Extinction of cocaine self-administration produces alterations in corticotropin releasing factor gene expression in the paraventricular nucleus of the hypothalamus

Molecular Brain Research 117 (2003) 160–167 www.elsevier.com / locate / molbrainres

Research report

Extinction of cocaine self-administration produces alterations in corticotropin releasing factor gene expression in the paraventricular nucleus of the hypothalamus ´ Oliva b , Javier Corchero c , Jose´ Antonio Crespo a , Jorge Manzanares b , Jose´ Marıa a ´ Carmen Garcıa-Lecumberri , Emilio Ambrosio a , * a

´ , Facultad de Psicologıa ´ , Universidad Nacional de Educacion ´ a Distancia ( UNED), Madrid 28040, Spain Departamento de Psicobiologıa b ´ , Hospital Doce de Octubre, Av. Cordoba ´ s /n, Madrid 28041, Spain Servicio de Psiquiatria y Centro de Investigacion c ´ ´ Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s /n, Badajoz 06071, Departamento de Bioquımica y Biologıa Spain Accepted 15 July 2003

Abstract The long-term effect of cocaine self-administration on corticotropin releasing factor (CRF) mRNA content in the hypothalamic CRF-containing neurons has not yet been established. The purpose of this study was to examine the time course effects of the extinction of cocaine self-administration behavior on CRF gene expression in the paraventricular nucleus of the hypothalamus (PVN) using in situ hybridization histochemistry (IHHS). Seventy-two littermate male Lewis rats were randomly assigned in triads to one of three conditions: (a) contingent intravenous self-administration of 1 mg / kg / injection of cocaine (CONT), (b) non-contingent injections of either 1 mg / kg / injection of cocaine (NONCONT) or (c) saline yoked (SALINE) to the intake of the self-administering subject. The self-administering rats were trained to self-administer cocaine under a fixed ratio 5 (FR5) schedule of reinforcement for a minimum of 3 weeks. After stable baseline levels of drug intake had been reached, saline was substituted for drug. Following this first extinction period, cocaine self-administration was reinstated for an additional period of 2 weeks. Immediately after cessation of the last session of cocaine self-administration (Day 0) and 1, 5 and 10 days after the second extinction period, animal brains in each triad were removed to be processed for IHHS. CRF mRNA levels in the PVN were significantly lower in the NONCONT cocaine group at Day 0 compared to CONT or SALINE groups. On Day 1, hypothalamic CRF gene expression significantly decreased in the CONT cocaine group with respect to the SALINE group, but there were no differences between the cocaine groups or among the NONCONT cocaine and SALINE groups. After 5 and 10 days of extinction, no differences were found in CRF mRNA content in the PVN between the three conditions of this study. These results suggest that, after the extinction of cocaine self-administration, changes in hypothalamic CRF gene expression are differentially affected depending upon the type of cocaine administration, and that the stages of cocaine withdrawal might not be associated with enduring changes in hypothalamic CRF mRNA levels.  2003 Elsevier B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Drugs of abuse: cocaine Keywords: Extinction; Cocaine self-administration; CRF mRNA; Gene expression; Withdrawal

1. Introduction Cocaine is among the most widely abused central drug stimulants. Research concerning the physiological and *Corresponding author. Tel.: 134-91-398-7974; fax: 34-91-398-6287. E-mail address: [email protected] (E. Ambrosio). 0169-328X / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00316-4

behavioral effects of cocaine has gained impetus in the last few decades as cocaine abuse constitutes a major societal problem. Although cocaine abuse is associated with a wide range of psychiatric and other medical disorders, at the present time there are no suitable medications for the treatment of cocaine abuse and dependence. Studies on the neurobiology of cocaine abuse suggest that cocaine affects

