Cocaine and the Changing Brain
Regulation Of The Dopamine Transporter And Cocaine Sensitization
Nancy R. Zahniser, Ph.D.
University of Colorado School of Medicine
Our particular interest is in determining how changes in dopamine transporter (DAT) function may contribute to cocaine sensitization (Zahniser et al. 1995). The DAT is critical in determining the concentration of extracellular dopamine (DA) and, thus, overall dopaminergic tone. Cocaine blocks all three neuronal membrane monoamine transporters with approximately equal affinity. By blocking the DAT, cocaine allows released DA to persist in the extracellular space, prolongs DA receptor stimulation, and results in behavioral activation.
Repeated, intermittent administration of cocaine often results in enhanced locomotor responsiveness or behavioral sensitization (Kalivas and Stewart 1991). This phenomenon is not unique to cocaine; other psychomotor stimulants, some other classes of drugs, and stress all can induce behavioral sensitization. One of the hallmarks of behavioral sensitization is its persistence. Sensitized behavioral responses can be observed in response to a challenge dose of cocaine administered days to months after withdrawal from repeated drug treatment. However, it should be noted that, at least with the cocaine treatment and withdrawal regimens that we have used, some of the rats (25 to 50 percent) do not exhibit behavioral sensitization (Cass et al. 1993a). These "nonsensitized" rats generally demonstrate relatively more activation than the "sensitized" rats to the initial cocaine injection.
Behavioral sensitization likely reflects synaptic plasticity and, in fact, shares some properties with long-term potentiation, a more well-known type of synaptic plasticity. Many changes associated with psychomotor stimulant-induced behavioral sensitization-including neurochemical, electrophysiological, molecular, and morphological changes-have been identified. Besides being of interest to basic neuroscientists, behavioral sensitization may have clinical relevance. Several lines of evidence (Robinson and Berridge 1993) suggest that sensitization plays a role in human cocaine abuse. In particular, behavioral sensitization may contribute to the intense craving, high relapse rate, and paranoid psychosis associated with cocaine abuse.
Do Changes In DAT Function Contribute To Expression Of Cocaine Sensitization?
Since cocaine directly interacts with the DAT, we hypothesized that repeated cocaine administration might induce long-lasting changes in the activity of the DAT and that these changes might contribute to the expression of behavioral sensitization. Whereas changes at DA neuronal cell bodies are critical for initiation of sensitization, changes in the brain regions containing the DA neuronal terminals are thought to mediate the persistence, or expression, of sensitization (Kalivas and Stewart 1991). Thus, we focused on the DAT in dorsal striatum and nucleus accumbens. To address our hypothesis, Wayne Cass, in collaboration with Greg Gerhardt, developed an electrochemical method using high-speed chronoamperometry for measuring in vivo clearance of exogenously applied DA. We carried out a number of studies to demonstrate that DA clearance measured with this method reflects the activity of the DAT (Cass et al. 1993b).
To measure exogenous DA clearance, electrode-pipette assemblies were constructed, calibrated in vitro, and then implanted under stereotaxic control into dorsal striatum and core of the nucleus accumbens of urethane-anesthetized rats. The electrode-pipette assembly consisted of a glass micropipette glued a fixed distance (~300 &m) from a single, Nafion-coated carbon fiber electrochemical electrode. Pressure ejection of finite volumes of DA (200 &M barrel concentration, 12-200 nl) once every 5 minutes resulted in transient and reproducible DA signals that rose rapidly to approximately 2 &M and then decayed within 0.5 to 1.5 minutes. DA clearance rate was determined from the slope of the initial linear portion of the decay curve, that is, the slope between the points when the signal had decayed by 20 percent and 60 percent from its maximal amplitude. The density of DATs in dorsal striatum is 40 percent higher than in nucleus accumbens, and consistent with this observation, baseline clearance rates were 80 percent faster in dorsal striatum (164 Å 29 nM/s, n = 15) than in nucleus accumbens (90 Å 14 nM/s). DA clearance rates were measured in rats that received a single acute injection of either saline (1 ml/kg, i.p.) or cocaine (10 or 20 mg/kg, i.p.). The effect of the 10 mg/kg dose did not differ from that of saline in either brain region. However, clearance rate only in nucleus accumbens decreased to a greater extent in response to the 20 mg/kg dose of cocaine compared with the 10 mg/kg dose (see figure 1). Thus, the nucleus accumbens with its lower density of DATs was more sensitive to the effects of cocaine, suggesting that there may be "spare" transporters in the dorsal striatum. We have also observed a greater sensitivity of nucleus accumbens, versus dorsal striatum, with local application of cocaine (Cass et al. 1993b).
