Rafael Maldonado

Absence of opiate rewarding effects in mice lacking dopamine D2 receptors

Dopamine receptors have been implicated in the behavioural response to drugs of abuse. These responses are mediated particularly by the mesolimbic dopaminergic pathway arising in the ventral tegmental area and projecting to the limbic system. The rewarding properties of opiates and the somatic expression of morphine abstinence have been related to changes in mesolimbic dopaminergic activity that could constitute the neural substrate for opioid addiction. These adaptive responses to repeated morphine administration have been investigated in mice with a genetic disruption of the dopaminergic D2 receptors. Although the behavioural expression of morphine withdrawal was unchanged in these mice, a total suppression of morphine rewarding properties was observed in a place-preference test. This effect is specific to the drug, as mice lacking D2 receptors behaved the same as wild-type mice when food is used as reward. We conclude that the D2 receptor plays a crucial role in the motivational component of drug addiction.

Disruption of the κ‐opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective κ‐agonist U‐50,488H and attenuates morphine withdrawal

μ‐, δ‐ and κ‐opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood‐altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the κ‐opioid receptor (KOR) to opioid function in vivo, we have generated KOR‐deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic κ‐agonist U‐50,488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR‐encoded receptor plays a modulatory role in specific aspects of opioid function.

Reduction of Morphine Abstinence in Mice with a Mutation in the Gene Encoding CREB

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 α and Δ isoforms of the cAMP-responsive element-binding protein (CREB). In CREBαΔ 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.

Participation of noradrenergic pathways in the expression of opiate withdrawal: biochemical and pharmacological evidence.

Several lines of biochemical and pharmacological evidence provide support for the involvement of the noradrenergic system in the expression of the somatic symptoms of opiate withdrawal. Early studies reported changes in brain noradrenaline and metabolite levels during opiate dependence. The significance of these changes has been clarified in recent microdialysis studies indicating that acute morphine decreases the extraneuronal levels of noradrenaline, whereas an increase in release of the neurotransmitter occurs during opiate withdrawal in several brain areas. Changes in the sensitivity and density of alpha 2- and beta-adrenoceptors have also been reported, probably as a consequence of the decreased presynaptic noradrenergic activity induced during morphine dependence. In addition, the administration of alpha 2-agonists, such as clonidine, or beta-antagonists, such as propranolol, has been reported to attenuate some manifestations of opiate withdrawal. The noradrenergic structure mediating the expression of opioid abstinence seems to be the locus coeruleus. However, the activation of the locus coeruleus during morphine withdrawal seems to be primarily due to the afferent projections containing excitatory amino acids and derived from the nucleus paragigantocellularis, although intrinsic modifications, consisting of an up-regulation of the cAMP pathway, seem also to be involved in this activation. The participation of the mesolimbic dopaminergic system in opiate dependence and its relation with the changes produced in the noradrenergic system are also discussed.

Behavioural and biochemical evidence for signs of abstinence in mice chronically treated with Δ-9-tetrahydrocannabinol

Tolerance and dependence induced by chronic Δ‐9‐tetrahydrocannabinol (THC) administration were investigated in mice. The effects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20u2003mgu2003kg−1 THC twice daily). A rapid tolerance to the acute effects was observed from the second THC administration. The selective CB‐1 receptor antagonist SRu2003141716A (10u2003mgu2003kg−1) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SRu2003141716A 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. 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 specifically observed in the cerebellum of these mice. The motivational effects 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 (20u2003mgu2003kg−1), without observing place preference at any of the doses used. This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quantification of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.

D1 dopamine receptors in the nucleus accumbens modulate cocaine self-administration in the rat

Previous work using systemic injections of dopamine receptor antagonists has established that dopamine D1 receptors may have a role in cocaine self-administration. The purpose of the present study was to test the hypothesis that these effects were mediated by dopamine D1 receptors in the region of the nucleus accumbens. Animals were trained to perform operant responses to self-administer cocaine via an IV catheter on a fixed-ratio 5 (FR 5) schedule of reinforcement. SCH23390, a selective D1 dopamine antagonist, significantly increased the self-administration of cocaine when injected into the nucleus accumbens. This increase in self-administration is thought to reflect decreases in the magnitude of the reinforcer, similar to the increase observed when the dose of cocaine is reduced. Similar doses of SCH23390 injected into the posterior caudate nucleus failed to alter cocaine self-administration. These data suggest that D1 receptors in the nucleus accumbens are important for the reinforcing properties of cocaine.