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key elements of the brain reward system, particularly mesocorticolimbic dopamine pathways [58]. Cocaine stimulates the hypothalamic–pituitary–adrenal (HPA) axis in several species. Indeed, cocaine administration results in increased plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in rats [2] as well as ACTH and cortisol levels in rhesus monkeys [6,45] and humans [1,22,33,54]. It appears that these effects of cocaine do not occur at the level of the pituitary [41] or the adrenal gland [29], but rather it has been suggested that the actions of cocaine on the HPA axis are centrally mediated via a CRF mechanism [35,41–43]. CRF, a 41 amino acid polypeptide, is the main hypophysiotropic factor regulating ACTH and b-endorphin secretion [10,11,37,51]. Several receptor subtypes for CRF have been described and cloned [7], showing a great homology between species. The distribution of brain CRF overlaps regions that are important for mediating the reinforcing effects of cocaine [10,11]. Central administration of CRF produces a wide variety of behavioral effects, an increase in arousal and / or ‘anxiogenic-like’ effects being the most characteristic [3,12,27,49,52]. Consistent with these actions, central administration of CRF antagonist such as a-helical CRF ( 9 – 41) or D-Phe CRF ( 12 – 41 ) blocks the behavioral effects induced by CRF or stress exposure [4,5,23,25,28,34,53]. These behavioral actions are centrally mediated and independent of the HPA axis [4,15,23]. Clinical reports indicate that chronic cocaine abusers show signs and symptoms following cessation of cocaine intake comprising an abstinence syndrome [20,47,55]. Major symptoms experienced by individuals following cocaine withdrawal are dysphoria, anxiety, depression, anergia, severe agitation and restlessness [19,20,56]. These signs and symptoms may be important motivational factors for cocaine relapse [19,31]. It is well known that CRF plays a role in the response to several stressors [12,26] and in the development of anxiety and depression [36]. Since anxiety and depression may occur following cocaine withdrawal, it has been suggested that endogenous CRF may mediate, at least in part, some of the effects induced by cocaine withdrawal [26]. Supporting this idea, it has been shown that cocaine withdrawal produces a profound increase in CRF release in the amygdala 12 h after a 12-h session of cocaine self-administration [38], and that protracted withdrawal from cocaine is associated with alterations in CRF-like immunoreactivity in the amygdala and prefrontal cortex [61]. In addition, it has been reported that chronic cocaine administration decreases brain immunoreactive CRF in the hypothalamus, basal forebrain and amygdala 48 h after the last dose of cocaine [44] and decreases hypothalamic CRF mRNA [60]. However, while these data suggest an involvement of CRF extrahypothalamic levels in the mediation of ‘stress-like’ responses after cessation of cocaine intake, Zhou et al. [60] did not find any alteration in CRF mRNA expression in the hypothalamus 10 days after chronic cocaine treatment, and

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Zorrilla et al. [61] did not find CRF-like immunoreactivity changes in the hypothalamus after protracted cocaine withdrawal. At present, however, the effect of cocaine self-administration behavior and its extinction on hypothalamic CRF mRNA content is not known. Therefore, the aim of the present work was to examine the time course effects in CRF gene expression in the paraventricular nucleus of the hypothalamus after cocaine self-administration and extinction. We included in our behavioural experimental design a yoked-box procedure that involves the use of triads to assess relative differences in hypothalamic CRF gene expression related to contingent versus noncontingent cocaine administration [13]. In previous works, we used this same behavioural experimental design to analyze the effect of contingent vs. noncontingent cocaine administration on proenkephalin (PENK) [8] and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) [9] gene expression in slices adjacent to those of this study. In contrast to the present results, which show transient changes in the hypothalamic CRF mRNA content, we found more long-lasting neuroadaptative changes in PENK and NMDAR1 gene expression of contingent cocaine administration animals. These results suggest that longterm neuroadaptations to chronic cocaine administration could arise not only from the pharmacological actions of cocaine on neurobiological systems, but also by an interaction between its pharmacological action and the animal’s environment [13,14,24,50,57]. Indeed, it has been suggested that, in humans, the classical conditioning of the pharmacological actions of cocaine with environmental stimuli plays an important role in the long-term addictive potential of this drug and in the liability to relapse in recovering cocaine addicts [16].

2. Materials and methods

2.1. Animals Adult male Lewis rats (Criffa, France) weighing approximately 300–350 g at the beginning of their training were used. All animals were experimentally naive, housed individually in a temperature-controlled room (23 8C) with a 12-h light–dark cycle (08:00–20:00 lights on) and given free access to Purina laboratory chow and tap water prior to initiation of the experiments. Animals used in this study were maintained in facilities according to European Union Laboratory Animal Care Rules.

