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.

Cross-talk between protein kinase A and mitogen-activated protein kinases signalling in the adaptive changes observed during morphine withdrawal in the heart.

Our previous studies have shown that morphine withdrawal induced an increase in the expression of protein kinase (PK) A and mitogen-activated extracellular kinase (MAPK) pathways in the heart during morphine withdrawal. The purpose of the present study was to evaluate the interaction between PKA and extracellular signal-regulated kinase (ERK) signaling pathways mediating the cardiac adaptive changes observed after naloxone administration to morphine-dependent rats. Dependence on morphine was induced by a 7-day subcutaneous implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (2 mg/kg). ERK1/2 and tyrosine hydroxylase (TH) phosphorylation was determined by quantitative blot immunolabeling using phosphorylation state-specific antibodies. Naloxone-induced morphine withdrawal activates ERK1/2 and phosphorylates TH at Ser31 in the right and left ventricle, with an increase in the mean arterial blood pressure and heart rate. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor, was infused, concomitantly with morphine, it diminished the expression of ERK1/2. In contrast, the infusion of calphostin C (a PKC inhibitor) did not modify the morphine withdrawal-induced activation of ERK1/2. The ability of morphine withdrawal to activate ERK that phosphorylates TH at Ser31 was reduced by HA-1004. The present findings demonstrate that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation (phosphorylation) of TH.

The involvement of CRF1 receptor within the basolateral amygdala and dentate gyrus in the naloxone-induced conditioned place aversion in morphine-dependent mice.

Drug withdrawal-associated aversive memories trigger relapse to drug-seeking behavior. Corticotrophin-releasing factor (CRF) is an important mediator of the reinforcing properties of drugs of abuse. However, the involvement of CRF1 receptor (CRF1R) in aversive memory induced by opiate withdrawal has yet to be elucidated. We used the conditioned-place aversion (CPA) paradigm to evaluate the role of CRF1R on opiate withdrawal memory acquisition, along with plasticity-related processes that occur after CPA within the basolateral amygdala (BLA) and dentate gyrus (DG). Male mice were rendered dependent on morphine and injected acutely with naloxone before paired to confinement in a naloxone-associated compartment. The CPA scores as well as the number of TH-positive neurons (in the NTS-A2 noradrenergic cell group), and the expression of the transcription factors Arc and pCREB (in the BLA and DG) were measured with and without CRF1R blockade. Mice subjected to conditioned naloxone-induced morphine withdrawal robustly expressed CPA. Pre-treatment with the selective CRF1R antagonist CP-154,526 before naloxone conditioning session impaired morphine withdrawal-induced aversive memory acquisition. CP-154,526 also antagonized the enhanced number of TH-positive neurons in the NTS-A2 that was seen after CPA. Increased Arc expression and Arc-pCREB co-localization were seen in the BLA after CPA, which was not modified by CP-154,526. In the DG, CPA was accompanied by a decrease of Arc expression and no changes in Arc-pCREB co-localization, whereas pre-treatment with CP-154,526 induced an increase in both parameters. These results indicate that CRF-CRF1R pathway could be a critical factor governing opiate withdrawal memory storage and retrieval and might suggest a role for TH-NA pathway in the effects of withdrawal on memory. Our results might indicate that the blockade of CRF1R could represent a promising pharmacological treatment strategy approach for the attenuation of the relapse to drug-seeking/taking behavior triggered by opiate withdrawal-associated aversive memories.

Activation of the ERK signalling pathway contributes to the adaptive changes in rat hearts during naloxone-induced 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.

Activation of c-fos expression in hypothalamic nuclei by mu- and kappa-receptor agonists: correlation with catecholaminergic activity in the hypothalamic paraventricular nucleus.

