Introduction

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Repeated administration of psychomotor stimulants, such as cocaine and amphetamine, has been used as an experimental paradigm to model the progression of behavioral and neurochemical changes leading to compulsive drug use in addicts. Repeated stimulant administration often results in behavioral sensitization, a progressive increase in responsiveness to drug. Sensitization in rats is manifested as augmented locomotor activity and stereotypic behaviors, as well as self-administration and drug-seeking behaviors (Robinson and Berridge, 1993; Covington and Miczek, 2001; De Vries et al., 2001; Vezina et al., 2002). Thus, it is important to understand the neurobiological changes induced by repeated stimulant administration because they likely contribute to intensification of drug motivation and/or craving that promote relapse in addicts (Robinson and Berridge, 1993).

Cocaine potentiates dopamine (DA) signaling in mesocorticolimbic reward pathways by inhibiting uptake of DA by the DA transporter (DAT). With repeated administration, the responsiveness of DA systems to stimulants is enhanced in behaviorally sensitized rats (Vanderschuren and Kalivas, 2000). Transient increases in somatodendritic DA release and spontaneous firing activity of DA neurons in the ventral tegmental area have been associated with the induction of behavioral sensitization, whereas long-lasting alterations within DA neuronal terminal fields have been associated with its expression (Vanderschuren and Kalivas, 2000; Nestler, 2001; Everitt and Wolf, 2002). Initially, reduced sensitivity of inhibitory DA D₂-autoreceptors may result in the potentiated cocaine-induced increases in extracellular DA concentrations in nucleus accumbens (NAc; Vanderschuren and Kalivas, 2000). However, the D₂-autoreceptor changes seem to be transient, whereas the ability of a cocaine challenge to produce a greater augmentation in NAc DA levels persists. This suggests that other mechanisms must underlie the long-lasting changes in DA neuronal function.

Because cocaine blocks DAT, repeated cocaine may regulate DAT expression/activity. However, whether it does is controversial (Izenwasser and Cox, 1990; Ng et al., 1991; Cass et al., 1993a; Masserano et al., 1994; Meiergerd et al., 1994; Zahniser et al., 1995; Kuhar and Pilotte, 1996; Zahniser and Doolen, 2001; Chefer and Shippenberg, 2002). Discrepancies may result from differences in strains/individuals, drug administration/withdrawal paradigms, and/or assays used. We have combined high-speed chronoamperometry with local applications of DA to measure dynamic changes in extracellular DA concentrations after acute and repeated cocaine administration (Cass et al., 1993a; Zahniser et al., 1999; Sabeti et al., 2002b). Using this approach in striatum of drug-naïve rats, we have demonstrated that the decay or clearance of locally applied DA primarily reflects in vivo DAT activity and that diffusion makes a more minor contribution to cessation of the DA signals (Cass et al., 1993b; Sabeti et al., 2002a,b). Furthermore, we observed cocaine-induced changes consistent with behavioral sensitization: greater cocaine-induced inhibition of exogenous DA clearance in NAc of anesthetized rats withdrawn from repeated cocaine administration, compared with rats given the same dose acutely (Cass et al., 1993a). This finding was consistent with reports of decreased DAT binding sites (but see Cass et al., 1993a; Pilotte et al., 1994; Wilson et al., 1994; Boulay et al., 1996; Letchworth et al., 1999) and increased cocaine potency (Lee et al., 1998) after repeated cocaine treatment. However, because the rats were anesthetized for the clearance measurements (Cass et al., 1993a), it was impossible to relate cocaine-induced regulation of DAT function with individual behavioral changes. This relationship was further complicated by the fact that not all male Sprague-Dawley rats exhibit behavioral sensitization with repeated cocaine administration (Cass et al., 1993a).

Greater locomotor activity in a novel environment predicts enhanced vulnerability to behavioral sensitization induced by repeated amphetamine (Bardo et al., 1996; Piazza and Le Moal, 1996; Cools and Gingras, 1998), although this relationship may not generalize to cocaine (Djano and Martin-Iverson, 2000; Sutton et al., 2000). High-novelty responding rats also exhibit higher DA release (Bardo et al., 1996; Piazza and Le Moal, 1996; Cools and Gingras, 1998) and firing rates of ventral tegmental area DA neurons (Marinelli and White, 2000). We have found that greater acute inhibition of DA clearance is correlated with high initial locomotor responsiveness to cocaine (Sabeti et al., 2002b). However, it is unclear whether this association extends to cocaine-induced locomotor sensitization.

