Increase in A2A receptors in the nucleus accumbens after extended cocaine self-administration and its disappearance after cocaine withdrawal

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Increase in A2A receptors in the nucleus accumbens after extended cocaine self-administration and its disappearance after cocaine withdrawal Daniel Marcellino a,c,⁎, David C.S. Roberts b , Gemma Navarro c , Malgorzata Filip d , Luigi Agnati e , Carme Lluís c , Rafael Franco c , Kjell Fuxe a a

Department of Neuroscience, Karolinska Institutet, 17177 Stockholm, Sweden Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA c IDIBAPS (Insitut d'Investigació Biomédica August Pi i Sunyer) and Department of Biochemistry and Molecular Biology,University of Barcelona, 08028 Barcelona, Spain d Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, 31343 Krakow, Poland e Department of Biomedical Sciences, University of Modena, 41100 Modena, Italy b

A R T I C LE I N FO

AB S T R A C T

Article history:

Effects of extended cocaine self-administration and its withdrawal have been studied on A2A

Accepted 18 January 2007

and D2 receptor binding characteristics and expression in the nucleus accumbens and the

Available online 28 January 2007

anterior and posterior dorsal striatum of the rat (Rattus norvegicus). Biochemical binding techniques have been used with the D2-like receptor antagonist radioligand [3H]-Raclopride

Keywords:

and the A2A receptor antagonist radioligand [3H]-ZM 241385 and immunoblots to study their

Adenosine A2A receptor

expression. A substantial and significant increase in functional A2A, but not in functional D2

Dopamine D2 receptor

receptors, was observed in the nucleus accumbens immediately following 10 days of cocaine

Receptor–receptor interaction

self-administration which returned to normal levels after 7 days of drug withdrawal. In

Cocaine self-administration

contrast, in the posterior dorsal striatum significant reductions in A2A expression were

Cocaine withdrawal

observed immediately after cocaine self-administration which was associated with a trend

Striatum

for a reduction of the A2A receptor antagonist binding sites. In cocaine withdrawal groups, significant increases in the density and Kd value of D2-like antagonist binding sites were observed in the nucleus accumbens in the absence of changes in D2 expression, suggesting an up-regulation of D3 receptors in this region after cocaine withdrawal. A2A receptor increases in the nucleus accumbens induced by cocaine may represent a compensatory upregulation to counteract cocaine-induced increases in D2 signaling and D3 signaling which is in line with its disappearance in the 7-day withdrawal group displaying increased reinforcing efficacy of cocaine. A2A agonists may therefore represent cocaine antagonist drugs to be used in treatment of cocaine addiction acting inter alia by antagonizing signaling in accumbens A2A/D2 and A2A/D3 heteromers. © 2007 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +46 8 315721. E-mail address: [email protected] (D. Marcellino). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.01.079

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1.

Introduction

Recent results suggest that adenosine A2A receptors are involved in the locomotor and sensitizing effects of cocaine (Filip et al., 2006). The pharmacological analysis indicated that A2A receptors exert antagonistic effects on cocaine selfadministration, locomotor activity, conditioned activity and conditioned place preference in rats mediated via D2 receptors and is in agreement with previous work (see Adams et al., 2001; Kita et al., 1999; Caine et al., 1999). This underlines the role of D2 receptors in mediating the rewarding actions of cocaine which is based on the increased extracellular levels of DA induced by cocaine's inhibition of the DA reuptake transporter (Andrews and Lucki, 2001). This is likely to be based on the existence of A2A/D2 heteromeric complexes in the ventral striatum (Hillion et al., 2002; Canals et al., 2003; Nakata et al., 2005, Fuxe et al., 2005) where A2A receptors inhibit D2 recognition of dopamine and D2 agonists and

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transduction that leads to reduced D2 mediated signaling in the mesolimbic DAnergic pathway onto the ventral striatopallidal GABA pathway (Anden et al., 1966). The mesolimbic DA afferents directly regulate the activity of the ventral striato-pallidal GABA pathway by participating in its local circuits where D2 receptors are located predominantly on the neck of the dendritic spines (Greengard, 2001). In line with this view, the initiation of cocaine self-administration in rats is reduced by the A2A agonist 5′-N-ethylcarboxamido-adenosine (NECA) (Knapp et al., 2001) and A2A activation via the A2A agonist CGS 21680 attenuates brain stimulation reward and cocaine withdrawal (Baldo et al., 1999). Furthermore, the discovered A2A agonist properties of Polygala tenuifolia root extracts may be responsible for the ability of this extract to diminish cocaine-induced conditioned place preference (Shin et al., 2004). Both A1 and A2A receptors have been reported to be involved in the discriminative-stimulus effects of cocaine since either antagonist produced high levels of cocaine-lever

Fig. 1 – Effects of an extended cocaine self-administration history and its withdrawal on the density of adenosine A2A receptors in the nucleus accumbens. Bmax values (expressed as fmol/mg protein) were deduced from saturation curves of A2A antagonist [3H]-ZM 241385 binding (A) to nucleus accumbens membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and withdrawal from extended daily access to cocaine (1-day; 7-day). Representative saturation curves of the specific A2A antagonist binding in control and the 0-day group are shown. Radioligand binding experiments were performed as indicated in Experimental procedures. Values are means ± SEM (n = 6). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In A, (ANOVA F(3,15) = 23.34, P < 0.0001); in B, (ANOVA F(3,15) = 4.673, P = 0.0169). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in A, ***P < 0.001, **P < 0.01, *P < 0.05 vs. control and §§ P < 0.01 vs. 0-day; in B, *P < 0.05 vs. control.

