and Reith, 2003). Thus, LCRs and HCRs could differ in steady-state expression of cell surface DATs, DA uptake kinetics and/or cocaine-induced DAT trafficking. To begin to explore possible differences in functional DAT expression between LCRs and HCRs, we recently measured ex vivo [3H]DA uptake and [3H]WIN 35,428 binding in dSTR synaptosomes prepared from rats at the time of maximal cocaine-induced behavioral activation (Briegleb et al., 2004). In these studies, cocaine-treated HCRs exhibited higher uptake of [3H]DA than LCRs, whereas total DAT number was similar in the two groups, suggesting a greater number of surface DATs in dSTR of HCRs. In addition, regardless of the LCR/HCR classification, there was a significant, positive correlation between the levels of [3H]DA uptake and cocaine-induced locomotor activity in individual rats. Furthermore, 1 week after their initial classification when cocaine-induced locomotor activity no longer differed between LCRs and HCRs, [3H]DA uptake was also similar. Thus, it appears that a difference in basal and/or cocaine-induced surface expression of DATs may contribute to the individual differences observed in cocaine-induced activation of male Sprague–Dawley rats. Interestingly, we also observed that individual differences in behavioral activation and DAT activity following acute amphetamine administration were not as pronounced as those with cocaine. Although the underlying mechanisms for the differential effects of cocaine and amphetamine are unknown, it is intriguing that not only their mechan-

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isms of action and dependency on DA release differ, but also their manner in which they rapidly regulate DAT surface expression differ (see below). Normalizing [3H]DA uptake by the total number of 3 [ H]WIN 35,428 binding sites is an indirect measure of DAT cell surface expression. Therefore, it will be important in the future to use a direct method to confirm basal/cocaine-induced differences in DAT trafficking in LCRs and HCRs. For example, PICK 1-induced re-distribution of DAT to the plasma membrane has been detected with an ectodomain antibody that recognizes the second extracellular loop of DAT (Torres et al., 2001). Alternatively, several groups have recently measured changes in synaptosomal cell surface DATs by labeling the surface transporters with a membraneimpermeant biotin, followed by quantification of total extracts and surface fractions by western blot analysis (Chi and Reith, 2003; Salvatore et al., 2003). It will also be important to compare potential differences in surface DAT expression in NAc and mPFC, with those in dSTR. Ideally, surface expression of DAT in core versus shell subregions of NAc should be compared, as well. Although both locomotor activation and drug reinforcement involve the mesocorticolimbic DA pathway, these behaviors can be dissociated (e.g. Rocha, 2003). Thus, it will also be important to investigate whether our observations about the role of DAT in individual differences in cocaine-induced activation extend to the rewarding/reinforcing effects of cocaine. We plan to do this by determining if a similar relationship exists between individual differences in DAT function and cocaine-induced conditioned place preference and/or self-administration. In Fig. 1, we illustrate how transient cocaine-induced DAT up-regulation in mesolimbic DA neurons might contribute to cocaine psychomotor activation and addiction.

4. Short-term, rapid regulation of DAT As mentioned above, cocaine exposure can rapidly up-regulate surface DAT expression. In fact, in the past few years it has been shown that DAT uptake activity can be regulated within minutes by changes in membrane potential or exposure to DAT substrates and inhibitors, ligands interacting with presynaptic receptors, signaling molecules that activate/inhibit kinases and phosphatases, and other diffusible second messengers (Zahniser and Doolen, 2001; Gulley and Zahniser, 2003; Kahlig and Galli, 2003; Mortensen and Amara, 2003; Torres et al., 2003b). An obvious mechanism by which DAT activity could be rapidly regulated would be a conformational change in the transporter that, in turn, alters its kinetic activation and/or affinity for DA, Na+ and/or Cl . Certain kinases regulate other members of this Na+/Cl -dependent

