Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochernistry
Differences in Dopamine Clearance and Diffusion in Rat Striatum and Nucleus Accumbens Following Systemic Cocaine Administration *Wayne A. Cass, *?$Greg A. Gerhardt, *R. Dayne Mayfield, *Pamela Curella, and *$Nancy R. Zahniser Departments of *Pharmacology and ?Psychiatry and $Neuroscience Training Program, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.
Abstract: Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCI (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopa-
mine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine. Key Words: Cocaine-Dopamine transporter-Striatum-Nucleus acuptake-Dopamine cumbens-In vivo electrochemistry-Behavior. Cass W. A. et al. Differences in dopamine clearance and diffusion in rat striatum and nucleus accumbens following systemic cocaine administration. J. Neurochern. 59, 259-266 ( 1 992).
Cocaine is a psychomotor stimulant whose neurochemical actions include blocking the high-affinity reuptake of dopamine (DA), norepinephrine, and serotonin back into monoamine nerve terminals (Ritz et al., 1987). Present evidence indicates that some of the behavioral effects (increased locomotor activity, stereotypies)and reinforcing properties of cocaine involve its effects on DA systems. In rats, psychomotor stimulants increase locomotor activity primarily by activating mesolimbic DA pathways, whereas the nigrostriatal DA system is more involved in producing stereotyped behaviors (Costal1 and Naylor, 1977; Delfs et al., 1990). The reinforcing properties of co-
caine appear to involve increased activity in the mesolimbic and mesocortical DA pathways (Wise and Bozarth, 1987; Kuhar et al., 1991). This cocaine-induced enhancement of activity in DA systems is likely due to the ability of cocaine to increase synaptic DA concentrations. Studies using in vivo microdialysis have demonstrated that a single systemic injection of cocaine increases extracellular DA concentrations in both striatum (Church et al., 1987; Carboni et al., 1989; Di Paolo et al., 1989; Hurd and Ungerstedt, 1989; Akimoto et al., 1990) and nucleus accumbens (NAc) (Bradberry and Roth, 1989; Carboni et al., 1989; Moghaddam and Bunney, 1989;Ka-
Received September 12, 199 1; revised manuscript received December 10, 199 I ; accepted December 3 1, I99 1. Address correspondence and reprint requests to Dr. W. A. Cass at Department of Pharmacology, C-236, University of Colorado Health SciencesCenter, 4200 East 9th Avenue, Denver, CO 80262, USA.
Abbreviations used: DA, dopamine; NAc, nucleus accumbens; TwB0,time for the signal to decay in amplitude beginning at the time point when the signal had declined by 40% of its amplitude until it had decreased by 80%of its amplitude.
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h a s and Duffy, 1990). A peripheral injection of cocaine also causes concurrent increases in the DA oxidation current measured with in vivo chronoamperometry in NAc and in the rate of self-stimulation (Phillips et al., 1989). Local application of cocaine in the striatum produces similar results: It augments the extracellular concentration of DA detected with in vivo microdialysis under basal conditions (Nomikos et al., 1990) as well as the DA concentration detected with in vivo electrochemistry in response to potassium stimulation (Gerhardt et al., 1988). An intriguing finding from one of the in vivo microdialysis studies was that peripheral administration of cocaine to freely moving rats increases extracellular DA concentrations in the NAc to a greater extent than in the striatum (Carboni et al., 1989). This preferential increase in the NAc could be due to the ability of cocaine to inhibit DA uptake and/or to increase DA release to a greater extent in the NAc. In the present study we used in vivo electrochemistry to measure directly the clearance and diffusion of exogenously applied DA in the striatum and NAc to determine if a differential effect of cocaine on DA uptake could explain this observation. The relationship of the electrochemical data obtained in anesthetized rats to the effects produced by cocaine in unanesthetized animals was investigated using behavioral measurements. Quantitative autoradiography with [3H]mazindolwas used to define further the locus of the differential effect of cocaine on the DA transporter, and these results were compared with tissue levels of DA in the striatum and NAc. MATERIALS AND METHODS Animals Male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE. U.S.A.) weighing 200-350 g were used for all experiments. They were housed in groups of four to six under a 12-h light-dark cycle with food and water freely available.
In vivo electrochemistry Rats were anesthetized with urethane (1.25- 1.5 g/kg of body weight) and prepared for electrochemistry as previously described (Gratton et al., 1989). A micropipette/Nafion-coated carbon fiber electrode assembly (Gerhardt et al., 1987;Gratton et al., 1989) was lowered into the dorsal stnatum (1-1.5 mm anterior to bregma, 2.2 mm lateral from midline, and 4-4.5 mm below the dura) or NAc (1.5 mm anterior, 2.2 mm lateral, and 6.5-7 mm below the dura). The micropipette (tip diameter, 10-1 5 pm; positioned 300 k 30 pm from the electrode tip) contained 200 @A4 DA (Sigma Chemical Co., St. Louis, MO, U.S.A.)and 100 p M ascorbic acid in 0.9% NaCl (pH 7.4). The carbon fiber electrodes each contained one to three fibers (fiber diameter, 30 pm; exposed length of 100 pm) sealed in a glass capillary. They were calibrated as previously described (Gerhardt et al., 1987; Gratton et al., 1989). The electrodes were coated with Nafion to give a selectivity for DA over ascorbic acid that averaged 1.064 f 224: 1 (Gerhardt et al., 1984, 1987;
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Mitchell and Gratton, 199 1). All electrodes exhibited linearity for calibration with 1-8 pM DA; correlation coefficients ranged from 0.9994 to I .OOOO. The sensitivity of the electrodes to DA was 26,553 f 2,564 digital counts per 1 pA4 change in DA concentration. Based on the measured detection limit of the electrodes, the amplitude of the signal had to reach 46 f 14 nM DA to achieve a signal-to-noise ratio of 3.0. DA was pressure-ejected (25-100 nl, 5-30 psi for 2-5 sec) at 5-min intervals into the striatum or NAc. The amount ejected was monitored by measuring the volume of fluid displaced in the micropipette using a dissection microscope and was based on previous calculations that there is 246 nl of solution in a I-mm segment of the micropipette. Highspeed chronoamperometric electrochemical measurements (IVEC-5 system; Medical Systems Corp., Greenvale, NY, U.S.A.)were continuously made at 5 Hz and averaged to 1 Hz (oxidation potential, 0.55 V for 100 ms versus a Ag/ AgCl reference electrode; resting potential, 0.0 V for 100 ms; oxidation and reduction currents digitally integrated during the last 90 ms of each 100-ms pulse) as previously described (Gerhardt et al., 1989h; Gratton et al., 1989). After a steady baseline was achieved and the DA signals were reproducible, rats were injected with either saline ( 1 ml/kg, i.p.1 or cocaine-HCI (Sigma) in saline (15 or 20 mg/ kg, i.p.). DA was pressure-ejected at 5-min intervals for an additional 65 min. Each animal received only one cocaine injection. Some control animals received two saline injections so that recordings from dorsal striatum and NAc were made in the same animal. At the end of the experiments, brains were fixed by immersion in 10% neutral buffered formalin. The brains were later frozen, sectioned, and stained with cresyl violet acetate to verify electrode placement. Only animals in which the recording electrodes were positioned in the dorsal striaturn or NAc were included in the results.
Behavior Locomotor and stereotypic activities were determined according to the method ofBarret al. (1983) with minor modifications. Behaviors were rated by an experienced observer who was unaware of the drug treatment. One day before behavioral testing the rats were placed in a cylindrical wire mesh chamber (diameter, 30 cm; height, 38 cm), allowed to habituate for I h, then given a saline injection ( 1 ml/kg, i.p.), and kept in the chamber for an additional hour. The next day the animals were placed in the chambers and given 1 h to habituate, and then behavioral rating was begun. Each animal was rated for 2 min at 10-min intervals beginning 10 min before injection of saline or cocaine-HCl(l5 or 20 mg/kg, i.p.) and continuing for 60 min after injection. Locomotor activity (quadrant crossing) was rated as the number of times the head and forepaws crossed a quadrant boundary during each 2-min rating period. Stereotypic activity (head bobbing) was defined as occurrence of repetitive up-and-down or side-to-side head movement (not including movements due to breathing or grooming) and represents the number of 10-s intervals during each 2-min rating period in which head bobbing occurred (the maximal possible score per rating period was 12).