To determine whether long-lasting adaptations in DAT function contribute to cocaine-induced locomotor sensitization, we simultaneously recorded behavior and DA clearance in NAc of freely moving rats over a 2-week period. We then 1) compared the time course of changes in behavior and DA clearance and 2) correlated individual variations in the magnitude of sensitization with initial cocaine-induced locomotor activation and with basal and cocaine-induced changes in DA clearance.

	Materials and Methods

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Animals. Outbred male Sprague-Dawley rats were obtained from Charles Rivers Laboratory (Sasco, Omaha, NE). Before surgeries, rats were housed no more than six per cage with a 12-h light/dark cycle and unrestricted access to food and water. To habituate rats to handling and the insertion of injector tubing on experimental days, rats were handled for 1 to 2 days before surgery and before each recording session. After surgery, rats were housed individually. All animal care procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center.

Stereotaxic Implantation of Recording Microelectrode/Microcannulae Assemblies. Procedures for the construction and implantation of recording microelectrode/microcannulae assemblies have been previously described in detail (Gerhardt et al., 1999; Sabeti et al., 2002a,b). Briefly, microelectrodes were fabricated from a single carbon fiber (fiber diameter, 30 µm; exposed length, 150-300 µm; Textron Systems, Wilmington, MA) that was coated with Nafion (5% solution; Aldrich Chemical Co., Milwaukee, WI). They were calibrated in vitro as described previously. Microelectrodes displayed a DA over ascorbic acid selectivity of 1000:1 and linear responses to DA (1-6 µM) in vitro. Each microelectrode was assembled onto two stainless steel guide microcannulae, which permitted the delivery of KCl or DA from injectors that were inserted before each recording session. Injectors were fabricated from fused silica tubing (40 µm i.d. × 150 µm o.d.; Polymicro Technologies, Phoenix, AZ). Insertion of the injector through the guide positioned the tip of the injector precisely within 250 to 350 µm from the exposed tip of the carbon fiber microelectrode.

Treatment Protocol and Timeline of Simultaneous Behavioral and Electrochemical Recordings. Three to five days after implantation of the microelectrode/microcannulae assembly, each rat was transferred from its home cage to an open field activity apparatus (16 × 16 × 15 inches; San Diego Instruments, San Diego, CA; enclosed inside a 2 × 2 × 2-foot Faraday cage). On this day (day 0), initial behavioral and electrochemical responses in the activity apparatus and to a saline injection (1 ml/kg i.p.) were examined in all of the rats. Subsequently, rats were divided into two groups (Table 1). On days 1 to 6, once daily injections of either saline (control group) or ()-cocaine HCl (10 mg/kg i.p.; experimental group) were administered. On day 7, both groups were injected with cocaine. Rats in the control group continued to be injected with cocaine for five more days and then were injected with saline on the 7th day. After a 7-day withdrawal in the experimental group, cocaine was administered on day 15. All injections were given in the activity apparatus. The data on days 0 and 1 from rats in the experimental group have been previously presented (Sabeti et al., 2002b) and are included in the mean values reported here.

Behavioral Data Acquisition. Automated recordings of locomotor activity were obtained in the open field apparatus using a single photo beam frame (eight beams per dimension) near the base of the apparatus. Consecutive beam interruptions were converted to distance traveled (centimeters) per 5-min period. Head/limb stereotypy was defined as repetitive head movements, including head bobs and side-to-side head sways, or back and forth repetitive forelimb movements. The frequency of stereotypy was determined by observation and expressed as the fraction of time during each 15-min interval in which the behavior was exhibited, as previously described in detail (Sabeti et al., 2002b). Rearing was the cumulative number of times within each 15-min interval during which both forepaws were lifted and then at least one forepaw was placed back onto the floor.