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Fig. 1. Mechanisms by which transient cocaine-induced DAT up-regulation in mesolimbic DA neurons could contribute to cocaine psychomotor activation and addiction. Cocaine competitively inhibits DA uptake by somatodendritic DATs in the ventral tegmental area (VTA) and axon terminal DATs in the NAc, thereby increasing extracellular concentrations of DA (top panel). Extracellular DA stimulates postsynaptic DA D1 and D2 receptors (D1Rs and D2Rs, respectively) in NAc, reducing firing of GABAergic output neurons and ultimately resulting in increased activation and reward. These psychomotor stimulant effects are somewhat counter-balanced because DA also activates presynaptic D2Rs localized on DA neurons (‘autoreceptors’). Autoreceptors reduce DA neuron firing, release and synthesis, as well as accelerate uptake. If cocaine rapidly up-regulates cell surface expression of DATs but its concentration is insufficient to inhibit a significant number of these newly expressed transporters, then DA neuron firing would be increased because of effects in the VTA but postsynaptic D1R and D2R activation might still be reduced because of effects in the NAc (bottom panel). Depending on the net effect in VTA and NAc, cocaine-induce DAT up-regulation could directly contribute to increased DA-mediated stimulation. Alternatively, if the net effect is to attenuate DA stimulation, this could represent a compensatory response to limit activation and reward. However, even if this is the case, shortening the duration of stimulation could still enhance the addiction potential of cocaine by sharpening the cocaine-induced spike of DA.

transporter gene family, e.g. GAT1 and NET, in a trafficking-independent manner by altering transporter kinetics (Deken et al., 2000; Apparsundaram et al., 2001). This is also true for protein kinase C-mediated activation of DAT reverse transport (Gnegy, 2003). Nonetheless, regulation of DAT, similar to that of other members of this gene family, appears to occur

largely by changes in trafficking of the transporter and thereby its cell surface expression (Fig. 2). At first, not only the dynamic regulation of DAT, but also the trafficking-dependent mechanism, came as a surprise. Now, however, we know from studies in model systems that DATs are constitutively internalized and recycled/trafficked back to the plasma

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Fig. 2. Proposed dynamic constitutive and drug-induced regulation of DAT cell surface expression in a DA neuron. DAT is exported from the endoplasmic reticulum (ER) as an oligomer and maintains this oligomeric state in other cellular compartments. Although DAT is excluded from the active zones of synapses where DA is released from synaptic vesicles, it efficiently removes DA from the extracellular space and limits the interaction of DA with its families of postsynaptic D1 and presynaptic/postsynaptic D2 receptors. The recruitment of DAT to the presynaptic membrane may require DAT interactions with specific retention complexes (RC). DAT is constitutively internalized from the plasma membrane via clathrin-coated vesicles (CCVs) to the endosomes, from where it is recycled back to the plasma membrane. Trafficking of DAT provides a ready mechanism for dynamic regulation of transporter capacity. DAT substrates like amphetamines and DA rapidly and transiently reduce DAT cell surface expression, thereby diminishing DA uptake. In contrast, DAT inhibitors like cocaine, as well as activation of presumed presynaptic D2 autoreceptors, result in the opposite regulation, thereby accelerating DA uptake. One of the challenges for the future is to understand how this drug-induced regulation contributes to the addictive properties of stimulants.

membrane at a relatively rapid rate. For example, in rat pheochromcytoma PC12 cells, the half-life of DAT at the plasma membrane is estimated to be ~13 min (Loder and Melikian, 2003). This relatively rapid redistribution of DATs provides a mechanism by which the cell surface expression and activity of DATs can be quickly and finely regulated. The details regarding DAT trafficking in the brain remain to be elucidated (see next section). Whether rapid regulation of transporters, in turn, provides a physiologically relevant mechanism by which the compliment of functional transporters can be regulated in parallel with neurotransmitter release also remains to be investigated. Interestingly, Deken and colleagues (2003) recently reported results supporting this idea for GAT1. They found that intracellular GAT1 is associated with a class of vesicles distinct from neurotransmitter synaptic vesicles. The GAT1-containing vesicles undergo slower recycling than synaptic vesicles; however, similar to synaptic vesicles, their recycling is dependent on depolarization and Ca2+ concentration. In contrast to the rapid up-regulation of DAT expression induced by inhibitors like cocaine,

substrates like amphetamine rapidly down-regulate DAT surface expression (Saunders et al., 2000; Fig. 2). Following acute systemic injection of amphetamine, methamphetamine or methyllenedioxymethamphetamine (MDMA), a rapid and reversible reduction in DAT activity occurs in brain, as assessed ex vivo with [3H]DA uptake into striatal synaptosomes (Metzger et al., 1998; Fleckenstein et al., 1999). Similar results were observed with [3H]serotonin uptake by SERT (Fleckenstein et al., 1999). However, relatively high doses are required for this regulation. In vitro exposure of striatal synaptosomes to methamphetamine produces similar effects (Sandoval et al., 2001). The identification of several DAT-associated currents has facilitated DAT regulation studies by providing a realtime measure of DAT activity (Sonders et al., 1997). Using this approach, we observed down-regulation of DAT currents and surface binding sites following brief exposure of Xenopus lavis oocytes expressing the human DAT to amphetamine, DA or tyramine (Gulley et al., 2002). The mechanism by which substrates induce DAT down-regulation appears to be complex because this regulation is attenuated in both brain and

