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Background and Purpose: The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [noradrenergic activity, induction of the hypothalamic-pituitary-adrenocortical (HPA) axis and activation of heat shock proteins (Hsps)]. Corticotropin-releasing factor (CRF) pathways are important mediators in the negative symptoms of opioid withdrawal. We performed a series of experiments to characterize the role of the CRF₁ receptor in the response of stress systems to morphine withdrawal and its effect in the heart using genetically engineered mice lacking functional CRF₁ receptors., Experimental Approach: Wild-type and CRF₁ receptor-knockout mice were treated with increasing doses of morphine. Precipitated withdrawal was induced by naloxone. Plasma adrenocorticotropic hormone (ACTH) and corticosterone levels, the expression of myocardial Hsp27, Hsp27 phosphorylated at Ser⁸², membrane (MB)- COMT, soluble (S)-COMT protein and NA turnover were evaluated by RIA, immunoblotting and HPLC., Key Results: During morphine withdrawal we observed an enhancement of NA turnover in parallel with an increase in mean arterial blood pressure (MAP) and heart rate (HR) in wild-type mice. In addition, naloxone-precipitated morphine withdrawal induced an activation of HPA axis and Hsp27. The principal finding of the present study was that plasma ACTH and corticosterone levels, MB-COMT, S-COMT, NA turnover, and Hsp27 expression and activation observed during morphine withdrawal were significantly inhibited in the CRF₁ receptor-knockout mice., Conclusion and Implications: Our results demonstrate that CRF/CRF₁ receptor activation may contribute to stress-induced cardiovascular dysfunction after naloxone-precipitated morphine withdrawal and suggest that CRF/CRF₁ receptor pathways could contribute to cardiovascular disease associated with opioid addiction., (© 2013 The British Pharmacological Society.)

Background and Purpose: Recent evidence suggests that glucocorticoid receptor (GR) is a major molecular substrate of addictive properties of drugs of abuse. Hence, we performed a series of experiments to further characterize the role of GR signalling in opiate withdrawal-induced physical signs of dependence, enhanced noradrenaline (NA) turnover in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) phosphorylation (activation) as well as GR expression in the nucleus of the solitary tract noradrenergic cell group (NTS-A₂)., Experimental Approach: The role of GR signalling was assessed by i.p. pretreatment of the selective GR antagonist, mifepristone. Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with mifepristone or vehicle 30 min before naloxone and physical signs of abstinence, NA turnover, TH activation, GR expression and the hypothalamus-pituitary-adrenocortical axis activity were measured using HPLC, immunoblotting and RIA., Key Results: Mifepristone alleviated the somatic signs of naloxone-induced opiate withdrawal. Mifepristone attenuated the increase in the NA metabolite, 3-methoxy-4-hydroxyphenylethylen glycol (MHPG), in the PVN, and the enhanced NA turnover observed in morphine-withdrawn rats. Mifepristone antagonized the TH phosphorylation at Ser³¹ and the expression of c-Fos expression induced by morphine withdrawal. Finally, naloxone-precipitated morphine withdrawal induced up-regulation of GR in the NTS., Conclusions and Implications: These results suggest that the physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by GR signalling. Overall, the present data suggest that drugs targeting the GR may ameliorate stress and aversive effects associated with opiate withdrawal., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background and Purpose: Recent evidence suggests that corticotropin-releasing factor (CRF) receptor signalling is involved in modulating the negative symptoms of opiate withdrawal. In this study, a series of experiments were performed to further characterize the role of CRF-type 2 receptor (CRF₂) signalling in opiate withdrawal-induced physical signs of dependence, hypothalamus-pituitary-adrenal (HPA) axis activation, enhanced noradrenaline (NA) turnover in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) phosphorylation (activation), as well as CRF₂ expression in the nucleus of the solitary tract-A₂ noradrenergic cell group (NTS-A₂)., Experimental Approach: The contribution of CRF₂ signalling in opiate withdrawal was assessed by i.c.v. infusion of the selective CRF₂ antagonist, antisauvagine-30 (AS-30). Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with AS-30 or saline 10 min before naloxone and the physical signs of abstinence, the HPA axis activity, NA turnover, TH activation and CRF₂ expression were measured using immunoblotting, RIA, HPLC and immunohistochemistry., Key Results: Rats pretreated with AS-30 showed decreased levels of somatic signs of naloxone-induced opiate withdrawal, but the corticosterone response was not modified. AS-30 attenuated the increased production of the NA metabolite, 3-methoxy-4-hydroxyphenylglycol, as well as the enhanced NA turnover observed in morphine-withdrawn rats. Finally, AS-30 antagonized the TH phosphorylation at Serine40 induced by morphine withdrawal., Conclusions and Implications: These results suggest that physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by CRF₂ signalling. Furthermore, they indicate a marginal role for the HPA axis in CRF₂-mediation of opiate withdrawal., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Background and Purpose: Our previous studies have shown that morphine withdrawal induced hyperactivity of cardiac noradrenergic pathways. The purpose of the present study was to evaluate the effects of morphine withdrawal on site-specific phosphorylation of TH in the heart., Experimental Approach: Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets in rats. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg kg(-1)). TH phosphorylation was determined by quantitative blot immunolabelling using phosphorylation state-specific antibodies., Key Results: Naloxone-induced morphine withdrawal induced phosphorylation of TH at serine (Ser)40 and Ser31 in the right ventricle, associated with both an increase in total TH levels and an enhancement of TH activity. When HA-1004 (PK A inhibitor) was infused, concomitantly with morphine, it diminished the increase in noradrenaline turnover, total TH levels and TH phosphorylation at Ser40 in morphine-withdrawn rats. In contrast, the infusion of calphostin C (PKC inhibitor), did not modify the morphine withdrawal-induced increase in noradrenaline turnover and total TH levels. In addition, we show that the ability of morphine withdrawal to stimulate phosphorylation at Ser31 was reduced by SL327, an inhibitor of ERK 1/2 activation., Conclusions and Implications: The present findings demonstrate that the enhancement of total TH levels and the increased phosphorylation state of TH during morphine withdrawal were dependent on PKA and ERK activities and suggest that these transduction pathways might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal.