[3H1Mazindol binding Quantitative autoradiographic analysis of binding of [3H]mazindol (Dupont-New England Nuclear, Boston,
COCAINE AND DOPAMINE TRANSPORTER MA, U.S.A.) in rat dorsal striatum and NAc was carried out as described by Marshall et al. (1990) with the following changes. Sections 10 pm thick were used [from 1.2 to 1.6 mm anterior to bregma according to the atlas of Paxinos and Watson (1986)l. The concentration of [3H]mazindol used for competition curves was 10 nM, and that for saturation curves was 1-30 nM. GBR 12909 { l-[2-[bis(4-fluorophenyl)methoxy]ethyl]d-(3-phenylpropyl)piperazine; 1 pM, a gift from Novo Industri A/S, Bagsvaerd, Denmark} was used to define nonspecific binding. Two 5-min washes in ice-cold buffer followed the incubations. The computerbased analysis was carried out as previously described (Pens et al., 1990).
A
DA levels in the dorsal striatum and NAc were determined by HPLC with electrochemical detection. The NAc was dissected from a coronal slice as described by Horn et al. (1974). Dorsal striatum was taken from the same slice. The samples were weighed (approximate tissue weights: dorsal striaturn, 20 mg: NAc, I5 mg) and sonicated in 1 ml of 0.1 A4 perchloric acid. After filtration the samples were injected into an HPLC system. The HPLC conditions and calculations were as previously described (Gerhardt et al., 1989~). DA levels are expressed as micrograms per gram wet weight of tissue.
Data analysis Two parameters were examined from the electrochemistry experiments: the maximal amplitude ofthe signal resulting from the ejection of DA and the time for the signal to decay in amplitude beginning at the time point when the signal had declined by 40% of its amplitude until it had see Fig. 1). For decreased by 80% of its amplitude ( T40-80; analysis, the values for these parameters at 5 min before injection of cocaine or saline and immediately following injection were averaged to obtain a baseline value. The values of the time points were then calculated as percent change from baseline. Electrochemistry data were analyzed using two-factor analysis of variance with repeated measures followed by Newman-Keuls post hoc comparisons. Behavioral data were analyzed with repeated-measures analysis of variance after being transformed to common logarithms to meet the assumptions of analysis of variance (Sokal and Rohlf, 198l ) . Post hoc comparisons were made using univariate F tests. Parameters from the binding experiments were determined by iterative curve fitting (GraphPADSoftware, San Diego, CA, U.S.A.). Binding and biochemical data were analyzed with paired I tests. All data are given as mean f SEM values, where n indicates the number of rats.
RESULTS In vivo electrochemistry The ejection of a finite amount of DA into the striatum or NAc at 5-min intervals produced a transient and reproducible electrochemical signal (Fig. I ). In saline-injected controls, the amplitude of the signals (range, 0.35-3.16 p M ) significantly decreased by 2636% ( p < 0.05) during the recording sessions (Fig. 2), whereas the T40-80interval (range, 5-54 s) remained constant (Fig. 3). The time for the signals to decrease
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FIG. 1. Reproducibility of electrochemical signals from local application of DA in the dorsal striatum (A) and NAc (6) of the rat. A constant amount of DA was pressure-ejected(asterisks) at 5-min intervals, and the resulting oxidation signals for three consecutive applications are shown for both regions. The oxidation currents are shown as 1-s averages from a 5-Hz recording rate and are converted directly to rnicromolar concentrations by using calibration factors determined in vitro. Also shown in 6 are the time points used to calculate the T.+*,, values.
by 50% oftheir amplitude was greater in the NAc than in the striatum: dorsal striatum. 19.3 f 2.4 s; NAc, 30.3 +_ 3.9 s (n = 12, p < 0.05). The systemic administration of cocaine produced differential effects in the striatum and NAc. A dose of 15 mg/kg of cocaine-HCI had no effect on the amplitude of the DA signal in the striatum, but the signal increased 55.4% compared with saline controls in the time course increased in both NAc (Fig. 2). The T40-80 areas at this dose (Fig. 3); however, the change was statistically significant only in the NAc. When 20 mg/ kg of cocaine was administered, changes occurred in both the striatum and NAc. The amplitude and the T40-80 of the signals increased in both regions, although the increases were greater in the NAc (Figs. 2 and 3). The baseline remained stable in all animals during the recording sessions and was not altered in animals that received cocaine. Similarly, the rise times for the signals, a measure of the diffusion of the pressure-ejected DA from the micropipette to the electrode (Rice et al., 1985; Gerhardt et al., 1987), did not change during the course of the experiments in either saline- or cocaine-treated animals. Behavior We compared the time course of behaviors induced by a single injection of cocaine with the electrochemi-
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are fewer transporters in the NAc. The affinity of cocaine for the DA transporter was examined by constructing competition curves for cocaine inhibition of [3H]mazindolbinding. The affinity of cocaine for the transporter was not different between the two regions (Ki: dorsal striatum, 3.4 f 0.3 pM, NAc, 4.0 f 0.7 p M , n = 4; Fig. 5A). However, the density of [3H]mazindol binding sites did differ in the two regions. Binding was distributed in a dorsoventral gradient, with the highest density in the dorsal striatum and with the density in the NAc being 60% lower than in the dorsal striatum (BmaX: dorsal striatum, 1.64 4 0.2 1 pmol/mg of protein; NAc, 0.65 f 0.10 pmol/mg; n = 4,p < 0.05; Fig. 5B). The affinity of [3H]mazindol for the transporter was not different between the dorsal striatum and NAc (KD:dorsal striatum, 38.1 k 3.1 nM, NAc, 27.5 f 8.9 nM;n = 4). Similar to the differential distribution of [3H]mazindol binding sites, the
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FIG. 2. Time course for amplitude changes in DA clearance and diffusion in dorsal striatum (A) and NAc (B) of rats acutely treated with saline or cocaine at time 0. Values were calculated as percent change from baseline, where baseline was defined as the average peak height at the -5 and 0 time points. Data are mean +- SEM (bars) values (n = 4 per group). Two-factor analyses of variance with repeated measures for the time factor were used to analyze the data: dorsal striatum, all three groups (f = 5.16, df = 2,9, p < 0.05).and further analysis showed that the 20 mg/kg cocaine group was significantly different from the other two groups (p < 0.05); NAc, all three groups (f = 13.86, df = 2.9, p < 0.01), and further analysis showed that the saline group was significantly different from the other two groups ( p < 0.01) but that the two cocaine groups were not different from one another. ' p < 0.05 compared with saline controls at the same time point (Newman-Keuls post hoc comparisons).
cal responses measured in anesthetized rats. Acute cocaine treatment increased quadrant crossings (locomotor activity) as well as the occurrence of head bobbing (Fig. 4). The locomotor response to cocaine increased as a function of dose ( F = 8.7, df= 2,12, p < 0.01), with the peak effect occurring within 10-20 min postinjection. The increased locomotor activity declined to control levels within 40 min. The frequency of head bobbing also increased in response to acute cocaine treatment ( F = 6.1, df = 2,12, p < 0.05) and showed a similar time course; however, this effect was not dose related. [3H]Mazindolbinding and tissue DA levels Two possible explanations for the electrochemical data are that the affinity of the transporter for cocaine is greater in the NAc than in the striatum or that there
J. Neurochem.. Vol. 59, No. I . 1992
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FIG. 3. Time course for T,,-, changes in DA clearance and diffusion in dorsal striatum (A) and NAc (6) of rats acutely treated with saline or cocaine at time 0. Values were calculated as in Fig. 2. Data are mean +- SEM (bars)values (n = 4 per group). Two-factor analyses of variance with repeated measures for the time factor were used to analyze the data: dorsal striatum, all three groups (f = 21.22, df = 2,9, p < 0.01), and further analysis showed that the 20 mg/kg cocaine group was significantly different from the other two groups ( p < 0.01) but that the 15 mg/kg cocaine group was not significantlydifferent from the saline group (p = 0.06); NAc, all three groups (F = 35.09, df = 2,9, p < O.OOl), and further analysis showed that each group was significantly different from the other two groups ( p .c 0.05). *p < 0.05compared with saline controls at the same time point (Newman-Keuls post hoc comparisons).