2.2. Surgery Experimentally naive subjects were surgically prepared with an i.v. catheter placed in the jugular vein. Polyvinylchloride tubing (0.064 i.d.) was implanted in the right jugular vein approximately at the level of the atrium under ketamine and diazepam anesthesia. The catheter was

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passed s.c. and exited in the midscapular region. The catheter 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 given at least 7 days to recover.

2.3. Apparatus Twelve operant chambers (Coulburn Instruments, USA) were used for cocaine self-administration studies. Two levers designed to register a response when 3.0 g of force was applied were placed 14 cm apart on the front wall of the chamber. A microliter injection pump (Harvard 22) was used to deliver i.v. saline or drug injections to the rat. Drug delivery, operant data acquisition and storage were accomplished using IBM computers (Med Associates, USA).

2.4. Experimental procedure Cocaine-reinforced behavior was studied according to a procedure described previously [8]. Briefly, before surgical implantation of the i.v. catheter, animals were trained to press a lever for food reinforcement under a FR5 schedule of reinforcement. Initially, a single lever press on the left-hand lever resulted in the delivery of a food pellet (45 mg, Noyes Pellets, UK) and turned on a stimulus light above the lever. After the response was initiated, the response requirement to food delivery was raised in increments to 5 and a programmed 30-s time out (TO) period in which responses had no programmed consequences followed each food pellet delivery (FR5:TO 30 s). When behavior was maintained under the FR5 schedule of food-reinforced behavior, the catheter was surgically implanted as described. After the post-operative period, 72 littermate male Lewis rats were randomly assigned in triads to one of three conditions: (a) contingent i.v. self-administration of 1 mg / kg / injection of cocaine (CONT) and (b) noncontingent i.v. injections of either 1 mg / kg of cocaine (NONCONT) or (c) saline (SALINE) yoked to the intake of the selfadministering subject. Initially, substitution of food delivery by cocaine began under a FR1 schedule of reinforcement and was subsequently raised to FR5. In this case, 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 selfadminister 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 established (less than 10% variability in the number of injections for 5 sequential days), saline was substituted for 4 days in the operant chambers. After this first extinction period, cocaine self-administration behavior was reinstated and maintained for 2 weeks. Saline substitution was carried out again for 1, 5, and 10 days in

the operant chambers. Immediately after the last day of cocaine self-administration, in which the animals reached the stability criterion (Day 0), and after 1-day (Day 1), 5-day (Day 5) and 10-day (Day 10) periods of extinction, animal brains of each triad were removed to be processed for ISHH. The total number of subjects in each group of triads was 18 (CONT56, NONCONT56, SALINE56) and the total number of brains removed was 72 [four groups of triads (Day 0, Day 1, Day 5 and Day 10)318]. The basal hypothalamic CRF mRNA content of Lewis rats has been reported not to be different from those of other laboratory rat strains [21,30,39].

2.5. In situ hybridization histochemistry Brain sections (six slides / level; two sections / slide) were cut at 20 mm at the PVN level, according to the Paxinos and Watson Atlas. The sections were mounted onto gelatin-coated slides and stored at 280 8C until the day of the assay. ISHH was performed as described previously [59] using 48-base synthetic oligonucleotide probes complementary to CRF (NEN-Dupont, NEP-554, Madrid, Spain). The oligonucleotide probe was labelled using terminal deoxytransferase (Boehringer, Madrid, Spain) to add a 35 S-labeled deoxyATP (1000 Ci mmol 21 ; Amersham, Madrid, Spain) tail to the 39 end of the probes. The probe (in 50 ml of hybridization buffer) was applied to each section and left overnight at 37 8C for hybridization. Following hybridization, sections were washed four times for 15 min each in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2 (13 saline sodium citrate, SSC) at 55 8C, followed by two 30-min washes in 13 SSC at room temperature, one brief water dip and were then blown dry with air. The dried slides were apposed to Kodak BioMax MR-1 film (Amersham) for 7 days. Two slides per PVN level (two slices / slide; two measurements / slice) for each animal were analyzed with a Macintosh computer using the public domain NIH Image program. Previous experiments in our laboratory have found that the selected times of exposure to film in the PVN brain region, and under our experimental conditions (oligonucleotide probe, radioactivity added to each slide, incubation conditions, type of film selected), renders a hybridization signal, the grey levels of which are linear with the optical density, according to the NIH Image Program. Therefore, optical densities were calculated from the uncalibrated mode of the Image Program by subtracting from each measurement the corresponding background and expressed in grey scale values. The background measurement was taken from an area of the slice with the lowest nonspecific hybridization signal and subtracted from the hybridization signal measurement on the same slice. Measurements were pooled from brain sections and the values were averaged. Results are presented considering mean control values as 100%. Additional brain sections were co-hybridized with a 100-fold excess of cold probes or with RNAse to assess the specificity of the

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signal. As expected, no hybridization signal was detected in these sections (data not shown).