Background and Purpose: We have previously demonstrated that morphine withdrawal induced hyperactivity of the heart by activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline turnover and c-Fos expression. The extracellular signal-regulated kinase (ERK) has been implicated in drug addiction, but its role in activation of the heart during morphine dependence remains poorly understood. Here, we have looked for activation of ERK during morphine withdrawal and if this activation induced gene expression., Experimental Approach: Dependence on morphine was induced by s.c. implantation of morphine pellets for 7 days. Morphine withdrawal was precipitated on day 8 by injection of naloxone (2 mg kg(-1), s.c.). ERK1/2, their phosphorylated forms and c-Fos were measured by western blotting and immunohistochemistry of cardiac tissue., Key Results: Naloxone-induced morphine withdrawal activated ERK1/2 and increased c-Fos expression in cardiac tissues. c-Fos expression was blocked by SL327, a drug that prevents ERK activation., Conclusions and Implications: These results indicate that signalling through the ERKs is necessary for morphine withdrawal-induced hyperactivity of the heart and suggest that this pathway may also be involved in activation of immediate-early genes in both cytosolic and nuclear effector mechanisms that have the potential to bring about long-term changes in the heart.

1. In the present work, we have studied the expression of Fos during acute and chronic administration of the kappa-opioid receptor agonist U-50488H and after U-5088H withdrawal in the rat hypothalamic paraventricular nucleus (PVN). Fos production was also studied in brainstem regions that innervate the PVN: the A(2) cell group of the nucleus of solitary tract (NTS-A(2)) and the A(1) cell group of the ventrolateral medulla (VLM-A(1)), combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons after acute U-50488H administration. 2. For acute experiments, male rats were treated with saline i.p. for 4 days. On day 5, rats were given saline or U-50488H (15 mg x kg(-1), i.p.). Other groups of rats were rendered tolerant/dependent on U-50488H by injecting the drug twice daily (15 mg x kg(-1), i.p.) for 4 days. Control animals received saline i.p. on the same time schedule. On day 5, rats were treated with vehicle i.p., with U-50488H (15 mg x kg(-1)) or with the selective kappa opioid-receptor antagonist nor-binaltorphimine (Nor-BNI, 5 mg x kg(-1), i.p.). 3. Using immunohistochemical staining of Fos, present results indicate that acute administration of U-50488H produced an increase in Fos expression in the PVN and in the noradrenergic A(1) and A(2) cell groups. Moreover, when double-label immunohistochemistry was used to identify Fos and catecholaminergic-positive neurons in the brainstem, it was found that catecholaminergic-positive neurons in the NTS and VLM showed a significant increase in Fos expression in response to acute U-50488H injection. Chronic application of U-50488H leads to the development of tolerance towards their effects on Fos expression in the PVN as well as in the NTS and VLM. However, administration of Nor-BNI to U-50488H-dependent rats did not induce any changes in Fos immunoreactivity in the PVN or in the brainstem. 4. These findings demonstrate that acute activation of kappa-opioid receptors results in different altered patterns of immediate-early gene expression in the PVN, which occurs concurrently with an increased activity of their inputs from the brainstem. Interestingly in contrast to morphine withdrawal, present results demonstrate that rats withdrawn from U-50488H did show no changes in Fos-immunoreactivity in the PVN, NTS or VLM, indicating the absence of dependence on the kappa-agonist under the present experimental conditions.

