Differential cocaine-induced modulation of glutamate and dopamine transporters after contingent and non-contingent administration

Neuropharmacology 55 (2008) 771–779

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Differential cocaine-induced modulation of glutamate and dopamine transporters after contingent and non-contingent administration Miguel Migue´ns a,1, Jose´ Antonio Crespo a,1, Nuria Del Olmo b, Alejandro Higuera-Matas a, Gonzalo L. Montoya a, Carmen Garcı´a-Lecumberri a, Emilio Ambrosio a, * a b

´n a Distancia (UNED), C/ Juan del Rosal n 10, 28040 Madrid, Spain Departamento de Psicobiologı´a, Facultad de Psicologı´a, Universidad Nacional de Educacio Laboratory of Pharmacology, University of San Pablo CEU, P.O. Box 67, 28660 Boadilla, Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 December 2007 Received in revised form 11 June 2008 Accepted 12 June 2008

Although dopamine and glutamate transmission has been implicated in cocaine dependence, the effects of the extinction of cocaine self-administration on protein transporters in both of these neurotransmitter systems remain unknown. We have used a yoked-box procedure to simultaneously test rats in triads, one rat that actively self-administered cocaine (CONT), while the other two received yoked injections of either cocaine (NON-CONT) or saline (SALINE). The brains in each triad were removed and processed for quantitative autoradiography immediately after the last session of cocaine self-administration (Day 0), or after 1, 5, or 10 days of extinction, and excitatory amino acid transporters (EAATs) and dopamine transporter (DAT) binding was examined. When compared to NON-CONT and SALINE animals, binding of radioligand to EAATs was significantly lower in the hippocampal CA1 field and the cerebellar cortex of CONT rats on Day 0, although it was significantly higher after 1 day of extinction in the infralimbic cortex. No differences in EAAT binding were observed after 5 or 10 days of extinction in any of the brain regions analyzed. In contrast and at all the time points of extinction, binding to DAT was significantly enhanced in CONT animals when compared to SALINE and NON-CONT rats in different forebrain and mesencephalic regions, including the nucleus accumbens, ventral tegmental area or caudate putamen. These results suggest that changes in protein transporter binding after cocaine self-administration and extinction are transient for EAAT while they are more enduring for DAT, and that they depend on the type of access to cocaine. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Cocaine DAT Glutamate transporter Self-administration Extinction Autoradiography

1. Introduction Studies on the neurobiology of cocaine abuse suggest that cocaine affects key elements in the brain reward system, particularly the mesocorticolimbic dopamine pathways (Wise, 1996). Moreover, the ability of cocaine to bind to dopamine and serotonin transporters is critical for its reinforcing effects (Ritz et al., 1987, 1988; Rocha et al., 1998; Uhl et al., 2002), although more recent data highlights the involvement of other neurotransmitter systems in cocaine addiction (such as the glutamatergic system, reviewed in Kalivas, 2004). Several studies with rodents have identified longterm neuroadaptation in the glutamatergic system after chronic cocaine administration and cocaine abstinence (Crespo et al., 2002; Churchill et al., 1999; Lu et al., 2003; Sutton et al., 2003). Indeed, altered ionotropic glutamate receptor subunits have been observed in the nucleus accumbens (Nacc) of cocaine abusers and non-

* Corresponding author. Tel.: þ34913987974; fax: þ34913986287. E-mail address: [email protected] (E. Ambrosio). 1 These authors contributed equally to this work. 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.06.042

human primates after long-term cocaine self-administration (Hemby et al., 2005). Microdialysis studies have demonstrated that behavioral sensitization to cocaine is related to the capacity of repeated doses of this drug to increase glutamate release in the Nacc and the ventral tegmental area (VTA; Pierce et al., 1996; Reid and Berger, 1996). Moreover, cocaine priming after the extinction of cocaine self-administration augments glutamate release in the Nacc against a background of reduced basal glutamate levels (Baker et al., 2003; McFarland et al., 2003; Miguens et al., 2008). The glutamatergic and dopaminergic systems coincide in relevant areas of the mesocorticolimbic reward circuit. In fact, in the Nacc, glutamate axons arising from prefrontal cortex, hippocampus, basolateral amygdala and periventricular thalamic nucleus are in direct axo-axonal apposition to dopaminergic afferents from the VTA (Berendse et al., 1992; Fallon and Moore, 1978; Wright et al., 1996). The convergence of both neurotransmitter systems may modulate the effects of cocaine and thus, it is important to analyze both glutamate and dopamine transporters together in a cocaine self-administration paradigm. It is generally accepted that most glutamate transport in the central nervous system, particularly that related to excitatory

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M. Migue´ns et al. / Neuropharmacology 55 (2008) 771–779