2.6. Statistical analyses Analysis of the CRF gene expression data was performed using a two-way analysis of variance (ANOVA) with the type of cocaine administration (CONT, NONCONT or SALINE) serving as one independent variable and the PVN brain area serving as the other independent variable. Subsequently, a one-way ANOVA followed by Student Newman Keul’s test was performed for day of extinction (0, 1, 5 and 10), with the type of cocaine administration (CONT, NONCONT or SALINE) serving as the independent variable. Differences were considered significant if the probability of error was less than 5%.

3. Results The behavioral data for the contingent, noncontingent and saline groups are depicted in Fig. 1. These data correspond to the response of the contingent group, since the noncontingent and saline groups did not have the possibility of responding: in both groups the subjects received cocaine (1 mg / kg / injection) or saline passively

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when the contingent animals self-administered the drug. The behavioral data presented in Fig. 1 have been divided into four consecutive phases: phase 1 corresponds to the Acquisition and maintenance of cocaine i.v. self-administration behavior; phase 2 corresponds to the First Extinction of cocaine i.v. self-administration behavior; phase 3 corresponds to Reacquisition; and phase 4 corresponds to the Second Extinction. It is clear from the figure that cocaine maintained a robust response compared to the response under saline (first and second extinction phases). The last 15 days of phase 1 (acquisition and maintenance) were similar to the 15 days of phase 3 (reacquisition). In both phases of acquisition the number of cocaine injections / hour was clearly greater than the number of saline injections / hour. Both extinction phases were very similar, suggesting that extinction occurred in all subjects in a very consistent manner. This behavioral response pattern suggests that cocaine served as a positive reinforcer under our experimental conditions. A two-way ANOVA was performed to analyze the differences in CRF gene expression in the hypothalamic PVN during the extinction of cocaine self-administration behavior, considering the type of cocaine administration and this brain region as independent variables. There was a significant main effect of type of administration (F(4,52)5 18.30, P,0.001) on hypothalamic CRF gene expression

Fig. 1. Cocaine self-administration behavior and its extinction in contingent, noncontingent and saline groups. The behavioral data correspond to the response of cocaine (1 mg / kg / infusion) contingent self-administering animals because noncontingent and saline subjects received cocaine (1 mg / kg / infusion) or saline passively when the contingent animals self-administered. The behavioral responses are presented divided into four consecutive phases: phase 1 corresponds to the Acquisition and maintenance of cocaine i.v. self-administration behavior; phase 2 corresponds to the First Extinction of cocaine i.v. self-administration behavior; phase 3 corresponds to Reacquisition; and phase 4 corresponds to the Second Extinction. When animals have reached a stable cocaine self-administration behavior (less than 10% variability in the number of injections for 5 sequential days) during the acquisition and maintenance phase, saline substitution of the first extinction period was carried out. Subsequently, subjects reacquired cocaine self-administration behavior and were maintained self-administering for 2 weeks. When the animals again reached the stability criterion, a second extinction period started for 10 days. Cocaine maintained a robust response compared to the response under saline (first and second extinction phases). The response during the last 15 days of the Acquisition phase is very similar to that of the 15 days of the Reacquisition phase. In both acquisition phases the number of cocaine injections / hour was clearly greater than the number of saline injections / hour. Also, both extinction phases were similar and the extinction of cocaine self-administration behavior occurred in all subjects in a very consistent manner. On days 0 (last day of cocaine i.v. self-administration), 1, 5 and 10 of the second extinction phase, the brain of the triads (n518 in each triad; Contingent, n56; Noncontingent, n56; Saline, n56) were removed to be processed for ISHH. See details in the text. Ordinates: number of cocaine or saline injections per hour in 2-h sessions of cocaine or saline i.v. self-administration behavior. Abscissas: consecutive phases and sessions of cocaine or saline i.v. self-administration behavior.