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expression systems by selective inhibitors of protein kinase C (PKC; Sandoval et al., 2001; Gulley et al., 2002) or activation of phosphatidylinositol 3-kinase (PI 3-kinase; Carvelli et al., 2002). A conventional isoform of PKC—a, bI, bII, and/or c—mediates this downregulation in oocytes (Doolen and Zahniser, 2002). PKC phosphorylates the N-terminus of DAT (Foster et al., 2002). Interestingly, however, N-terminal phosphorylation is not required for amphetamine-induced DAT internalization but is required for amphetamine to reverse the transporter and thereby release DA (Gra˚na¨s et al., 2003; Khoshbouei et al., 2004). Clearly, more research is needed to clarify how substrates reduce DAT cell surface expression. Given the consistent reports of substrate-induced down-regulation of DAT, we were puzzled why we had not observed this when using electrochemical recording to measure in vivo DA clearance to assess DAT activity. In all of our studies over the years we had found that DA clearance signals were relatively stable when a constant low amount of DA was locally applied into dSTR or NAc once every 5 min. To investigate this question, we applied DA at more frequent intervals and found that applications of DA every 2 or 3 min do indeed increase and prolong DA clearance signals in dSTR in a reversible manner, consistent with a transient loss of transporter activity (Gulley et al., 2002). However, surprisingly, similar changes are not observed in NAc. Likewise, systemic methamphetamine administration down-regulates DAT when measured ex vivo in synaptosomes prepared from dSTR, but not NAc (Kokoshka et al., 1999). We do not know the explanation for these intriguing regional differences. There are fewer DATs in NAc than in dSTR, but there is only one DAT gene and one gene product. These results suggest that DAT trafficking and its regulation may differ between dSTR and NAc, perhaps due to different retention complexes. This possibility will be very interesting to explore in the future. Like substrates, membrane depolarization also rapidly and transiently reduces DAT velocity (Sonders et al., 1997). In contrast, both hyperpolarization and interaction of DA with dopaminergic D2 receptors accelerate DA uptake, although these two phenomena appear to be unrelated (Mayfield and Zahniser, 2001). Several lines of evidence support D2 receptor-mediated regulation of DAT, including our results showing reduced in vivo clearance of DA in dSTR of D2 receptor knock-out mice and a pertussis toxin-sensitive, D2 receptor-mediated increase in DAT activity and surface binding in DAT-expressing oocytes (Dickinson et al., 1999; Mayfield and Zahniser, 2001). However, this regulation may be very transient because it has not been observed with [3H]DA uptake experiments in either synaptosomes or cultured DA neurons but has been seen with electrochemical measurement of DA

flux in synaptosomes (see Gulley and Zahniser, 2003). The positive results in synaptosomes and oocytes suggest that DA could regulate DAT trafficking, at least in part, by activating presynaptic D2 autoreceptors localized on DA neurons. Several other presynaptic receptors on DA neurons may also regulate DAT. Activation of glutamatergic mGlu5 receptors reduces DAT activity, whereas activation of GABAB and jopioid receptors increases function (see Gulley and Zahniser, 2003). Together, these observations suggest a complex rapid regulation of DAT by DA (Fig. 2). It is not clear if all of these mechanisms are engaged by synaptic release of DA, or even if they are, whether they would oppose one another or occur sequentially. However, one scenario based on the latter idea would be that depolarization would elicit release of DA, as well as transiently lower DAT velocity by reducing DAT cell surface expression. Also, by being a substrate, DA would further reduce DAT cell surface expression by a PKCdependent mechanism. Both of these mechanisms would allow the released DA to diffuse across the synaptic cleft and interact with its postsynaptic receptors, initiating signaling cascades. Subsequently, by interaction with presynaptic D2 autoreceptors, DA would not only increase DAT surface expression and velocity of uptake, but also inhibit further stimulation-evoked release of DA, thereby eliciting mechanisms that would work in concert to limit prolonged stimulation of postsynaptic DA receptors. Since cocaine can transiently up-regulate DAT expression, as well as increase extracellular DA by DAT inhibition, the net result of DAT regulation by cocaine may be even more complex than that for DA. These questions are likely to be resolved by using a combination of approaches, such as electrophysiological recording of DAT-associated currents and real-time imaging techniques in cultured DA neurons (Ingram et al., 2002). Growth factors like brain-derived and glial-derived growth factors, as well as insulin, may also regulate DAT expression via activation of protein kinase cascades, initiated by protein tyrosine kinase activation. Insulin receptors are expressed on DA neurons (Figlewicz et al., 2003). Streptozotocin treatment of rats, which results in chronic hypoinsulinemia, regulates DAT function and reduces amphetamine, but not cocaine, self-administration (Galici et al., 2003). Growth factormediated regulation could also be particularly critical during brain development. Furthermore, it will be important to understand this type of regulation because of the utility of primary neuronal and organotypic slice cultures as model brain systems for cellular and molecular investigations. Inhibitors of protein tyrosine kinases, but not a selective inhibitor of Src tyrosine kinases, down-regulate DAT activity and cell surface expression in DAT-expressing oocytes (Doolen