transmission, is mediated by the high-affinity, sodium-dependent excitatory amino acid transporters (EAATs; Danbolt, 2001). In addition, EAATs play a functional role in preserving the local integrity of excitatory synaptic transmission (Marcaggi and Attwell, 2004) and they have been proposed as mediators of synaptic plasticity (Levenson et al., 2002; Pita-Almenar et al., 2006). However, there are no studies on the effects of cocaine self-administration and its extinction on EAATs. In contrast, human studies have shown that dopamine transport is elevated in cocaine users (Mash et al., 2002) and that the level of DAT occupancy correlates with self-reported euphoric states after cocaine intake (Volkow et al., 1997, 1996a,b). In addition, several rodent studies have identified altered DAT levels after withdrawal of passive cocaine administration (Hitri and Wyatt, 1993; Pilotte et al., 1994; Sharpe et al., 1991). Similarly, cocaine self-administration studies in the rat and monkey have also found alterations in DAT within different brain areas (Ben-Shahar et al., 2006; Letchworth et al., 2001; Wilson and Kish, 1996; Wilson et al., 1994a,b). Nevertheless, the interpretation of animal results is confusing given that the mentioned reports diverge in terms of the duration of the treatment, the access to cocaine, the time of withdrawal and even the radioligand employed. In addition, there are no studies on dopamine transporter levels after extinguishing cocaine self-administration at different time points. Therefore, we set out to examine how binding to EAATs and DAT is modulated during the extinction of cocaine self-administration behavior using a yoked-box procedure. This experimental design involves the use of triads to assess differences related to contingent versus non-contingent presentation of environmental events (Smith and Dworkin, 1986). EAAT and DAT levels were measured by quantitative autoradiography in slices adjacent to those analyzed in a previous study (Crespo et al., 2002) from several rat brain regions on the last day of cocaine self-administration (Day 0) and on extinction Day 1, 5, and 10. 2. Materials and methods 2.1. Animals Adult male Lewis rats (Harlan Interfauna Ibe´rica, Barcelona, Spain) were used in this study that weighed approximately 300–350 g at the beginning of the experiments. All animals were experimentally naı¨ve, and they were housed individually in a temperature-controlled room (23  C) with a 12-h light–dark cycle (08:00–20:00 lights on) with free access to Purina laboratory feed and tap water prior to the initiation of the experiments. The animals used in this study were maintained and handled in full compliance with European Union Laboratory Animal Care Rules (86/ 609/EEC Directive). 2.2. Surgery The animals were prepared with an i.v. catheter, surgically implanting polyvinylchloride tubing (0.064 i.d.) in the right jugular vein approximately at the level of the atrium under ketamine (40 mg/kg) and diazepam (10 mg/kg) anaesthesia. The catheter was passed s.c. and exited in the midscapular region, and it was then passed through a spring tether system (Alice King, Chatham, USA) that was mounted to the skull of the rat with dental cement. All subjects were housed individually following surgery and allowed to recover for at least 7 days. The catheter patency was tested with the barbiturate anesthetic methohexital (10 mg/kg, i.v.) and it was assumed to remain unblocked if the rat immediately lost consciousness. 2.3. Apparatus Twelve operant chambers (Coulbourn Instruments, USA) were used for cocaine self-administration studies. The chambers had two levers placed 14 cm apart on the front wall of the chamber that were designed to register a response when 3.0 g of force was applied. A microliter injection pump (Harvard 22) was used to deliver i.v. saline or cocaine injections to the rat. Drug delivery, operant data acquisition and storage were accomplished using IBM computers (Med Associates, USA).

maintained under this schedule, the catheter was surgically implanted as indicated. After the post-operative period, 72 male littermates were randomly assigned in triads to one of the three conditions: (a) contingent i.v. self-administration of 1 mg/ kg/injection of cocaine (CONT) and (b) non-contingent i.v. 1 mg/kg of cocaine (NONCONT) or (c) saline (SALINE) yoked to the intake of the self-administering subject. Initially, substitution of food delivery by cocaine began under a FR1 schedule of reinforcement and was subsequently raised to FR5, a programmed 30-s time out in which responses had no programmed consequences followed each cocaine injection (FR5:TO 30 s). Animals were allowed to self-administer cocaine in daily 2-h sessions between 9:00 and 14:00 h, 7 days a week for a minimum of 3 weeks. After stable behavior was reached (less than 10% variability in the number of injections on 5 sequential days), saline was substituted for 4 days in the operant chambers. After this first extinction period, cocaine self-administration was reinstated and maintained for 2 weeks. Saline substitution was then carried out again for 1, 5, and 10 days in the operant chambers. Immediately after the last day of cocaine selfadministration, in which the animals reached the stability criterion (Day 0), or after 1 (Day 1), 5 (Day 5) and 10 (Day 10) days of extinction, the brains of each member of the triad were removed and processed for quantitative autoradiography. The total number of subjects in each group of the triad was 18 (CONT ¼ 6, NON-CONT ¼ 6, SALINE ¼ 6) and the total number of brains removed was 72 [four groups of triads (Day 0, Day 1, Day 5 and Day 10)  18]. 2.5. Quantitative autoradiography Brain coronal sections (20 mm) were cut at the level of the Nacc, thalamus, ventral tegmental area (VTA), raphe nuclei (RN) and locus coeruleus (LC), according to the atlas of Paxinos and Watson (1998). The sections were mounted onto gelatincoated slides and stored at 80  C until the day of the assays. All the sections used in the present work were adjacent to those analyzed in a previous study (Crespo et al., 2002). 2.6. Glutamate transporter autoradiographic assay The protocol used to analyze the glutamate transporter followed the method described by Takamoto et al. (2002). Briefly, brain sections mounted on the slides were preincubated at 25  C in a buffer containing 120 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 5 mM NaH2PO4/Na2HPO4 at pH 7.35, and then with 20 nM of L-[2,3-3H]-aspartic acid (39 Ci/mmol; Amersham Biosciences/GE Healthcare, Spain) at the same temperature and in the same buffer. This labelling was performed either in the presence or absence of 10 mM unlabelled L-glutamate to determine total and non-specific binding, respectively. After incubation with the radioligand, the slides were briefly washed four times in the same buffer and subsequently in distilled water at 0–4  C. Finally, the sections were dried under a stream of cool dried air. It should be noted that this protocol does not reveal the identity of individual transporters involved in glutamate transport because this ligand binds to all of the different EAAT subtypes (Robinson and Dowd, 1997; Vandenberg, 1998). 2.7. Dopamine transporter autoradiographic assay The DAT was assayed using the protocol described by Canfield et al. (1990) with few modifications. In brief, slide-mounted brain sections were preincubated for 20 min in phosphate buffered saline (PBS) 50 mM at 0–4  C (pH 7.4) containing 50 mM NaCl to remove any residual cocaine that might be present, and they were then incubated for 2 h in the same buffer containing 10 nM of [N-methyl-3H]-WIN 35,428 (86 Ci/mmol; NEN Perkin Elmer, Spain). The incubation was performed in either the presence or absence of 100 mM of unlabelled cocaine to determine total and non-specific binding, respectively. After incubation, the slides were washed twice (1 min) in cold PBS and they were then rinsed briefly twice in distilled water before drying under a stream of cool dry air. 2.8. Densitometry of autoradiograms The slides were exposed to tritium-sensitive film ([3H]-Hyperfilm, Amersham Biosciences/GE Healthcare, Spain) in standard X-ray cassettes for a period of 8–10 weeks at 4  C. At the end of this period, the films were developed for 5 min at 20  C in a Kodak D-19 developer, fixed for 10 min and finally rinsed in water and air dried. Autoradiograms were analyzed on a PC computer using the Scion Image program (Scion Corporation, Frederick, MA, USA). Density measurements were calculated for each animal from two slides per region (two slices/slide; two measurements/slice in consecutive brain sections) and they were transformed to concentrations (nCi/mg of tissue equivalent) using tritium-labelled microscale standards (Amersham Biosciences/GE Helathcare, Spain). Specific binding was determined by subtracting non-specific binding from total binding. Finally, the fmol/mg tissue equivalent values were calculated.