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selection in substitution tests and produced a leftward shift in the cocaine dose–response curve (Justinova et al., 2003). Furthermore, the A2A receptor antagonist CGS 15943 reinstates cocaine-seeking behavior and maintains self-administration in baboons (Weerts and Griffiths, 2003). In contrast, inactivation of A2A receptors reduces the reinforcing efficacy of cocaine (Soria et al., 2006) and attenuates amphetamine behavioral sensitization (Chen et al., 2003). Therefore there is a need to neurochemically clarify the role of A2A and D2 receptors within the nucleus accumbens and dorsal striatum in animal models of cocaine addiction. In the present experiments, A2A and D2 proteins and binding characteristics were investigated in rats (Rattus norvegicus) after cocaine self-administration. A self-administration protocol was used which has previously been shown to produce sensitization of the reinforcing effects of cocaine (Morgan et al., 2002a). The model incorporates two conditions found to be necessary for sensitization: extended access to cocaine and a deprivation period (see Morgan and Roberts,

2004). Rats were given access to cocaine throughout the day– night cycle for 10 days using a discrete trials procedure which limits drug intake to a maximum of 4 injections per hour. After access to cocaine on this regimen, rats have been shown to respond to higher break points for cocaine reinforcement when tested on a progressive ratio schedule. Interestingly, this effect is not observed immediately after extended access, but requires a 7-day drug deprivation period. Thus, A2A and D2 receptors were investigated 1 or 7 days after extended access to intravenous cocaine self-administration.

2.

Results

2.1.

Cocaine self-administration

Thirty-six animals completed 10 days of cocaine selfadministration under the discrete trials procedure. The animals were randomly assigned to one of three groups (0, 1 and 7

Fig. 2 – Effects of an extended cocaine self-administration history and its withdrawal on the density of dopamine D2-like receptors in the nucleus accumbens. Bmax values (expressed as fmol/mg protein) were deduced from saturation curves of D2-like antagonist [3H]-Raclopride binding (A) to nucleus accumbens membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and withdrawal from extended daily access to cocaine (1-day; 7-day). Representative saturation curves of the specific D2-like antagonist binding sites in control and the 7-day group are shown. Radioligand binding experiments were performed as indicated in Experimental procedures. In C, the ratio of D2-like Bmax/A2A Bmax in the nucleus accumbens is reported. Values are means ± SEM (n = 6). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In A, (ANOVA F(3,15) = 7.826, P = 0.0022); in B, (ANOVA F(3,15) = 5.219, P = 0.0115); in C, (ANOVA F(3,15) = 7.218, P = 0.0032). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in A, **P < 0.01 vs. control; in B, *P < 0.05 vs. control; in C, *P < 0.05 vs. control and 1-day group, §§P < 0.01 vs. 7-day group.

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Days Off). The pattern of self-administration was virtually identical to that previously described (see Morgan et al., 2002a). During the first day, rats self-administered cocaine (1.5 mg/kg/injection) during almost every trial, averaging 90.2 injections out of a maximum of 96 trial opportunities. On days 2–10 cocaine self-administration stabilized at a lower level with animals taking approximately 50 injections/day for the duration of the experiment. The total average (±SEM) intake per animal for the 10-day period was 890 ± 20.0 mg/kg.

2.2.