High-Speed Chronoamperometry in Freely Moving Rats. Upon transfer to the activity apparatus, the rat was connected to a miniature potentiostat headstage/tether (RAT HAT; Quanteon, L.L.C., Lexington, KY) via a four-pin telephone connector mounted on the rat's head. This allowed the rat free movement within the activity apparatus (Gerhardt et al., 1999; Sabeti et al., 2002a,b). The headstage/tether was linked to an IVEC-10/FAST-12 electrochemical recording system (Quanteon, L.L.C.). Continuous 100-ms square-wave potential pulses (0.0 to +0.55 V versus Ag/AgCl reference) were applied at 5 Hz to the recording microelectrode. A stable background oxidation signal was established in the absence of exogenous DA and set to zero. Subsequently, exogenous DA (40-300 pmol in saline containing 100 µM ascorbic acid, pH 7.4 adjusted with sodium hydroxide) was applied at 5-min intervals into NAc, using a microprocessor-controlled syringe-pump (1.01 µl/s; Stoelting Co., Wood Dale, IL; Gerhardt et al., 1999; Sabeti et al., 2002a,b). During each recording session, the DA ejection volume was initially adjusted for each rat to evoke baseline peak DA signal amplitude (A_max) responses within a range of 0.3 to 1.2 µM. Once three reproducible (i.e., within 20%) A_max responses were elicited by the same ejection volume, DA was applied once every 5 min at this constant amount throughout the remainder of the recording session for that day.

k(tt₀)

Statistical Analysis. Data are expressed as mean values + S.E.M. Statistical analyses, including one- and two-way analysis of variance (ANOVA) and Pearson correlation analysis, were performed using either SigmaStat (SPSS Science, Chicago, IL) or Prism (GraphPad Software, Inc., San Diego, CA) software. A level of p < 0.05 was considered statistically significant.

Chemicals and Drugs. Dopamine and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). ()-Cocaine HCl was obtained from the National Institute on Drug Abuse (Research Triangle Institute International, Research Triangle Park, NC).

	Results

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Individual Differences in Cocaine-Induced Behavioral Sensitization Are Associated with Initial Locomotor Responsiveness to Cocaine. Previously we demonstrated that outbred male Sprague-Dawley rats can be effectively divided into two subgroups of cocaine responders, namely, low or high cocaine responders (LCRs or HCRs, respectively), using the median split of locomotor activity during the initial 30 min after an acute i.p. injection of 10 mg/kg cocaine (Sabeti et al., 2002b). In the follow-up longitudinal cocaine study reported here, a subset of rats from this previously characterized population, the group that had electrochemical microelectrode/microcannulae assemblies implanted chronically in NAc, continued to receive daily cocaine treatment. Thus, these rats received an injection of saline (1 ml/kg i.p.) on day 0 and daily injections of cocaine (10 mg/kg i.p.) on days 1 to 6 (experimental group; Table 1). Rats were subsequently challenged with an i.p. injection of the same dose of cocaine following a 24-h withdrawal (i.e., day 7) and a 7-day withdrawal (i.e., day 15) from the daily cocaine treatment.