Destruction of the locus coeruleus decreases physical signs of opiate withdrawal

The purpose of the present study was to investigate the role of the locus coeruleus in the development of opiate dependence. Two groups of rats each were subjected to either a electrolytic lesion of the locus coeruleus or a sham lesion. All animals were implanted with an intracerebroventricular (i.c.v.) cannula, and made physically dependent by subcutaneous insertion of two 75-mg morphine (base) pellets. Abstinence was precipitated by i.c.v. administration of methylnaloxonium (31-1,000 ng) 72 h after pellet implantation. Methylnaloxonium administered intracerebroventricularly induced a withdrawal syndrome characterized by the appearance of teeth chattering, mastication, rearing, wet dog shakes, jumping, piloerection, hyperactivity, ptosis and eye twitch. Withdrawal observed in the electrolytic lesion groups was less severe than in sham group. The presence of mastication, rearing, piloerection, hyperactivity, ptosis and eye twitch was significantly lower. These results support the hypothesis that the locus coeruleus has an important role in the expression of the physical signs of opiate dependence.

Precipitation of morphine withdrawal syndrome in rats by administration of mu-, delta- and kappa-selective opioid antagonists

The acute effects of opioid drugs are generally hypothesized to be mediated by multiple receptors, for which three types of binding sites have been established. In order to evaluate the selective participation of each type of opioid receptor in opiate withdrawal, the opiate withdrawal syndrome, precipitated by the intraventricular acute administration of mu-, delta- and kappa-selective opioid antagonists was investigated. After implantation of the cannula into the lateral ventricle, rats were made physically dependent by subcutaneous insertion of two 75-mg pellets of morphine (base). D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (5-5000 ng), a mu-selective opioid antagonist, naltrindole (62-2000 ng), a delta-selective antagonist or nor-binaltorphimine (nor-BNI) (600-20,000 ng), a kappa-selective antagonist, were administered 72 hr after implantation of the pellets. All three drugs elicited some signs of morphine withdrawal but they differed in both their potency and their efficacy. The most efficacious and the most potent was CTAP, eliciting 8 of the 14 withdrawal signs at doses of 5-5000 ng. Nor-BNI was less efficacious and less potent, eliciting a significant increase in 5 of the 14 withdrawal signs in a dose range of 600-20,000 ng. Naltrindole was the least potent and least efficacious of the three drugs, eliciting a significant increase of only 2 withdrawal signs after intraventricular administration of 2000 ng. In a second experiment, the withdrawal syndrome was precipitated by the combined administration of CTAP+naltrindole or CTAP+nor-BNI. The severity of withdrawal, obtained with these two combinations, was similar to that observed with CTAP alone. These results support the importance of the mu receptor in the expression of central opiate dependence and suggest a minor role for delta and kappa receptors.

Implication of endogenous opioid system in the learned helplessness model of depression.

The involvement of opioid system on the learned helplessness model of depression was investigated. Animals preexposed to inescapable shocks were treated with either Met-enkephalin, Leu-enkephalin, morphine, imipramine, naloxone, RB 38A (a mixed inhibitor of enkephalin degrading enzymes), or RB 38B (a selective inhibitor of neutral endopeptidase EC 3.4.24.11). Stimulation of opioid system by either opioid agonists or enkephalin catabolism inhibitors reversed the escape deficit induced by shock pretreatment. In contrast, administration of naloxone potentiated the effect of inescapable shocks. Imipramine reduced the number of escape failures in this test, and this effect was antagonized by naloxone. These results point to the involvement of the endogenous opioid system in this model of depression.

Involvement of δ-opioid receptors in the effects induced by endogenous enkephalins on learned helplessness model

Pharmacological, neurochemical and behavioural findings support a possible role of endogenous opioids in clinical depression. There is evidence from animal studies that delta-opioid receptors are involved in several behavioural responses to opioids, including motivational activities. In the present study, the mixed enkephalin catabolism inhibitor, RB 101 (N(R,S)-2-benzyl-3[(S)-(2-amino-4-methylthiobutyldithio]-1-oxoprop yl)-L-phenylalanine benzyl ester) (1.25, 2.5 and 5 mg/kg), induced a dose-dependent antidepressant-like effect in a learned helplessness model. Thus, RB 101 reversed escape deficits in rats previously subjected to inescapable shocks, suggesting the involvement of endogenous enkephalins in depression. Similar effects were observed after administration of the selective delta-opioid receptor agonist, BUBU (Tyr-D.Ser-(O-tert-butyl)-Gly-Phe-Leu-Thr(O-Tet-butyl-OH) (1 and 2 mg/kg). Moreover, RB 101 effects were antagonized by administration of naltrindole (NTI) (0.1 mg/kg), which points to a preferential involvement of delta-opioid receptors in this enkephalin-controlled behaviour. As RB 101 has been reported to be almost devoid of opiate-related side-effects, it could represent a promising alternative in the treatment of depressive patients who are unresponsive to, or intolerant of, classical antidepressants.