Increase of morphine withdrawal in mice lacking A 2a receptors and no changes in CB 1 /A 2a double knockout mice Fernando Berrendero, 1 Anna Castan Äe Â, 1 Catherine Ledent, 2 Marc Parmentier, 2 Rafael Maldonado 1 and Olga Valverde 1 1 Laboratori de Neurofarmacologia, Facultat de Cie Áncies de la Salut i de la Vida, Universitat Pompeu Fabra, C/Doctor Aiguader 80, 08003 Barcelona, Spain 2 IRIBHN. Universite  libre de Bruxelles, N-1070 Bruxelles, Belgium Keywords:A 2a adenosine receptors, CB 1 cannabinoid receptors, emotional-like responses, knockout mice, opioid withdrawal Abstract CB 1 cannabinoid and A 2a adenosine receptors are highly expressed in the central nervous system where they modulate numerous physiological processes including emotional behaviour and the responses of several drugs of abuse. To investigate the contribution of these receptors in emotional-like responses and opioid dependence we have generated CB 1 /A 2a double de®cient mice (CB = 1 =A = 2a ). The spontaneous locomotor activity was reduced in double knockout as compared to wild-type animals. Emotional-like responses of CB = 1 =A = 2a mice were investigated using the elevated plus-maze and the lit-dark box. Mutant mice exhibited an increased level of anxiety in both behavioural models. The speci®c involvement of CB 1 and A 2a receptors in morphine dependence was evaluated by using A 2a knockout mice and CB 1 /A 2a double mutant mice. The severity of naloxone-precipitated morphine withdrawal syndrome was signi®cantly increased in the absence of A 2a adenosine receptors whereas no modi®cations were observed in the double knockout mice. These results suggest that both receptors participate in the control of emotional behaviour and seem to play an opposite role in the expression of opioid physical dependence. Introduction Adenosine is an important inhibitory neuromodulator in the central nervous system which induces its physiological effects by acting on four different adenosine receptor subtypes, A 1 ,A 2a ,A 2b and A 3 (Olah & Stiles, 1995). A 1 and A 3 receptor activation inhibits adenylate cyc- lase activity whereas A 2a and A 2b receptors are positively coupled to this enzyme. The highest levels of A 2a receptors are found in the striatum where they are coexpressed with postsynaptic D 2 dopamine receptors in GABAergic striatopallidal neurons, and regulate proen- kephalin gene expression (Fink et al., 1992; Schiffmann & Vander- haeghen, 1993). The speci®c function of A 2a receptors has been investigated by using A 2a knockout mice (Ledent et al., 1997; Chen et al., 1999), and an important increase in basal levels of anxiety was observed in these mutant mice. Adenosine has been suggested to regulate different responses induced by opioids. Thus, the antinoci- ceptive effects of morphine in the spinal cord seem to be mediated at least in part by release of endogenous adenosine and subsequent activation of A 1 and A 2 receptors (Sweeney et al., 1987; Sweeney et al., 1991). Adenosine also participates in opioid dependence as the blockade of adenosine metabolism by adenosine kinase inhibitors decreases the severity of morphine withdrawal (Kaplan & Coyle, 1998). In agreement, adenosine agonists inhibit the expression of morphine withdrawal while adenosine antagonists increase the inci- dence of withdrawal signs (Kaplan & Sears, 1996; Salem & Hope, 1997). However, the low speci®city of adenosine antagonists makes it dif®cult to clarify the participation of the different adenosine receptor subtypes in opioid dependence. On the other hand, the cannabinoid system represents another im- portant inhibitory neuromodulator acting in the central nervous system through the stimulation of CB 1 cannabinoid receptors (Matsuda etal., 1990). Cannabinoid receptor stimulation reduces adenylyl cyclase activity also through activation of G proteins. The CB 1 receptor has been reported to be responsible for the addictive properties of canna- binoids, including rewarding effects and physical dependence (Ledent et al., 1999), and bi-directional interactions between cannabinoid and opioid systems in these processes have been recently con®rmed (reviews in Manzanares et al., 1999; Maldonado & Rodrõ Âguez de Fonseca, 2002). Thus, the severity of opioid withdrawal is reduced in mice lacking the CB 1 cannabinoid receptor (Ledent et al., 1999), whereas cannabinoid withdrawal syndrome is attenuated in preproen- kephalin-de®cient mice (Valverde et al., 2000a). CB 1 cannabinoid receptors seem to be also involved in the regulation of emotional behaviour as an increase in the basal level of anxiety has been reported in CB 1 knockout mice (Martin et al., 2002). The aim of the present study was to investigate the speci®c role of CB 1 cannabinoid receptors and A 2a adenosine receptors in emotional- like responses and opioid dependence by disrupting these genes in mice. For this purpose, basal levels of locomotor activity and anxiety- like behaviour were analyzed in CB = 1 =A = 2a mice and wild-type animals. In addition, the contribution of CB 1 and A 2a receptors on the expression of the opioid withdrawal syndrome was evaluated follow- ing chronic morphine treatment in mice lacking A 2a receptors (Ledent et al., 1997) and CB = 1 =A = 2a mice. These behavioural responses were compared with the results obtained in A 2a and CB 1 single mutant mice in the same paradigms. All the studies were performed under identical experimental conditions in order to make possible a direct comparison of double vs. single knockout mice. European Journal of Neuroscience, Vol. 17, pp. 315±324, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02439.x Correspondence: Dr Olga Valverde, as above. E-mail: ovalverde@imim.es Received 28 October 2002, accepted 6 November 2002