Sl Workshop W.l Molecular mechanisms W.1.01 Study of the neurobiological mechanisms of cannabinoid dependence by using knockout mice R. Maldonado. Laboratori de Neurofarmacologia, Facultat de Ciencih de la Salut i de la Vida, Universitat Pompeu Fabra, 08003 Barcelona, Spain The neurobiological mechanisms involved in cannabinoid dependence have been investigated by using a variety of pharmacological and biochemical approaches. These studies have associated several neuroanatomical and neurochemical mechanisms with different components of camrabinoid addictive processes [ 11. However, progress has accelerated dramatically in the last few years by novel research tools that have been provided by the recent advances in molecular biology techniques. Particularly, the generation of knockout mice by homologous recombination has been extremely useful since it allows to remove the expression of specific genes encoding proteins that could be involved in the biological responses of cannabinoids [2]. Recent studies using knockout mice deficient in the different opioid receptors or opioid peptide precursors have provided new highlights to clarify the involvement of the endogenous opioid system in cannabinoid antinociception. Thus, antinociceptive responses induced by a high dose of delta9-tetrahydrocannabinol (THC) (20 mg/kg) in the tail immersion and hot plate tests were not modified in knockout animals deficient in mu-, delta- or kappa-opioid receptors [3]. Therefore, the suppression of a single opioid receptor was not enough to alter this acute cannabinoid response. However, an attenuation of THC-induced antinociception in the tail immersion test was observed in knockout mice deficient in the pre-proenkephalin gene [4], but derivatives from proenkephalin are not selective agonists of any opioid receptor. THC-induced antinociception in the tail immersion test was also reduced in pro-dynorphin knockout mice, whereas the effects of THC in the hot plate test remained unaffected in these animals [5]. Most of the peptides derived from pro-dynorphin are preferential agonists of the kappa opioid receptor but are not selective compounds, and can therefore activate other opioid receptors. New data about the mechanisms involved in cannabi- noid tolerance and dependence have been also provided by using knockout mice deficient in the different components of the endogenous opioid system. The development of tolerance to the hypothermic effects of THC was not modified in knockout mice lacking the pre-proenkephalin gene. However these knockout mice showed a decrease in the development of tolerance to THC antinociceptive effects and a slight attenuation of tolerance to THC-induced hypolocomotion [4]. The development of tolerance to the different pharmacological responses of THC was not significantly modified in knockout mice deficient in the pro-dynorphin gene [5]. THC tolerance has been also investigated in knockout mice deficient in the different opioid receptors [3]. Thus, the development of tolerance to THC-induced hypothermia, hypolocomotion and antinociception was not modified in knockout mice lacking mu or delta opioid receptor. Kappa opioid receptor knockout mice showed a slight decreased tolerance to THC hypolocomotor effects. However, the development of tolerance to THC-induced antinociception and hypothermia was not significantly modified in kappa knockout mice [3]. These results indicate that the suppression of a single opioid receptor has not significant consequences on the development of cannabinoid tolerance. Opioid peptide derivatives from pro-dynorphin gene do not seem to participate in cannabinoid tolerance. However, peptide derivatives from pre-proenkephalin gene participate in the development of tolerance to antinociceptive effects. On the other hand, the severity of SR 1417 16A-precipitated cannabinoid withdrawal was decreased in THC-dependent knockout mice lacking the pre-proenkephalin gene [4], but was not modified in knockout mice deficient in the prodynorphin gene [5]. The behavioral expression of SR 1417 16A-precipitated THC withdrawal was also evaluated in knockout mice lacking the different opioid receptors. Cannabinoid withdrawal syndrome precipitated by SR 141716A administration was not modified in mu-, delta- or kappa-opioid receptor knockout mice chronically treated with 20 mg/kg (twice daily) of THC [3]. Another recent study has reported a decrease in the severity of cannabinoid withdrawal syndrome in mu knockout mice chronically treated once daily with 30 and 100 mg/kg