Introduction

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Although cocaine binds to dopamine, serotonin, and norepinephrine transporters with similar affinities, many of the stimulant and reinforcing effects of cocaine in rodents and nonhuman primates have been attributed to its action at the dopamine transporter (DAT) protein (Ritz et al., 1987; Bergman et al., 1989; Kuhar et al., 1991; Wise et al., 1996; Wilcox et al., 1999). By binding to DAT, cocaine prevents the reuptake of extrasynaptic dopamine, thus increasing levels of this neurotransmitter in the synapse, and thereby influencing behavior (Di Chiara and Imperato, 1988; Kalivas and Duffy, 1990). In rodents, repeated exposure to cocaine can alter levels of DAT mRNA and protein, although the nature of these changes has been inconsistent and seems to be influenced by many factors, including route of administration, dose, and withdrawal period (Kuhar and Pilotte, 1996).

Chronic cocaine treatment produced decreases in DAT mRNA levels in the ventral tegmental area (VTA) and substantia nigra in several studies (Xia et al., 1992; Burchett and Bannon, 1997; Letchworth et al., 1997). In contrast, DAT protein binding was unaltered in the VTA and substantia nigra, but increased in the anterior nucleus accumbens following chronic cocaine treatment (Letchworth et al., 1997). Some studies have found a short-term increase followed by a decrease in DAT binding in the nucleus accumbens after repeated cocaine treatment (Pilotte et al., 1996).

Withdrawal from repeated cocaine treatment has also been found to induce changes in DAT mRNA and protein levels. Again, these reported changes have been inconsistent. A 10- to 14-day withdrawal period after repeated cocaine administration resulted in a decrease in binding to DAT protein in the nucleus accumbens but not in the striatum (Cerruti et al., 1994; Boulay et al., 1996) or in the substantia nigra (Xia et al., 1992). Yet another study found increased DAT binding in the striatum, but not in the nucleus accumbens, after 3 days of withdrawal from repeated cocaine (Claye et al., 1995). After withdrawal from chronic cocaine, DAT mRNA levels in the VTA were decreased in one study (Cerruti et al., 1994) but increased (Arroyo et al., 2000) in another study and unchanged in a third study (Maggos et al., 1997). DAT mRNA levels were unchanged in the substantia nigra (Xia et al., 1992; Arroyo et al., 2000) after withdrawal from chronic cocaine treatment.

The level of DAT protein at any given time is due to the balance between the synthesis and degradation rates of the protein. Previously, in our laboratory, we developed an approach to determine the turnover kinetics of the DAT protein in the rat striatum and nucleus accumbens using the irreversible DAT ligand, RTI-76 [3-(3-p-chlorophenyl)tropan-2-carboxylic acid p-isothiocyanatophenylethyl ester hydrochloride] (Fleckenstein et al., 1996), and determined that the half-life of this protein is 2 to 3 days in both brain regions (Kimmel et al., 2000, 2001). In a previous study, acute (3 days) treatment with cocaine increased the half-life of DAT in both the striatum and the nucleus accumbens (Kimmel et al., 2001). In the present study, we determined the DAT synthesis rate (r) and degradation rate constant (k) in the rat striatum and nucleus accumbens following a 10-day withdrawal period from 10 days of cocaine administration. In addition, to correlate the observed biochemical changes with behavioral changes, we determined the locomotor-stimulating effects of cocaine in rats pretreated with saline or cocaine for 10 days followed by a 10-day withdrawal period.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (225-250 g; Charles River, Raleigh, NC) were grouped three per cage with food and water available at all times. Animals were housed in a colony room with a 12-h light/dark cycle. The animals were maintained in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH/85-23), and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Emory University.

Cocaine Pretreatment, Withdrawal, and RTI-76 Administration. Animals (n = 80) were treated once per day with a single injection of 20 mg/kg cocaine i.p. or saline i.p. for 10 consecutive days. Following these 10 days of cocaine or saline pretreatment, all animals were left untreated for another 10 days. On the 11th day following the final cocaine or saline injection, animals were anesthetized with 400 mg/kg chloral hydrate (i.p.) and placed in a stereotaxic frame. All rats were given a single unilateral injection of either 100 nmol of RTI-76 or saline in the right lateral ventricle. Stereotaxic coordinates used relative to bregma were: anteroposterior = 0.8, mediolateral = 1.4, dorsoventral = 4.0. A 25-µl Hamilton syringe was used to inject 10 µl of the RTI-76 solution over a 1-min period. Upon completion of the injection, the needle was kept in place for 3 min to minimize the back flow of the solution. Since RTI-76 is a light- and temperature-sensitive compound, all manipulations were done under low-light conditions, and the containers were wrapped in foil and kept on ice. Animals were allowed to recover under a warming lamp until they regained consciousness and then returned to their home cages.