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and Zahniser, 2001). Conversely, insulin activates PI 3kinase and increases [3H]DA uptake and cell surface expression (Carvelli et al., 2002). Recently, we observed marked increases in [3H]DA uptake following in vitro exposure of rat dSTR synaptosomes to brain-derived growth factor and attenuation of this up-regulation by selective protein tyrosine kinase inhibitors (B.R. Hoover and N.R. Zahniser, unpublished observations). Together, these results raise the intriguing possibility that not only the natural substrate DA, but also endogenous growth factors and hormones, may be constantly fine-tuning the cell surface expression of DAT in DA neurons. It will be important to determine whether all of these endogenous factors utilize the same, or different, cellular mechanisms to regulate DAT activity by its trafficking.

5. DAT trafficking Effective and specific targeting of DAT to the synapses of dopaminergic neurons is required for the function of DAT in terminating DA signaling. This targeting involves transport of newly synthesized DAT from the endoplasmic reticulum (ER) through the Golgi complex, where DAT acquires N-glycosylation at the extracellular loops, and to the plasma membrane (Fig. 2). Electron microscopic studies demonstrated that DAT is localized in the plasma membrane of mesocorticolimbic DA neurons mostly in intermediate and distal dendrites, as well as in axons adjacent to the active zone of the synapse (Nirenberg et al., 1997a and 1997b). Concentration of DAT in these extra- or perisynaptic areas of DA neurons may require DAT interaction with specific retention complexes. By directly binding to DAT at the plasma membrane, these complexes may not only concentrate DAT at the synapse but also restrict or slow down DAT endocytosis. For example, proteins like PICK1 could be a part of such retention complexes (Torres et al., 2001). Whether the surface expression of DAT in neurons can be rapidly regulated by endocytosis has not been demonstrated directly. In fact, most of our knowledge about DAT endocytosis and other pathways of DAT trafficking has been derived from experiments with non-neuronal cells expressing transfected DAT. To analyze DAT trafficking, we generated fusion proteins of DAT with yellow (YFP) and cyan fluorescent proteins (CFP). CFP- and YFP-DAT displayed normal DA transport characteristics and biochemical properties when transiently or stably expressed in mammalian cells (Sorkina et al., 2003). Therefore, these fusion proteins were used to study various steps of DAT trafficking in living cells.