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largely governed by processes other than diffusion, such as neuronal DA uptake. Local application of DA uptake blockers increases both the amplitude and the time course of the DA signal resulting from either local pressure-ejection of DA or potassium-stimulated overflow of DA (Gerhardt et al., 1987; Friedemann and Gerhardt, 1992). As shown here, systemic administration of cocaine produces similar effects (Figs. 2 and 3). Although a major influence in eliminating the DA signal is likely to be high-affinity uptake (Horn, 1979), it is possible that other cellular mechanisms may also be involved in cessation of the DA signal. For example, enzymes such as monoamine oxidase and catechol-0-methyltransferase could alter the apparent decline of the DA signals by forming compounds, i.e., 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, and/or homovanillic acid, that are not readily detected by the Nafion-coated electrodes with the applied potentials used in this study. Low-affinity uptake may also contribute. Future studies are needed to ascertain how much of the clearance of locally applied DA is truly mediated through these other cellular processes. In addition, it has been noted that as high-affinity DA uptake is inhibited, the decline of the electrochemical response appears to become more diffusion controlled (Ger-
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FIG. 4. Behavioral effects of acute cocaine treatment. Rats were administered saline (1 ml/kg, i.p.) or cocaine (15 or 20 mg/kg, i.p.) (arrow) and rated for locomotor activity (quadrant crossing; A) and stereotyped head bobbing (6) as described in Materials and Methods. Data are mean f SEM (bars) values (n = 5 per group). The variance of the control group was small; therefore, these data were transformed to common logarithms before analysis to meet the assumptions of analysis of variance (Sokal and Rohlf. 1981). Post hoc comparisons indicated that for both behaviors the values for the 15 and 20 mg/kg cocaine groups were significantly different ( p i0.05) from the saline group only at 10, 20, and 30 min postinjection.
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concentration of DA in the dorsal striatum was 12.2 0.1 pg/g of tissue, whereas in the NAc it was 44% lower (6.8 +- 0.4pg/g of tissue; n = 4,p < 0.01).
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DISCUSSION
For the in vivo electrochemistry studies, we used a local DA application paradigm to eliminate possible contributions from release and, in this way, to confine our focus largely to uptake processes. However, a fraction of the exogenously applied DA would likely have been removed by metabolic processes and diffusion. Previous studies have shown that the rising portion of the electrochemical response is largely composed of a diffusion process (Rice et al., 1985; Gerhardt et al., 1987).The rise time of such signals appears to be very reproducible, provided that the distance between the ejection source and the electrochemical electrode is kept constant (Gerhardt et al., 1986, 1987, 19896). However, the cessation of such signals appears to be
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FIG. 5. Cocaine competition curves (A) and saturation curves (6) for [3H]mazindol binding to dorsal striatum and NAc. Competition curves were generated using 10 nM [3H]mazindol, and saturation curves were generated using 1-30 nM [3H]mazindol; nonspecific binding was defined with 1 pM GBR 12909. Values were determined by quantitative autoradiographic analysis and iterative curve fitting (GraphPAD),and data are mean -t SEM (bars) values (n = 4 per region). K, values for cocaine were not different between the two regions, whereas B,,, values were significantly lower in the NAc.
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hardt et al., 1987). For these reasons, the terminology “clearance and diffusion” seems to be the most appropriate to describe the response that is measured with this paradigm. Differences in the amplitude and T40-80 responses were observed in both saline- and cocaine-treated animals. During the course of the experiments, the amplitude of the electrochemical signals in control rats decreased by 26-36%, whereas the T40-80value remained constant. Recalibration of the electrodes after the experiments indicated that there had been up to 15% loss in sensitivity during the experiments. Although this could have contributed to the amplitude decrease, it cannot be the complete explanation. It is possible that, owing to the constant application of DA. the efficiency of the DA transporter could have increased during the experiment and that this contributed to the lowering of signal amplitude as well. In addition, under baseline conditions, the time course for the T40-80 signal was generally longer in the NAc compared with the striatum, similar to results reported by Stamford et al. ( 1988), likely indicating less efficient uptake processes in the NAc. We investigated the T40-80 portion of the signals because this portion of the curve appears to be more sensitive to changes in high-affinity uptake than the signal amplitude (Friedemann, 1992). Our observations were also consistent with this conclusion (Figs. 2 and 3). In response to cocaine administration, the percent change in the T40-80 was greater than the change in amplitude, and valin contrast with the amplitude changes, the T40-80 ues had not returned to baseline by the end of the experiments. Behavioral experiments were carried out to determine whether the time course for the changes in the electrochemical signals measured in anesthetized rats corresponded to the time course for changes in locomotion and stereotypies. Increases in the amplitude of the DA signal occurred in parallel with the behavioral changes, peaking within the first 20 min and then declining toward baseline during the next 20-30 min (Figs. 2 and 4). In contrast, the time course for the changes in T4,-,, did not mirror those for the behavioral changes (Figs. 3 and 4). The T4,-,, remained elevated at the end of the experiment, whereas the behaviors were not significantly different from control animals. It is possible that compensatory mechanisms may allow the behaviors to return to baseline despite the elevated neurochemical signals. Alternatively, other behaviors not monitored in this study may be related to the prolongation of the DA signal. In any case, the effects on the T40-80 interval represent a relatively long-term effect of cocaine administration. One possible explanation for the differential in vivo electrochemistry results in NAc and striatum is that after acute peripheral administration of cocaine, drug concentrations are higher in the NAc than in the stria-
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tum. Although we cannot rule out this possibility, it appears unlikely because Hurd et al. (1988) found no difference between striatal and NAc cocaine levels in rats given two intravenous injections of cocaine. In contrast, in animals treated repeatedly with cocaine, enhanced drug levels are found in cerebral cortex and NAc relative to acutely treated animals (Reith et al., 1987; Pettit et al., 1990). However, it remains to be established whether cocaine levels are increased to a similar extent in both striatum and NAc in response to repeated cocaine administration. An alternative explanation for our results, which has not been ruled out, could be uptake of the exogenously applied DA by serotonin terminals. The serotonin transporter has a higher affinity for cocaine than the DA transporter (Ritz et al., 1987),and the greater density of serotonin terminals in the NAc, compared with that in the dorsal striatum, could account for the greater effect of cocaine in the NAc. Other possible explanations for our findings include a higher affinity of the DA transporter for cocaine in the NAc or fewer transporters for cocaine to inhibit in the NAc. We used [3H]mazindol, which binds to a recognition site on the DA transporter (Javitch et al., 1984), to investigate these two possibilities. The affinity of cocaine for the transporter was not different between the two regions (Fig. 5A), eliminating this first possibility. Other groups have also found that the affinity of cocaine for the DA transporter is the same in the striatum and NAc (Boja and Kuhar, 1989; Izenwasser et al., 1990); however, it has also been reported that the DA transporter in striatum is more sensitive to the effects of cocaine than the transporter in NAc (Missale et al., 1985). In agreement with others who have measured radioligand binding and [3H]DA accumulation (Marshall et al., 1990; Richfield, 1991), we found that the density of DA transporters was 60% lower in the NAc than in the dorsal striatum (Fig. 5B). Similarly, the concentration of DA was 44% lower in the NAc. Again, in the literature some groups have reported findings in agreement with ours (Horn et al., 1974; Bacopoulos and Bhatnagar, 1977; Cass et al., 1989); however, others have found no differences in DA concentrations in striatum and NAc (Beal and Martin, 1985; Marshall et al., 1990). It is possible that differences in dissection technique could account for these discrepancies. Taken together, our results suggest that the NAc is less densely innervated by DA fibers than the dorsal striaturn and that correspondingly there are fewer DA transporters for cocaine to inhibit in the NAc. The present results support the conclusions of Carboni et al. (1989) that the NAc is more sensitive to the effects of an acute, peripheral injection of cocaine than the striaturn. Although we did not examine release processes, our results suggest that this increased sensitivity could be due to a differential effect on DA uptake instead of on release. Lending further support
COCAINE AND DOPAMINE TRANSPORTER
for the major effect of cocaine being inhibition of uptake, Nicolaysen and Justice (1988), using mathematical modeling, have reported that systemic cocaine administration in rats likely causes little or no augmentation of DA release. The greater sensitivity of the NAc to cocaine, compared with dorsal striatum, appears to be due to fewer DA transporter complexes for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine. Acknowledgment: This work was supported by U.S. Public Health Service grants DA042 16, AA07464, AG06434, AGO044 I , and NS09 199.