2.4. Experimental procedure 2.9. Statistical analyses Cocaine-reinforced behavior was described previously (Crespo et al., 2002). Briefly, before surgical implantation of the i.v. catheter, animals were trained to press a lever for food under a FR5 schedule of reinforcement. When the behavior was

Analysis of the DAT and EAAT levels was performed using a two-way multivariate analysis of variance (MANOVA) with the different regions serving as

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dependent variables, and the type of administration (CONT, NON-CONT or SALINE) and the day of extinction (0, 1, 5, 10) as the between-subjects factors. For all multivariate analyses, we used Pillai’s trace as our test statistic. Tukey’s post-hoc tests were performed when appropriate and differences were considered significant if the probability of error was less than 5%.

3. Results 3.1. Behavioral A detailed description of the behavioral results can be found in Crespo et al. (2002). Briefly, the CONT animals earned a greater number of cocaine injections in the acquisition and reacquisition phases when compared to the saline injections of the extinction phases. The averaged number of cocaine injections per session during the acquisition and reacquisition phases was 16.7  2.4, and the mean of the total dose administered was 669.6 mg/kg. During the second extinction period the animals rapidly extinguished cocaine self-administration behavior. The averaged number of saline injections was 4.6  1.1 in the day 1, 2.5  0.9 in the day 5, and 2.1  0.9 in the day 10. This response pattern suggests that cocaine served as a positive reinforcer under our experimental conditions. In contrast, the NON-CONT and SALINE groups were unable to respond and the subjects in both groups received cocaine (1 mg/kg/injection) or saline (0.9% NaCl) passively when the contingent animals self-administered the drug. 3.2. Glutamate transporters The two-way MANOVA indicates a multivariate significant effect of the type of administration  day of extinction interaction on EAATs binding (Pillai’s Trace, V ¼ 5.24; F300,96 ¼ 2.22, p < 0.001). In addition, the analysis revealed statistically significant univariate effects in CA1 field of the hippocampus, the cerebellar cortex and the infralimbic subregion of the medial prefrontal cortex (F values are shown in Table 1). A decrease in L-[2,3-3H]-aspartic acid binding was observed on Day 0 in the CA1 field of the hippocampus and in the cerebellar cortex (Fig. 1) of CONT rats when compared to SALINE and NON-CONT animals (p < 0.05). However, the levels of binding in CONT animals were not significantly different to those of SALINE or NON-CONT animals in these regions during the extinction of cocaine self-administration (Days 1, 5 and 10). On Day 1, L-[2,3-3H]-aspartic acid binding levels were significantly higher in the infralimbic cortex subregion of the medial prefrontal cortex of CONT rats (Fig. 1) when compared to SALINE and NON-CONT animals (p < 0.05). Representative coronal autoradiograms of total binding of L-[2,3-3H]-aspartic acid to EAATs in the conditions where we found statistically significant differences are shown in Fig. 2. The specific binding values of L-[2,3-3H]-aspartic acid to EAATs in different brain regions where statistically significant differences were not found are presented in Tables 2–5.

Table 1 ANOVA’s analysis of specific L-[2,3-3H]-aspartic acid binding to EAATs Brain area

Type of administration (T)

Extinction day (D)

TD

IL

N.S

CA1

N.S

Cb

F2,60 ¼ 4.47 P < 0.05

F3,60 ¼ 8.92 p < 0.001 F3,60 ¼ 3.64 p < 0.05 F3,60 ¼ 3.23 p < 0.05

F6,60 ¼ 2.94 p < 0.05 F6,60 ¼ 3.37 p < 0.01 F6,60 ¼ 2.43 p < 0.05

Brain areas: infralimbic cortex (IL), CA1 field of the hippocampus (CA1), cerebellar cortex (Cb).

Fig. 1. Time course of the effects of extinction of cocaine self-administration on EAATs’ binding in the hippocampal CA1 field, the infralimbic cortex (IL), and the cerebellar cortex of the rat brain of SALINE, CONT, and NON-CONT animals. Data represent the mean  s.e.m of [3H]-L-aspartate specific binding expressed as fmol/mg of equivalent tissue. *p < 0.05 and **p < 0.01 indicate a significant difference from SALINE control subjects; þp < 0.05 indicates a significant difference when compared to NON-CONT subjects.