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DAT export from the ER is the first sorting step of transporter targeting in neurons. Proper folding of newly synthesized DAT and effective ER export depend not only on the integrity of the transmembrane domains of the transporter but also require carboxylterminal sequences of DAT (Sorkina et al., 2003; Torres et al., 2003a; Miranda et al., in press). DAT mutants lacking the last 3–5 amino acid residues are very slowly delivered to the cell surface due to retention in the ER (Sorkina et al., 2003; Torres et al., 2003a). The site-directed mutagenesis of the carboxylterminal tail of DAT revealed the importance of Gly585, Lys590 and Asp600 for the efficient exit of DAT from the ER (Miranda et al., in press). Coexpression of ER-exit deficient mutants with wild-type DAT results in reduced cell surface expression of the wild-type DAT (Sorkina et al., 2003; Torres et al., 2003a; Miranda et al., in press). These data suggest that the wild-type and mutant DATs can form complexes in the ER and that hetero-oligomers containing mutant DATs are not capable of efficient exit from the ER. To test directly whether DAT is an oligomer in the ER, we co-expressed CFP- and YFP-tagged DAT and examined their interactions using fluorescence resonance energy transfer (FRET) in living cells. FRET microscopic analysis revealed that DAT is oligomerized in the ER and is maintained in an oligomeric state in the plasma membrane and other cellular compartments (Sorkina et al., 2003; Fig. 2). These data are consistent with the model whereby oligomerization is necessary for the efficient exit of DAT from the ER (Torres et al., 2003a). Oligomerization of DAT may also play an important role in the DA transport activity of DAT and in the inhibition of this activity by cocaine (Hastrup et al., 2003). Elucidation of the mechanisms of rapid DAT endocytic trafficking at neuronal synapses requires addressing several questions: (1) what are the kinetics of constitutive endocytosis and recycling? (2) can signaling by DAT substrates, DAT inhibitors and presynaptic receptors influence these processes? (3) what are the molecular signals in the DAT molecule that control constitutive and induced internalization and recycling? (4) what are the proteins that interact with DAT and control DAT endocytic trafficking? To date, attempts to address these issues have been made mostly in nonneuronal expression systems. These studies revealed different rates of constitutive endocytosis in different cell models, such as MDCK cells (Daniels and Amara, 1999) and PC12 cells (Loder and Melikian, 2003). We have used porcine aortic endothelial (PAE), and human HeLa and HEK293 cells as DAT expression systems. In all of these cells, a pool of untagged DAT or YFP/CFP-tagged DAT was present in the early endosomes under steady-state conditions, indicative of constitutive internalization. However, more detailed

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kinetics studies are necessary to demonstrate that endosomal transporters have been internalized from the plasma membrane, rather than delivered to endosomal compartments on their way from the ER and Golgi. The proper measurement of the specific rates of DAT internalization and recycling is, however, complicated because methods for measurement of surface DAT expression are limited by conventional biotinylation techniques. Radioligand binding cannot be used to distinguish surface from intracellular DATs in intact cells (except oocytes) with significant levels of intracellular DATs. Furthermore, DAT antibodies that recognize the extracellular sequence(s) of DAT in living cells are not available. Subcellular fractionation techniques do not allow quantitative separation of plasma membrane and early endosomal cargo, nor are they sensitive enough to perform rapid analysis of internalization and recycling kinetics. Given these constraints, DAT endocytosis has been detected primarily through the observation of downregulation of surface DAT and accumulation of intracellular DAT in cells induced by either in vitro activation of PKC (Daniels and Amara, 1999; Gra˚na¨s et al., 2003) or incubation with amphetamine (Saunders et al., 2000). Using live-cell imaging, we have demonstrated the accumulation of YFP- and CFP-DAT in early endosomes in cells treated with phorbol ester or amphetamine (Sorkina et al., 2003). Interestingly, within the endosomal compartments, DAT was excluded from the subdomains containing active Rab5 and hepatocyte growth factor receptor substrate (HRS), which are proteins responsible for endosome fusion and sorting to lysosomes, respectively. However, it remains unclear whether PKC- or amphetamine-dependent endosomal accumulation of DAT is due to accelerated internalization or retarded recycling of constitutively internalized DAT, or both (Loder and Melikian, 2003). A dominant-negative mutant of dynamin inhibits both PKC- and amphetamine-dependent down-regulation of DAT, suggesting that down-regulation in both cases is mediated either by clathrincoated pits or caveolae (Daniels and Amara, 1999; Saunders et al., 2000). In order to dissect the process of DAT internalization, we have recently developed molecular tools such as a series of small interfering RNAs (siRNAs) targeted to 13 proteins involved in clathrinmediated endocytosis (Huang et al., in press). Analysis of the effects of these siRNAs on DAT endocytosis is currently in progress in our laboratory. The application of the molecular tools for the analysis of DAT endocytosis in DA neurons by biochemical and live-cell microscopy methods requires development of an in vitro experimental model. Although embryonic and postnatal primary cultures of midbrain neurons can be used for single cell analyses of endocytosis, biochemical analysis of endocytosis has not been conduc-

ted in these cultures. On the other hand, whereas biochemical experiments can be performed using synaptosomal membrane preparations, this experimental model does not allow analysis of DAT trafficking by live-cell microscopy or the use of genetically encoded inhibitors of endocytosis. Perhaps organotypic nigrostriatal slice co-cultures will be an appropriate experimental model (e.g. Plenz and Kitai, 1998); slice co-cultures not only might allow various types of analysis, but also would be expected to possess a set of relevant regulatory components that would enable study of DAT regulation and trafficking under physiological conditions.