Administration of the preferential mu-opioid receptor agonist, morphine, and selective K-opioid receptor agonists elicits activation of the hypothalamus-pituitary-adrenocortical axis, although the site or the molecular mechanisms for these effects have not been determined. The expression ofFos, the protein product of the c-fos protooncogene, has been widely used as an anatomical marker of monitoring neuronal activity. In the present study we evaluated 1) the effects of the mu-opioid receptor agonist, morphine, and those of the selective K-opioid receptor agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl-]benzeneacet amide methane sulfonate (U-50,488H), administration on the expression of Fos in hypothalamic nuclei; and 2) the possible modification of the activity of noradrenergic neurons known to send afferent projections to the paraventricular nucleus (PVN), the site of CRF neurons involved in initiating ACTH secretion. Using immunohistochemical staining of Fos, the present results indicate that acute treatment with either morphine or U-50,488H induces marked Fos immunoreactivity within the hypothalamus, including the medial parvicellular PVN and supraoptic and suprachiasmatic nuclei. Pretreatment with naloxone attenuated the effect of morphine, whereas nor-binaltorphimine, a selective kappa-opioid receptor antagonist, abolished the effect of U-50,488H on Fos induction. Correspondingly, morphine and U-50,488H injection increased the production of the cerebral noradrenaline metabolite 3-methoxy-4-hydroxyphenylethylene glycol as well as noradrenaline turnover in the PVN. These effects were antagonized by naloxone and nor-bin-altorphimine, respectively. All of these findings are discussed in terms of specific events that couple opioid-induced activation of the hypothalamus-pituitary-adrenocortical axis and noradrenergic activity with changes in gene expression in selective hypothalamic nuclei.

Effects of U-50,488H withdrawal on catecholaminergic neurones of the rat ventricle.

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.

Quantitative analysis of corticotropin-releasing factor and arginine vasopressin mRNA in the hypothalamus during chronic morphine treatment in rats: an in situ hybridization study.

The content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) increases during chronic morphine treatment. Because these experiments cannot distinguish between increased synthesis or reduced release, the present study measured changes in CRF and AVP mRNAs in the PVN by in situ hybridization. Concomitantly, changes in noradrenaline turnover in the PVN and changes in plasma corticosterone release were determined. Male rats were implanted with placebo (naive) or morphine pellets for 7 days. On day 7, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg, i.p.). Acute morphine injection did not change the total size of the labelled area for CRF mRNA in the PVN of naive or morphine-pelleted rats, indicating that the number of CRF-containing neurones was unchanged. On the other hand, in rats chronically treated with morphine, the intensity of labelling for CRF mRNA was significantly reduced, suggesting a decrease in the synthesis of CRF. In placebo rats, injection of saline or morphine did not affect the surface hybridized for AVP mRNA. By contrast, in the morphine-group injected with saline, there was a significant reduction in the number of labelled neurones, measured by the size of labelled area. Similarly, there was a decrease in intensity of AVP mRNA expression in the parvocellular and magnocellular neurones of the PVN in the morphine-group injected with saline, suggesting a decreased synthesis of AVP in these neurones. In parallel with the decrease in the expression of CRF and AVP mRNAs in the PVN, there was a pronounced decrease in noradrenaline turnover and in the release of corticosterone in the morphine-pelleted rats. In conclusion, present results show that, in addition to modifications in corticosterone secretion and in noradrenaline turnover, chronic morphine administration produces a reduction in the synthesis of CRF and AVP.

Effect of chronic administration of dihydropyridine Ca2+ channel ligands on sufentanil-induced tolerance to mu- and kappa-opioid agonists in the guinea pig ileum myenteric plexus.