Nucleus accumbens

The A2A and D2 saturation binding experiments are summarized in Figs. 1 and 2 from the four experimental groups studied using the A2A antagonist ([3H]-ZM 241385) and D2-like antagonist ([3H]-Raclopride) radioligands. There is a significant rise in the Bmax value of the [3H]-ZM 241385 binding sites in membranes from the nucleus accumbens in the group with a cocaine self-administration history without withdrawal versus the control group and the 1-day and 7-day cocaine withdrawal groups (Fig. 1A). During the withdrawal period, there is a gradual decline of the Bmax values towards the values found in the control group. Furthermore, the Kd value of the [3H]-ZM 241385 binding sites was significantly increased in the cocaine self-administration group without withdrawal versus controls and the 1-day and 7-day withdrawal groups (Fig. 1B). As seen in Fig. 2A and in Fig. 2B, the [3H]-Raclopride binding sites were significantly altered in the two cocaine withdrawal groups as seen from the significant increases in Bmax and Kd values in these two groups versus control. The D2like/A2A ratio of the Bmax values (Fig. 2C) became significantly reduced in the cocaine self-administration group (0-day) versus the control group and the 7-day withdrawal group, which recovered to control values. [3H]-ZM 241385 saturation binding curves were also performed in membranes prepared from the nucleus accumbens from animals outside the experimental groups in the presence or absence of a high Fig. 3 – Expression pattern of adenosine A2A and dopamine D2 receptors in the nucleus accumbens from rats with extended cocaine self-administration history and its withdrawal. Nucleus accumbens membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and its withdrawal (1-day; 7-day) were analyzed by Western blot as indicated in Experimental procedures. A representative image is given in each case. The intensity of the immunoreactive bands of five independent experiments was measured by densitometric scanning and normalized by using tubulin as a control protein (bar graph). The results are presented as % of the immunoreactivity detected in the nucleus accumbens of the control group of animals (mean ± SEM). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In A, (ANOVA F(3,12) = 15.50, P = 0.0002); in B, (ANOVA F(3,12) = 5.497, P = 0.0131). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in A, ***P < 0.001 vs. control and §§P < 0.01 vs. the 1-day and 7-day cocaine withdrawal groups, in B, *P < 0.05 vs. the 7-day cocaine withdrawal group.

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concentration of cocaine (140 μM) where no significant changes were noted in either the Kd (1.0 ± 0.45 vs. 0.95 ± 0.31 nM; n = 3) value or the Bmax values (121.8 ± 23.9 vs. 118.8 ± 19.3 fmol/mg protein; n = 3). Western blot analysis also showed a rise of the A2A protein expression in the nucleus accumbens in the group with a cocaine self-administration history without withdrawal versus controls and the 7-day withdrawal group (Fig. 3A). However, the nucleus accumbens D2 immunoreactivity was not increased in the 7-day withdrawal group (Fig. 3B) in spite of the demonstrated increases in the density of D2-like binding sites in this group (Fig. 2A) suggesting that the rise in D2-like binding density represented an increase in D3 receptor densities which also bind [3H]-Raclopride with a high affinity (Kd 3.5 nM, Seeman and Van Tol, 1994). Instead, the D2

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immunoreactivity was slightly but significantly reduced in the accumbens of the 0-day group and the 1-day withdrawal group (Fig. 3B). Similar to the D2-like/A2A ratio of the Bmax values, the D2/A2A ratios of the Western blot IR values were significantly reduced in the 0-day cocaine self-administration group versus controls and versus the 7-day withdrawal group, which recovered to control values (ANOVA F(3,12) = 7.160, P < 0.05, data not shown).

2.3.

Anterior and posterior parts of the dorsal striatum

As seen in Figs. 4 and 5, no significant changes in the Bmax (Fig. 4A) and Kd (Fig. 4B) values of the [3H]-ZM 241385 binding sites were found in the anterior dorsal striatum of any experimental group, while a trend for reduction in the Bmax values of the [3H]-ZM 241385 binding sites was found in the posterior dorsal striatum in the group with a cocaine self-administration history without withdrawal (Fig. 4C). The Kd values of the [3H]-ZM 241385 binding sites were significantly reduced in the

posterior dorsal striatum in the cocaine 1-day withdrawal group versus the control group (Fig. 4D). No changes were found in the Bmax values of the [3H]-Raclopride binding sites in any of the experimental groups either in the anterior (Fig. 5A) or the posterior (Fig. 5C) dorsal striatum with only a trend for a reduction of the Bmax values in the latter region (Fig. 5C) of the group with cocaine self-administration history without withdrawal versus control that appeared to persist in the two cocaine withdrawal groups. Furthermore, the Kd values (Figs. 5B, D) of [3H]-Raclopride binding sites or the D2-like/A2A Bmax ratios (data not shown) were not significantly changed among the experimental groups in the anterior and posterior dorsal striatum. In the immunoblot analysis, a significant increase in the intensity in the A2A IR band appeared in the anterior dorsal striatum of the cocaine group without withdrawal versus the control and the two cocaine withdrawal groups (Fig. 6A) with lack of changes in the D2 IR band in any experimental group (Fig. 6B). This led to a significant reduction in the ratio of D2/

Fig. 4 – Effects of an extended cocaine self-administration history and its withdrawal on the density and antagonist affinity of adenosine A2A receptors in both the anterior and posterior dorsal striatum. Bmax values (expressed as fmol/mg protein) and Kd values (expressed in nM) were deduced from saturation curves of A2A antagonist [3H]-ZM 241385 binding to anterior dorsal (A, B) or posterior dorsal (C, D) striatum membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and its withdrawal (1-day; 7-day). Radioligand binding experiments were performed as indicated in Experimental procedures. Values are means ± SEM (n = 6). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In D, (ANOVA F(3,12) = 15.50, P = 0.0002). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in D, *P < 0.05 vs. control.

