Naloxone-induced conditioned place aversion score and extinction period are higher in C57BL/6J morphine-dependent mice than in Swiss: Role of HPA axis.

Intense associative memories develop between drug-paired contextual cues and the drug withdrawal associated aversive feeling. They have been suggested to contribute to the high rate of relapse. Our study was aimed to elucidate the involvement of hypothalamic-pituitary-adrenocortical (HPA) axis activity in the expression and extinction of aversive memory in Swiss and C57BL/6J (B6) mice. The animals were rendered dependent on morphine by i.p. injection of increasing doses of morphine (10-60 mg/kg). The negative state associated with naloxone (1 mg/kg s.c.) precipitated morphine withdrawal was examined by using conditioned place aversion (CPA) paradigm. B6 mice obtained a higher aversion score and took longer to extinguish the aversive memory than Swiss mice. In addition, corticosterone levels were increased after CPA expression. Moreover, corticosterone levels were decreased during CPA extinction in Swiss mice without changes in B6 mice. Pre-treatment with the selective CRF1 receptor antagonist CP-154,526 before naloxone, impaired morphine-withdrawal aversive memory acquisition and decreased the extinction period. CP-154,526 also antagonized the increased levels of corticosterone observed after CPA expression in Swiss mice, without any changes in B6 mice. These results indicate that HPA axis could be a critical factor governing opioid withdrawal memory storage and retrieval, but in a strain or stock-specific manner. The differences observed between Swiss and B6 mice suggest that the treatment of addictive disorders should consider different individual predisposition to associate the aversive learning with the context.

Cardiac adverse effects of naloxone-precipitated morphine withdrawal on right ventricle: role of corticotropin-releasing factor (CRF) 1 receptor.

Opioid addiction is associated with cardiovascular disease. However, mechanisms linking opioid addiction and cardiovascular disease remain unclear. This study investigated the role of corticotropin-releasing factor (CRF) 1 receptor in mediating somatic signs and the behavioural states produced during withdrawal from morphine dependence. Furthermore, it studied the efficacy of CRF1 receptor antagonist, CP-154,526 to prevent the cardiac sympathetic activity induced by morphine withdrawal. In addition, tyrosine hydroxylase (TH) phosphorylation pathways were evaluated. Like stress, morphine withdrawal induced an increase in the hypothalamic-pituitary-adrenal (HPA) axis activity and an enhancement of noradrenaline (NA) turnover. Pre-treatment with CRF1 receptor antagonist significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropic hormone (ACTH) levels, NA turnover and TH phosphorylation at Ser31 in the right ventricle. In addition, CP-154,526 reduced the phosphorylation of extracellular signal-regulated kinase (ERK) after naloxone-precipitated morphine withdrawal. In addition, CP-154,526 attenuated the increases in body weight loss during morphine treatment and suppressed some of morphine withdrawal signs. Altogether, these results support the idea that cardiac sympathetic pathways are activated in response to naloxone-precipitated morphine withdrawal suggesting that treatment with a CRF1 receptor antagonist before morphine withdrawal would prevent the development of stress-induced behavioural and autonomic dysfunction in opioid addicts.

Morphine withdrawal activates hypothalamic-pituitary-adrenal axis and heat shock protein 27 in the left ventricle: the role of extracellular signal-regulated kinase.

The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [e.g., noradrenergic activity, induction of the hypothalamo-pituitary-adrenocortical (HPA) axis, and the expression and activation of heat shock proteins (Hsps)]. The present study investigated the role of extracellular signal-regulated protein kinase (ERK) and ß-adrenoceptor on the response of stress systems to morphine withdrawal by the administration of [amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327), a selective inhibitor of ERK activation, or propranolol (a ß-adrenoceptor antagonist). Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by the injection of naloxone (2 mg/kg s.c.). Plasma concentrations of adrenocorticotropin and corticosterone were determined by radioimmunoassay; noradrenaline (NA) turnover in left ventricle was determined by high-performance liquid chromatography; and catechol-O-methyl transferase (COMT) and Hsp27 expression and phosphorylation at Ser82 were determined by quantitative blot immunolabeling. Morphine-withdrawn rats showed an increase of NA turnover and COMT expression in parallel with an enhancement of adrenocorticotropin and plasma corticosterone concentrations. In addition, we observed an enhancement of Hsp27 expression and phosphorylation. Pretreatment with SL327 or propranolol significantly reduced morphine withdrawal-induced increases of plasma adrenocorticotropin and Hsp27 phosphorylation at Ser82 without any changes in plasma corticosterone levels. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of HPA axis in parallel with an enhancement of Hsp27 expression and Hsp27 phosphorylation at Ser82 and suggest a role for ß-adrenoceptors and ERK pathways in mediating morphine-withdrawal activation of the HPA axis and cellular stress response.

Naloxone-precipitated morphine withdrawal evokes phosphorylation of heat shock protein 27 in rat heart through extracellular signal-regulated kinase.

Heat shock protein 27 (Hsp27) is a well-known stress response protein that becomes phosphorylated through extracellular signal-regulated kinase (ERK). Different drugs of abuse, such as morphine and/or its withdrawal, induce severe stress situations. In this study, we investigated Hsp27 and phospho-Hsp27 expression during morphine dependence and withdrawal and evaluated the involvement of ERK in the phosphorylation of Hsp27 in the rat right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg/kg, s.c.). ERK1/2, Hsp27 and phospho-Hsp27 at Ser15 were determined by quantitative blot immunolabeling using specific antibodies. Hsp27 expression was increased 30, 60, 90 and 120 min (144.5±14.2%, P<0.0001; 128.9±4.6%, P=0.04; 177.4±12.7, P<0.0001; and 136.2±11.0%, P=0.042, respectively) after saline injection to rats dependent on morphine. Naloxone-precipitated morphine withdrawal also increased the phosphorylation of Hsp27 at Ser15 at those time points (146.8±19.8%, P=0.034; 143.9±17.9%, P=0.032; 161.2±33.3%, P=0.029; and 152.2±25.5%, P=0.008, respectively). However, there were no changes in Hsp27 phosphorylation in the morphine dependent group injected with saline. In addition, there was an increase in the phosphorylation of ERK 60 min after naloxone injection in morphine dependent rats (pERK1: 116.3±4.2%, P=0.015 and pERK2: 117.2±1.5%, P=0.05). Pretreatment with SL327, an inhibitor of ERK phosphorylation, decreased activation (phosphorylation) of both ERK and Hsp27 (pERK1: 4.5±3.6%, P<0.0001; pERK2: 42.3±3.3%, P<0.0001; and pHsp27: 97.6±1.5%, P=0.008), suggesting that ERK activation triggers Hsp27 phosphorylation. The present findings demonstrate that morphine withdrawal is capable of inducing the activation of Hsp27 in the heart and suggest that phosphorylation of Hsp27 is closely linked to and also dependent on the ERK pathway.