6. Summary and future directions During the past few years, it has become clear that not only is cell surface expression of DAT regulated, but also that it can be regulated rapidly. The dynamic regulation mediated by DA could potentially be complex because of its opposing regulatory effects as a DAT substrate and D2 receptor agonist. It is also provocative that brief exposure to stimulants can result either in transient down-regulation (amphetamines) or up-regulation (cocaine) of DAT expression. Our work suggests that differences in DAT basal expression and/ or regulation help to explain the differential individual responsiveness of rats to cocaine, both in terms of their initial locomotor stimulation and cocaine-induced locomotor sensitization. Knowing if VTA DA neuronal firing and release differs between low and high cocaine responding phenotypes might help explain differential baseline DAT expression and/or drug-induced DAT regulation. We have also begun to identify mechanisms critical for constitutive DAT trafficking. To date, we have focused on the NAc core because it is crucial for cocaine-induced locomotor activation, whereas the shell subregion of NAc is more likely involved in the drug rewarding/reinforcing effects. Thus, future studies need to focus on the potential contribution of DAT regulation in NAc shell to behaviors like cocaine-induced conditioned place preference and self-administration. Inbred animals with differing steady-state levels of DAT expression would be helpful for exploring contributions from basal DAT activity to stimulant-induced activation and reward. DAT has been over-expressed by 20%–30% in transgenic mice by using the promoter for the catecholamine synthetic enzyme tyrosine hydroxylase to drive expression of a rat DAT cDNA variant in DA neurons (Donovan et al., 1999). These mice habituate to a novel environment more quickly and show enhanced reward. However, except at high doses, their cocaine-induced locomotor activity is similar to wild-type mice. Mice with a DAT knockdown mutation resulting in only

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10% of normal DAT have been used to study the effects of chronically elevated extracellular DA concentrations on food intake and reward (Pecin˜a et al., 2003). The Cre-loxP system and an adeno-associated viral vector have been used to focally delete the adenosine A1 receptor in hippocampus of adult mice (Scammell et al., 2003). This approach could be used as well to eliminate DAT in a neuron-specific manner. Other challenges for the future are to understand how the dynamic constitutive and drug-induced DAT regulation occurs in the brain, how it contributes to normal neurotransmission and how it plays a role in the addictive properties of stimulants. To meet these challenges, electron microscopic studies before and after exposure to psychostimulants would be useful. As already mentioned, we also need to develop in vitro DA neuronal model systems that will allow both single cell and biochemical analyses of DAT endocytosis. In addition to mammalian models, C. elegans, Drosophila and zebrafish possess DATs and provide genetically tractable models that may aid in dissecting relevant pathways (Jayanthi et al., 1998; Andretic et al., 1999; Holzschuh et al., 2001; Po¨rzgen et al., 2001; Rothenfluh and Heberlein, 2002; Nass and Blakely, 2003). Using all of these systems, we next need to identify specific regulators of DAT endocytosis. Then, rather than targeting general endocytosis proteins, we could use siRNAs or other inhibitors of these specific regulators to analyze their impact on the function of DAT in the brain. We also need to be able to identify DA neurons and visualize DAT in these neurons, both in vitro and in vivo. An ectodomain DAT antibody that recognizes DAT in living cells would be extremely helpful. Expression of green FP (GFP) under control of tyrosine hydroxylase has been used to generate transgenic mice with GFP-labeled DA neurons (Sawamoto et al., 2001). This approach allows visualization and enrichment of isolated live DA neurons and could be modified to generate mice with fluorescently tagged DAT. Knock-in mice expressing GAT1-GFP protein fusions have already been generated and found useful for measuring surface density of these transporters at presynaptic structures in several brain regions (Chiu et al., 2002). Such novel approaches should help us to better understand how DAT regulation contributes to normal neurotransmission and to the addictive properties of stimulants. Acknowledgements We thank the members of our labs for their many valuable contributions to this work and NIDA for generous support (DA R37 DA04216, R01 DA14204 and K05 DA15050).

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