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Fig. 5 – Effects of an extended cocaine self-administration history and its withdrawal on the density and antagonist affinity of dopamine D2-like receptors in both the anterior and posterior dorsal striatum. Bmax values (expressed as fmol/mg protein) and Kd values (expressed in nM) were deduced from saturation curves of D2-like antagonist [3H]-Raclopride binding to anterior (A, B) or posterior (C, D) dorsal striatum membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and its withdrawal (1-day; 7-day). Radioligand binding experiments were performed as indicated in Experimental procedures. Values are means ± SEM (n = 6). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding no significant effect of withdrawal time.

A2A Western blot IR values in the 0-day group versus the control animals, similar to findings in the nucleus accumbens (ANOVA F(3,12) = 4.275, P < 0.05, data not shown). In contrast, in the posterior dorsal striatum a significant reduction appeared in the A2A IR band of the cocaine group without withdrawal, which persisted in the two cocaine withdrawal groups (Fig. 7A) with lack of changes in the D2 IR bands in any experimental group (Fig. 7B). Therefore, a significant increase in the ratio of D2/A2A Western blot IR values was found in the 7-day withdrawal group versus the control animals (ANOVA F(3,9) = 6.239, P < 0.05, data not shown). In both the prefrontal cortex and in the hippocampal formation, the A2A IR remained undetectable in the three experimental groups and D2 IR was unchanged (data not shown).

3.

Discussion

Pharmacological data have shown an important modulation of the behavioral effects of cocaine by A2A receptor mechanisms (see Introduction). For example, A2A antagonists can

reinstate cocaine self-administration (Weerts and Griffiths, 2003) whereas A2A agonists can attenuate the reinforcing effects of cocaine (Knapp et al., 2001). Importantly, Filip et al. (2006) have shown that stimulation of adenosine A2A receptors protected against both the development and expression of sensitization to the locomotor stimulant effects of cocaine. With the demonstration of A2A/D2 heteromeric complexes in the striatum (Hillion et al., 2002; Canals et al., 2003; Nakata et al., 2005, Fuxe et al., 2005) we hypothesized that A2A/D2 receptor interactions may be involved in cocaine addiction. Here we used a self-administration procedure, which has been shown to produce sensitization to the reinforcing effects of cocaine, to investigate whether A2A and D2 receptor systems are altered in a time course which matches changes in the motivation to self-administer cocaine. The biochemical results in the nucleus accumbens from the A2A antagonist radioligand binding analysis and from the A2A immunoblots are in agreement and give evidence for an increased density of functional A2A receptors in the nucleus accumbens immediately following 10 days of cocaine self-administration. In contrast, the increase in A2A immunoreactivity in the anterior

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dorsal striatum was not associated with an increase in the density of A2A antagonist binding sites, indicating that the increase in A2A expression in the anterior dorsal striatum does not reflect an increase in the density of functional A2A receptors. However, the reduction of A2A receptor IR in the posterior dorsal striatum was associated with a trend for a reduction of A2A antagonist binding sites suggesting a reduction of functional A2A receptors in rats with the present type of cocaine self-administration history. No changes were found in the prefrontal cortex and hippocampal regions. This increase in the accumbens A 2A receptors may represent a compensatory up-regulation to counteract the cocaine-induced enhancement of D2 signaling in view of the existence of antagonistic A2A/D2 interactions (see below and Fuxe et al., 2005). This likely enhancement of A2A signaling by

cocaine in the 0-day group does not involve direct effects of cocaine on the A2A receptors since cocaine added in vitro does not alter the binding characteristics of the A2A antagonist binding sites in accumbens membrane preparations. In rats from the 7-day cocaine withdrawal group, which show an increase in the reinforcing efficacy of cocaine (Morgan et al., 2002a,b) this rise of accumbens A2A receptor density has decreased to control levels. The reduction of A2A receptors in the posterior dorsal striatum as observed in the Western blots largely persists in the cocaine withdrawal period tested; however, there was only a trend for a reduction of A2A receptor binding density in the 0day group. Thus, it is not clear, but certainly possible, that the functional receptor A2A density is reduced. In the dorsal striatum, this differential regulation by cocaine in the posterior versus the anterior part may be related to the relative absence of A2A receptors stimulating glutamate release in the posterior striatum (Blum et al., 2003). This may lead to a dominance of D2-like receptors inhibiting glutamate release from the corticostriatal terminals by the D2 receptor activation through the cocaine-induced increases in extracellular levels of DA. It is postulated that an intact glutamate transmission is required for the compensatory up-regulation of the A2A receptors to take place in the striato-pallidal GABA neurons after the present cocaine self-administration history. A reduced glutamate transmission via especially mGluR5 and NMDA receptors reduces the activity of calcium/calmodulindependent kinases and extracellular signal-regulated kinases (see review by Ferre et al., 2003) and thus the subsequent phosphorylation of the transcription factor CREB, which binds to and activates the A2A receptor promoter (Chiang et al., 2005). Therefore, the antagonistic A2A/D2 interaction may not be increased in this region but, in contrast, reduced in view of the postulated decrease in glutamate synaptic activity causing a reduction of A2A gene expression. This reduction may result in enhanced D2 signaling and contribute to the addictive properties of cocaine. However, in the present study, no change in the density or the affinity of functional D2 receptors was Fig. 6 – Expression pattern of adenosine A2A and dopamine D2 receptors in the anterior dorsal striatum from rats with extended cocaine self-administration history and its withdrawal. Anterior dorsal striatum membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and its withdrawal (1-day; 7-day) were analyzed by Western blot as indicated in Experimental procedures. A representative image is given in each case. The intensity of the immunoreactive bands of five independent experiments for A2AR detection and six independent experiments for D2R detection was measured by densitometric scanning and normalized by using tubulin as a control protein (bar graph). The results are presented as % of the immunoreactivity detected in the anterior dorsal striatum of the control group of animals (mean ± SEM). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In A, (ANOVA F(3,12) = 10.76, P = 0.0010). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in A, **P < 0.01 vs. all other groups.