Preparation of Membranes. Rats (n = 3-4) were rapidly decapitated at different time points after RTI-76 injection (1, 2, 3, 4, and 7 days). Brains were removed immediately and placed in cold saline. The olfactory tubercles were removed with the aid of fine forceps, exposing the diagonal bands of Broca. A razor blade was used to cut away the frontal cortex and to dissect the nucleus accumbens from both hemispheres. The pair of nucleus accumbens was pooled for each animal, for a total weight of 10 to 20 mg. Fine forceps were used to remove the right striatum, weighing 20 to 30 mg. All tissues were removed rapidly and frozen on dry ice, and then stored at 80°C until assayed.

[³H]GBR12935 Binding. After the final centrifugation, the tissue pellet was resuspended in buffer for a final concentration of 5 mg of wet tissue weight/ml. Polystyrene test tubes were filled with 1.5 ml of buffer (50 mM Tris-HCl, pH 7.7 at 24°C, containing 120 mM NaCl and 0.01% bovine serum albumin), 100 µl of 5 µM mazindol (final concentration) or buffer, 400 µl of radioligand, and 40 µl of tissue suspension for a total volume of 2.04 ml. Tubes were incubated at room temperature for 60 min, and the incubations were terminated by rapid filtration over Whatman GF/B filters that were pretreated with 0.05% polyethylenimine. The filters were rinsed three times with 4 ml of ice-cold buffer, and the radioactivity remaining on the filters was measured by conventional liquid spectrometry.

Drugs. RTI-76 was prepared as previously reported (Carroll et al., 1992). Cocaine hydrochloride was obtained from the National Institute of Drug Abuse, National Institutes of Health (Bethesda, MD). [³H]GBR12935 was obtained from PerkinElmer Life Sciences (Boston, MA). Bovine serum albumin, chloral hydrate, mazindol hydrochloride, polyethylenimine, and potassium chloride were obtained from Sigma-Aldrich (St. Louis, MO). Sodium chloride was obtained from Fisher Scientific (Pittsburgh, PA). Tris hydrochloride and Tris base were obtained from EM Science (Gibbstown, NJ). Cocaine hydrochloride, chloral hydrate, and RTI-76 were dissolved in 0.9% saline, and compounds used in the binding assay were dissolved in the appropriate buffer.

Binding Data Curve Fitting. B_max values were determined in each binding assay by fitting a nonlinear least-squares analysis and by a linear Scatchard plot, both giving similar results. Time course data were analyzed using GB-STAT v. 6.5 (Dynamic Microsystems Inc., Silver Spring, MD) to perform a two-way analysis of variance (systemic drug treatment versus time). Tukey's post hoc test was used to determine differences between groups.

Calculation of Receptor Kinetic Parameters. The half-life of recovery of [³H]GBR12935 binding to DAT in the striatum and the nucleus accumbens following inactivation by RTI-76 was determined, assuming a zero-order transporter production rate and a first-order transporter degradation rate. The repopulation kinetics of a transporter after irreversible inactivation can be defined by the monoexponential equation: B_t = (r/k)(1  e^kt (eq. 1) (Mauger et al., 1982; Sladeczek and Bockaert, 1983). Here, B_t is the transporter concentration (fmol/mg protein) at time t, r is the transporter production rate (fmol/mg protein/h), and k is the transporter degradation rate constant (h¹). As time nears infinity, transporter recovery reaches steady-state levels (B_ss). The term e^kt approaches 0, so that eq. 1 may be restated as: B_t = B_ss = r/k (eq. 2). B_t and B_ss are derived from pooled data at each time point.

kt

Locomotor Activity. Locomotor activity was measured using eight Digiscan animal activity monitors (AccuScan Instruments, Inc., Columbus, OH). Rats (250-274 g) received 0.9% saline (i.p.) (n = 8) or 20 mg/kg cocaine (i.p.) (n = 8) once daily for 10 consecutive days. Immediately after injection, animals were placed individually inside a polycarbonate cage (16.5 × 16 × 12 inches), contained inside of an activity monitor. Locomotor activity was monitored by infrared light beam sensors (eight beams per side) located on two opposing sides of the monitor. Activity was measured with a Digiscan analyzer and saved to a data file for subsequent analyses. Data were collected in 10-min intervals for 180 min. During the 10-day withdrawal period, animals did not receive any injections, but they were placed into the testing chambers and behavior was recorded for 180 min as described above. All rats were habituated to the locomotor activity chamber for at least 5 days before testing.