DA clearance was determined following withdrawal from repeated cocaine administration (Cass et al. 1993a). Rats received once-daily intraperitoneal injections of either saline or cocaine (10 mg/kg) for 1 week. Treatment was withheld for 1 week, and then a challenge dose of either saline or cocaine (10 mg/kg), respectively, was administered while the rats were anesthetized during the in vivo electrochemical recording session on day 15. Saline challenge, following repeated saline injection and withdrawal, did not alter DA clearance rate in nucleus accumbens. In contrast, the 10 mg/kg challenge dose of cocaine significantly attenuated clearance in the nucleus accumbens of the rats that had been withdrawn from repeated cocaine treatment (see figure 1). In fact, the 10 mg/kg cocaine challenge dose decreased DA clearance rate in the repeatedly treated/withdrawn rats to an extent similar to that of the acute 20 mg/kg dose in naive rats (figure 1). This result is consistent with cocaine-induced behavioral sensitization; diminished DA clearance would be expected to result in higher extracellular concentrations of DA and greater behavioral activation.
Conclusions and Future Directions. By analogy to our initial findings in dorsal striatum and nucleus accumbens discussed above, one explanation for a greater sensitivity of the DAT to the challenge dose of cocaine in the nucleus accumbens of rats treated repeatedly and withdrawn from cocaine would be a decreased density of DATs. Several hours after the conclusion of the in vivo electrochemical experiments, the rats were sacrificed, and quantitative autoradiography with [3H]mazindol was used to study the DATs. No differences were detected in the density of DATs or in the affinity of cocaine for the DAT in nucleus accumbens (Cass et al. 1993a). However, several other groups have observed decreased densities of DATs selectively in nucleus accumbens following 10 to 60 days of withdrawal from repeated cocaine administration (Kuhar and Pilotte 1996). Furthermore, this is one of the most persistent neurochemical alterations, with a duration similar to that of behavioral sensitization, that has been identified following repeated cocaine administration (Kuhar and Pilotte 1996).
||Figure 1. Enhanced inhibition of DA clearance in nucleus accumbens of rats given a challenge dose of cocaine following withdrawal from repeated cocaine treatment compared with acute administration of cocaine. Groups of rats were injected i.p. with the dose of cocaine indicated. The repeated cocaine treatment regimen is indicated below the graph; the challenge dose (C) was administered on day 15. Mean values ± SEM are shown for n = 4-5.
Greater cocaine-induced inhibition of DA clearance was observed in the nucleus accumbens of all rats withdrawn from repeated cocaine treatment when compared with either administration of saline or acute administration of the same dose of cocaine. This result is consistent with cocaine-induced behavioral sensitization. However, since the rats were anesthetized during the electrochemical experiment when the challenge dose of cocaine was administered, it was impossible to determine whether these rats exhibited behavioral sensitization in response to the challenge dose. Their behavior had been monitored on the first and last days of the repeated treatment, and only about half of the rats exhibited behavioral sensitization (Cass et al. 1993a). Therefore, to test our hypothesis further we need to measure DA clearance and behavior concomitantly. Greg Gerhardt, Charlie Ksir, and Chad Pivik have developed the technology to measure DA clearance in the freely behaving rat. The electrode-pipette assembly is fabricated from fused silica tubing, and the headstage has been miniaturized. An acute injection of cocaine induces a simultaneous change in both locomotor activity (measured with a computer-based activity chamber) and exogenous DA clearance in nucleus accumbens. The altered clearance appears to be somewhat longer lasting than the altered behavior, as observed by other groups using in vivo microdialysis to measure extracellular endogenous DA concentrations in freely behaving rats. Shelly Dickinson has recently established this methodology in my laboratory and is currently conducting experiments to test the long-term stability of the in vivo exogenous DA clearance measurements in the chronically implanted rats. This approach can be used to determine whether the cocaine-induced alterations in DAT function are associated with expression of behavioral sensitization.
By What Mechanisms Are DAT Activity And Expression Regulated And Modulated?