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observed in the dorsal striatum of any experimental group, which does not give support to an enhancement of D2 signaling by an increased number of receptors. Dorsal striatum is involved in habitual behavior and in cue-elicited cocaine craving (Robbins and Everitt, 2002; Vanderschuren et al., 2005; Wong et al., 2006) However, the A2A receptor reduction is not altered by cocaine withdrawal and therefore not primarily involved in the increased reinforcing efficacy of cocaine after a 7-day withdrawal (Morgan et al., 2002a,b, 2004). The above results are the major findings of the present paper. They indicate that cocaine self-administration may be associated with an up-regulation of A2A receptors in the nucleus accumbens, a critical region for early responses to reward and for drug addiction (Robbins and Everitt, 2002;

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Kalivas et al., 2005). In view of the existence of antagonistic A2A/D2 receptor interactions in both the ventral and dorsal striatum, an interaction that takes place mainly in the striatopallidal GABA neurons (see Fuxe et al., 2005), a compensatory up-regulation of A2A receptor expression and increased A2A signaling may develop. This may counteract the increased D2 signaling produced via the cocaine-induced increases in extracellular DA levels (see Andrews and Lucki, 2001). In agreement with this view it is also known that there exists an atypical cAMP-response element (CRE) site in the core promoter of the A2A receptor gene (Chiang et al., 2005) and CREB reduces cocaine reward in the nucleus accumbens (Carlezon et al., 1998; Mattson et al., 2005). In line with this view, the compensatory A2A up-regulation had disappeared in the group with a 7-day withdrawal from cocaine self-administration previously determined to be associated with an increased reinforcing efficacy of cocaine (Morgan et al., 2002a, b). It is likely that the reduction of the A2A antagonistic brake on D2 signaling after a 7-day withdrawal period may contribute to the increases in the reinforcing efficacy of cocaine as measured by a progressive ratio schedule (Morgan et al., 2002a,b). Furthermore, no A2A receptor up-regulation is observed after withdrawal from a chronic escalating dose “binge” cocaine administration (Bailey et al., 2005). Taken together, a possible increase in CREB phosphorylation produced by cocaine self-administration may cause increases in A2A receptor gene expression and result in the increased antagonism of D2 receptor signaling through the antagonistic A2A/D2 receptor interactions at both the membrane and cytoplasmic level. However, the observed A2A up-regulation may also operate via other mechanisms to counteract the D2 inhibition of the striato-pallidal GABA neurons including the increased formation of A2A homomers, thereby increasing the excitability of these neurons (Antonelli et al., 2006). Another mechanism may also be the ability of a persistent D2 activation, from increased extracellular DA levels, to sensitize A2A receptor

Fig. 7 – Expression pattern of adenosine A2A and dopamine D2 receptors in the posterior dorsal striatum from rats with extended cocaine self-administration history and its withdrawal. Posterior dorsal striatum membranes from cocaine naive rats (control), rats with extended cocaine self-administration history (0-day group) and its withdrawal (1-day; 7-day) were analyzed by Western blot as indicated in Experimental procedures. A representative image is given in each case. The intensity of the immunoreactive bands of four independent experiments for A2AR detection and five independent experiments for D2R detection was measured by densitometric scanning and normalized by using tubulin as a control protein (bar graph). The results are presented as % of the immunoreactivity detected in the posterior dorsal striatum of the control group of animals (mean ± SEM). Data were analyzed using one-way analysis of variance (ANOVA) with repeated measures yielding a significant effect of withdrawal time. In A, (ANOVA F(3,9) = 19.10, P = 0.0003). Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test and yielded in A, **P < 0.01 and ***P < 0.001 vs. control.