3.3. Dopamine transporter The two-way MANOVA indicates a multivariate significant effect of the type of administration  day of extinction interaction on [N-methyl-3H]-WIN 35,428 binding to DATs (Pillai’s Trace, V ¼ 1.31; F36,360 ¼ 1.87, p < 0.005). In addition, the analysis revealed statistically significant univariate effects on different brain regions related to the brain reward system (F values are shown in Table 6). An overall view of the time course of [N-methyl-3H]-WIN 35,428 binding to DAT following extinction of cocaine self-administration is shown in Fig. 3. Representative coronal autoradiograms of [N-methyl-3H]-WIN 35,428 binding to DAT at the level of dorsal striatum and nucleus accumbens in each experimental condition are presented in Fig. 4. The specific binding of [N-methyl-3H]-WIN 35,428 to DAT were assayed in the different brain regions related to the brain reward system across the different extinction periods, and the data were expressed as fmol/mg of tissue equivalent. After

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Fig. 2. Representative coronal autoradiograms of total [3H]-L-aspartate binding to EAATs at the level of hippocampus (A), cerebellar cortex (B) and infralimbic cortex (C), in the conditions days where statistically significant differences were observed. Measurements of the cerebellar cortex were taken from the molecular layer at 10.00 mm anteroposterior relative to Bregma.

cocaine self-administration (Day 0) CONT animals showed elevated [N-methyl-3H]-WIN 35,428 binding levels compared to those found in SALINE and NON-CONT rats in most of the analyzed regions. This elevation was progressively increased in the absence of cocaine and persisted after 10 days. However, NON-CONT animals only showed increased DAT binding levels in VTA and Nacc (Shell) after 1 day, and in VTA, CPu and mPFC after 10 days of cocaine abstinence when compared with SALINE animals. 4. Discussion In the present work, changes in glutamate and dopamine transporters have been found after cocaine self-administration and during the extinction of this behavior, indicating that continuous administration of this drug involves neuroadaptation of both transmission systems. First, we show that binding to EAATs diminishes in the CA1 subfield of the hippocampus and the cerebellar cortex after long-term chronic cocaine self-administration. By contrast, the binding to EAATs augments after extinction of this behavior for 1 day, but only in the infralimbic portion of the medial prefrontal cortex. No other differences in EAAT binding levels were found in any of the brain regions analyzed. However, it is worth mentioning that the radioligand employed in this study does not enable us to discriminate between the different subtypes of EAATs involved in glutamate transport. Therefore, the failure to observe effects in some areas could be explained if the changes in the expression of a particular subtype of EAAT were masked by the expression of another. Interestingly, the neuroadaptive changes in EAAT binding only appeared in the CONT animals, in agreement with several studies that show differential neurochemical effects depending upon the contingent versus non-contingent administration of the drug (Crespo et al., 2003, 2001a, 2002; Hemby et al., 1995; McFarland et al., 2003). However, these EAAT changes were reversible since they were no longer present after 5 and 10 days of extinction. It is tempting to speculate that the diminished binding to EAATs in the CA1 field of the hippocampus and the cerebellar cortex could be due to a down-regulation of such proteins, as a consequence of a possible reduction in basal extracellular glutamate in these brain areas after chronic cocaine administration. In this respect, we

recently showed that after 20 days of chronic cocaine selfadministration, extracellular glutamate levels diminish in the core of the Nacc (Miguens et al., 2008). Decreased basal accumbal extracellular glutamate has also been found after repeated cocaine injections (Pierce et al., 1996) and after a long withdrawal period following chronic cocaine self-administration (Baker et al., 2003; McFarland et al., 2003). Thus, it is possible that chronic cocaine selfadministration could also result in lower extracellular glutamate levels in brain areas other than the Nacc. In turn, this decrease could down-regulate EAATs establishing a mechanism to maintain the concentration of synaptic glutamate within a narrow range. However, the reduced basal extracellular glutamate levels found after prolonged periods of cocaine withdrawal could depend on factors independent of the Naþ dependent EAATs. In this sense, it has been shown that a reduction in the activity of the glutamate– cystine exchanger could be at least partially responsible for the diminished glutamate levels in the Nacc when cocaine selfadministration ceases (Baker et al., 2002). The decrease we observe in binding to EAATs in the CA1 field also suggests that glutamate transmission in this area might play a role in maintaining chronic cocaine self-administration. Indeed, the stimulation of hippocampal glutamatergic fibres, but not of dopaminergic neurons in the medial forebrain bundle, evokes cocaine-seeking behavior that depends on VTA glutamate (Vorel et al., 2001). Similarly, we recently found cocaine self-administration to facilitate the generation of hippocampal long-term potentiation (LTP), a phenomenon that is largely dependent on NMDA glutamate receptors. Significantly, this LTP enhancement is maintained even after three weeks of extinction (Del Olmo et al., 2006). Our data also point to a role of cerebellar EAATs in the action of cocaine. Several functional neuroimaging studies in humans have found cerebellar activation in the presence of contextual cues associated with cocaine, and this activity is correlated with craving (Bonson et al., 2002; Grant et al., 1996; Kilts et al., 2001; Wang et al., 1999). In addition, it has been shown that activation of the cerebellum occurs when there is expectation of cocaine consumption (Volkow et al., 2003), and the cerebellar vermis is thought to be involved in the acute and persistent effects of cocaine (Anderson et al., 2006). Moreover, cerebellar deficits were recently reported to contribute to the neuropsychological deficits and motor dysfunction frequently observed in cocaine-dependent subjects (Sim et al.,

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Table 2 [2,3-3H]-L-aspartate binding in the rat brain during the extinction of cocaine selfadministration (cortical regions)

Table 3 [2,3-3H]-L-aspartate binding in the rat brain during the extinction of cocaine selfadministration (striatum, hippocampus and thalamus)

Brain area Group

Brain area

Cerebral cortex Cg CONT NON-CONT SALINE PrL CONT NON-CONT SALINE Pir CONT NON-CONT SALINE M1 CONT NON-CONT SALINE M2 CONT NON-CONT SALINE DP CONT NON-CONT SALINE VO CONT NON-CONT SALINE Au CONT NON-CONT SALINE Ent CONT NON-CONT SALINE RSA CONT NON-CONT SALINE RSG CONT NON-CONT SALINE V1 CONT NON-CONT SALINE

