Despite tremendous efforts in the search for safe, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Opioids exert their pharmacological actions through three opioid-receptor classes, mu, delta and kappa, whose genes have been cloned. Genetic approaches are now available to delineate the contribution of each receptor in opioid function in vivo. Here we disrupt the mu-opioid-receptor gene in mice by homologous recombination and find that there are no overt behavioural abnormalities or major compensatory changes within the opioid system in these animals. Investigation of the behavioural effects of morphine reveals that a lack of mu receptors abolishes the analgesic effect of morphine, as well as place-preference activity and physical dependence. We observed no behavioural responses related to delta- or kappa-receptor activation with morphine, although these receptors are present and bind opioid ligands. We conclude that the mu-opioid-receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component of the opioid system for morphine action.

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Three distinct classes of drugs: dopaminergic agonists (such as D-amphetamine), serotonergic agonists (such as LSD), and glutamatergic antagonists (such as PCP) all induce psychotomimetic states in experimental animals that closely resemble schizophrenia symptoms in humans. Here we implicate a common signaling pathway in mediating these effects. In this pathway, dopamine- and an adenosine 3',5'-monophosphate (cAMP)-regulated phospho-protein of 32 kilodaltons (DARPP-32) is phosphorylated or dephosphorylated at three sites, in a pattern predicted to cause a synergistic inhibition of protein phosphatase-1 and concomitant regulation of its downstream effector proteins glycogen synthesis kinase-3 (GSK-3), cAMP response element-binding protein (CREB), and c-Fos. In mice with a genetic deletion of DARPP-32 or with point mutations in phosphorylation sites of DARPP-32, the effects of D-amphetamine, LSD, and PCP on two behavioral parameters-sensorimotor gating and repetitive movements-were strongly attenuated.

1 In order to explore potential therapeutic implications of cannabinoid antagonists, the eects of the prototypical cannabinoid antagonist SR141716A on monoamine eux from the medial prefrontal cortex and the nucleus accumbens of the rat were investigated by in vivo microdialysis. 2 SR141716A moderately increased serotonin eux and concentrations of its metabolite 5-HIAA, both in the medial prefrontal cortex and the nucleus accumbens, and increased norepinephrine, dopamine and their metabolites in the medial prefrontal cortex. In contrast, it had no eect on norepinephrine, dopamine and their metabolites in the nucleus accumbens. 3 At the same doses, SR141716A increased acetylcholine eux in the medial prefrontal cortex, in agreement with previous studies; contrary to the eects in cortex, SR141716A had no eect on acetylcholine eux in the nucleus accumbens. 4 The ecacy of SR141716A in the psychostimulant-induced hyperlocomotion and the forced swimming paradigms was also explored in mice. SR141716A attenuated phenylcyclidine-and damphetamine-induced hyperlocomotion, without aecting locomotor activity when administered alone, and decreased immobility in the forced swimming test. 5 These results suggest that the cortical selectivity in the release of catecholamines, dopamine in particular, induced by the cannabinoid antagonist SR141716A, its procholinergic properties, together with its mild stimulatory eects on serotonin and norepinephrine eux make similar compounds unique candidates for the treatment of psychosis, aective and cognitive disorders.

1 Tolerance and dependence induced by chronic D-9-tetrahydrocannabinol (THC) administration were investigated in mice. The e ects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20 mg kg 71 THC twice daily). A rapid tolerance to the acute e ects was observed from the second THC administration. 2 The selective CB-1 receptor antagonist SR 141716A (10 mg kg 71 ) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SR 141716A administration precipitated several somatic signs that included wet dog shakes, frontpaw tremor, ataxia, hunched posture, tremor, ptosis, piloerection, decreased locomotor activity and mastication, which can be interpreted as being part of a withdrawal syndrome. 3 Brains were removed immediately after the behavioural measures and assayed for adenylyl cyclase activity. An increase in basal, forskolin and calcium/calmodulin stimulated adenylyl cyclase activities was speci®cally observed in the cerebellum of these mice. 4The motivational e ects of THC administration and withdrawal were evaluated by using the place conditioning paradigm. No conditioned change in preference to withdrawal associated environment was observed. In contrast, a conditioned place aversion was produced by the repeated pairing of THC (20 mg kg 71 ), without observing place preference at any of the doses used. 5 This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quanti®cation of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.