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signaling at the level of the adenylate cyclase via the release of βγ dimers from the activated trimeric G-proteins (Vortherms and Watts, 2004). Finally, it should be considered that the A2A receptor up-regulation and its disappearance upon cocaine withdrawal may have a modulatory influence on the reinforcing efficacy of cocaine by causing changes in the phosphorylation of actin binding proteins and thus changes in actin cycling of relevance for dendritic plasticity (Carlisle and Kennedy, 2005). In fact, withdrawal from cocaine produces increases in actin cycling in the nucleus accumbens which may modulate the cocaine-induced reinstatement of drug seeking (Toda et al., 2006; Kalivas et al., 2005) and the disappearance of the A2A up-regulation upon cocaine withdrawal may contribute to such events. The significant reduction in the affinity of the A 2A antagonist sites of the nucleus accumbens observed in the group with a cocaine self-administration history without withdrawal may be caused by the fact that the increased density of A2A receptors was found in this group in this region. This may reflect the increased formation of A2A homomers or formation of novel A2A/D2 heteromers with different stoichiometry and topography of the participating A2A and D2 receptors. This may result in altered allosteric interactions between the two receptors and thus to altered development of positive and negative cooperativity of possible high order A2A/ D2 heteromers formed, so called receptor mosaics (Agnati et al., 2003, 2007; Franco et al., 2003, 2005; Fuxe et al., 2003, 2005). Such mechanisms may also be involved in causing the increase in the affinity of the A2A antagonist binding sites in the posterior dorsal striatum of the cocaine 1-day withdrawal group. However, in this region, the allosteric modulations in the receptor mosaics go in the opposite direction, with an enhancement of affinity. The present D2-like receptor binding experiments in the nucleus accumbens as well as the results from the D2 immunoblot analysis from this region show an increase in the density and Kd values of the [3H]-Raclopride binding sites in the absence of an increase in the amount of D2 IR in the cocaine 1-day and 7-day withdrawal groups. These findings can be explained by an increase in the density of nucleus accumbens D3 receptors after cocaine withdrawal, which are known to have a slightly lower affinity for Raclopride versus the D2 receptors (Seeman and Van Tol, 1994). In our present findings, the postulated increase in D3 receptor density deduced from increases in [3H]-Raclopride binding is based on recently published data by Neisewander et al. (2004) who have in fact demonstrated increases in D3 receptor binding in striatal sub-regions in rats after a cocaine challenge at various time points after cocaine self-administration. This probable rise of D3 receptors, after cocaine withdrawal, has been associated with an increase in cocaine-seeking behavior (Tran-Nguyen et al., 1998). Thus, the present findings likely indicate an increase of D3 receptor signaling in the nucleus accumbens after cocaine withdrawal and may contribute to the increased reinforcing efficacy of cocaine in such states since D3 receptor antagonists may reduce cocaine addiction (Vorel et al., 2002; see Joyce and Millan, 2005). As to mechanistic aspects of persistent increases in D3 receptor expression after chronic cocaine, indications exist that phasic cocaine-induced increases in BDNF release may be involved in

causing this increase in D3 expression associated with cocaine conditioning (Le Foll et al., 2005). Adaptive increases in D3 receptors and an up-regulation of D3 mRNA have also been observed in reward circuits of human cocaine fatalities (Staley and Mash, 1996; Segal et al., 1997). It is known that A2A/D3 heteromers may exist in the nucleus accumbens where A2A activation antagonizes D3 signaling (Torvinen et al., 2005). Thus, the reduction of the D2-like/A2A Bmax ratio in the nucleus accumbens may reflect not only the probable increase of A2A signaling over D2 signaling but also over the D3 signaling in the nucleus accumbens in the present cocaine self-administrating group without withdrawal. Therefore, a reduced antagonistic A2A/D3 interaction in the nucleus accumbens may also contribute to the increased motivation to self-administer cocaine in the 7-day withdrawal group in the present study. The slight reduction of accumbens D2 immunoreactivity in the 0-day group and the 1-day withdrawal group was also not found in the [3H]-Raclopride binding experiments. Again such a change may be masked by the postulated rise of D3 receptor binding sites in the accumbens. No significant effects on D2-like binding characteristics and D2 expression in the dorsal striatum were observed in the present study following 10 days of high cocaine intake. This is a significant finding and indicates that changes in D2 receptor expression and recognition in this region are not involved in the adaptive responses to cocaine reward in this model to a major degree. Recently biphasic changes in D2 receptors in the nucleus accumbens core have been noted after withdrawal from brief daily access to intravenous cocaine (Ben-Shahar et al., 2007). It is likely that the dose and duration of exposure account for this difference. Interestingly, chronic cocaine selfadministration in rhesus monkeys involving periods of several to many months causes reduction of striatal D2 receptor densities as shown with in vitro receptor autoradiography (Moore et al., 1998; Nader et al., 2002). Imaging studies have also shown lower D2 receptor availability in human cocaine addicts compared to non-drug using control subjects (Volkow et al., 1993; Martinez et al., 2004). Similarly, PET imaging (Morgan et al., 2002a,b; Nader et al., 2006) has shown reduced D2 availability in non-human primates during chronic cocaine self-administration in monkeys persisting during 1 year of abstinence. A persistent down-regulation of striatal D2 receptors may contribute to such findings and involve D2 internalization after the chronic cocaine self-administration. It is therefore possible that the trends in the present study for a reduction of D2 receptor and A2A receptor densities in the posterior dorsal striatum with a significant reduction of A2A expression observed after an extended cocaine self-administration history and its withdrawal can be explained by a cointernalization of A2A/D2 receptor heteromers in this region. Taken together, the present findings give neurochemical evidence that the present extended cocaine self-administration procedure gives rise to an up-regulation of functional A2A receptors in the nucleus accumbens. Its disappearance during the withdrawal period may help to explain the increased reinforcing efficacy of cocaine in the 7-day withdrawal group which could involve increased signaling via accumbens D2 and D3 receptors. The results indicate a putative role of antagonistic A2A/D2 interactions and possible antagonistic A2A/D3 interactions in the nucleus accumbens in the prevention of