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Baseline and cocaine-stimulated locomotor activity of individual rats in the experimental group (Table 1) on day 1 reveals differential initial responsiveness to cocaine. Horizontal lines in the scatter plots represent the median responses during the 30 min preceding (baseline) and the 30 min after injection of cocaine (10 mg/kg i.p.). Subsequently, individual rats were identified as either LCRs (open circles) or HCRs (filled circles) based on the median split of locomotor responsiveness to the acute cocaine injection. The bar graphs illustrate the mean values ± S.E.M. of the cocaine-stimulated activity in LCRs and HCRs and demonstrate the effectiveness of the median-split procedure for subdividing the rats into two groups of cocaine responders.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Differences in induction, expression, and persistence of cocaine-induced locomotor sensitization in LCRs (A) and HCRs (B). Baseline, saline- or cocaine-induced locomotor activity was recorded in the rats characterized as LCRs or HCRs in Fig. 1 (Table 1, experimental group). Saline-induced activity was recorded on day 0. Cocaine-induced activity was recorded on days 1, 3, 5, and 7 during the 7-day regimen of once-daily cocaine administration and after a 7-day withdrawal (day 15). Left, time courses of baseline and saline- or cocaine-induced locomotor activity during the 5-min recording intervals. Arrows indicate the time at which saline (1 ml/kg i.p.) or cocaine (10 mg/kg i.p.) was injected. Two-way ANOVAs, with both treatment day and time as the repeated measures, were used to analyze the activity during the 60 min after cocaine injection (see Results). Right, comparison of peak cocaine-stimulated locomotor responses. Activity was averaged during the first 30 min post-treatment. Because no significant differences existed in peak responses between days 1 and 3 or between days 5 and 7, these data sets were collapsed to represent the induction and expression phases of locomotor sensitization, respectively. Data are mean values ± S.E.M. (n = 10, LCRs; n = 7, HCRs). Significant differences reflect post hoc Bonferroni's multiple t test comparisons. ***, p < 0.001 versus days 1 to 3 cocaine-induced response. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus day 0 saline-induced response.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Magnitude of cocaine-induced locomotor sensitization in individual LCR rats correlates inversely with their initial locomotor responsiveness to an acute cocaine injection. "Sensitization score" is the ratio of day7/day1 activity induced in the first 30 min after cocaine injection (Fig. 2A). The linear regression fit () and the 95% confidence interval (-) are shown. A sensitization score of 1 (horizontal dashed line) would indicate a lack of locomotor sensitization, whereas scores above 1 indicate increasing magnitudes of locomotor sensitization.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Time course of cocaine-induced changes in stereotypic behaviors and rearing during repeated cocaine administration and after withdrawal in LCRs (A) and HCRs (B). The head/limb stereotypies and rearing were scored by observation (see Materials and Methods) in the same rats and during the same periods as the locomotor activity presented in Fig. 2. Behaviors were summed for 15-min intervals during the 30 min of baseline (B1 and B2) and 60 min of saline- or cocaine-induced responses (1-4). Data are mean values ± S.E.M. for LCR (n = 10) and HCR (n = 7) rats. For statistical analysis, cocaine-induced behaviors were averaged for days 1 and 3 and for days 5 and 7 and compared with day 15, after a 7-day withdrawal. A two-way ANOVA (treatment days1-3,5-7,15 × time1,2,3,4) revealed a significant effect of days on rearing in HCRs (F2,72 = 13.7, p < 0.0001).