Data Analysis. The locomotor stimulant effects of saline, cocaine, or withdrawal were analyzed by a two-factor repeated measures analysis of variance, followed by a Tukey's post hoc test for multiple pairwise comparisons. Differences were considered statistically significant if p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

As described under Materials and Methods, four groups of animals were prepared. The two main groups were pretreated with either cocaine or saline (i.p.) for 10 days and subjected to a withdrawal period. Each main group was then subdivided into and treated with either RTI-76 or saline (i.c.v.). Three to four animals were taken from each group at 1, 2, 3, 4, or 7 days after i.c.v. treatment for measurement of DAT binding (B_max).

One day after i.c.v. treatment with RTI-76, DAT binding was reduced (Figs. 1 and 2) and binding gradually returned to control levels over the seven-day period. Treatment with i.c.v. saline had no effect on DAT binding, as expected. Linear transformation of the recovery data (Figs. 3 and 4) as described under Materials and Methods indicated that the half-life of the DAT protein in saline-pretreated animals was 2.1 days in the striatum and 2.2 days in the nucleus accumbens (Tables 1 and 2). These values are similar to our earlier findings (Kimmel et al., 2000, 2001).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   A, time course of recovery of [3H]GBR12935 binding of DAT in the striatum after saline pretreatment and withdrawal followed by an i.c.v. injection of 100 nmol of RTI-76. There was a significant difference between the saline- and RTI-76-treated animals [F(1,42) = 56.01, p < 0.0001]. A significant effect of time was observed [F(4,42) = 6.75, p = 0.0003], as well as a significant interaction between i.c.v. treatment and time [F(4,42) = 4.72, p = 0.0031]. B, time course of recovery of [3H]GBR12935 binding of DAT in the striatum after cocaine pretreatment and withdrawal followed by an i.c.v. injection of 100 nmol of RTI-76. Points represent the mean ± standard error of three to six rats at each time point. There was a significant difference between the saline- and RTI-76-treated animals [F(1,34) = 22.77, p < 0.0001]. A significant effect of time was not observed [F(4,34) = 2.3, N.S.], but there was a significant interaction between i.c.v. treatment and time [F(4,34) = 3.17, p = 0.0026]. Asterisks denote binding lower than saline-treated group, , p < 0.01.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   A, time course of recovery of [3H]GBR12935 binding of DAT in the nucleus accumbens after saline pretreatment and withdrawal followed by an i.c.v. injection of 100 nmol of RTI-76. There was a significant difference between the saline- and RTI-76-treated animals [F(1,35) = 25.18, p < 0.0001]. A significant effect of time was not observed [F(4,35) = 1.0, N.S.]), but there was a significant interaction between i.c.v. treatment and time [F(4,35) = 3.30, p = 0.0021]. B, time course of recovery of [3H]GBR12935 binding of DAT in the nucleus accumbens after cocaine pretreatment and withdrawal followed by an i.c.v. injection of 100 nmol of RTI-76. Points represent the mean ± standard error of three to six rats at each time point. There was a significant difference between the saline- and RTI-76-treated animals [F(1,34) = 19.02, p < 0.0001]. A significant effect of time was observed [F(4,34) = 4.54, p = 0.005], but there was no significant interaction between i.c.v. treatment and time [F(4,34) = 1.49, N.S.]. Asterisks denote binding lower than saline-treated group, , p < 0.01 and , p < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Semilogarithmic plot of the time course of recovery of [3H]GBR12935 binding of DAT in striatum after pretreatment and withdrawal from saline or cocaine. The correlation coefficients for the saline-pretreated animals and the cocaine-pretreated animals were 0.96 and 1.00, respectively. See text for details.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Semilogarithmic plot of the time course of recovery of [3H]GBR12935 binding of DAT in nucleus accumbens after pretreatment and withdrawal from saline or cocaine. The correlation coefficients for the saline-pretreated animals and the cocaine-pretreated animals were 0.99 and 1.00, respectively. See text for details.