REFERENCES Akimoto K., Hamamura T., Kazahaya Y., Akiyama K., and Otsuki S. (1990) Enhanced extracellular dopamine level may be the fundamental neuropharmacological basis of cross-behavioral sensitization between methamphetamine and cocaine-an in vivo dialysis study in freely moving rats. Brain Res. 507, 344346. Bacopoulos N. G. and Bhatnagar R. K. (1 977) Correlation between tyrosine hydroxylase activity and catecholamine concentration or turnover in brain regions. J. Neurochem. 29,639-643. Barr G. A., Sharpless N. S., Cooper S., Schiff S. R., Paredes W., and Bridger W. H. (1 983) Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy. Lifi Sci. 33, 1314-1351. Beal M. F. and Martin J. B. (1985) Topographical dopamine and serotonin distribution and turnover in rat striatum. Brain Res. 358, 10-15. Boja J. W. and Kuhar M. J. (1989) [’HICocaine binding and inhibition of [3H]dopamine uptake is similar in both the rat striatum and nucleus accumbens. Eur. J. Pharmacol. 173,215-217. Bradbeny C. W. and Roth R. H. ( 1 989) Cocaine increases extracelMar dopamine in rat nucleus accumbens and ventral tegmental area as shown by in vivo microdialysis. Neurosci. Letl. 103, 97-102. Carboni E., lmperato A., Perezzani L., and Di Chiara G. (1989) Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience 28, 653-66 I. Cass W. A,, Bowman J. P.. and Elmund J. K. (1989) Behavior, striatal and nucleus accumbens field potential patterns and dopamine levels in ratsgiven amphetamine continuously. Neuropharmacology 28,2 17-227. Church W. H., Justice J. B. Jr., and Byrd L. D. (1987) Extracellular dopamine in rat striatum following uptake inhibition by cocaine, nomifensine and benztropine. Eur. J. Pharmacol. 139, 345-348. Costall B. and Naylor R. J. (1977) Mesolimbic and extrapyramidal sites for the mediation of stereotyped behavior patterns and hyperactivity by amphetamine and apomorphine in the rat, in Advances in Behavioral Biolog.y, Vol. 21: Cocaine and Other Stimulants (Ellinwood E. H. Jr. and Kilbey M. M., eds), pp. 47-76. Plenum Press, New York. Delfs J. M., Schreiber L., and Kelley A. E. (1990) Microinjection of cocaine into the nucleus accumbens elicits locomotor activation in the rat. J. Neurosci. 10, 303-310. Di Paolo T., Rouillard C., Morissette M., Levesque D., and Bedard P. J. (1989) Endocrine and neurochemical actions ofcocaine. Can. J. Physiol. Pharmacol. 67, 1 177- 1 I8 I. Friedemann M. N. (1992) In vivo electrochemical studies of dopamine diffusion and clearance in the sfriatum of young and aged Fischer 344 rats. Age 15, 23-28. Friedemann M. N. and Gerhardt G. A. (1992) Regional effects of
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aging on dopaminergic function in the Fischer 344 rat. Neurobiol. Aging (in press). Gerhardt G. A., Oke A. F., Nagy G., Moghaddam B., and Adams R. N. (1984) Nafion-coated electrodes with high selectivity for CNS electrochemistry. Brain Res. 290, 390-395. Gerhardt G. A., Rose G. M., and Hoffer B. J. (1986) Release of monoamines from striatum of rat and mouse evoked by local application of potassium: evaluation of a new in vivo electrochemical technique. J. Neurochem. 46,842-850. Gerhardt G. A., Pang K., and Rose G. M. (1987) I n vivo electrochemical demonstration of the presynaptic actions of phencyclidine in rat caudate nucleus. J. Pharmacol. Exp. Ther. 241, 7 14-721. Gerhardt G. A., Gratton A., and Rose G. M. (1 988) In vivo electrochemical studies of the effects of cocaine on dopamine nerve terminals in the rat neostriatum. Physiol. Bohemoslov. 37, 249-257. Gerhardt G. A., Dwoskin L. P., and Zahniser N. R. (1989~)Outflow and overflow of picogram levels of endogenous dopamine and DOPAC from rat striatal slices: improved methodology for studies of stimulus-evoked release and metabolism. J. Neurosci. Methods 26, 2 17-227. Gerhardt G. A., Friedemann M., Brodie M. S., Vickroy T. W., Gratton A. P., Hoffer B. J., and RoseG. M. (1989b)The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study. Brain Res. 499, 157-163. Gratton A., Hoffer B. J., and Gerhardt G. A. (1989) I n vivo electrochemical studies of monoamine release in the medial prefrontal cortex of the rat. Neuroscience 29, 57-64. Horn A. S. ( 1979) Characteristics of dopamine uptake, in The Neurohiology of Dopamine (Horn A. S., Korf J., and Westerink B. H. C., eds), pp. 2 17-235. Academic Press, New York. Horn A. S., Cuello A. C., and Miller R. J. (1974) Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase activity. J. Neurochem. 22,265-270. Hurd Y. L. and Ungerstedt U. (1 989) Cocaine: an in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum. Synapse 3,48-54. Hurd Y. L., Kehr H., and Ungerstedt U. (1988) In vivo microdialysis as a technique to monitor drug transport: correlation of extracellular cocaine levels and dopamine overflow in the rat brain. J. Neurochem. 51, 1314-1316. Izenwasser S., Werling L. L., and Cox B. M. (1990) Comparison of the effects of cocaine and other inhibitors of dopamine uptake in rat striatum, nucleus accumbens, olfactory tubercle, and medial prefrontal cortex. Brain Res. 520, 303-309. Javitch J. A., Blaustein R. O., and Snyder S. H. (1984) [3H]Mazindol binding associated with neuronal dopamine and norepinephrine uptake sites. Mol. Pharmacol. 26, 35-44. Kalivas P. W. and Duffy P. (1990) Effect ofacute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse 5,48-58. Kuhar M. J., Ritz M. C., and Boja J. W. (1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299-302. Marshall J. F., O’Dell S. J., Navarrete R., and Rosenstein A. J. ( 1990) Dopamine high-affinity transport site topography in rat brain: major differences between dorsal and ventral striatum. Neuroscience 37, 11-2 I . Missale C., Castelletti L., Govoni S., Spano P. F., Trabucchi M., and Hanbauer I. ( 1985)Dopamine uptake is differentiallyregulated in rat striatum and nucleus accumbens. J. Neurochern. 4 5 5 1-56. Mitchell J. B. and Gratton A. (1991) Opioid modulation and sensitization of dopamine release elicited by sexually relevant stimuli: a high speed chronoamperometric study in freely behaving rats. Brain Res. 551, 20-27. Moghaddam B. and Bunney B. S. (1 989) Differential effect of co-
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caine on extracellulardopamine levels in rat medial prefrontal cortex and nucleus accumbens: comparison to amphetamine. S-vnapse 4, 156- 16 1. Nicolaysen L. C. and Justice J. B. Jr. (1988) Effects of cocaine on release and uptake of dopamine in vivo: differentiation by mathematical modeling. Pharmacol. Biochem. Behav. 31, 327-335. Nomikos G. G., Damsma G., Wenkstern D., and Fibiger H. C . (1990) In vivo characterization of locally applied dopamine uptake inhibitors by stnatal microdialysis. Synapse 6, 106112. Paxinos G. and Watson C. ( 1 986) The Rat Brain in Stereotaxic Coordinates,2nd ed. Academic Press, New York. Pens J., Boyson S. J., Cass W. A., Curella P., Dwoskin L. P., Larson G., Lin L.-H., Yasuda R. P., and Zahniser N. R. ( I 990) Persistence of neurochemical changes in dopamine systems after repeated cocaine administration. J. Pharmacol. Exp. Ther. 253, 38-44. Pettit H. O., Pan H.-T., Parsons L. H., and Justice J. B. Jr. (1990) Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration. J. Neurochem. 55,798-804. Phillips A. G., Blaha C. D., and Fibiger H. C. (1989) Neurochemi-
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cal correlatesof brain-stimulation reward measured by ex vivo and in vivo analyses. Neurosci. Biobehav. Rev. 13,99-104. Reith M. E. A., Benuck M., and Lajtha A. (1987) Cocaine disposition in the brain after continuous or intermittent treatment and locomotor stimulation in mice. J. Pharrnacol. Exp. Ther. 243,281-287. Rice M. E., Gerhardt G. A., Hied P. M., Nagy G., and Adams R. N. ( 1985) Diffusion coefficients of neurotransmitters and their metabolites in brain extracellularfluid space. Neuroscience 15, 891-902. Richfield E. K. (1991) Quantitative autoradiography of the dopamine uptake complex in rat brain using [3H]GBR 12935: binding characteristics. Brain Res. 540, 1-13. Ritz M. C., Lamb R. J., Goldberg S. R., and Kuhar M. J. (1987) Cocaine receptors on dopamine transporters are related to selfadministration of cocaine. Science 237, I2 19- 1223. Sokal R. R. and Rohlf F. J. (1981) Biometry: The Principles and Practice of Statistics in Biological Research, 2nd ed., pp. 400453. W. H. Freeman and Co., San Francisco. Stamford J. A., Kruk Z. L., Palij P., and Millar J. (1988) Diffusion and uptake of dopamine in rat caudate and nucleus accumbens compared using fast cyclic voltammetry. Brain Res. 448, 381-385. Wise R. A. and Bozarth M. A. (1987) A psychomotor stimulant theory of addiction. Psychol. Rev. 94,469-492.
Differences in Dopamine Clearance and Diffusion in Rat Striatum and Nucleus Accumbens Following Systemic Cocaine Administration *Wayne A. Cass, *?$Greg A. Gerhardt, *R. Dayne Mayfield, *Pamela Curella, and *$Nancy R. Zahniser Departments of *Pharmacology and ?Psychiatry and $Neuroscience Training Program, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.