1. In the present work we have studied in the heart the expression of Fos, the protein product of the c-fos proto-oncogene and the adaptive changes in noradrenergic neurons after naloxone or nor-binaltorphimine (nor-BNI) administration to morphine or U-50,488H pretreated rats. 2. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received saline s.c., naloxone (5 mg kg(-1) s.c.) or nor-BNI (5 mg kg(-1) i.p.). Other groups of rats were rendered tolerant/dependent on U-50,488H by injecting the drug twice daily (15 mg kg(-1) i.p.) for 4 days. Control animals received saline. On day 5 the animals were injected with vehicle i.p. or nor-BNI (5 mg kg(-1) i.p.). 3. Using immunohistochemical staining of Fos, present results indicate that morphine withdrawal induced marked Fos immunoreactivity (Fos-IR) within the cardiomyocyte nuclei. Moreover, Western blots analysis revealed a peak expression of c-fos in right and left ventricle after naloxone induced withdrawal in parallel with an increase in noradrenaline (NA) turnover. 4. However, after nor-BNI administration to rats chronically treated with U-50,488H, we found a decrease in the NA turnover. In addition, the administration of nor-BNI to rats chronically treated with U-50,488H or morphine did not induce modifications in the Fos-IR, in the heart. 5. These results demonstrated that morphine withdrawal induces the expression of Fos protein, as well as an enhancement of noradrenergic activity in the heart. In contrast to morphine U-50,488 withdrawal produces no changes in Fos-IR in parallel with a decrease in NA turnover, indicating that the kappa-opioid receptors are not involved in the molecular adaptive mechanisms responsible for the development of opioid dependence in the heart.

Different data support a role for brainstem noradrenergic inputs to the hypothalamic paraventricular nucleus (PVN) in the control of hypothalamus - pituitary - adrenocortical (HPA) axis. However, little is known regarding the functional adaptive changes of noradrenergic afferent innervating the PVN and supraoptic nucleus (SON) during chronic opioid exposure and upon morphine withdrawal. Here we have studied the expression of Fos after administration of morphine and during morphine withdrawal in the rat hypothalamic PVN and SON. Fos production was also studied in brainstem regions that innervate hypothalamic nuclei: the nucleus of solitary tract (NTS - A2) and the ventrolateral medulla (VLM - A1) and combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons during morphine withdrawal. Male rats were implanted with s.c. placebo or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received an injection of saline i.p., morphine i.p., saline s.c. or naloxone s.c. Acute morphine administration produced an increase in Fos expression at hypothalamic nuclei and in the brainstem regions, and tolerance developed towards this effect. Precipitated morphine withdrawal induced marked Fos immunoreactivity within the PVN and SON. Concomitantly, numerous neurons in the brainstem were stimulated by morphine withdrawal. Moreover, catecholaminergic-positive neurons in the brainstem showed a significant increase in Fos expression in response to morphine withdrawal. These findings demonstrate that chronic activation of opioid receptors results in altered patterns of immediate-early genes (IEG) expression in the PVN and SON, which occurs concurrently with an increased activity of their inputs from the brainstem.

1. In the present study the changes in noradrenaline (NA) and dopamine (DA) content and turnover during naloxone-induced withdrawal were analysed in the right ventricle of rats chronically treated with the kappa-agonist U-50,488H. 2. Rats were rendered tolerant by administration of U-50,488H twice a day for 4 days. On the day of death the animals were injected with saline or naloxone (3 mg kg(-1), s.c.) to precipitate a withdrawal syndrome. 3. After naloxone administration to U-50,488H- treated rats we found neither behaviour signs of physical dependence nor changes in the tissue content of noradrenaline (NA). However, naloxone induced a decrease in both cardiac normetanephrine (NM) levels and NA turnover. 4. Similarly, naloxone enhanced the dopamine content and decreased the 3,4-dihydroxyphenylacetic acid (DOPAC) concentration and dopamine turnover. 5. Importantly and in contrast to mu-agonists, the present results demonstrate that U-50,488H withdrawal produced a decrease in the NA and dopamine turnover, without behavioural signs of physical dependence.