The present investigation was aimed at elucidating if the entry of Ca2+ plays a role in the development of tolerance to mu- and kappa-opioid agonists in the guinea pig ileum myenteric plexus. For this purpose, the influence of the L-type Ca2+ channel modulators nimodipine (Ca2+ blocker) and Bay K 8644 (Ca2+ activator) on the expression of tolerance to the inhibitory effects of mu- and kappa-opioid agonists in the ileum of guinea pigs rendered tolerant to sufentanil was investigated. Chronic perfusion of guinea pigs with nimodipine (2 micrograms/microliter/h for 7 days) or Bay K 8644 (0.5 microgram/microliter/h for 7 days) did not cause any modification of the height of contractions induced by electrical stimulation of the myenteric plexus-longitudinal muscle (MPLM) strip from naive animals. Tolerance to sufentanil (a selective mu-agonist) was induced by s.c. implantation of osmotic minipumps for 7 days, which deliver at 2 micrograms/microliter/h. Control groups received saline. Tolerance to sufentanil as well as to U-50,488H (selective kappa-agonist) was observed following chronic treatment with sufentanil and was revealed as a rightward shift of the concentration-response curves. Chronic perfusion of guinea pigs with the Ca2+ antagonist nimodipine concurrently with chronic sufentanil, markedly blocked the expression of tolerance to sufentanil, as well as the cross-tolerance between sufentanil and U-50,488H. On the contrary, when guinea pigs were perfused with the Ca2+ agonist Bay K 8644 concurrently with sufentanil, it enhanced the magnitude of tolerance to both sufentanil and U-50,488H. These results suggest that, in guinea pig ileum, chronic exposure to opioids may involve the activation of L-type Ca2+ channel, which would indicate that intracellular Ca2+ may be one of the final pathways through which myenteric neurons adapt to the chronic opioid exposure.

Effects of chronic treatment with atypical neuroleptics on the biosynthesis and release of opioid peptides in guinea-pig ileum.

The guinea-pig ileum myenteric plexus is known to contain opioid peptides, which can be released by electric stimulation at high frequency. Haloperidol, a classic neuroleptic drug, increases the biosynthesis and release of endogenous opioid peptides from the myenteric plexus. In the present work we have examined the effects of chronic treatment with sulpiride and clozapine, two atypical neuroleptics, on the release of these peptides in the myenteric plexus of guinea-pig ileum. Both neuroleptics, administered over a period of 7 days, produced an increase of the inhibitory response obtained by electrical stimulation at 10 Hz of the ileum myenteric plexus-longitudinal muscle preparation. The inhibitory response was reversed by the specific opioid antagonist naloxone, which suggest that the increase in the inhibitory response produced by blocking the dopaminergic receptors is mediated by an increase in the release of opioid peptides. When sulpiride- or clozapine-treated guinea-pigs received cycloheximide (an inhibitor of peptide biosynthesis) there was a significant decrease of the inhibitory response, which indicates that neuroleptics produced an increase of the synthesis of opioid peptides in the ileum myenteric plexus. These results reveal a possible influence of the dopaminergic system on the biological turnover of these peptides.

Effects of morphine and U-50,488H on neurochemical activity of the hypothalamic noradrenergic neurons and pituitary-adrenal response.

We examined the ability of morphine and U-50,488 H administered acutely to the rat to modify the hypothalamic noradrenaline (NA) content and turnover as well as corticosterone release. Hypothalamic NA and its metabolite 3-methoxy-4-hydroxy-phenylethylen glycol (MHPG) were determined by high-performance liquid chromatography (HPLC). Plasma corticosterone was measured by radioimmunoassay (RIA). The administration of morphine (30 mg/kg i.p.) induced a decrease in the hypothalamic content of NA, with an enhancement in the MHPG and the MHPG/NA ratio. Similarly, the administration of U-50,488 H (15 mg/kg i.p.) produced an increase in the MHPG and MHPG/NA ratio without any changes in the NA content. The present results also show that corticosterone secretion was increased after acute morphine or U-50,488 H, in parallel with an enhanced NA turnover and strongly suggest that noradrenergic neurons have stimulatory effects on the HPA axis activity, reflecting an indirect action of opioid on the axis through hypothalamic catecholaminergic systems.

Chronic naloxone-induced supersensitivity affects neither tolerance to nor physical dependence on morphine at hypothalamus-pituitary-adrenocortical axis.