Abstract: Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCI (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopa-
mine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine. Key Words: Cocaine-Dopamine transporter-Striatum-Nucleus acuptake-Dopamine cumbens-In vivo electrochemistry-Behavior. Cass W. A. et al. Differences in dopamine clearance and diffusion in rat striatum and nucleus accumbens following systemic cocaine administration. J. Neurochern. 59, 259-266 ( 1 992).
Cocaine is a psychomotor stimulant whose neurochemical actions include blocking the high-affinity reuptake of dopamine (DA), norepinephrine, and serotonin back into monoamine nerve terminals (Ritz et al., 1987). Present evidence indicates that some of the behavioral effects (increased locomotor activity, stereotypies)and reinforcing properties of cocaine involve its effects on DA systems. In rats, psychomotor stimulants increase locomotor activity primarily by activating mesolimbic DA pathways, whereas the nigrostriatal DA system is more involved in producing stereotyped behaviors (Costal1 and Naylor, 1977; Delfs et al., 1990). The reinforcing properties of co-
caine appear to involve increased activity in the mesolimbic and mesocortical DA pathways (Wise and Bozarth, 1987; Kuhar et al., 1991). This cocaine-induced enhancement of activity in DA systems is likely due to the ability of cocaine to increase synaptic DA concentrations. Studies using in vivo microdialysis have demonstrated that a single systemic injection of cocaine increases extracellular DA concentrations in both striatum (Church et al., 1987; Carboni et al., 1989; Di Paolo et al., 1989; Hurd and Ungerstedt, 1989; Akimoto et al., 1990) and nucleus accumbens (NAc) (Bradberry and Roth, 1989; Carboni et al., 1989; Moghaddam and Bunney, 1989;Ka-
Received September 12, 199 1; revised manuscript received December 10, 199 I ; accepted December 3 1, I99 1. Address correspondence and reprint requests to Dr. W. A. Cass at Department of Pharmacology, C-236, University of Colorado Health SciencesCenter, 4200 East 9th Avenue, Denver, CO 80262, USA.
Abbreviations used: DA, dopamine; NAc, nucleus accumbens; TwB0,time for the signal to decay in amplitude beginning at the time point when the signal had declined by 40% of its amplitude until it had decreased by 80%of its amplitude.
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h a s and Duffy, 1990). A peripheral injection of cocaine also causes concurrent increases in the DA oxidation current measured with in vivo chronoamperometry in NAc and in the rate of self-stimulation (Phillips et al., 1989). Local application of cocaine in the striatum produces similar results: It augments the extracellular concentration of DA detected with in vivo microdialysis under basal conditions (Nomikos et al., 1990) as well as the DA concentration detected with in vivo electrochemistry in response to potassium stimulation (Gerhardt et al., 1988). An intriguing finding from one of the in vivo microdialysis studies was that peripheral administration of cocaine to freely moving rats increases extracellular DA concentrations in the NAc to a greater extent than in the striatum (Carboni et al., 1989). This preferential increase in the NAc could be due to the ability of cocaine to inhibit DA uptake and/or to increase DA release to a greater extent in the NAc. In the present study we used in vivo electrochemistry to measure directly the clearance and diffusion of exogenously applied DA in the striatum and NAc to determine if a differential effect of cocaine on DA uptake could explain this observation. The relationship of the electrochemical data obtained in anesthetized rats to the effects produced by cocaine in unanesthetized animals was investigated using behavioral measurements. Quantitative autoradiography with [3H]mazindolwas used to define further the locus of the differential effect of cocaine on the DA transporter, and these results were compared with tissue levels of DA in the striatum and NAc. MATERIALS AND METHODS Animals Male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE. U.S.A.) weighing 200-350 g were used for all experiments. They were housed in groups of four to six under a 12-h light-dark cycle with food and water freely available.
In vivo electrochemistry Rats were anesthetized with urethane (1.25- 1.5 g/kg of body weight) and prepared for electrochemistry as previously described (Gratton et al., 1989). A micropipette/Nafion-coated carbon fiber electrode assembly (Gerhardt et al., 1987;Gratton et al., 1989) was lowered into the dorsal stnatum (1-1.5 mm anterior to bregma, 2.2 mm lateral from midline, and 4-4.5 mm below the dura) or NAc (1.5 mm anterior, 2.2 mm lateral, and 6.5-7 mm below the dura). The micropipette (tip diameter, 10-1 5 pm; positioned 300 k 30 pm from the electrode tip) contained 200 @A4 DA (Sigma Chemical Co., St. Louis, MO, U.S.A.)and 100 p M ascorbic acid in 0.9% NaCl (pH 7.4). The carbon fiber electrodes each contained one to three fibers (fiber diameter, 30 pm; exposed length of 100 pm) sealed in a glass capillary. They were calibrated as previously described (Gerhardt et al., 1987; Gratton et al., 1989). The electrodes were coated with Nafion to give a selectivity for DA over ascorbic acid that averaged 1.064 f 224: 1 (Gerhardt et al., 1984, 1987;
-
Mitchell and Gratton, 199 1). All electrodes exhibited linearity for calibration with 1-8 pM DA; correlation coefficients ranged from 0.9994 to I .OOOO. The sensitivity of the electrodes to DA was 26,553 f 2,564 digital counts per 1 pA4 change in DA concentration. Based on the measured detection limit of the electrodes, the amplitude of the signal had to reach 46 f 14 nM DA to achieve a signal-to-noise ratio of 3.0. DA was pressure-ejected (25-100 nl, 5-30 psi for 2-5 sec) at 5-min intervals into the striatum or NAc. The amount ejected was monitored by measuring the volume of fluid displaced in the micropipette using a dissection microscope and was based on previous calculations that there is 246 nl of solution in a I-mm segment of the micropipette. Highspeed chronoamperometric electrochemical measurements (IVEC-5 system; Medical Systems Corp., Greenvale, NY, U.S.A.)were continuously made at 5 Hz and averaged to 1 Hz (oxidation potential, 0.55 V for 100 ms versus a Ag/ AgCl reference electrode; resting potential, 0.0 V for 100 ms; oxidation and reduction currents digitally integrated during the last 90 ms of each 100-ms pulse) as previously described (Gerhardt et al., 1989h; Gratton et al., 1989). After a steady baseline was achieved and the DA signals were reproducible, rats were injected with either saline ( 1 ml/kg, i.p.1 or cocaine-HCI (Sigma) in saline (15 or 20 mg/ kg, i.p.). DA was pressure-ejected at 5-min intervals for an additional 65 min. Each animal received only one cocaine injection. Some control animals received two saline injections so that recordings from dorsal striatum and NAc were made in the same animal. At the end of the experiments, brains were fixed by immersion in 10% neutral buffered formalin. The brains were later frozen, sectioned, and stained with cresyl violet acetate to verify electrode placement. Only animals in which the recording electrodes were positioned in the dorsal striaturn or NAc were included in the results.
Behavior Locomotor and stereotypic activities were determined according to the method ofBarret al. (1983) with minor modifications. Behaviors were rated by an experienced observer who was unaware of the drug treatment. One day before behavioral testing the rats were placed in a cylindrical wire mesh chamber (diameter, 30 cm; height, 38 cm), allowed to habituate for I h, then given a saline injection ( 1 ml/kg, i.p.), and kept in the chamber for an additional hour. The next day the animals were placed in the chambers and given 1 h to habituate, and then behavioral rating was begun. Each animal was rated for 2 min at 10-min intervals beginning 10 min before injection of saline or cocaine-HCl(l5 or 20 mg/kg, i.p.) and continuing for 60 min after injection. Locomotor activity (quadrant crossing) was rated as the number of times the head and forepaws crossed a quadrant boundary during each 2-min rating period. Stereotypic activity (head bobbing) was defined as occurrence of repetitive up-and-down or side-to-side head movement (not including movements due to breathing or grooming) and represents the number of 10-s intervals during each 2-min rating period in which head bobbing occurred (the maximal possible score per rating period was 12).
[3H1Mazindol binding Quantitative autoradiographic analysis of binding of [3H]mazindol (Dupont-New England Nuclear, Boston,
COCAINE AND DOPAMINE TRANSPORTER MA, U.S.A.) in rat dorsal striatum and NAc was carried out as described by Marshall et al. (1990) with the following changes. Sections 10 pm thick were used [from 1.2 to 1.6 mm anterior to bregma according to the atlas of Paxinos and Watson (1986)l. The concentration of [3H]mazindol used for competition curves was 10 nM, and that for saturation curves was 1-30 nM. GBR 12909 { l-[2-[bis(4-fluorophenyl)methoxy]ethyl]d-(3-phenylpropyl)piperazine; 1 pM, a gift from Novo Industri A/S, Bagsvaerd, Denmark} was used to define nonspecific binding. Two 5-min washes in ice-cold buffer followed the incubations. The computerbased analysis was carried out as previously described (Pens et al., 1990).
