Olga Valverde

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.

Δ9-tetrahydrocannabinol releases and facilitates the effects of endogenous enkephalins: reduction in morphine withdrawal syndrome without change in rewarding effect

Recent studies have suggested that cannabinoids might initiate the consumption of other highly addictive substances, such as opiates. In this work, we show that acute administration of Δ9‐tetrahydrocannabinol in mice facilitates the antinociceptive and antidepressant‐like responses elicited by the endogenous enkephalins protected from their degradation by RBu2003101, a complete inhibitor of enkephalin catabolism. This emphasizes the existence of a physiological interaction between endogenous opioid and cannabinoid systems. Accordingly, Δ9‐tetrahydrocannabinol increased the release of Met‐enkephalin‐like material in the nucleus accumbens of awake and freely moving rats measured by microdialysis. In addition, this cannabinoid agonist displaced the in vivo[3H]diprenorphine binding to opioid receptors in total mouse brain. The repetitive pretreatment during 3u2003weeks of Δ9‐tetrahydrocannabinol in mice treated chronically with morphine significantly reduces the naloxone‐induced withdrawal syndrome. However, this repetitive administration of Δ9‐tetrahydrocannabinol did not modify or even decrease the rewarding responses produced by morphine in the place preference paradigm. Taken together, these behavioural and biochemical results demonstrate the existence of a direct link between endogenous opioid and cannabinoid systems. However, chronic use of high doses of cannabinoids does not seem to potentiate the psychic dependence to opioids.

The attenuation of morphine-conditioned place preference following chronic mild stress is reversed by a CCKB receptor antagonist

Abstract Chronic exposure to mild unpredictable stress has been found to abolish the acquisition of preference for a distinctive environment paired with morphine, whereas morphine induced conditioning place preference in non-stressed rats. Chronic treatment for 21 days with the tricyclic antidepressant imipramine reversed the motivational effects produced by chronic mild stress, and animals showed a place preference for the morphine-paired compartment. When the CCKB receptor antagonist PD-134,308 was co-administered with morphine in stressed animals during the conditioning period, the preference for the morphine-paired compartment was also re-established. The CCKB receptor antagonist given alone did not induce rewarding effects in this paradigm. These findings indicate that the administration of a CCKB receptor antagonist reversed the effects of chronic mild stress on opiate rewarding properties.

Protein kinases in the locus coeruleus and periaqueductal gray matter are involved in the expression of opiate withdrawal

The aim of this study was to evaluate the role played in the behavioral expression of morphine withdrawal syndrome by protein kinases in the locus coeruleus and the periaqueductal gray matter. Two different families of specific protein kinases have been investigated: serine/threonine and tyrosine kinases.Rats were implanted with cannulas into both the lateral ventricle and the locus coeruleus or the periaqueductal gray matter. Physical dependence was induced by chronic peripheral administration of morphine (from 7 to 30 mg/kg) and withdrawal syndrome was precipitated by injection of naloxone (2 μg) into the lateral ventricle. The administration of the serine/threonine kinase inhibitor 1-(5-isoquinolinylsulfonyl)2-methylpiperazine, H7 (1, 3, 10, and 30 nmol per side) into the locus coeruleus induced a strong attenuation of morphine withdrawal behavior. Signs related to the motor component of abstinence, such as jumping, rearing, and hyperactivity, were the most severely reduced. However, this effect was not dose-dependent, and the response was almost the same with all the doses used. A similar attenuation was observed after the injection of H7 (1, 3, and 10 nmol per side) into the periaqueductal gray matter, but in this case motor signs were less strongly reduced and a larger number of signs were modified, mainly when using the highest dose. The administration of the tyrosine kinase inhibitor 2-hydroxy-5-[N-[(2,5-dihydroxyphenyl)methyl] amino]-benzoic acid 3-phenylpropyl ester, KB23 (0.3, 1, and 3 nmol per side) into the locus coeruleus or the periaqueductal gray matter had no effect on the withdrawal syndrome behavior, except on teeth chattering.These results suggest that, in the locus coeruleus and in the periaqueductal gray matter, serine/threonine kinases are implicated in the behavioral expression of morphine abstinence. In these brain structures, tyrosine kinases appear not to be involved.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation

&NA; RB101 (Symbol is a recently developed full inhibitor of the enkephalin‐catabolizing enzymes able to cross the blood‐brain barrier, whereas RB38A ((Symbol) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freunds complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non‐inflamed paws. RB101 (10 and 20 mg/kg i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 &mgr;g) blocked the effect of morphine only in non‐inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues. Symbol. No caption available Symbol. No caption available

Pain-suppressive effects on various nociceptive stimuli (thermal, chemical, electrical and inflammatory) of the first orally active enkephalin-metabolizing enzyme inhibitor RB 120

&NA; RB 101 (N‐((R,S)‐2‐benzyl‐3[(S)(2‐amino‐4‐methylthio)butyldithio]‐1‐oxopropyl)‐l‐phenylalanine benzyl ester) is a full inhibitor of the enkephalin‐catabolizing enzymes, which induces strong naloxone‐reversible antinociceptive responses after i.v. or i.p. administration, but is only slightly active after oral administration. Chemical modifications were introduced on this compound, resulting in molecules such as RB 120 (N‐((S)‐2‐benzyl‐3[(S)(2‐amino‐4‐methylthio)butyldithio]‐1‐oxopropyl)‐l‐alanine benzyl ester), which was selected for a complete study, after oral administration, in various assays commonly used to select analgesics: mouse hot plate test, rat tail‐flick test, electrical stimulation of the tail in rats, paw pressure test on inflamed paws in rats, acetic acid‐induced writhing test and the formalin test in mice. RB 120 induced potent dose‐dependent antinociceptive responses in all these tests after oral administration. The differences in antinociceptive effects induced by RB 120 in the various assays is probably related to the amount of enkephalins released and to the efficiency of peptidase inactivation in particular brain regions implicated in the control of a given nociceptive input. The goal of discovering orally active analgesics endowed with a potency similar to that of morphine but devoid of its major side‐effects, seems now to have been reached with mixed neutral endopeptidase/aminopeptidase N (NEP/APN) inhibitors, although these compounds have yet to be evaluated in clinical trials.