Chronic morphine administration induces an up-regulation of several components of the cyclic adenosine 5'-monophosphate (cAMP) signal transduction cascade. The behavioral and biochemical consequences of opiate withdrawal were investigated in mice with a genetic disruption of the alpha and Delta isoforms of the cAMP-responsive element-binding protein (CREB). In CREBalphadelta mutant mice the main symptoms of morphine withdrawal were strongly attenuated. No change in opioid binding sites or in morphine-induced analgesia was observed in these mutant mice, and the increase of adenylyl cyclase activity and immediate early gene expression after morphine withdrawal was normal. Thus, CREB-dependent gene transcription is a factor in the onset of behavioral manifestations of opiate dependence.

Fluoxetine (Prozac) is the most widely prescribed medication for the treatment of depression. Nevertheless, little is known about the molecular basis of its clinical efficacy, apart from the fact that fluoxetine increases the synaptic availability of serotonin. Here we show that, in vivo, fluoxetine, given either acutely or chronically, regulates the phosphorylation state of dopamine-and cAMPregulated phosphoprotein of M r 32,000 (DARPP-32) at multiple sites in prefrontal cortex, hippocampus, and striatum. Acute administration of fluoxetine increases phosphorylation of DARPP-32 at the protein kinase A site, Thr-34, and at the casein kinase-1 site, Ser-137, and decreases phosphorylation at the cyclin-dependent kinase 5 site, Thr-75. Each of these changes contributes, through distinct signaling pathways, to increased inhibition of protein phosphatase-1, a major serine͞threonine protein phosphatase in the brain. Fluoxetine also increases phosphorylation of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 at Ser-831 and Ser-845. Both the fluoxetinemediated increase in AMPA receptor phosphorylation at Ser-845-GluR1 and the beneficial responsiveness to fluoxetine in an animal test of antidepressant efficacy were strongly reduced in DARPP-32 knockout mice, indicating a critical role for this phosphoprotein in the antidepressant actions of fluoxetine. Mice chronically treated with fluoxetine had increased levels of DARPP-32 mRNA and protein and a decreased ability to increase phospho-Ser-137-DARPP-32 and phospho-Ser-831-GluR1. These chronic changes may be relevant to the delayed onset of therapeutic efficacy of fluoxetine.A gents that enhance serotonergic neurotransmission, such as serotonin reuptake inhibitors (e.g., fluoxetine), and monoamine oxidase inhibitors (e.g., moclobemide), are effective as antidepressants. Although these agents immediately increase the synaptic availability of serotonin, there is a temporal delay in the onset of their beneficial actions. Recent studies in experimental animals have focused on understanding the effects of various antidepressant agents on signal transduction pathways in neurons located in brain regions thought to be implicated in depression (1, 2). In particular, it has been shown that treatment with various antidepressants, including fluoxetine, enhances the efficacy of the cAMP-dependent protein kinase A (PKA) pathway at several different levels in the frontal cortex and hippocampus (1). The possibility that the cAMP͞PKA system is involved in the action(s) of antidepressants is strongly supported by evidence from basic and clinical studies showing that cAMP phosphodiesterase inhibitors, such as rolipram, have antidepressant efficacy (3, 4).One major target protein for the cAMP͞PKA pathway is dopamine-and cAMP-regulated phosphoprotein of M r 32,000 ref. 5). DARPP-32 is enriched in striatum and the olfactory tubercle (6). Moderate to high levels of DARPP-32 also are found in several extrastriatal regions, such as cerebral cortex, hippocampu...

Dopaminergic dysfunction is an important pathogenetic factor for brain pathologies such as Parkinson's disease, ADHD, schizophrenia, and addiction as well as for metabolic disorders and anorexia. Dopaminergic neurons projecting from the midbrain to forebrain regions, such as the nucleus accumbens and the prefrontal cortex, regulate motor and cognitive functions and coordinate the patterned response of the organism to sensory, affective, and rewarding stimuli. In this study, we showed that dopaminergic neurotransmission is highly dependent on M4 cholinergic muscarinic receptor function. Using in vivo microdialysis, we found elevated dopamine (DA) basal values and enhanced DA response to psychostimulants in the nucleus accumbens of M4 knockout mice. We also demonstrated impaired homeostatic control of cholinergic activity that leads to increased basal acetylcholine efflux in the midbrain of these animals. Thus, loss of M4 muscarinic receptor control of cholinergic function effectuates a state of dopaminergic hyperexcitability. This may be responsible for pathological conditions, in which appetitive motivation as well as affective and cognitive processing is impaired. We propose that M4 receptor agonists could represent an innovative strategy for the treatment of pathologies associated with hyperdopaminergia.