However, pretreatment with cocaine and a subsequent withdrawal reduced the DAT protein half-life in the striatum by more than 50% to 0.94 day. The DAT protein half-life in the nucleus accumbens was unchanged. The corresponding degradation rate constant (k) in the striatum, which determines the half-life, was increased. This is reflected in the more rapid recovery after RIT-76 in Fig. 1 and by the steeper slope calculated in Fig. 3. The synthesis rate (r) in the striatum was also increased in the cocaine-pretreated group. The net effect of increasing both k and r was that the B_max at full recovery, i.e., 7 days after RTI-76 administration, was not changed in cocaine-pretreated animals compared with saline-pretreated animals (Figs. 1 and 2). Thus, pretreatment with cocaine and withdrawal changed the intracellular dynamics of the transporter protein without altering its overall levels to any significant extent. Because the animals that received saline both i.c.v. and systemically had similar B_max values at all the time points after i.c.v. treatment (Figs. 1 and 2), the changes observed in B_max after cocaine and RTI-76 must be due to the administration of the drugs rather than to the handling, the systemic or i.c.v. injections or the surgeries. Also, as previously reported, K_d values were unchanged in all of the treatment groups.

Because the above findings suggested a persistent change in DAT proteins in striatal dopamine neurons during withdrawal, we tested behavioral effects both during and after cocaine administration. A separate group of animals were pretreated either with saline (n = 8) or with cocaine (n = 8) daily for 10 days, and their locomotor activity was measured for 3 h each day. There was a significant effect of drug treatment [F(1,14) = 87.1, p < 0.0001] and of treatment day [F(20,335) = 9.4, p < 0.001] and of the interaction [F(20,335) = 7.8, p < 0.001]. Cocaine-pretreated animals exhibited an initial sensitization to the locomotor effects of cocaine between treatment days 1 and 2 (Fig. 5), and this response leveled off during treatment days 3 to 10. Saline-pretreated animals did not show any sensitization to their testing environment during the treatment period. Cocaine-pretreated animals showed significantly higher activity than did the saline-treated animals [F(1,29) = 293.7, p < 0.0001] during the 10-day treatment period as expected.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Horizontal locomotor activity was measured in animals pretreated with either saline or 20 mg/kg cocaine once daily for 10 consecutive days. Animals that received cocaine during the initial 10-day period exhibited significantly higher activity than did animals that received saline during this time period [F(1,28) = 318.5, p < 0.0001]. Following pretreatment with saline or cocaine, animals were withdrawn from drug treatment for 10 days. On the 11th day of withdrawal, all animals received a single challenge injection of 20 mg/kg cocaine. Animals that had received cocaine previously exhibited greater horizontal activity than those that received saline (p < 0.05). Asterisks denote activity significantly higher than saline control group for that treatment or withdrawal day, , p < 0.05. Dagger denotes activity significantly lower than all other cocaine treatments, +, p < 0.05.

During the 10-day withdrawal period, cocaine-pretreated animals exhibited slightly higher locomotor activity than did the saline-pretreated rats (Fig. 5) [F(1,29) = 4.74, p = 0.038]. However, post hoc tests did not reveal any significant differences between the two pretreatment groups on any specific withdrawal day. After the 10th day of withdrawal, both groups received a single challenge injection of 20 mg/kg cocaine (Fig. 5A). Cocaine-pretreated animals exhibited a higher response to this challenge dose of cocaine than did the saline-pretreated animals, but this response was also not statistically different from locomotor activity measured after cocaine injection during days 2 to 10. After this challenge dose of cocaine, the saline-pretreated animals showed an increase in locomotor activity comparable with that of the first day of treatment in the cocaine-pretreated animals.

	Discussion

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The most striking finding of this study is that repeated cocaine pretreatment followed by withdrawal altered DAT protein synthesis and degradation rates in the striatum, but not in the nucleus accumbens. The nucleus accumbens, as part of the mesolimbic dopamine system, is thought to be important in psychomotor stimulant-induced locomotor activity (Kelly and Iversen, 1976; Mogenson and Nielsen, 1984; Clarke et al., 1988; Delfs et al., 1990) and in the reinforcing effects of cocaine (Nestler, 1994). The striatum, part of the nigrostriatal system, is thought to have a role in motor activity, such as stereotypy (Alexander and Crutcher, 1990; Cote and Crutcher, 1991) but also in rewarded "habit" learning (Everitt and Wolf, 2002; Kelley and Berridge, 2002). That we observed a difference in response of DAT after cocaine pretreatment and withdrawal in these two brain regions emphasizes that, despite their similarities, these are two distinct brain regions with distinct functions and connectivity.