The CCKB antagonist PD-134,308 facilitates rewarding effects of endogenous enkephalins but does not induce place preference in rats

The interaction between cholecystokinin and endogenous opioid systems on rewarding responses was examined. Motivational effects induced by peripheral administration of a complete inhibitor of enkephalin catabolism, RB 101 or the CCKB antagonist PD-134,308, and by both compounds in combination were evaluated in the conditioned place preference test in rats. RB 101 (5, 10, 20, 40 and 80 mg/kg, IP, and 20 mg/kg, IV) given alone produced a bell-shaped dose-effect function. A significant increase of the preference for the drug-associated compartment was only observed at doses of 10 and 20 mg/kg (IP). The effect observed with morphine was stronger, and all the doses used of this compound (1.25, 2.5 and 5 mg/kg, SC) were found to be active. These results suggest that the inhibitor of enkephalin catabolism has weak rewarding properties. Pretreatment with the CCKB antagonist PD-134,308 (0.1, 0.3, 1 and 3 mg/kg, IP) alone failed to produce a reliable aversion or preference on the paradigm studied. When PD-134,308 (0.3 mg/kg, IP) was coadministered with a subthreshold dose of morphine (0.6 mg/kg, SC) or RB 101 (5 mg/kg, IP), a conditioned place preference was observed, indicating that the CCKB antagonist facilitated the motivational responses induced by endogenous enkephalins as compared to morphine. This suggests that endogenous cholecystokinin, acting through CCKB receptors, modulates the rewarding effects of endogenous enkephalins.

Protein Kinases in the Rat Nucleus Accumbens are Involved in the Aversive Component of Opiate Withdrawal

The specific participation of protein kinases in the expression of the somatic signs of morphine withdrawal has been previously demonstrated, suggesting that changes in intracellular signalling systems are involved in opioid addiction. In the present study, the involvement of protein kinases in the aversive/dysphoric effects of morphine abstinence has been investigated in the nucleus accumbens, because of the critical role played by the mesolimbic system in the rewarding effects of opioids. Rats were chronically treated with morphine, twice a day for 5 days, with doses progressively increased from 5 to 30 mg/kg (i‐p.). In addition, microinjections into the nucleus accumbens of the serine‐threonine kinase inhibitors H7 or H8 (1 or 10 nmol per side) or saline once daily were also given, both in control and in morphine‐treated animals. After these chronic treatments, withdrawal syndrome was induced by naloxone administration (0.1 mg/kg, s. c.), and the motivational component of morphine abstinence was studied using the place aversion paradigm. When administered at the highest dose (10 nmol), H7 and H8 strongly reduced the place aversion induced by naloxone in morphine dependent animals. Protein kinase inhibitors did not induce significant behavioural responses in non‐dependent animals. Chronic morphine treatment induced a selective up‐regulation of adenylate cyclase activity in the amygdala, without affecting other brain regions. The morphine‐increased adenylate cyclase activity in amygdala was reversed by the chronic intra‐accumbens microinjection of H7 or H8. These results suggest that serine‐threonine kinases in the nucleus accumbens play an important role in the emotional/dysphoric properties which characterize opiate withdrawal.

Similar involvement of several brain areas in the antinociception of endogenous and exogenous opioids

The complete inhibitor of the enkephalin degrading enzymes, RB 101, N-{(R,S)-2-benzyl-3[(S)-(2-amino-4-methylthio)butyldithio]-1- oxopropyl}-L-phenylalanine benzyl ester, which crosses the blood-brain barrier, induced antinociceptive effects similar to those of exogenous opiates. The almost complete absence of tolerance and dependence after chronic administration of RB 101 is therefore due to limited stimulation of opioid receptors by protected endogenous enkephalins. In order to clarify the mechanisms involved in these response, we have investigated the participation of several brain structures in the antinociceptive effects induced by systemic administration of morphine or RB 101. Rats were implanted with bilateral cannulae into the ventro-basal thalamus, central amygdala and periaqueductal gray matter, or with a cannula into the raphe magnus nucleus. The antinociceptive responses induced by systemic morphine or RB 101 were measured by using the tail-electrical stimulation test, where three different thresholds were determined: motor response, vocalization and vocalization post-discharge. The ability of the opioid receptor antagonist methylnaloxonium to block these antinociceptive responses was evaluated after local injection into the different brain structures. The blockade of morphine- and RB 101-induced antinociception was similar, and was stronger when methylnaloxonium was injected into the periaqueductal gray matter and raphe magnus nucleus than when it was injected into the ventro-basal thalamus and amygdala. These results suggest that brain structures related to the control of pain seem to be the same for the antinociception induced by exogenous opiates and endogenous opioids.