Individual Differences in Cocaine-Induced Locomotor Sensitization Are Associated with Differential Cocaine-Induced Modulations in DA Clearance in NAc. Simultaneous with the behavioral measurements, changes in DA clearance in NAc of all the rats were continuously monitored by high-speed chronoamperometry on days 0, 1, 3, 5, and 7 (Fig. 5). Additionally, the effects of the 7-day withdrawal and challenge by cocaine were evaluated on DA clearance in a subset of these rats whose microelectrode/microcannulae assemblies remained patent on day 15 (n = 7, LCRs; n = 4, HCRs). A constant amount of DA (40-300 pmol; subsaturating relative to the maximal clearance rates measured in vivo; Sabeti et al., 2002a) was applied at the recording site every 5 min. These DA applications evoked reproducible oxidation signals that were measured over a 30-min recording interval to obtain the predrug baseline value on each recording day (Fig. 5). Changes over baseline in the A_max and k parameters of the DA signal were used as indices of altered DAT function (Fig. 5). On each recording day amounts of DA locally applied were initially adjusted for each rat so that similar baseline A_max responses were achieved. Thus, mean baseline A_max values did not differ between LCRs and HCRs; these averaged 0.8 ± 0.4 and 0.7 ± 0.1 µM, respectively, over the 2-week period in which rats were treated repeatedly and then withdrawn from cocaine. The amounts of DA applied relative to A_max values at baseline were averaged across days 1 and 3 and across days 5 and 7 to correspond to the induction and expression phases of locomotor sensitization, respectively, in the LCRs (see above). Initially (i.e., days 1-3) HCRs required 3-fold significantly greater amounts of DA than LCRs to elicit similar A_max responses (Fig. 6A). This finding is in agreement with our previous observations on day 1 in the same LCR and HCR rats (Sabeti et al., 2002b). Furthermore, although DA applications in LCRs did not differ significantly across the repeated treatment days or after withdrawal, in HCRs significantly lower amounts were necessary on days 5 to 7 than on days 1 to 3 to achieve comparable A_max responses (Fig. 6A). Despite the differences in the amounts of DA applied, predrug baseline k values (0.016-0.023 s¹) were not significantly different between LCRs and HCRs across the repeated cocaine treatment days and after withdrawal (Fig. 6B). This finding is in agreement with our observation that k is independent of applied DA within the relatively narrow range of DA applied here, whereas higher amounts of exogenous DA modulate the k for DA clearance as expected by active uptake kinetics (Sabeti et al., 2002a). Furthermore, predrug baseline k values were not significantly correlated with initial locomotor responsiveness to cocaine in individual rats (p = 0.673).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Representative time course of the effects of a challenge injection of cocaine (10 mg/kg i.p., arrow) on high-speed chronoamperometric recordings of exogenous DA signals in NAc of a rat that had been treated with cocaine (10 mg/kg/day i.p.) for 6 days before this cocaine challenge on day 7. Oxidation currents were evoked by local application of DA (80 pmol) at the recording site at 5-min intervals, averaged across 1-s bins and converted to concentrations based on in vitro electrode calibration. Note the reproducibility of the DA signals during the 30 min of predrug baseline recording and the transient increase after administration of cocaine. Inset, two representative signals evoked by local applications of DA (arrow) in this rat are shown on an expanded time scale to illustrate the increase above baseline in Amax, and the decrease in k, 10 min after the cocaine challenge. See Materials and Methods for details. Increased Amax and decreased k values are indicative of reduced DAT function.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Baseline DA clearance parameters in NAc of LCRs and HCRs across repeated cocaine treatment days and after a 7-day withdrawal. Data are mean values ± S.E.M. of five to six reproducible predrug DA signals in LCRs (open columns; n = 10, days 1-3 and 5-7; n = 7, day 15) and HCRs (closed columns; n = 7, days 1-3 and 5-7; n = 4, day 15). Data were collapsed across days 1 and 3 and 5 and 7 to correspond to the induction and expression phases of locomotor sensitization, respectively. A, DA ejection volumes are indicated in arbitrary units relative to the baseline Amax responses evoked in each individual rat. B, baseline k reflects the efficiency of exogenous DA clearance in the absence of cocaine. Mean Amax responses were similar in all rats (0.6-0.8 µM). *, p < 0.05 versus the time-matched value in LCRs. #, p < 0.05 versus day 1 to 3 value.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Time course comparisons of baseline and cocaine-induced changes in DA signal Amax during repeated cocaine administration and after withdrawal in LCRs (A) and HCRs (B). Electrochemical data were recorded simultaneously with behavior in the same rats shown in Figs. 2 and 4 (n = 10, LCRs; n = 7, HCRs), with the exception of day 15 in which electrochemical data were obtained from a subset of these same animals (n = 7, LCRs; n = 4, HCRs). Data from days 1 and 3, and likewise from days 5 and 7, were collapsed to correspond to the induction and expression phases of locomotor sensitization, respectively (Fig. 2A). Arrows indicate time of the i.p. injections of saline (1 ml/kg) or cocaine (10 mg/kg). Left, time courses for repeated treatment effects on baseline and cocaine-induced changes in Amax are shown for the 5-min recording intervals. Two-way ANOVAs, with time as the only repeated measure, were performed on cocaine-induced changes in Amax across 0 to 60 min (see Results). Right, graphs summarize the peak effects on Amax during the first 30 min post-treatment. Significant differences reflect post hoc Bonferroni's multiple t test comparisons. **, p < 0.01 versus the days 1 to 3 cocaine-induced response. #, p < 0.05; ##, p < 0.01 versus the day 0 saline-induced response.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Time course comparisons of baseline and cocaine-induced changes in DA signal k during repeated cocaine administration and after withdrawal in LCR (A) and HCR (B) rats. See Fig. 7 for experimental details and groups. Arrows indicate time of the i.p. injections of saline (1 ml/kg) or cocaine (10 mg/kg). Left, time courses for repeated treatment effects on baseline and cocaine-induced changes in k are shown for the 5-min recording intervals. Two-way ANOVAs, with time as the only repeated measures, were performed on cocaine-induced changes in k across 0 to 60 min (see Results). Right, bar graphs summarize the peak effects on k during the first 30 min post-treatment. Significant differences reflect post hoc Bonferroni's multiple t test comparisons. **, p < 0.01 versus the averaged days 1 to 3 cocaine-induced effect. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus the day 0 saline-induced effect.