Earlier studies in this laboratory also found differences in the effects of systemic pretreatment with various dopamine receptor agonists and antagonists on the DAT half-life in the striatum and nucleus accumbens of rats (Kimmel et al., 2001) and the results of the present study can be examined in light of those earlier findings. In the rat striatum, systemic pretreatment with D2 dopamine receptor agonists caused a decrease in the DAT half-life, while D1 dopamine receptor agonists were without effect (Kimmel et al., 2001). Here, we observed a similar decrease in DAT half-life in the striatum, suggesting that D2 dopamine receptors or their signaling pathways may be active under these conditions of cocaine withdrawal. We previously found that pretreatment with D1 and D2 dopamine receptor agonists both increased DAT half-life in the nucleus accumbens. Thus, in the present study, the observed lack of change in DAT turnover is not explained by the activation of either D1 dopamine receptors alone or D2 dopamine receptors alone. Our current results suggest that these receptors may have a low level of activity under the withdrawal conditions studied. The mechanism for the lack of change in DAT turnover in the nucleus accumbens remains to be elucidated.

Several studies have shown changes in dopaminergic function following intermittent cocaine treatment and withdrawal. In rats, withdrawal from self-administered and experimenter-administered cocaine resulted in an increased response to the locomotor depressant effects of dopamine receptor antagonists, suggesting a possible withdrawal-induced deficit in dopaminergic function in the basal ganglia (Baldo et al., 1999). Repeated intermittent treatment with cocaine for 14 days followed by a 7-day withdrawal period resulted in a functional subsensitivity of release-modulating dopamine D2 autoreceptors in the caudate (Jones et al., 1996) and in the nucleus accumbens (Davidson et al., 2000) of rats. If the dopamine autoreceptors are not as sensitive to extrasynaptic dopamine levels, then this could lead to increased stimulation of postsynaptic dopamine receptors, resulting in the observed decrease in DAT turnover in the striatum by the mechanism described above. In the present study, we focused on a single schedule of cocaine administration and withdrawal. Different withdrawal periods from cocaine may very well produce different changes in DAT kinetics, similar to the different changes observed in dopamine neurons after different cocaine withdrawal periods (Kuhar and Pilotte, 1996).

Somewhat surprisingly, changes were found in the striatum and not in the accumbens, whereas changes in DAT protein levels have been observed in the accumbens but not in the striatum following treatment with cocaine (Pilotte et al., 1994, 1996; Wilson et al., 1994; Letchworth et al., 1997). However, some of the previous studies used a different strain of rats (Lewis versus Sprague-Dawley) and a different route of cocaine administration (continuous i.v. versus intermittent i.p.) from that in the present study (Pilotte et al., 1994, 1996). Our present data showed sustained changes in locomotor activity during withdrawal from repeated cocaine pretreatment (Fig. 5), supporting changes in striatal DAT, a brain region involved in locomotor effects in rodents and other mammals.

The changes observed in the striatum following cocaine treatment and withdrawal were an increase in both r and k. The mechanism(s) underlying these changes in r and k are unknown at present. However, alterations in r and k presumably reflect changes in cellular processes such as protein synthesis, mRNA stabilization, DAT insertion into and retrieval from the cell membrane, and others.

These data also confirm those previously reported, suggesting that the half-life of DAT in the striatum and nucleus accumbens is about 2 days (Kimmel et al., 2000, 2001). An additional important finding is that repeated handling of the animals and systemic injections of saline prior to i.c.v. administration of RTI-76 did not alter DAT protein kinetics. The present data suggest that this treatment design does not produce any long-term side effects in and of itself; the observed changes in the cocaine-pretreated animals are most likely due to the repeated exposure to the drug and subsequent withdrawal, not due to the physical manipulations involved.

One question that remains to be addressed is that of the effect of repeated, daily administration of cocaine without withdrawal upon DAT protein kinetics. A review of the literature reveals discrepancies in the effects of repeated cocaine treatment upon DAT levels (see the introduction). In addition, it is important to elucidate what aspect of cocaine binding to DAT causes these subsequent changes in DAT protein kinetics. Although cocaine binds directly to DAT, many of its effects are due to increases in synaptic dopamine, which then binds to pre- and postsynaptic dopamine receptors.

In summary, repeated daily pretreatment with cocaine followed by withdrawal results in a trend toward sensitization to the locomotor effects of a subsequent cocaine challenge and a change in DAT protein turnover in the striatum of the rat. Although the nucleus accumbens has been associated with sensitization to the locomotor effects of cocaine in rats (Pierce and Kalivas, 1995; Filip and Siwanowicz, 2001; Todtenkopf et al., 2002), DAT kinetics in this brain region were not altered by cocaine pretreatment and withdrawal in this study. However, the present results indicate that the effects of cocaine are long-lasting and that these biochemical changes may be partly responsible for the withdrawal symptoms observed in cocaine addicts.