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during the extinction of cocaine self-administration behavior. The CRF mRNA content of the NONCONT cocaine administration group was reduced compared to the CONT and SALINE groups. An overall view of the time course effects of the extinction of cocaine self-administration on CRF mRNA levels in the PVN is shown in Fig. 2A. Extinction from cocaine self-administration produces a decrease in CRF mRNA levels in CONT and NONCONT animals in the hypothalamic PVN when compared with each corresponding SALINE group. No statistical differences were found between the different time points of extinction of cocaine self-administration in the SALINE group in this brain region. A detailed one-way ANOVA analysis revealed that CRF

mRNA levels in the PVN were different on the last day of the self-administration session (Day 0) and during the days of the extinction period (Days 1, 5 and 10). Hypothalamic CRF mRNA levels in the NONCONT cocaine group significantly decreased when compared to the CONT cocaine and SALINE group at Day 0 (F(2,12)560.67, P,0.001). No statistical differences were found between the CONT and SALINE groups. On Day 1 of extinction, CRF mRNA levels in the CONT cocaine group decreased (F(2,11)55.19, P,0.05) when compared to the SALINE group. In the NONCONT cocaine group, however, the apparent decrease observed with respect to the SALINE group did not reach statistical significance (P,0.09). No differences were found between the CONT and NON-

Fig. 2. (A) Time course effects of the extinction of cocaine self-administration on CRF mRNA levels in the paraventricular hypothalamic nucleus (PVN) of the rat brain of SALINE, CONTINGENT and NONCONTINGENT groups. The experimental design is described in Fig. 1 and in the Materials and methods section. Symbols represent the means and the vertical lines 61 S.E.M. of the CRF mRNA levels (arbitrary units of optical density) in five to six rats. *Values from CONTINGENT or NONCONTINGENT animals that are significantly different (P,0.05) from the SALINE group. (B) Representative autoradiograms of coronal brain sections at the level of the paraventricular hypothalamic nucleus (PVN) of the time course effects of the extinction of cocaine self-administration on CRF mRNA levels in SALINE, CONTINGENT and NONCONTINGENT groups. Slides were apposed to film (Kodak, Biomax, MR-1) for 7 days. Experimental design as indicated in Fig. 1 and in the Materials and methods section. Bar: 1 mm.

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CONT groups. No significant changes were found on Day 5 or Day 10 of extinction among the three groups of our study. Considering the time course of the hypothalamic CRF mRNA content of the CONT cocaine group throughout the entire extinction period, there was a significant reduction only on Day 1 compared to Day 0 (last day of selfadministration) (F(3,18)53.74, P,0.05). Although CRF mRNA levels in the NONCONT cocaine group increased slightly across the extinction sessions, a significant effect was reached only on Day 10 (F(3,18)55.189, P,0.05) with respect to Day 0. No differences were found between the remaining extinction sessions. Fig. 2B shows representative autoradiograms of the groups SALINE, NONCONT and CONT used to analyze CRF mRNA levels in the PVN. It is clear that the hybridization signals for the PVN of NONCONT animals on Day 0 and for CONT on Day 1 are lower than the signal found for the SALINE group. The optical densities of the hybridization signal in these autoradiograms were measured and the values are similar to the mean values of the respective experimental groups.

4. Discussion The results of this study show that long-term cocaine self-administration and its cessation alters CRF gene expression in the PVN, the major brain region containing CRF neurosecretory cells. However, the type of cocaine administration seems to be an important variable for regulating CRF gene expression in the PVN. While longterm cocaine self-administration did not change CRF mRNA levels in the PVN, passive cocaine administration in yoked subjects dramatically reduced CRF content compared to self-administered and yoked saline animals. In our experimental design, cocaine self-administering subjects are able to regulate and control their own cocaine intake. In contrast, yoked cocaine animals are not able to control their intake. Thus, these results suggest that environmental events associated with cocaine self-administration could be important factors affecting hypothalamic CRF mRNA levels. The profound decrease in CRF gene expression in the PVN of passively chronic cocaine administered animals found in our study is in agreement with the results of Zhou et al. [60]. These authors found a decrease in hypothalamic CRF mRNA content, as measured by solution hybridization / Rnase protection assay, after 14 days of chronic cocaine administration (3315 mg / kg). In the same work, the authors did not find alterations in CRF mRNA levels in the amygdala [60]. However, after 6 weeks of chronic binge cocaine administration, CRF mRNA levels in extrahypothalamic regions such as the hippocampus and dentate gyrus were decreased [32]. Thus, although only a few studies have been carried out, our data and those of