Day 0

Day 1

57.19  13.4 82.18  3.5 75.62  10.0 67.56  1.6 59.44  6.4 67.07  6.5 66.42  11.7 88.16  3.5 89.87  15.6 73.77  2.3 71.61  4.9 75.65  6.8 67.45  8.4 106.37  10.0 68.88  11.4 89.65  10.5 84.70  9.2 83.81  7.1 45.42  7.7 70.03  7.0 60.85  10.3 63.19  1.1 44.86  4.5 52.41  3.9 49.24  9.8 71.30  6.3 62.04  9.9 64.29  2.6 45.68  4.9 53.17  4.1 63.35  10.3 85.94  4.2 82.48  11.1 80.58  3.0 71.50  7.6 72.86  4.3 87.59  11.8 95.70  8.0 91.63  10.2 93.75  7.3 80.43  8.3 82.89  5.4 77.18  6.6 90.10  3.3 97.79  9.4 87.71  6.8 76.55  9.9 80.96  4.8 57.04  2.0 82.21  4.4 57.49  5.6 69.69  13.4 62.76  2.4 73.13  8.4 42.42  5.86 61.39  9.81 46.44  5.82 60.62  9.39 60.70  3.72 65.30  7.27 44.35  3.2 66.50  9.5 49.80  7.3 63.11  9.4 63.85  3.0 69.92  9.8 56.47  3.0 75.52  9.8 69.88  4.2 76.07  8.1 72.15  5.2 80.35  6.0

Day 5

Day 10

67.41  7.6 83.61  10.1 45.43  12.3 76.90  13.7 70.15  15.1 63.30  5.7 69.87  8.5 88.80  9.0 47.07  12.3 84.84  16.1 73.84  13.5 71.85  6.8 80.80  8.7 89.62  10.2 64.80  18.4 67.91  5.8 68.42  14.0 87.20  7.4 52.08  8.5 61.84  9.8 36.72  9.9 66.55  9.5 54.71  10.4 50.96  10.8 53.22  7.5 67.24  9.4 39.17  11.7 63.18  8.7 54.66  10.1 52.37  5.6 61.29  10.6 79.67  10.4 55.34  10.0 84.18  11.4 71.70  11.3 66.75  5.2 80.25  11.0 91.37  9.2 62.07  15.0 94.65  10.4 78.54  13.7 79.07  9.4 88.50  13.8 93.03  11.5 63.13  7.7 92.59  19.5 87.91  21.5 69.91  6.2 73.19  16.1 87.52  9.5 57.17  13.0 81.97  13.5 80.96  17.9 69.98  7.6 68.72  11.50 94.71  13.58 49.59  9.51 73.72  7.22 65.33  17.41 64.36  8.65 75.11  12.7 93.90  20.0 49.40  9.1 73.73  8.1 72.99  19.9 67.12  9.4 79.74  15.2 98.31  11.9 66.03  10.2 101.78  8.1 82.51  17.0 79.83  7.0

The data represent the mean  SEM expressed as fmol/mg of tissue equivalent in different cortical brain regions: medial prefrontal cortex (mPFC), cingulate cortex (Cg), prelimbic cortex (PrL), piriform cortex (Pir), primary motor cortex (M1), secondary motor cortex (M2), dorsal peduncular cortex (DP), ventral orbital cortex (VO), primary auditory cortex (Au), entorhinal cortex (Ent), retrosplenial agranular cortex (RSA), retrosplenial granular cortex (RSG), primary visual cortex (V1).

2007). Together these data suggest a role for the cerebellum in the activity of cocaine, and although there is no data currently available regarding human glutamate transporters, our results indicate that the maintenance of cocaine self-administration could involve a reduction in cerebellar EAATs. Increased EAAT binding in the infralimbic portion of the medial prefrontal cortex was also observed 1 day after extinction. The implication of the medial prefrontal cortex in cocaine dependence has been established in several human and animal studies, although the role of its different subareas is not clear. The medial prefrontal cortex is an heterogeneous region that can be divided into at least three different areas with different efferent and afferent projections: the infralimbic, the prelimbic, and the anterior cingulate areas (Groenewegen et al., 1990; Van Eden and Uylings, 1985; Tzschentke and Schmidt, 2000). Selective inactivation of the dorsomedial prefrontal cortex, but not the infralimbic cortex, attenuates cue, stress and priming induced reinstatement of extinguished cocaine-seeking behavior (Capriles et al., 2003; McLaughlin and See, 2003). Moreover, lesions of the prelimbic but not of the infralimbic cortex seem to be relevant in cocaine sensitization (Tzschentke, 2000). By contrast, the expression of Zif268 in the infralimbic cortex after 14 days of withdrawal in animals that received contingent cocaine, reflects a decrease in neuronal depolarization or intracellular activation of second messengers

Group

Striatum Nacc (Core) CONT NON-CONT SALINE Nacc (Shell) CONT NON-CONT SALINE Tu CONT NON-CONT SALINE CPu CONT NON-CONT SALINE VP CONT NON-CONT SALINE Hippocampus DG CONT NON-CONT SALINE CA2 CONT NON-CONT SALINE CA3 CONT NON-CONT SALINE S CONT NON-CONT SALINE Thalamus PV

IMD

Re

NGM

Day 0 65.09  10.7 67.51  9.5 70.05  6.7 56.34  6.1 72.64  5.8 59.26  4.1 62.55  9.6 55.84  10.4 60.26  9.6 46.94  7.4 56.35  2.6 58.05  4.1 28.60  6.5 30.24  5.4 37.42  5.0

Day 1 78.35  6.3 88.22  5.4 73.00  4.3 72.89  3.4 66.04  1.6 59.73  2.6 81.77  6.9 71.19  3.6 62.68  6.1 76.08  6.9 69.15  4.7 58.19  2.8 60.47  6.8 51.94  3.3 45.12  3.1

Day 5 67.20  9.9 52.0  13.8 71.86  8.8 56.76  3.1 58.22  8.6 55.77  4.3 53.65  7.9 47.83  14.0 51.10  11.7 53.84  9.6 40.32  12.0 51.04  6.8 33.03  5.9 34.95  10.6 41.61  7.6