This study reports the endocrine effects of chronic mu-blockade induced by naloxone on morphine tolerance and withdrawal at hypothalamus-pituitary-adrenocortical (HPA) axis level. Naloxone (0.5 mg/kg/h) or vehicle (1 microliter/h) were infused s.c. to Sprague-Dawley rats via osmotic minipumps for 7 days, concomitantly with morphine or placebo pellets for 7-8 days. In opiate-naive rats, the mu-preferring opioid agonist morphine (30 mg/kg) increased plasma corticosterone in a partial but significant naloxone-reversible manner. In vehicle-perfused rats, chronic morphine treatment produced tolerance to its neuroendocrine effect, while the development of morphine tolerance was antagonized in the naloxone-treated group. An enhancement of plasma corticosterone levels after acute morphine (30 mg/kg) occurred 24 h after removal of chronic naloxone treatment in vehicle-perfused rats, as a functional index of supersensitivity to the neuroendocrine effects of the mu agonist. By contrast, 24 h after naloxone removal, rats implanted with morphine pellets were significantly less sensitive to acute morphine (tolerance) than its control-placebo group. Substantial elevation of plasma corticosterone, accompanied by motor and behavioural signs, was observed after acute naloxone injection (1 mg/kg) to tolerant rats 24 h after naloxone-pumps removal, which indicates withdrawal. No endocrine, motor or behavioural signs appeared in the naloxone group with pumps in place. These results indicated that morphine desensitizes mu-opioid receptors that were probably upregulated by chronic naloxone in presence of chronic agonist administration, and suggest that opioid tolerance/dependence as well as opioid supersensitivity simultaneously and independently can occur at mu-opioid receptors mediating HPA function.

Effects of chronic U-50,488H treatment on the isolated right atrium of the rat.

The aim of the present investigation was to determine if chronic activation of kappa-opioid receptor induces development of tolerance and dependence to kappa-opioid agonists on the isolated right atrium of the rat. Tolerance to the kappa-agonist was induced by chronic administration of U-50,488H, a selective kappa-agonist (15 mg/kg i.p. twice a day for 4 days). The rats were killed on day 5. Tolerance to U-50,488H was observed after its chronic administration and was revealed as a rightward shift of the concentration-response curve; it was accompanied by a decrease in the maximum response and in the slope. Withdrawal to the kappa-agonist was induced by administration of Mr-2266 (preferentially kappa-antagonist) or nor-binaltorphimine (nor-bni; selective kappa-antagonist) to the organ bath. The administration of the kappa-antagonists Mr-2266 or nor-bni to preparations from tolerant rats in the organ bath induced an increase in auricular contraction frequency. In contrast, the administration of the kappa-antagonists to preparations from vehicle-treated rats induced a decrease in auricular contraction frequency. These findings demonstrate that the hearts of rats that had received chronic U-50,488H treatment develop tolerance to the cardiac effects of U-50,488H and exhibit excitatory reactions to kappa-antagonist's precipitated withdrawal.

Changes in hypothalamic oxytocin levels during morphine tolerance.

The role of hypothalamic oxytocin neurons in the hypothalamus-pituitary-adrenal (HPA) axis adaptation during opioid tolerance has not been explored. In this study the modification of oxytocin levels in different hypothalamic nuclei was determined after acute or chronic morphine exposure. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. In morphine-tolerant rats, there was a decrease in the oxytocin content in the median eminence (ME) and in the supraoptic nucleus (SO) after acute injection of saline or morphine. No modifications were seen in the paraventricular nucleus (PVN). The present study demonstrates that chronic morphine administration alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine responses to opioids.

Pituitary-adrenocortical response to acute and chronic administration of U-50,488H in the rat.

K-opioid substances have been shown to stimulate and/or depress the HPA activity. The objective of this study was to determine the effects of the acute and chronic administration of U-50,488H, a k-opioid receptor agonist, on the pituitary-adrenocortical activity in the rat. The acute administration of U-50,488H (25 mg/kg i.p.) produced a hypothermic effect and an increase in plasma levels of B-END-LI and cortisol, effects which were prevented by naloxone (3 mg/kg s.c.). Chronic administration of U-50,488H twice a day for 4 days resulted in a decrease in basal plasma levels of B-END-LI and cortisol and in the development of tolerance to its neuroendocrine and hypothermic effects. In rats made tolerant to U-50,488H, naloxone precipitated hypothermia (which is an index of opiate dependence in rats), whereas no changes in plasma B-END-LI and cortisol levels were seen. These data suggest that k receptors may be involved in the regulation of pituitary-adrenocortical activity in physiological conditions and during opiate abuse. On the other hand, U-50,488H induced only negligible dependence in rats, which was not morphine-like.