The idea that regulation of DAT activity and expression might contribute to cocaine-induced behavioral sensitization leads to curiosity about the mechanisms that might be involved generally in DAT regulation. In 1993 I collaborated with Susan Amara, Mark Sonders, and Mike Kavanaugh at the Vollum Institute, Oregon Health Sciences University, to show that the human DAT (hDAT) expressed in Xenopus laevis oocytes is electrogenic (Sonders et al. 1997). As expected based on other investigators' conclusion that DAT cotransports two Na+ ions, one Clå ion, and one DA+ molecule, we found (using a two-electrode voltage clamp) small, but measurable and concentration- dependent, inward currents induced by DA and other DAT substrates. The transport-associated currents were voltage dependent, increasing at more hyperpolarized potentials. Somewhat more unexpected, however, was the observation that DAT blockers, like cocaine, induced small outward currents in hDAT-expressing oocytes. This was shown to be due to blockade of an inward cation leak associated with hDAT expression. Blockade of this leak current by substrates was also apparent at more depolarized holding potentials (>0 mV) and under conditions when transport was abolished, such as substitution of Na+ by Li+ in the buffer. An important prediction from these observations was that DA uptake by the hDAT should also be voltage dependent. Si-Jia Zhu, working in my laboratory, measured uptake in the hDAT-expressing oocytes under voltage clamp and found that this was indeed the case. Hyperpolarization increased the velocity of the transporter, whereas depolarization decreased velocity (Sonders et al. 1997). A change of 30 mV altered DA uptake by 25 percent. These results suggest one mechanism, changes in membrane potential, that may transiently regulate the activity of the DAT.
In order to determine whether activity of the DAT in the brain is also regulated by membrane potential, Alex Hoffman, Greg Gerhardt, Carl Lupica, and I have begun to carry out electrochemical measurements in rat brain slices containing the substantia nigra pars compacta (SNc) (Zahniser et al. 1998). Characterization experiments indicate that the DAT is still the major determinant of exogenous DA clearance in the SNc. The patch-clamp method was used to monitor changes in the membrane potential of SNc neurons in the hemisphere opposite to that where DA clearance was measured. Neurons were identified as being DA-like by their characteristically long-duration action potentials. As our first approach to alter membrane potential, the effect of increasing the concentration of KCl in the superfusion buffer was investigated. Tetrodotoxin (0.5 &M) was included throughout these experiments to block voltage-gated sodium channels and to ensure that any effects of KCl on DA clearance were due to direct voltage changes and not to changes in Na+ gradients. Elevating the KCl concentration to 30 mM resulted in a 30-mV depolarization and decreased DA clearance by 35 percent. Although preliminary, these results suggest that DAT velocity in the rat brain, similar to that of the hDAT expressed in oocytes, is transiently reduced by membrane depolarization. Future experiments will use different strategies to hyperpolarize, as well as to depolarize, membrane potential. The idea that the DAT is voltage dependent is also interesting because it provides a potential mechanism by which presynaptic receptors, such as D2 DA autoreceptors, could influence DAT activity. Several groups have demonstrated that D2 receptor ligands can modulate rat striatal DA clearance measured both in vitro and in vivo (Cass and Gerhardt 1994). Their results demonstrate that activation of D2 receptors decreases, whereas antagonism of these receptors increases, DA clearance rate. This is exactly what we would have predicted based on the fact that D2 receptors open potassium channels, which hyperpolarize the membrane. Shelly Dickinson has employed another approach to address the question of D2 DA receptor/DAT interactions. She has measured in vivo DA clearance in dorsal striatum of the D2 receptor knockout mice produced by Malcolm Low, David Grandy, Michele Kelly, and Marcelo Rubinstein of the Vollum Institute. She found that a significantly larger volume of DA had to be ejected in the wildtype than in the knockout mice to produce a maximal signal amplitude of 2-3&M. The DA clearance rate was also higher in the wildtype mice. These results are consistent with the idea that the exogenous DA that we normally apply to measure clearance is interacting with D2 receptors, accelerating DAT velocity, and thereby increasing DA clearance. In the knockout mice, DAT clearance is attenuated because they lack D2 receptors. Thus, our results again support the idea that release of DA and activation of D2 autoreceptors produce two effects that would reduce extracellular concentrations of DA-inhibition of further DA release and acceleration of DA uptake. Dayne Mayfield is currently investigating the effects of coexpressing the D2 DA receptor and appropriate potassium channels, along with the hDAT, in oocytes, since in this system the contribution of changes in membrane potential to D2 receptor/DAT interactions can be evaluated.