Conditioning Does Not Contribute Significantly to Sensitized Behavioral and Electrochemical Responses to Cocaine Measured Here. To control for the passage of time and any potential conditioned responses to the repeated injections of cocaine and/or local applications of DA, another group of rats with electrochemical assemblies chronically implanted in NAc received repeated saline injections for 7 days before an acute cocaine challenge injection (Table 1, control group). Furthermore, this same group subsequently received cocaine injections on five additional days, followed by a saline challenge injection on the 7th day (Table 1). Data collected in the experimental group on the day 7 cocaine challenge were averaged across the LCR and HCR responses and reanalyzed for significant differences from the control group. These comparisons are summarized in Fig. 9. Baseline locomotor activity during the 30 min immediately preceding either the cocaine or saline challenge injections did not differ significantly between the treatment groups, confirming that differences in locomotor responsiveness to the challenges were not due to differences in levels of baseline activity per se. The cocaine challenge on day 7 significantly increased locomotor activity above baseline in both the saline-pretreated control group and cocaine-pretreated experimental group. However, this increase was significantly greater (by 80%) in the cocaine-pretreated, compared with saline-pretreated, rats. This result confirmed that, independent of the LCR/HCR classification, the repeated cocaine regimen used here was effective in inducing locomotor sensitization. In contrast with the cocaine challenge after repeated saline treatment in the control group, the saline challenge after repeated cocaine treatment in these same rats did not increase locomotor activity above baseline. Thus, there was no apparent conditioned locomotor response to this particular repeated cocaine regimen.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Only male Sprague-Dawley rats treated repeatedly with cocaine exhibit locomotor sensitization to a subsequent cocaine challenge. The effects of repeated treatment with either saline (S) or cocaine (C) on baseline and saline or cocaine challenge-induced locomotor activity are shown. The first and third pairs of columns represent the control group and the second pair of columns represents the experimental group (Table 1). Locomotor activity is the cumulative distance traveled (centimeters) in the 30 min immediately preceding (baseline) and the 30 min after the cocaine or saline challenge injection. Data are mean values ± S.E.M. A two-way ANOVA revealed a significant effect of treatment (F2,68 = 18.16, p < 0.001) and time (baseline versus challenge; F1,68 = 21.03, p < 0.001). Significant effects indicated reflect post hoc Bonferroni's multiple t test comparisons. +, p < 0.05; +++, p < 0.001 versus the respective baseline. **, p < 0.01 versus the daily saline with a cocaine challenge. ###, p < 0.001 versus the daily cocaine with a saline challenge.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Only rats treated repeatedly with cocaine exhibit inhibition of DA clearance to a subsequent cocaine challenge. The effects of repeated treatment with either saline (S) or cocaine (C) on cocaine- or saline-induced changes in DA signal Amax (A) and k (B) parameters in NAc are shown. Data are mean values ± S.E.M. for the same rats in which locomotor responses were recorded simultaneously and reported in Fig. 9. For each rat, the DA clearance signal parameters were averaged across the first 30 min after the "challenge" injection of either cocaine (10 mg/kg) or saline (1 ml/kg) and expressed relative to its baseline value (average of five to six signal parameters immediately preceding the challenge injection). A one-way ANOVA revealed a significant effect of treatment on Amax (F2,36 = 11.042, p < 0.001) and k (F2,36 = 27.941, p < 0.001). Significant differences indicated reflect posthoc Bonferroni's multiple t tests comparisons. *, p < 0.05; **, p < 0.01 versus the daily saline with a cocaine challenge. #, p < 0.05; ###, p < 0.001 versus the daily cocaine with a saline challenge.