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development of cocaine addiction. They open up the possibility that A2A agonists can represent cocaine antagonists to be used in the prevention of cocaine addiction.

4.

Experimental procedures

4.1.

Drugs

Cocaine hydrochloride (provided by the National Institute on Drug Abuse) was dissolved in sterile 0.9% saline and passed through a micro-filter for cocaine self-administering experiments in animals or into (IB) incubation buffer (20 mM Tris– HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM), EDTA (1 mM) and DTT (1 mM)) for the A2A receptor antagonist [3H]ZM 241385 saturation binding experiments.

4.2.

Subjects and surgery

Male, Sprague–Dawley rats (R. norvegicus) (Harlan, IN) weighing approximately 350 g at the start of the experiments were used as subjects. Upon arrival at the facility and throughout the experiments, rats were maintained on a reverse light/dark cycle (lights on from 3 pm to 3 am; during the dark phase the cage was dimly lit). Food (Ralston, Purina, St. Louis, MO) and water were available ad libitum. After a minimum 3-day acclimation period, each rat was anesthetized with a combination of ketamine (100 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.). A chronic indwelling cannula was implanted into the right jugular vein and exited the skin on the dorsal surface in the region of the scapulae (Roberts and Goeders, 1989). Butorphanol tartrate (0.2 mg/kg, s.c.) was administered as a post-surgical analgesic. Tygon tubing was attached at the back, enclosed by a stainless steel protective spring tether, and connected to a counterbalanced, fluid swivel (Instech Laboratories, Inc., Plymouth Meeting, PA, USA) mounted above the experimental chamber, which allowed free movement within the chamber. Tubing connected the swivel to an infusion pump (Razel Scientific Instruments, Inc., Stanford, CT, USA). The experimental chambers were 30 × 30 × 30 cm in size and served as both housing and testing chambers.

4.3.

Cocaine self-administration training

Cocaine self-administration training began 3–5 days following surgery, and these sessions started at 10 am throughout the study. At the beginning of each daily session a retractable lever extended into the cage to signal session onset. Each lever press response resulted in the delivery of approximately 0.1 ml of 5 mg/ml cocaine (cocaine hydrochloride was obtained from the National Institute on Drug Abuse, Rockville, MD; cocaine was dissolved in sterile 0.9% saline and passed through a micro-filter, and the dosages expressed as the salt weight). Infusion times were based on body weight, lasted for approximately 5 s and delivered a dose of 1.5 mg/kg cocaine. Concurrent with the start of each injection, the lever was retracted for 20 s and a stimulus light was activated to signal this 20-s post-response time-out period. Rats were initially exposed to a fixed ratio 1 (FR1) schedule of reinforcement. Daily training session continued

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until subjects self-administered 40 infusions/day for 5 days after schedule conditions were changed to a discrete trials procedure as described previously (Morgan et al., 2002a,b; Roberts et al., 2002). Briefly, animals were given access to cocaine during four discrete 10 min trials per hour (DT4), 24 h per day for 10 days. A trial began, every 15 min, with the introduction of a retractable lever into the cage. A single response resulted in a cocaine infusion that was signaled by the illumination of a stimulus light for 20 s. A trial was terminated and the lever retracted if a cocaine injection (1.5 mg/kg/injection) was collected or if 10 min had elapsed.

4.4.

Experimental groups

Following 10 days of self-administration on a discrete trials schedule (4 trials/h), animals were randomly assigned to one of three groups (n = 12). One group was sacrificed immediately at the end of the 10-day self-administration period when drug was still on board (0-Day off). The two remaining groups were sacrificed either the following day (1-Day off) or after 1 week of drug deprivation (7-Days off). Animals in the drug naive group (Control, n = 12) were anesthetized, implanted with an IV cannula and housed individually in cages identical to the self-administration groups but received no access to cocaine.

4.5.