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other authors seem to show that chronic passive cocaine administration decreases the CRF mRNA content of the hypothalamus, and decreases or does not alter this neuropeptide gene expression in extrahypothalamic brain areas. As far as we are aware, there is no previous work studying the effect of long-term cocaine self-administration on hypothalamic CRF mRNA levels. Therefore, in the present study we report that contingent cocaine self-administration does not alter CRF mRNA levels in the PVN. In contrast, the extinction of contingent self-administration produced a statistically significant decrease in CRF mRNA content in the PVN 24 h after the last cocaine self-administration session. Interestingly, this decrease was only detected in the self-administering group. The decrease of hypothalamic CRF mRNA levels in the contingent cocaine group during extinction is transitory, because CRF gene expression increased after 5 days of extinction and returned to saline values 10 days after the last cocaine self-administration session. Acute cocaine administration has been shown to produce increases in CRF gene expression in the PVN 5 h after an i.v. injection [40]. Similarly, acute cocaine administration (3315 mg / kg) increased CRF gene expression in the hypothalamus, amygdala and olfactory bulb [60]. In contrast, chronic cocaine administration decreased or did not alter the hypothalamic CRF mRNA and CRF extrahypothalamic content of several cerebral regions [32,60]. These results and others [46] suggest a potential up-regulation of CRF gene expression after acute cocaine injections that seems to be consistent with a role of CRF activity in the stimulatory effects of cocaine [46]. On the contrary, chronic cocaine administration would produce a compensatory down-regulation of CRF gene expression, resulting in inhibition of CRF activity [46]. In addition, it has been suggested that, after immediate cessation of chronic cocaine administration, an increase in the activity of CRF occurs that could be related, at least in part, to some of the early cocaine withdrawal symptoms, such as intense anxiety [46]. Although our noncontingent cocaine results may be consistent with this hypothesis, cocaine self-administration data do not support this. Indeed, we did not find a down-regulation of CRF gene expression in contingent cocaine animals and, after cessation of chronic contingent cocaine administration, a decrease in the activity of CRF-expressing neurons occurred instead of the suggested CRF hyperactivity. In addition, the contingent cocaine results suggest that neuroadaptative mechanisms regulating CRF-expressing PVN neurons during cocaine self-administration and extinction are not independent of the learning and memory processes involved in the selfadministration behavior paradigm. In summary, the neuroadaptative changes in CRF gene expression in the PVN after cessation of chronic cocaine administration are complex and depend upon the type of cocaine administration (contingent vs. noncontingent). The alterations produced in CRF gene expression in the PVN

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are transient since CRF mRNA levels returned to those of saline values 5 and 10 days after the last cocaine selfadministration session. Similar non-lasting chronic cocaine effects on hypothalamic CRF mRNA and CRF extrahypothalamic levels have already been described [32,60]. Certainly, more research is needed to define the complex involvement of the CRF neurotransmitter system in cocaine withdrawal because a role of extrahypothalamic CRF in the stress-induced reinstatement of cocaine seeking behavior has been demonstrated [17,18,48]. However, it seems that the actions of CRF on the HPA axis are not involved in the stress-induced reinstatement of cocaine self-administration [17,48], nor does cocaine withdrawal modify CRF-like immunoreactivity [61] and CRF mRNA content [60] in the hypothalamus. Taken together, the results of these studies and those of the present work suggest that the stages of cocaine withdrawal might not be associated with enduring changes in hypothalamic CRF levels.

Acknowledgements The authors are grateful to R. Ferrado for technical assistance. This research was supported by grants PM970027 and BSO2001-1099 to the UNED group. Cocaine ´ General de chlorhydrate was kindly provided by Direccion Estupefacientes (Spain).

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