	Discussion

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Whether DAT expression/activity is up- or down-regulated by repeated cocaine administration remains controversial (Zahniser et al., 1995; Kuhar and Pilotte, 1996; Zahniser and Doolen, 2001). Furthermore, the exact relationship between cocaine-induced adaptations in DAT function and changes in behavior has remained elusive. Previously, we found that DA clearance in NAc of anesthetized rats was more sensitive to cocaine inhibition after withdrawal from repeated cocaine administration (Cass et al., 1993a). Here, by monitoring the time course of cocaine-induced changes in behavior concomitantly with inhibition kinetics of DA clearance, we demonstrate the essential role of greater inhibition of DAT function to locomotor sensitization induced by repeated cocaine administration. Specifically, we found that the potential for expression of locomotor sensitization 1) can be predicted by the animal's initial locomotor responsiveness to an acute cocaine injection and 2) is confined to a subgroup of rats (LCRs) in which the efficiency to clear exogenous DA in NAc in the presence of cocaine diminishes with repeated administration.

Previously, we identified two distinct populations of cocaine responders using the median split of initial locomotor responses to acute low-dose cocaine in male Sprague-Dawley rats (Sabeti et al., 2002b). Subsequently, we have confirmed a trend for two components in this distribution (LCR component, mean 5,130 cm/30 min; S.D. = 2,140; proportion = 49%; HCR component, mean = 12,370; S.D. = 4,950; proportion = 51%; n = 32; p = 0.07; NOCOM program; Ott, 1979). Likewise, the distribution of A_max responses to acute cocaine in this larger population was bimodal (LCR component, mean 98% baseline; proportion = 58%; HCR component, mean 170% baseline; proportion = 42%; p < 0.01). Here, using a subset of these rats, we observed that differences in the initial locomotor responsiveness to cocaine accounted for nearly 47% of the variability in the magnitude of locomotor sensitization expressed by LCRs (Fig. 3). Furthermore, the LCR/HCR classification was predictive of the potential of individual rats to express cocaine-induced locomotor sensitization. LCRs exhibited minimal locomotor activation to the initial injection of cocaine but locomotor sensitization with repeated cocaine, whereas HCRs exhibited significant initial locomotor responsiveness to cocaine but failed to express locomotor sensitization with repeated administration. However, not all cocaine-induced behaviors were augmented in LCRs. Furthermore, HCRs exhibited sensitized cocaine-induced rearing responses. These observations, together with the finding that brain levels of cocaine are consistently increased in all male Sprague-Dawley rats receiving repeated i.p. injections of cocaine (Cass and Zahniser, 1993), suggest that pharmacokinetic differences can not satisfactorily explain the LCR versus HCR behavioral differences. It is also unlikely that increased rearing or head/limb stereotypy explained the lack of locomotor sensitization in HCRs because stereotypies were exhibited by LCRs to a similar extent on days 5 to 7 and did not preclude locomotor sensitization in these rats. However, we cannot rule out the possibility that a ceiling in locomotor activation precluded sensitization in HCRs because only the 10-mg/kg dose of cocaine was tested here. Dose-response studies would address whether HCRs are able to increase activity further and whether repeated cocaine administration shifts the dose-response relationship in LCRs, making cocaine a more potent or efficacious inhibitor of DAT. In any case, conditioned locomotor responses to the repeated injections were not apparent with our treatment paradigm, supporting earlier findings that the development of conditioned responses is not necessary for the expression of behavioral sensitization (Fraioli et al., 1999).

Greater activation in response to novelty, rather than to acute stimulant administration, has been used more often to predict enhanced vulnerability to psychostimulant-induced sensitization and self-administration (see Introduction). We observed the opposite relationship: HCRs exhibit higher spontaneous locomotor activity than LCRs during their initial exposure to the activity apparatus (i.e., day 0; Sabeti et al., 2002b) but did not express locomotor sensitization with repeated cocaine administration (this study). This lack of correspondence in expression of sensitization between our cocaine responders and the low- and high-novelty responders defined in the literature may reflect differences in experimental conditions. For instance, our rats underwent extensive handling and habituation to the testing environment before drug administration, factors previously documented to modulate drug responsiveness (Cools and Gingras, 1998; Fraioli et al., 1999; Tuinstra and Cools, 2000). Specifically, habituation can decrease the behavioral and NAc DA sensitivity to stimulants in high-novelty responders and increase sensitivity in low-novelty responders (Cools and Gingras, 1998; Tuinstra and Cools, 2000). These investigators have hypothesized that individual differences in the regulation of DA neurotransmission by neuroendocrine and/or noradrenergic systems underlie this reversal in sensitivity under various experimental conditions. On the other hand, the discrepancy may reflect differences underlying responsiveness to novelty and cocaine (Djano and Martin-Iverson, 2000; Sutton et al., 2000). The finding that unique provisional quantitative trait loci exist for novelty- versus cocaine-induced initial locomotor activity and sensitization (Phillips et al., 1998) further supports our hypothesis that HCRs may be phenotypically distinct from the high responders to novelty. Studies using self-administration and/or conditioned place preference paradigms are needed to define the relationship of the LCR/HCR phenotypes and cocaine reinforcement.