Day 10 79.97  4.2 73.06  8.2 67.60  4.8 64.51  5.4 64.47  3.5 59.64  3.7 66.39  8.0 54.55  7.2 68.27  7.9 62.90  8.3 59.17  5.4 57.34  4.8 49.98  5.9 46.44  6.2 50.45  5.0

89.67  7.1 92.04  5.8 89.86  15.9 91.10  10.8 112.01  9.0 79.31  10.7 74.46  5.9 99.12  12.6 108.21  12.9 97.18  4.2 93.35  20.7 77.96  9.5 59.77  6.8 97.79  11.6 87.43  20.0 100.82  10.2 99.46  16.4 79.66  4.9 76.81  10.2 83.70  5.4 111.38  12.5 107.17  9.6 112.09  18.2 99.12  6.7 63.91  9.4 82.18  10.4 87.83  20.8 89.17  11.7 96.15  12.1 67.22  5.7 66.03  8.8 72.01  3.6 112.52  11.6 101.03  10.7 99.99  17.0 91.09  8.3 45.21  3.4 80.87  10.2 76.75  19.2 88.09  9.0 51.95  7.8 61.42  11.8 53.84  7.6 87.24  7.7 65.54  4.9 73.59  5.8 86.10  19.2 63.16  3.5

CONT 84.63  11.0 NON-CONT 105.55  11.3 SALINE 107.95  13.6 CONT 84.42  11.1 NON-CONT 92.79  9.6 SALINE 94.35  14.8 CONT 68.19  6.3 NON-CONT 70.61  7.2 SALINE 58.76  13.0 CONT 39.83  4.6 NON-CONT 62.45  4.4 SALINE 58.99  6.8

83.50  14.1 74.31  5.9 93.10  6.1 78.06  13.5 70.32  7.4 80.43  3.3 63.85  10.5 62.19  7.2 59.95  4.0 49.58  8.2 42.50  1.8 58.89  4.1

69.89  13.2 66.02  0.7 75.42  17.0 49.42  13.1 57.78  5.3 65.50  13.0 41.45  9.7 42.25  5.8 59.96  12.6 44.37  11.3 43.50  6.7 57.56  8.4

95.95  10.1 84.21  17.1 83.94  13.2 75.54  6.8 74.25  14.7 70.94  11.1 71.58  4.8 58.07  5.6 54.62  9.5 44.98  9.9 41.91  3.0 52.39  4.6

The data represent the mean  SEM expressed as fmol/mg of tissue equivalent. Brain regions: nucleus accumbens core (Nacc (Core)), nucleus accumbens shell (Nacc (Shell)), olfactory tubercle (Tu), caudate putamen (striatum) (CPu), ventral pallidum (VP), dentate gyrus (DG), field CA2, CA3 of hippocampus (CA2, CA3, respectively), subiculum (S), paraventricular thalamic nucleus (PV), intermediodorsal thalamic nucleus (IMD), reuniens thalamic nucleus (Re), medial geniculate nucleus (NGM).

(Mutschler et al., 2000). We also found changes in the expression of the NMDAR1 receptor subunit in cortical regions that include the infralimbic and prefrontal cortices. Indeed, when compared to noncontingent and saline subjects, the expression of the R1 subunit of the NMDA receptor augmented in the prefrontal cortex after cocaine self-administration and progressively decreased during the time course of extinction (Crespo et al., 2002). Other studies also suggest a role for glutamate from the prefrontal cortex in cocaine dependence. Thus, inactivation of the glutamatergic projection from the prefrontal cortex to the Nacc by microinjection of the GABA receptor agonist baclofen prevents cocaine-induced reinstatement (McFarland et al., 2003). In addition, animals that were withdrawn from repeated daily cocaine administration for 1 and 7 days displayed an increase in cocaine-induced mPFC glutamate levels when compared to saline and acute control subjects (Williams and Steketee, 2004). Although all these data support an implication of medial prefrontal cortex glutamatergic transmission in cocaine dependence, there is no data on the role of glutamate

M. Migue´ns et al. / Neuropharmacology 55 (2008) 771–779

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Table 4 [2,3-3H]-L-aspartate binding in the rat brain during the extinction of cocaine selfadministration (hypothalamus, habenula and mesencephalon)

Table 5 [2,3-3H]-L-aspartate binding in the rat brain during the extinction of cocaine selfadministration (pons, raphe)

Brain area Group

Brain area

Day 0

Day 1

Day 5

Day 10

Hypothalamus and habenula SO CONT 79.63  7.0 86.36  7.7 72.85  13.0 91.75  11.6 NON-CONT 105.94  7.3 81.61  4.6 51.38  5.8 90.11  18.1 SALINE 77.06  13.4 79.26  4.3 82.23  22.1 69.91  7.6 PH CONT 66.86  7.7 62.27  10.5 44.61  10.5 56.11  7.2 NON-CONT 73.76  8.6 65.59  10.1 37.34  4.3 52.19  8.9 SALINE 63.32  9.7 60.64  3.3 59.93  12.7 52.64  10.1 DM CONT 95.92  8.7 96.82  11.8 73.65  12.1 85.65  8.0 NON-CONT 105.88  13.0 78.83  6.5 65.18  2.9 78.91  15.4 SALINE 104.10  15.3 95.86  9.6 92.76  16.1 79.15  13.2 VMH CONT 106.27  11.0 86.72  12.1 69.68  11.7 91.10  7.8 NON-CONT 121.0  15.8 77.41  5.7 64.18  3.5 83.42  14.2 SALINE 104.31  16.0 96.03  11.2 95.58  20.5 76.61  12.3 Arc CONT 95.10  10.7 77.10  17.8 67.84  9.7 87.49  9.9 NON-CONT 111.98  13.8 68.91  6.8 58.24  4.7 80.26  14.7 SALINE 91.58  13.9 84.55  10.3 78.47  23.2 73.44  13.1 MHb CONT 27.45  6.4 32.00  8.7 29.71  8.2 24.14  6.1 NON-CONT 38.32  4.3 39.63  5.1 27.09  3.7 25.73  8.8 SALINE 29.94  4.11 32.09  4.66 40.72  11.55 23.69  3.0 LHb CONT 25.46  4.5 30.52  11.7 22.11  4.6 21.84  4.6 NON-CONT 29.17  3.7 34.91  7.1 17.16  2.6 21.86  3.5 SALINE 24.95  4.3 23.12  2.7 24.70  5.5 18.45  3.3 Mesencephalon IP CONT NON-CONT SALINE IF CONT NON-CONT SALINE SNig CONT NON-CONT SALINE VTA CONT NON-CONT SALINE DMPAG CONT NON-CONT SALINE DLPAG CONT NON-CONT SALINE SuG CONT NON-CONT SALINE InG CONT NON-CONT SALINE