Phosphorylation of the DAT is perhaps the most obvious mechanism for regulation of its activity. The hDAT contains two consensus sites for phosphorylation by protein kinase C (PKC). Several groups-using rat striatal synaptosomes, primary mesencephalic cultures, or heterologous cell expression systems-have shown that DAT activity is sensitive to PKC activation (Zhu et al. 1997). Si-Jia Zhu used the oocyte expression system to investigate the mechanism by which PKC regulates the hDAT (Zhu et al. 1997). She found that bath application of the PKC activator phorbol 12-myristate 13-acetate (PMA) dose and time dependently diminished [3H]DA uptake in the hDAT-expressing oocytes. The IC50 was 22 nM, and 100 nM PMA produced a maximal effect. The effect of PMA (100 nM) appeared to be due to activation of PKC because it was partially reversed by the selective PKC inhibitor bisindolylmaleimide (1&M) and was not mimicked by the inactive PMA analog 4-ä-phorbol-12,13-didecanoate (400 nM). The reduction in [3H]DA uptake was due to decreased maximal velocity of transport (Vmax), with no change in transporter affinity. Exposure to 100 nM PMA for 30 minutes reduced Vmax by 69 Å 11 percent. Similarly, exposure to 100 nM PMA for 3 to 5 minutes, followed by 15 to 30 minutes of washing, reduced both the transport- associated and leak currents by 50 to 80 percent. Changes in oocyte cell surface area induced by PMA exposure were determined from capacitive transients. Baseline membrane capacitance did not differ among uninjected oocytes, hDAT-expressing oocytes, uninjected oocytes exposed to PMA, and hDAT-expressing oocytes exposed to PMA. Pretreatment for 3 to 5 minutes with 100 nM PMA produced a time-dependent decrease in membrane capacitance selectively in the hDAT-expressing oocytes; after 30 minutes of washing, the loss was approximately 40 percent. Saturation curves generated with [3H]mazindol in intact oocytes revealed a 78-percent decrease in the density of binding sites, with no change in affinity, following PMA pretreatment. However, no change in [3H]mazindol binding was observed in homogenates, which would contain both cell surface and internal membranes, from oocytes exposed to PMA. Taken together, these results suggest that PMA, via activation of PKC, alters cell surface trafficking of the hDAT, rather than converting the hDAT to a less active state.
Conclusions and Future Directions.
Our studies suggest several mechanisms by which DAT activity can be regulated. DAT velocity is transiently altered by changes in membrane potential and by activation of D2 DA receptors. Whether these two observations are related will require additional experimentation in the hDAT-expressing oocytes and rat brain slices. However, our results demonstrate that depolarization diminishes DAT activity, whereas hyperpolarization accelerates it. Our results also support the notion that increases in extracellular DA could produce two effects via activation of D2 autoreceptors, inhibition of further DA release and acceleration of DA uptake, that would work in concert to reduce extracellular DA and limit postsynaptic receptor activation.
Activation of PKC reduces hDAT activity in the oocyte expression system by inducing changes in membrane trafficking of the transporter. These observations raise the possibility that PKC-coupled presynaptic receptors, such as Class 1 metabotropic glutamate receptors, could mediate decreases in DAT expression that, in turn, would be expected to enhance and prolong DA neurotransmission. PKC-induced increases in endocytosis have been observed with three other Na+/Clå-dependent neurotransmitter transporters-GABA, taurine, and serotonin transporters-and the Na+/glucose cotransporter (Zhu et al. 1997). All of this evidence suggests that alterations in membrane trafficking of transporters is an effect common to PKC activation. Whether this mechanism, or another similar phosphorylation-dependent mechanism, mediates the persistent changes in DAT activity and/or expression induced by repeated cocaine administration remains to be explored in future experiments.
Many wonderful collaborators have participated in this work. They include Wayne Cass, Greg Gerhardt, Kelly Gillespie, Pam Curella, Dayne Mayfield, Karen Flach, Gaynor Larson, Si-Jia Zhu, Shelly Dickinson, Jilla Sabeti, Alex Hoffman, and Carl Lupica at the University of Colorado; Charlie Ksir and Chad Pivik at the University of Wyoming; and Susan Amara, Mark Sonders, Mike Kavanaugh, Malcolm Low, David Grandy, Michele Kelly, and Marcelo Rubinstein at the Vollum Institute, Oregon Health Sciences University. The work from my lab was supported by National Institutes of Health Grant No. DA-04216, career development award DA-00174, and postdoctoral fellowship DA-05706.
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