The temporal association between changes in behavior and DAT function in response to repeated cocaine (Figs. 2, 7, and 8) provides strong evidence that cocaine-induced regulation of DAT in NAc plays a critical role in the expression of locomotor sensitization to cocaine. This association reflected multiple recordings of exogenous DA clearance signals across the 7 days of repeated cocaine in the same individual rats. For example, initially on days 1 to 3, when locomotor sensitization was not yet expressed in LCRs, A_max and k parameters in NAc were not modulated by cocaine. The expression of cocaine-induced locomotor sensitization in LCRs on days 5 to 7 was, however, accompanied by potentiated A_max and reduced k, consistent with higher levels of extracellular DA and sensitized DA-related behaviors. Importantly, the sensitized effects of cocaine on behavior and DA clearance parameters in LCRs persisted after a 7-day withdrawal from repeated cocaine treatment, consistent with the long-lasting nature of sensitization. The absence of such regulation in HCRs and the control group ruled out a more general effect of the repeated DA applications on increased DAT sensitivity to cocaine. Overall, our findings are in agreement with reports of enhanced cocaine-induced inhibition of DA uptake after repeated cocaine administration (Izenwasser and Cox, 1990; Cass et al., 1993a; Lee et al., 1998; but see Ng et al., 1991; Masserano et al., 1994; Meiergerd et al., 1994; Chefer and Shippenberg, 2002).

The finding that cocaine-induced inhibition of DA clearance in LCRs was expressed only after 3 days of repeated cocaine administration strongly suggests that protein synthesis and/or recruitment of other systems was/were required for the long-term alterations in DAT sensitivity to cocaine in LCRs. On the other hand, LCR/HCR differences in the acute cocaine response may reflect intrinsic variations in more rapid, nongenomic mechanisms of DAT regulation in response to acute inhibition (i.e., cell surface trafficking; Daws et al., 2002; Little et al., 2002). Also, although the NAc is sufficient for mediating the initial locomotor response to acute cocaine (Delfs et al., 1990), a number of long-lasting neuroadaptations in NAc, as well as other DA projection sites, are necessary for the expression of stimulant-induced locomotor sensitization (Vanderschuren and Kalivas, 2000; Nestler, 2001; Everitt and Wolf, 2002). Whether and how glutamatergic and/or GABAergic systems are involved in the LCR/HCR differences in sensitization remains to be investigated. Furthermore, future studies will address whether sensitization in cocaine-induced rearing, as opposed to locomotor activity, in HCRs reflects alterations in DAT sensitivity to cocaine inhibition in the dorsal striatum, which plays an important role in stereotypic behaviors.

On day 1 greater amounts of exogenous DA were applied in NAc of HCRs than LCRs to achieve similar A_max responses (Sabeti et al., 2002b; present study). Here, we demonstrated that this group difference was surmounted after day 3, suggesting that the initial difference likely reflected higher baseline DA clearance capacity in NAc of HCRs than LCRs, rather than variability in injector locations. Although this is discordant with the observed higher initial sensitivity to cocaine inhibition, future kinetic experiments will address whether lower DAT affinity for DA might explain this apparent discrepancy. In contrast, in dorsal striatum equivalent amounts of DA were required on day 1 to achieve similar A_max responses in LCRs and HCRs (Sabeti et al., 2002b). This suggests that, by itself, differences in amounts of DA applied are unlikely to explain the absence of altered DAT sensitivity to cocaine inhibition over time, as observed here in HCRs. Two possible explanations for the decreased amounts of DA needed in NAc of HCRs over time are fewer functional uptake sites secondary to tissue damage and/or regulation resulting in reduced basal DAT function. Because the amount of DA needed in LCRs remained constant across time, the latter is the more likely explanation. Chefer and Shippenberg (2002) have reported such changes in behaviorally sensitized rats, namely a reduction in basal DAT function with no changes in cocaine-induced DAT inhibition. Interestingly, behavioral sensitization in this study was assessed by rating repetitive/stereotypic behaviors. Together, these findings underscore the importance of assessing sensitization by both locomotor activity and stereotypy to understand the relevance of DAT regulation to changes in behavioral responsiveness. Furthermore, our results, along with previous reports, support the hypothesis that cocaine-induced adaptations in DAT in the NAc are necessary for the expression of locomotor sensitization. Therefore, LCR/HCR rats may be useful models for further study of differential phenotypes for initial sensitivity and sensitization to cocaine.