A
DA levels in the dorsal striatum and NAc were determined by HPLC with electrochemical detection. The NAc was dissected from a coronal slice as described by Horn et al. (1974). Dorsal striatum was taken from the same slice. The samples were weighed (approximate tissue weights: dorsal striaturn, 20 mg: NAc, I5 mg) and sonicated in 1 ml of 0.1 A4 perchloric acid. After filtration the samples were injected into an HPLC system. The HPLC conditions and calculations were as previously described (Gerhardt et al., 1989~). DA levels are expressed as micrograms per gram wet weight of tissue.
Data analysis Two parameters were examined from the electrochemistry experiments: the maximal amplitude ofthe signal resulting from the ejection of DA and the time for the signal to decay in amplitude beginning at the time point when the signal had declined by 40% of its amplitude until it had see Fig. 1). For decreased by 80% of its amplitude ( T40-80; analysis, the values for these parameters at 5 min before injection of cocaine or saline and immediately following injection were averaged to obtain a baseline value. The values of the time points were then calculated as percent change from baseline. Electrochemistry data were analyzed using two-factor analysis of variance with repeated measures followed by Newman-Keuls post hoc comparisons. Behavioral data were analyzed with repeated-measures analysis of variance after being transformed to common logarithms to meet the assumptions of analysis of variance (Sokal and Rohlf, 198l ) . Post hoc comparisons were made using univariate F tests. Parameters from the binding experiments were determined by iterative curve fitting (GraphPADSoftware, San Diego, CA, U.S.A.). Binding and biochemical data were analyzed with paired I tests. All data are given as mean f SEM values, where n indicates the number of rats.
RESULTS In vivo electrochemistry The ejection of a finite amount of DA into the striatum or NAc at 5-min intervals produced a transient and reproducible electrochemical signal (Fig. I ). In saline-injected controls, the amplitude of the signals (range, 0.35-3.16 p M ) significantly decreased by 2636% ( p < 0.05) during the recording sessions (Fig. 2), whereas the T40-80interval (range, 5-54 s) remained constant (Fig. 3). The time for the signals to decrease
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FIG. 1. Reproducibility of electrochemical signals from local application of DA in the dorsal striatum (A) and NAc (6) of the rat. A constant amount of DA was pressure-ejected(asterisks) at 5-min intervals, and the resulting oxidation signals for three consecutive applications are shown for both regions. The oxidation currents are shown as 1-s averages from a 5-Hz recording rate and are converted directly to rnicromolar concentrations by using calibration factors determined in vitro. Also shown in 6 are the time points used to calculate the T.+*,, values.
by 50% oftheir amplitude was greater in the NAc than in the striatum: dorsal striatum. 19.3 f 2.4 s; NAc, 30.3 +_ 3.9 s (n = 12, p < 0.05). The systemic administration of cocaine produced differential effects in the striatum and NAc. A dose of 15 mg/kg of cocaine-HCI had no effect on the amplitude of the DA signal in the striatum, but the signal increased 55.4% compared with saline controls in the time course increased in both NAc (Fig. 2). The T40-80 areas at this dose (Fig. 3); however, the change was statistically significant only in the NAc. When 20 mg/ kg of cocaine was administered, changes occurred in both the striatum and NAc. The amplitude and the T40-80 of the signals increased in both regions, although the increases were greater in the NAc (Figs. 2 and 3). The baseline remained stable in all animals during the recording sessions and was not altered in animals that received cocaine. Similarly, the rise times for the signals, a measure of the diffusion of the pressure-ejected DA from the micropipette to the electrode (Rice et al., 1985; Gerhardt et al., 1987), did not change during the course of the experiments in either saline- or cocaine-treated animals. Behavior We compared the time course of behaviors induced by a single injection of cocaine with the electrochemi-
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are fewer transporters in the NAc. The affinity of cocaine for the DA transporter was examined by constructing competition curves for cocaine inhibition of [3H]mazindolbinding. The affinity of cocaine for the transporter was not different between the two regions (Ki: dorsal striatum, 3.4 f 0.3 pM, NAc, 4.0 f 0.7 p M , n = 4; Fig. 5A). However, the density of [3H]mazindol binding sites did differ in the two regions. Binding was distributed in a dorsoventral gradient, with the highest density in the dorsal striatum and with the density in the NAc being 60% lower than in the dorsal striatum (BmaX: dorsal striatum, 1.64 4 0.2 1 pmol/mg of protein; NAc, 0.65 f 0.10 pmol/mg; n = 4,p < 0.05; Fig. 5B). The affinity of [3H]mazindol for the transporter was not different between the dorsal striatum and NAc (KD:dorsal striatum, 38.1 k 3.1 nM, NAc, 27.5 f 8.9 nM;n = 4). Similar to the differential distribution of [3H]mazindol binding sites, the
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FIG. 2. Time course for amplitude changes in DA clearance and diffusion in dorsal striatum (A) and NAc (B) of rats acutely treated with saline or cocaine at time 0. Values were calculated as percent change from baseline, where baseline was defined as the average peak height at the -5 and 0 time points. Data are mean +- SEM (bars) values (n = 4 per group). Two-factor analyses of variance with repeated measures for the time factor were used to analyze the data: dorsal striatum, all three groups (f = 5.16, df = 2,9, p < 0.05).and further analysis showed that the 20 mg/kg cocaine group was significantly different from the other two groups (p < 0.05); NAc, all three groups (f = 13.86, df = 2.9, p < 0.01), and further analysis showed that the saline group was significantly different from the other two groups ( p < 0.01) but that the two cocaine groups were not different from one another. ' p < 0.05 compared with saline controls at the same time point (Newman-Keuls post hoc comparisons).
cal responses measured in anesthetized rats. Acute cocaine treatment increased quadrant crossings (locomotor activity) as well as the occurrence of head bobbing (Fig. 4). The locomotor response to cocaine increased as a function of dose ( F = 8.7, df= 2,12, p < 0.01), with the peak effect occurring within 10-20 min postinjection. The increased locomotor activity declined to control levels within 40 min. The frequency of head bobbing also increased in response to acute cocaine treatment ( F = 6.1, df = 2,12, p < 0.05) and showed a similar time course; however, this effect was not dose related. [3H]Mazindolbinding and tissue DA levels Two possible explanations for the electrochemical data are that the affinity of the transporter for cocaine is greater in the NAc than in the striatum or that there
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FIG. 3. Time course for T,,-, changes in DA clearance and diffusion in dorsal striatum (A) and NAc (6) of rats acutely treated with saline or cocaine at time 0. Values were calculated as in Fig. 2. Data are mean +- SEM (bars)values (n = 4 per group). Two-factor analyses of variance with repeated measures for the time factor were used to analyze the data: dorsal striatum, all three groups (f = 21.22, df = 2,9, p < 0.01), and further analysis showed that the 20 mg/kg cocaine group was significantly different from the other two groups ( p < 0.01) but that the 15 mg/kg cocaine group was not significantlydifferent from the saline group (p = 0.06); NAc, all three groups (F = 35.09, df = 2,9, p < O.OOl), and further analysis showed that each group was significantly different from the other two groups ( p .c 0.05). *p < 0.05compared with saline controls at the same time point (Newman-Keuls post hoc comparisons).
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largely governed by processes other than diffusion, such as neuronal DA uptake. Local application of DA uptake blockers increases both the amplitude and the time course of the DA signal resulting from either local pressure-ejection of DA or potassium-stimulated overflow of DA (Gerhardt et al., 1987; Friedemann and Gerhardt, 1992). As shown here, systemic administration of cocaine produces similar effects (Figs. 2 and 3). Although a major influence in eliminating the DA signal is likely to be high-affinity uptake (Horn, 1979), it is possible that other cellular mechanisms may also be involved in cessation of the DA signal. For example, enzymes such as monoamine oxidase and catechol-0-methyltransferase could alter the apparent decline of the DA signals by forming compounds, i.e., 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, and/or homovanillic acid, that are not readily detected by the Nafion-coated electrodes with the applied potentials used in this study. Low-affinity uptake may also contribute. Future studies are needed to ascertain how much of the clearance of locally applied DA is truly mediated through these other cellular processes. In addition, it has been noted that as high-affinity DA uptake is inhibited, the decline of the electrochemical response appears to become more diffusion controlled (Ger-
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FIG. 4. Behavioral effects of acute cocaine treatment. Rats were administered saline (1 ml/kg, i.p.) or cocaine (15 or 20 mg/kg, i.p.) (arrow) and rated for locomotor activity (quadrant crossing; A) and stereotyped head bobbing (6) as described in Materials and Methods. Data are mean f SEM (bars) values (n = 5 per group). The variance of the control group was small; therefore, these data were transformed to common logarithms before analysis to meet the assumptions of analysis of variance (Sokal and Rohlf. 1981). Post hoc comparisons indicated that for both behaviors the values for the 15 and 20 mg/kg cocaine groups were significantly different ( p i0.05) from the saline group only at 10, 20, and 30 min postinjection.