Dissection

After reaching a state of deep anesthesia (induced by a combination of ketamine and xylazine, 50 mg/kg of each), each animal was decapitated and the brain was quickly removed and chilled in ice cold saline. Coronal sections were made with the aide of a Rodent Brain Matrix (ASI Instruments, Warren, MI). The posterior striatum was dissected from a 3 mm thick section corresponding to bregma −2.3 to +0.7 (with respect to bregma, see Paxinos and Watson, 1986) and the anterior striatum and nucleus accumbens were dissected from a 2 mm slice corresponding to +2.7 to 0.7 mm. Samples were immediately frozen on dry ice and stored at −80 °C.

4.6.

Membrane preparation

Frozen tissue samples from two animals from each of the different groups of cocaine self-administering animals or control animals were homogenized in ice-cold preparation buffer (20 mM Tris–HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM) and EDTA (1 mM)) by sonication using a Bandelin Sonoplus GM 2070 sonicator. The membranes were precipitated by centrifugation at 47,000×g for 20 min and washed through rehomogenization in 10 ml of preparation buffer three sequential times. The final pellet was rehomogenized in 3 ml of the same buffer and an aliquot was taken to determine the protein concentration and for Western blot analysis. The concentration of total protein was determined by the modified Lowry method using bovine serum albumin as standard. Membranes were resuspended in incubation buffer (IB) (20 mM Tris–HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM), EDTA (1 mM) and DTT (1 mM)) and the suspension of membranes was subsequently used in the radioligand binding experiments.

218 4.7.

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Radioligand binding experiments

Saturation experiments with the D2-like receptor antagonist [3H]-Raclopride were carried out using 10 concentrations (0.2– 10 nM) of [3H]-Raclopride (60.1 Ci/mmol) by incubation for 90 min at 30 °C in IB (incubation buffer) and the incubation was terminated by rapid filtration through GF/B filters using a Brandel cell harvester with three washings of 5 ml of ice-cold washing buffer (20 mM Tris–HCl pH 7.4, containing NaCl (100 mM)). Nonspecific binding is defined by the binding in the presence of dopamine (1 mM) (see Torvinen et al., 2004). Saturation experiments with the A2A receptor antagonist [3H]ZM 241385 were carried out using 10 concentrations (0.02– 6.0 nM) of [3H]-ZM 241385 (27.4 Ci/mmol) by incubation for 90 min at 30 °C in IB (incubation buffer), and the incubation was terminated by rapid filtration through GF/B filters using a Brandel cell harvester with three washings of 5 ml of ice-cold washing buffer (20 mM Tris–HCl pH 7.4, containing NaCl (100 mM)). Nonspecific binding is defined by the binding in the presence of the A2A receptor antagonist MSX-3 (6 μM). [3H]-ZM 241385 saturation experiments were also performed in the presence or absence of 140 μM cocaine hydrochloride using 10 concentrations (0.02–6.0 nM) of [3H]-ZM 241385 (27.4 Ci/mmol) (see Uustare et al., 2005).

4.8.

Western blot

Membranes were solubilized in urea buffer 2× (urea 8 M, SDS 2%, DTT 200 mM) and incubated at 37 °C for 2 h. Twenty micrograms of protein was applied in each lane and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/ PAGE) was performed using 10% polyacrylamide gels. Proteins were immunoblotted to PVDF membranes (immobilion-P; Millipore) using semidry transfer system and developed with the enhanced SuperSignal chemiluminescence detection kit (Pierce Biotechnology). Receptors were detected with the following primary antibodies: mouse anti-adenosine receptor A2A (1:1000; UpState, Lake Placid, NY) and rabbit anti-dopamine D2 receptor (D2 receptor (rat) 246–316) (see Jansson et al., 1999) and the following horseradish peroxidase secondary antibodies: goat anti-rabbit IgG (1:30,000; Pierce Biotechnology) and rabbit antimouse IgG (1:2000; Dako A/S, Denmark). After striping, membranes were developed with mouse anti-β-tubulin (1:6000; Sigma-Aldrich, CO) followed with rabbit anti-mouse IgG to confirm equal loading of protein. Immunoreactive bands were developed with SuperSignal chemiluminescent detection kit (Pierce, Rockford, IL, USA). Quantitative analysis of detected bands was carried out by densitometric scanning and all values were normalized by using tubulin as a control protein (see Gimenez-Llort et al., 2007).

4.9.

Data analysis

All binding data were analyzed using the commercial program GraphPad PRISM 4.0 (GraphPad software, San Diego, USA). For statistical evaluation of the biochemical data, one-way analysis of variance (ANOVA) was used. Group differences after significant ANOVAs were measured by post hoc Bonferroni's Multiple Comparison test.

Acknowledgments This work was supported by a Grant from the Swedish Research Council (K. Fuxe) and by a Grant from the Spanish Ministry of Education and Science (SAF2005-00170 to C. Lluis and SAF2006-05481 to R. Franco). K. Fuxe and D. Roberts acknowledge NIDA (National Institute of Drug Abuse) as the supplier of the cocaine hydrochloride used in the present publication.

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