21.03  6.4 40.28  4.6 38.05  6.8 21.35  6.4 29.80  4.6 44.09  5.4 27.27  3.0 39.03  3.0 46.10  5.0 13.64  3.8 21.16  3.3 30.58  2.5 16.92  1.7 17.48  4.3 22.07  2.5 31.58  0.9 34.43  7.0 45.51  2.9 29.87  4.0 38.85  10.1 51.44  4.6 16.12  3.3 24.75  7.0 38.82  4.5

36.11  8.6 36.20  3.5 38.73  4.9 38.13  10.0 34.38  3.8 44.24  4.8 34.35  6.9 33.98  4.0 39.50  1.3 29.28  8.9 32.93  4.0 27.74  4.0 36.10  7.7 24.89  3.8 29.61  3.9 53.43  9.0 37.89  6.6 50.96  6.7 50.26  3.8 49.73  7.7 53.47  7.7 38.87  6.3 38.83  6.1 39.75  5.1

20.77  4.0 32.30  10.2 31.39  8.4 25.63  5.1 37.49  7.9 38.94  10.8 28.32  4.9 36.74  8.4 46.06  6.2 19.41  4.2 27.93  6.3 27.76  4.8 30.27  8.0 20.44  7.1 31.71  10.2 53.02  12.9 35.66  8.4 53.64  15.4 55.19  17.5 27.43  5.5 58.87  17.3 39.26  12.6 18.73  6.0 43.96  12.5

28.14  9.3 37.19  5.3 40.14  9.4 36.78  12.1 47.46  8.1 43.09  9.8 27.53  8.8 25.60  2.2 42.23  2.5 28.07  9.1 34.74  5.2 30.24  7.2 34.40  8.9 38.54  4.8 33.04  4.5 67.24  17.0 55.50  6.1 49.60  5.4 68.43  13.4 68.42  6.8 45.44  4.6 44.04  9.6 37.01  7.6 32.94  3.4

The data represent the mean  SEM expressed as fmol/mg of tissue equivalent. Brain regions: supraoptic nucleus (SO), posterior hypothalamic area (PH), dorsomedial hypothalamic nucleus (DM), ventromedial hypothalamic nucleus (VMH), arcuate hypothalamic nucleus (Arc), medial habenular nucleus (MHb), lateral habenular nucleus (LHb), interpeduncular nucleus (IP), interfascicular nucleus (IF), substantia nigra (SNig), ventral tegmental area (VTA), dorsomedial periaqueductal grey (DMPAG), dorsolateral periaqueductal gray (DLPAG), superficial grey layer of the superior colliculus (SuG), intermediate grey layer of the superior colliculus (InG).

transporters in this brain region. As far as we know, the present work is the first study to report changes in EAAT levels after the extinction of cocaine self-administration in this medial prefrontal cortex area. However, given the descriptive nature of this work, further research is needed to elucidate the functional significance of the changes in EAATs induced by cocaine in these areas. In slices adjacent to those employed in the EAAT study, we also analyzed the modulation of DAT binding. Contingent cocaine increased DAT binding in the CPu, Nacc (Core), Nacc (Shell) and VTA. In addition, when compared to saline and cocaine yoked animals, the binding to DATs remained high during the whole extinction period in most of the brain areas examined. It has been reported that DAT binding augmented and the expression of dopamine D2 receptors diminished after long-term chronic cocaine

Pons Pn

LC

LPB

MPB

Raphe MnR

DRD

DRV

Group

Day 0

Day 1

Day 5

Day 10

CONT NON-CONT SALINE CONT NON-CONT SALINE CONT NON-CONT SALINE CONT NON-CONT SALINE

13.05  2.4 22.54  4.7 18.27  0.6 38.62  9.1 51.13  14.0 45.34  5.7 19.75  5.2 44.28  10.9 31.14  5.7 21.23  5.1 31.69  7.0 27.43  3.4

30.77  6.9 19.83  4.3 22.01  3.1 62.83  11.2 51.66  7.4 48.03  3.0 32.22  7.7 30.28  5.8 29.87  2.5 36.56  9.4 25.14  3.4 27.88  1.4

23.00  6.4 21.98  8.6 22.83  4.5 58.60  16.3 35.69  9.3 44.58  12.6 41.93  12.0 20.50  7.8 30.44  7.3 25.72  6.5 16.98  5.5 22.17  4.9

27.24  8.2 29.80  7.3 23.28  6.0 51.52  19.2 59.13  11.3 44.65  6.9 28.78  11.3 21.15  4.7 21.08  4.7 34.94  15.1 23.24  5.2 28.05  5.2

CONT NON-CONT SALINE CONT NON-CONT SALINE CONT NON-CONT SALINE

15.18  3.2 18.80  2.7 26.62  2.3 29.22  1.8 28.39  5.4 29.21  2.8 19.70  2.7 18.73  3.0 21.33  1.6

34.21  10.3 19.01  3.1 30.31  4.1 48.12  10.9 26.60  5.1 37.84  4.9 46.34  10.6 24.53  4.4 30.16  3.5