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concentration of DA in the dorsal striatum was 12.2 0.1 pg/g of tissue, whereas in the NAc it was 44% lower (6.8 +- 0.4pg/g of tissue; n = 4,p < 0.01).
*
1
DISCUSSION
For the in vivo electrochemistry studies, we used a local DA application paradigm to eliminate possible contributions from release and, in this way, to confine our focus largely to uptake processes. However, a fraction of the exogenously applied DA would likely have been removed by metabolic processes and diffusion. Previous studies have shown that the rising portion of the electrochemical response is largely composed of a diffusion process (Rice et al., 1985; Gerhardt et al., 1987).The rise time of such signals appears to be very reproducible, provided that the distance between the ejection source and the electrochemical electrode is kept constant (Gerhardt et al., 1986, 1987, 19896). However, the cessation of such signals appears to be
200 100
i
m
0 0
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10 15 [3H-MAZINOOLl
20
25
30
(nM)
FIG. 5. Cocaine competition curves (A) and saturation curves (6) for [3H]mazindol binding to dorsal striatum and NAc. Competition curves were generated using 10 nM [3H]mazindol, and saturation curves were generated using 1-30 nM [3H]mazindol; nonspecific binding was defined with 1 pM GBR 12909. Values were determined by quantitative autoradiographic analysis and iterative curve fitting (GraphPAD),and data are mean -t SEM (bars) values (n = 4 per region). K, values for cocaine were not different between the two regions, whereas B,,, values were significantly lower in the NAc.
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hardt et al., 1987). For these reasons, the terminology “clearance and diffusion” seems to be the most appropriate to describe the response that is measured with this paradigm. Differences in the amplitude and T40-80 responses were observed in both saline- and cocaine-treated animals. During the course of the experiments, the amplitude of the electrochemical signals in control rats decreased by 26-36%, whereas the T40-80value remained constant. Recalibration of the electrodes after the experiments indicated that there had been up to 15% loss in sensitivity during the experiments. Although this could have contributed to the amplitude decrease, it cannot be the complete explanation. It is possible that, owing to the constant application of DA. the efficiency of the DA transporter could have increased during the experiment and that this contributed to the lowering of signal amplitude as well. In addition, under baseline conditions, the time course for the T40-80 signal was generally longer in the NAc compared with the striatum, similar to results reported by Stamford et al. ( 1988), likely indicating less efficient uptake processes in the NAc. We investigated the T40-80 portion of the signals because this portion of the curve appears to be more sensitive to changes in high-affinity uptake than the signal amplitude (Friedemann, 1992). Our observations were also consistent with this conclusion (Figs. 2 and 3). In response to cocaine administration, the percent change in the T40-80 was greater than the change in amplitude, and valin contrast with the amplitude changes, the T40-80 ues had not returned to baseline by the end of the experiments. Behavioral experiments were carried out to determine whether the time course for the changes in the electrochemical signals measured in anesthetized rats corresponded to the time course for changes in locomotion and stereotypies. Increases in the amplitude of the DA signal occurred in parallel with the behavioral changes, peaking within the first 20 min and then declining toward baseline during the next 20-30 min (Figs. 2 and 4). In contrast, the time course for the changes in T4,-,, did not mirror those for the behavioral changes (Figs. 3 and 4). The T4,-,, remained elevated at the end of the experiment, whereas the behaviors were not significantly different from control animals. It is possible that compensatory mechanisms may allow the behaviors to return to baseline despite the elevated neurochemical signals. Alternatively, other behaviors not monitored in this study may be related to the prolongation of the DA signal. In any case, the effects on the T40-80 interval represent a relatively long-term effect of cocaine administration. One possible explanation for the differential in vivo electrochemistry results in NAc and striatum is that after acute peripheral administration of cocaine, drug concentrations are higher in the NAc than in the stria-
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tum. Although we cannot rule out this possibility, it appears unlikely because Hurd et al. (1988) found no difference between striatal and NAc cocaine levels in rats given two intravenous injections of cocaine. In contrast, in animals treated repeatedly with cocaine, enhanced drug levels are found in cerebral cortex and NAc relative to acutely treated animals (Reith et al., 1987; Pettit et al., 1990). However, it remains to be established whether cocaine levels are increased to a similar extent in both striatum and NAc in response to repeated cocaine administration. An alternative explanation for our results, which has not been ruled out, could be uptake of the exogenously applied DA by serotonin terminals. The serotonin transporter has a higher affinity for cocaine than the DA transporter (Ritz et al., 1987),and the greater density of serotonin terminals in the NAc, compared with that in the dorsal striatum, could account for the greater effect of cocaine in the NAc. Other possible explanations for our findings include a higher affinity of the DA transporter for cocaine in the NAc or fewer transporters for cocaine to inhibit in the NAc. We used [3H]mazindol, which binds to a recognition site on the DA transporter (Javitch et al., 1984), to investigate these two possibilities. The affinity of cocaine for the transporter was not different between the two regions (Fig. 5A), eliminating this first possibility. Other groups have also found that the affinity of cocaine for the DA transporter is the same in the striatum and NAc (Boja and Kuhar, 1989; Izenwasser et al., 1990); however, it has also been reported that the DA transporter in striatum is more sensitive to the effects of cocaine than the transporter in NAc (Missale et al., 1985). In agreement with others who have measured radioligand binding and [3H]DA accumulation (Marshall et al., 1990; Richfield, 1991), we found that the density of DA transporters was 60% lower in the NAc than in the dorsal striatum (Fig. 5B). Similarly, the concentration of DA was 44% lower in the NAc. Again, in the literature some groups have reported findings in agreement with ours (Horn et al., 1974; Bacopoulos and Bhatnagar, 1977; Cass et al., 1989); however, others have found no differences in DA concentrations in striatum and NAc (Beal and Martin, 1985; Marshall et al., 1990). It is possible that differences in dissection technique could account for these discrepancies. Taken together, our results suggest that the NAc is less densely innervated by DA fibers than the dorsal striaturn and that correspondingly there are fewer DA transporters for cocaine to inhibit in the NAc. The present results support the conclusions of Carboni et al. (1989) that the NAc is more sensitive to the effects of an acute, peripheral injection of cocaine than the striaturn. Although we did not examine release processes, our results suggest that this increased sensitivity could be due to a differential effect on DA uptake instead of on release. Lending further support
COCAINE AND DOPAMINE TRANSPORTER
for the major effect of cocaine being inhibition of uptake, Nicolaysen and Justice (1988), using mathematical modeling, have reported that systemic cocaine administration in rats likely causes little or no augmentation of DA release. The greater sensitivity of the NAc to cocaine, compared with dorsal striatum, appears to be due to fewer DA transporter complexes for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine. Acknowledgment: This work was supported by U.S. Public Health Service grants DA042 16, AA07464, AG06434, AGO044 I , and NS09 199.