Prenatal stress alters the hypothalamic levels of methionine-enkephalin in pup rats.

We examined hypothalamic Methionine-enkephalin levels in offspring (postnatal day 10) from stressed female rats in different period of gestation: Group 1, restraint stress day 2 through 6; group 2, restraint stress day 7 through 11; group 3, restraint stress day 12 through day 16 and group 4, restraint stress day 2 through day 16. The hypothalamic levels of Methionine-enkephalin (134.37 +/- 6.19 pg/mg) in the offspring of stressed females from day 2 to day 6 of gestation were significantly (p < 0.001) higher than that obtained in the control group (100.66 +/- 10.13 pg/mg). Similar results were obtained in groups 2 and 3. However, in rat pups from females stressed during 15 days of gestation (group 4) the hypothalamic levels of Methionine-enkephalin (96.5 +/- 6.33 pg/mg) were similar to that obtained in the control group (101.5 +/- 5.15 pg/mg). These results suggest that acute prenatal stress alters the endogenous opioid activity in offspring with possible resultant effects on developmental processes.

Simultaneous changes in hypothalamic catecholamine levels and plasma corticosterone concentration in the rat after acute morphine and during tolerance.

The effects of morphine on plasma corticosterone and hypothalamic noradrenaline (NA) and dopamine (DA) content were studied in naive and in morphine-tolerant rats. Acutely administered morphine (30 mg/kg i.p.) significantly increased the plasma levels of corticosterone and significantly reduced the hypothalamic NA and DA content. In chronically morphine-treated rats (subcutaneously implanted with pellets for 7 days), a challenge dose of morphine (30 mg/kg intraperitoneally (i.p.)) did not modify the plasma corticosterone levels and inhibited the morphine-induced decreases in hypothalamic NA and DA content. These results suggest that: (1) In naive rats, the morphine-induced activation of hypothalamus-pituitary-adrenocortical (HPA) axis is mediated by catecholaminergic neurons in the hypothalamus; (2) In tolerant rats morphine did not modify the plasma corticosterone concentrations, presumably by attenuating hypothalamic noradrenergic and dopaminergic activity. (3) Hypothalamic catecholamines have a role in regulating the HPA axis during morphine tolerance.

Changes in oxytocin content in rat brain during morphine withdrawal.

In this study the modification in the oxytocin content in different hypothalamic nuclei during morphine withdrawal was analysed. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 7, groups of rats received an acute injection of saline s.c. (control) or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. After administration of naloxone to tolerant rats (withdrawal) an increase in the oxytocin content in the paraventricular nucleus (PVN) and median eminence (ME) was found. No changes were found in the arcuate nucleus (AN) and supraoptic nucleus (SON). Present data demonstrate that administration of naloxone to tolerant rats alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine response to opioid.

Effect of ACTH-like peptides on morphine-induced hypothermia in unrestrained guinea pigs.

Peripheral treatment with adrenocorticotropin (1-24) (ACTH1-24), at different doses and sequences, consistently antagonized the decrease in body temperature produced by morphine in the freely moving guinea pig, whereas adrenocorticotropin (4-10) (ACTH4-10), which lacks corticotrophic activity, was partially effective only when it was administered in a high dose 24 h prior to morphine. Centrally administered ACTH1-24 completely prevented the hypothermic effect of intracerebroventricularly (i.c.v.)-injected morphine. Likewise, the i.c.v. administration of ACTH4-10 was equally effective in blocking the i.c.v. morphine-induced hypothermia. Neither ACTH1-24 nor ACTH4-10 did produce changes in body temperature. These results suggest that peripherally administered ACTH1-24 antagonizes indirectly the actions of morphine through the release of adrenal corticosteroids, whereas centrally injected ACTH1-24 or ACTH4-10 act as direct antagonists of morphine effects through opioid receptors.