From the DEPARTMENT OF NEUROSCIENCE Karolinska Institutet, Stockholm, Sweden MOLECULAR MECHANISMS UNDERLYING THE ACTIONS OF PSYCHOACTIVE DRUGS IN THE BASAL GANGLIA: FOCUS ON CANNABINOIDS AND MORPHINE

This thesis is centered on the identification of the molecular mechanisms involved in the psychomotor effects of cannabinoids and morphine. These drugs share the ability of acting at the level of the basal ganglia, a group of subcortical structures involved in the control of locomotion, as well as in cognitive and motivational aspects of motor function. In Paper I and II, we have examined the involvement of the dopamineand cAMP-dependent phosphoprotein of 32 kDa (DARPP-32) in the motor depressant effect produced by activation of the neuronal CB1 receptor (CB1R). DARPP-32 is highly expressed in the medium spiny neurons of the striatum, which is the largest component of the basal ganglia. We found that administration of CP55,940, a selective CB1R agonist, or 9 -tetrahydrocannabinol ( 9 -THC), the active component of marihuana or hashish, increased the state of phosphorylation of DARPP-32 at the cAMP-dependent protein kinase site (PKA), Thr34. Similar increases were observed with AM404, a blocker of the reuptake of endogenous cannabinoids (e.g. anandamide and 2-arachidonyl glycerol), or URB597, an inhibitor of the enzyme fatty acid amide hydrolase (FAAH), which is responsible for the degradation of endocannabinoids. The motor depressant effect (catalepsy) produced by CP55,940, was attenuated by genetic inactivation of DARPP32. Point mutation of Thr34 on DARPP-32 produced a similar reduction in the effect of the CB1R agonist. Genetic inactivation either of dopamine D2 receptors (D2Rs) or of adenosine A2A receptors (A2ARs) reduced the phosphorylation of DARPP-32 at Thr34 and the motor depression produced by CP55,940. These data indicated that a considerable proportion of the psychomotor effect of cannabinoids is accounted for by a signaling cascade involving PKA-dependent phosphorylation of DARPP-32, achieved via CB1R-mediated modulation of D2R and A2AR transmission. In Paper III, we have examined the involvement of DARPP-32 in the shortand long-term effects of morphine. We found that acute administration of morphine increased DARPP-32 phosphorylation at Thr34 in both dorsal striatum and ventral striatum (nucleus accumbens). The ability of morphine to stimulate Thr34 phosphorylation was prevented by blockade of dopamine D1 receptors (D1Rs). Genetic inactivation of DARPP-32 or point mutation of Thr34 reduced the hyperlocomotor response to a single injection of morphine. In contrast, DARPP-32 mutant mice developed behavioral sensitization to morphine comparable to that of wild-type controls and displayed normal morphine conditioned place preference. These results demonstrated that dopamine D1R-mediated activation of the cAMP/DARPP-32 cascade in striatal MSNs is involved in the psychomotor action, but not in the rewarding properties, of morphine. Exposure to cues previously associated with intake of substances of abuse can promote drug related responses. In Paper IV, we have examined the effects of exposure to a drug-associated context on the psychomotor response to morphine. We found that the psychomotor sensitization produced by repeated administration of morphine was markedly increased in mice examined 4 weeks after the last drug injection. In addition, the withdrawal period was able to confer to the environment paired with morphine the ability to increase ERK phosphorylation in a specific compartment (i.e. the shell) of the nucleus accumbens. Using transgenic mice with enhanced green fluorescent protein (EGFP) expression under the control of the D1R (drd1a-EGFP) or D2R promoter (drd2-EGFP), we showed that context-dependent ERK phosphorylation was restricted to D1R-expressing MSNs. Furthermore, we found that this effect depended on D1R activation. This study showed that, following repeated morphine injections, a drug free period induced context-dependent phosphorylation of ERK in a discrete group of neurons within the nucleus accumbens shell. This activation was associated with enhanced psychomotor sensitization and could be implicated in context-elicited drug seeking induced by repeated exposure to drugs of abuse. LIST OF PUBLICATIONS I. Andersson M, Usiello A, Borgkvist A, Pozzi L, Dominguez C, Fienberg AA, Svenningsson P, Fredholm BB, Borrelli E, Greengard P, Fisone G. Cannabinoid action depends on phosphorylation of dopamineand cAMPregulated phosphoprotein of 32 kDa at the protein kinase A site in striatal projection neurons. J. Neurosci., 2005, 25(37):8432-8 II. Borgkvist A, Usiello A, Greengard P, Fisone G. Regulation of cAMP/DARPP32 by 9 -tetrahydrocannabinol. Neuropharmacology, 2008, 54(1):31-5 III. Borgkvist A, Usiello A, Greengard P, Fisone G. Activation of the cAMP/PKA/DARPP-32 signaling pathway is required for morphine psychomotor stimulation but not for morphine reward. Neuropsychopharmacology, 2007, 32(9):1995-2003 IV. Borgkvist A, Valjent E, Santini E, Hervé D, Girault JA, Fisone G. Delayed, contextand dopamine D1 receptor-dependent activation of ERK in morphinesensitized mice. Submitted Manuscript