REFERENCES Akimoto K., Hamamura T., Kazahaya Y., Akiyama K., and Otsuki S. (1990) Enhanced extracellular dopamine level may be the fundamental neuropharmacological basis of cross-behavioral sensitization between methamphetamine and cocaine-an in vivo dialysis study in freely moving rats. Brain Res. 507, 344346. Bacopoulos N. G. and Bhatnagar R. K. (1 977) Correlation between tyrosine hydroxylase activity and catecholamine concentration or turnover in brain regions. J. Neurochem. 29,639-643. Barr G. A., Sharpless N. S., Cooper S., Schiff S. R., Paredes W., and Bridger W. H. (1 983) Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy. Lifi Sci. 33, 1314-1351. Beal M. F. and Martin J. B. (1985) Topographical dopamine and serotonin distribution and turnover in rat striatum. Brain Res. 358, 10-15. Boja J. W. and Kuhar M. J. (1989) [’HICocaine binding and inhibition of [3H]dopamine uptake is similar in both the rat striatum and nucleus accumbens. Eur. J. Pharmacol. 173,215-217. Bradbeny C. W. and Roth R. H. ( 1 989) Cocaine increases extracelMar dopamine in rat nucleus accumbens and ventral tegmental area as shown by in vivo microdialysis. Neurosci. Letl. 103, 97-102. Carboni E., lmperato A., Perezzani L., and Di Chiara G. (1989) Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience 28, 653-66 I. Cass W. A,, Bowman J. P.. and Elmund J. K. (1989) Behavior, striatal and nucleus accumbens field potential patterns and dopamine levels in ratsgiven amphetamine continuously. Neuropharmacology 28,2 17-227. Church W. H., Justice J. B. Jr., and Byrd L. D. (1987) Extracellular dopamine in rat striatum following uptake inhibition by cocaine, nomifensine and benztropine. Eur. J. Pharmacol. 139, 345-348. Costall B. and Naylor R. J. (1977) Mesolimbic and extrapyramidal sites for the mediation of stereotyped behavior patterns and hyperactivity by amphetamine and apomorphine in the rat, in Advances in Behavioral Biolog.y, Vol. 21: Cocaine and Other Stimulants (Ellinwood E. H. Jr. and Kilbey M. M., eds), pp. 47-76. Plenum Press, New York. Delfs J. M., Schreiber L., and Kelley A. E. (1990) Microinjection of cocaine into the nucleus accumbens elicits locomotor activation in the rat. J. Neurosci. 10, 303-310. Di Paolo T., Rouillard C., Morissette M., Levesque D., and Bedard P. J. (1989) Endocrine and neurochemical actions ofcocaine. Can. J. Physiol. Pharmacol. 67, 1 177- 1 I8 I. Friedemann M. N. (1992) In vivo electrochemical studies of dopamine diffusion and clearance in the sfriatum of young and aged Fischer 344 rats. Age 15, 23-28. Friedemann M. N. and Gerhardt G. A. (1992) Regional effects of
265
aging on dopaminergic function in the Fischer 344 rat. Neurobiol. Aging (in press). Gerhardt G. A., Oke A. F., Nagy G., Moghaddam B., and Adams R. N. (1984) Nafion-coated electrodes with high selectivity for CNS electrochemistry. Brain Res. 290, 390-395. Gerhardt G. A., Rose G. M., and Hoffer B. J. (1986) Release of monoamines from striatum of rat and mouse evoked by local application of potassium: evaluation of a new in vivo electrochemical technique. J. Neurochem. 46,842-850. Gerhardt G. A., Pang K., and Rose G. M. (1987) I n vivo electrochemical demonstration of the presynaptic actions of phencyclidine in rat caudate nucleus. J. Pharmacol. Exp. Ther. 241, 7 14-721. Gerhardt G. A., Gratton A., and Rose G. M. (1 988) In vivo electrochemical studies of the effects of cocaine on dopamine nerve terminals in the rat neostriatum. Physiol. Bohemoslov. 37, 249-257. Gerhardt G. A., Dwoskin L. P., and Zahniser N. R. (1989~)Outflow and overflow of picogram levels of endogenous dopamine and DOPAC from rat striatal slices: improved methodology for studies of stimulus-evoked release and metabolism. J. Neurosci. Methods 26, 2 17-227. Gerhardt G. A., Friedemann M., Brodie M. S., Vickroy T. W., Gratton A. P., Hoffer B. J., and RoseG. M. (1989b)The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study. Brain Res. 499, 157-163. Gratton A., Hoffer B. J., and Gerhardt G. A. (1989) I n vivo electrochemical studies of monoamine release in the medial prefrontal cortex of the rat. Neuroscience 29, 57-64. Horn A. S. ( 1979) Characteristics of dopamine uptake, in The Neurohiology of Dopamine (Horn A. S., Korf J., and Westerink B. H. C., eds), pp. 2 17-235. Academic Press, New York. Horn A. S., Cuello A. C., and Miller R. J. (1974) Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase activity. J. Neurochem. 22,265-270. Hurd Y. L. and Ungerstedt U. (1 989) Cocaine: an in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum. Synapse 3,48-54. Hurd Y. L., Kehr H., and Ungerstedt U. (1988) In vivo microdialysis as a technique to monitor drug transport: correlation of extracellular cocaine levels and dopamine overflow in the rat brain. J. Neurochem. 51, 1314-1316. Izenwasser S., Werling L. L., and Cox B. M. (1990) Comparison of the effects of cocaine and other inhibitors of dopamine uptake in rat striatum, nucleus accumbens, olfactory tubercle, and medial prefrontal cortex. Brain Res. 520, 303-309. Javitch J. A., Blaustein R. O., and Snyder S. H. (1984) [3H]Mazindol binding associated with neuronal dopamine and norepinephrine uptake sites. Mol. Pharmacol. 26, 35-44. Kalivas P. W. and Duffy P. (1990) Effect ofacute and daily cocaine treatment on extracellular dopamine in the nucleus accumbens. Synapse 5,48-58. Kuhar M. J., Ritz M. C., and Boja J. W. (1991) The dopamine hypothesis of the reinforcing properties of cocaine. Trends Neurosci. 14, 299-302. Marshall J. F., O’Dell S. J., Navarrete R., and Rosenstein A. J. ( 1990) Dopamine high-affinity transport site topography in rat brain: major differences between dorsal and ventral striatum. Neuroscience 37, 11-2 I . Missale C., Castelletti L., Govoni S., Spano P. F., Trabucchi M., and Hanbauer I. ( 1985)Dopamine uptake is differentiallyregulated in rat striatum and nucleus accumbens. J. Neurochern. 4 5 5 1-56. Mitchell J. B. and Gratton A. (1991) Opioid modulation and sensitization of dopamine release elicited by sexually relevant stimuli: a high speed chronoamperometric study in freely behaving rats. Brain Res. 551, 20-27. Moghaddam B. and Bunney B. S. (1 989) Differential effect of co-
J. Neurochem.. Val. 59. No. I , 1992
266
W. A . CASS ET AL.
caine on extracellulardopamine levels in rat medial prefrontal cortex and nucleus accumbens: comparison to amphetamine. S-vnapse 4, 156- 16 1. Nicolaysen L. C. and Justice J. B. Jr. (1988) Effects of cocaine on release and uptake of dopamine in vivo: differentiation by mathematical modeling. Pharmacol. Biochem. Behav. 31, 327-335. Nomikos G. G., Damsma G., Wenkstern D., and Fibiger H. C . (1990) In vivo characterization of locally applied dopamine uptake inhibitors by stnatal microdialysis. Synapse 6, 106112. Paxinos G. and Watson C. ( 1 986) The Rat Brain in Stereotaxic Coordinates,2nd ed. Academic Press, New York. Pens J., Boyson S. J., Cass W. A., Curella P., Dwoskin L. P., Larson G., Lin L.-H., Yasuda R. P., and Zahniser N. R. ( I 990) Persistence of neurochemical changes in dopamine systems after repeated cocaine administration. J. Pharmacol. Exp. Ther. 253, 38-44. Pettit H. O., Pan H.-T., Parsons L. H., and Justice J. B. Jr. (1990) Extracellular concentrations of cocaine and dopamine are enhanced during chronic cocaine administration. J. Neurochem. 55,798-804. Phillips A. G., Blaha C. D., and Fibiger H. C. (1989) Neurochemi-
J. Neurochem.. Vol. 59. No. I . 1992
cal correlatesof brain-stimulation reward measured by ex vivo and in vivo analyses. Neurosci. Biobehav. Rev. 13,99-104. Reith M. E. A., Benuck M., and Lajtha A. (1987) Cocaine disposition in the brain after continuous or intermittent treatment and locomotor stimulation in mice. J. Pharrnacol. Exp. Ther. 243,281-287. Rice M. E., Gerhardt G. A., Hied P. M., Nagy G., and Adams R. N. ( 1985) Diffusion coefficients of neurotransmitters and their metabolites in brain extracellularfluid space. Neuroscience 15, 891-902. Richfield E. K. (1991) Quantitative autoradiography of the dopamine uptake complex in rat brain using [3H]GBR 12935: binding characteristics. Brain Res. 540, 1-13. Ritz M. C., Lamb R. J., Goldberg S. R., and Kuhar M. J. (1987) Cocaine receptors on dopamine transporters are related to selfadministration of cocaine. Science 237, I2 19- 1223. Sokal R. R. and Rohlf F. J. (1981) Biometry: The Principles and Practice of Statistics in Biological Research, 2nd ed., pp. 400453. W. H. Freeman and Co., San Francisco. Stamford J. A., Kruk Z. L., Palij P., and Millar J. (1988) Diffusion and uptake of dopamine in rat caudate and nucleus accumbens compared using fast cyclic voltammetry. Brain Res. 448, 381-385. Wise R. A. and Bozarth M. A. (1987) A psychomotor stimulant theory of addiction. Psychol. Rev. 94,469-492.