25.43  6.8 18.45  7.7 35.17  9.9 40.97  9.8 27.80  8.0 43.40  13.2 38.57  9.9 27.77  8.9 33.06  9.8

32.33  7.7 30.50  4.4 26.71  3.4 40.51  8.6 43.76  6.8 38.35  4.7 33.06  6.3 36.84  6.2 34.13  3.5

The data represent the mean  SEM expressed as fmol/mg of tissue equivalent. Brain regions: pontine nuclei (Pn), locus coeruleus (LC), lateral parabrachial nucleus (LPB), medial parabrachial nucleus (MPB), median raphe nucleus (MnR), dorsal raphe nucleus-dorsal part (DRD), dorsal raphe nucleus-ventral part (DRV).

self-administration in monkeys (Porrino et al., 2004). According to this, we have found a decrease in D2 receptor binding that persisted even 10 days after the extinction (Crespo et al., 2001b). The fact that the increment in the DAT protein binding is accompanied by a decrease in the D2 receptor binding suggests an elevation of the dopamine transmission, but in the absence of cocaine, increased DAT would lead to a greater clearance of synaptic dopamine and reduced dopamine neurotransmission. However, no changes in basal extracellular dopamine levels were reported in the Nacc after a long period of cocaine self-administration extinction (McFarland et al., 2003). An important question that needs to be addressed is the differential role played by withdrawal and extinction in the neuroadaptive changes in EAATs and DATs reported here. It must be noted that the effects caused by withdrawal from cocaine selfadministration and those due to extinction cannot be distinguished using the experimental design applied here (given that selfadministered animals were exposed to both). However, the data

Table 6 ANOVA’s results of specific binding of [N-methyl-3H]-WIN 35,428 to DATs Brain area

Type of administration (T)

Day of extinction (D)

TD

CPu

F2,60 ¼ 107.09 p < 0.001 F2,60 ¼ 60.85 p < 0.001 F2,60 ¼ 117.20 P < 0.001 F2,60 ¼ 92.02 p < 0.001 F2,60 ¼ 21.58 p < 0.001 F2,60 ¼ 30.62 p < 0.001

F3,60 ¼ 6.38 p ¼ 0.001 N.S

F6,60 ¼ 7.58 P < 0.001 F6,60 ¼ 4.25 P ¼ 0.001 F6,60 ¼ 5.64 P ¼ 0.001 F6,60 ¼ 9.97 P < 0.001 F6,60 ¼ 2.30 p < 0.05 F6,60 ¼ 2.29 p < 0.05

Nacc (Core) Nacc (Shell) mPFC SNig (Whole) VTA

F3,60 ¼ 6.75 p ¼ 0.001 F3,60 ¼ 10.02 p < 0.001 F3,60 ¼ 6.43 p ¼ 0.001 N.S

Brain areas: caudate putamen (CPu), nucleus accumbens core (Nacc (Core)), nucleus accumbens shell (Nacc (Shell)), medial prefrontal cortex (mPFC), ventral tegmental area (VTA), substantia nigra-whole (SNig (Whole)).

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Fig. 3. Time course of the extinction of cocaine self-administration on DAT levels in the caudate putamen (CPu), medial prefrontal cortex (mPFC), nucleus accumbens core (NaccCore), nucleus accumbens shell (NaccShell), ventral tegmental area (VTA) and substantia nigra (SNigwhole) in the brain of SALINE, CONT, and NON-CONT rats. Data represent the mean  s.e.m of [N-methyl-3H]-WIN 35,428 specific binding expressed as fmol/mg of equivalent tissue. *p < 0.05 and **p < 0.01 indicate a significant difference when compared to SALINE yoked control subjects and þp < 0.05 indicates a significant difference when compared to yoked NON-CONT subjects.

available clearly establishes a specific role for extinction in neuroadaptation when compared to withdrawal from chronic drug self-administration. Indeed, it has been reported that extinction training reverses neuroadaptation associated with cocaine withdrawal, whereas other changes arise as a consequence of extinction (see Self et al. (2004) for a review). Therefore, we cannot rule out the influence of both extinction and withdrawal in the neuroadaptation of EAATs and DATs observed here. Further studies will be needed to specifically address this issue by comparing selfadministered animals undergoing extinction with others suffering withdrawal. Finally, we found no major changes in cocaine yoked animals (NON-CONT) and since instrumental learning is absent in these animals, we suggest that the neuroadaptive response observed in EAATs and DATs could reflect a complex interaction between the pharmacological effects of the drug and experimental learning during cocaine self-administration and extinction. Our results might shed new light on the role of neurotransmitter transporters in cocaine self-administration. Indeed, in studies

carried out on serotonin transporter (SERT) and norepinephrine transporter (NET) knockout (KO) mice in the conditioning place preference model, suppression of DAT and SERT is required to eliminate the place preference associated with cocaine. Alternatively, NET appears to be responsible for the cocaine-induced aversive effects (Uhl et al., 2002). Furthermore, SERT compensated for DAT function in DAT null mice (Rocha et al., 1998), suggesting that the actions of cocaine on DAT alone cannot explain the reinforcing effects of this drug. As far as we are aware, this is the first evidence that EAATs are involved in cocaine self-administration and in the extinction of this behavior. We also show that the modulation of the dopamine transporter after extinguishing cocaine self-administration is clearly more widespread and enduring than that of the glutamatergic system. Moreover, it is tempting to speculate that the symptomatic neuroadaptive changes in both neurotransmitter transporters might participate early in cocaine withdrawal. Indeed, the dopaminergic and other neurotransmitter protein transporters

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M. Migue´ns et al. / Neuropharmacology 55 (2008) 771–779

Fig. 4. Representative coronal autoradiograms of total [N-methyl-3H]-WIN 35,428 binding to DATs at the level of the dorsal striatum and nucleus accumbens from rats in each experimental group.

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