Characterization of the signal transduction pathways mediating morphine withdrawal-stimulated c-fos expression in hypothalamic nuclei.

The transcription factor, Fos, is considered as a functional marker of activated neurons. We have shown previously that acute administration of morphine induces the expression of Fos in hypothalamic nuclei associated with control of the hypothalamus-pituitary-adrenocortex axis, such as the paraventricular nucleus and the supraoptic nucleus. In the current study, we examined the role of protein kinase A, protein kinase C and Ca2+ entry through L-type Ca2+ channels in naloxone-precipitated Fos expression in the paraventricular and supraoptic nuclei. After 7 days of morphine treatment, we did not observe any modification in Fos production. However, when opioid withdrawal was precipitated with naloxone a dramatic increase in Fos immunoreactivity was observed in the parvocellular division of the paraventricular nucleus and in the supraoptic nucleus. Chronic co-administration of chelerythrine (a selective protein kinase C inhibitor acting at its catalytic domain) with morphine did not affect the increase in Fos expression observed in nuclei from morphine withdrawn rats. In addition, infusion of calphostin C (another protein kinase C inhibitor, which interacts with its regulatory domain) did not modify the morphine withdrawal-induced expression of Fos. In contrast, when the selective protein kinase A inhibitor, N-(2'guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), was infused it greatly diminished the increased Fos production observed in morphine-withdrawn rats. Furthermore, chronic infusion of the selective L-type Ca2+ channel antagonist, nimodipine, significantly inhibited the enhancement of Fos induction in the paraventricular and supraoptic nuclei from morphine-withdrawn animals. Taken together, these data might indicate that protein kinase A activity is necessary for the expression of Fos during morphine withdrawal and that an up-regulated Ca2+ system might contribute to the activation of Fos. The present findings suggest that protein kinase A and Ca2+ influx through L-type Ca2+ channels might contribute to the activation of neuroendocrine cells in the paraventricular and supraoptic nuclei.

Neurochemical activity of noradrenergic neurons and pituitary-adrenal response after naloxone-induced withdrawal: the role of calcium channels.

The influence of the L-type calcium channel antagonist nimodipine on both the activity of noradrenergic neurons projecting to the hypothalamus and the pituitary-adrenal response during morphine tolerance and withdrawal was analysed. Tissue concentration of hypothalamic noradrenaline (NA) and its metabolite 3-methoxy-4-hydroxy-phenylethylen-glycol (MHPG) were determined by high-pressure liquid chromatography. Plasma corticosterone concentration (a marker of pituitary-adrenal activity) was measured by radioimmunoassay. Rats rendered tolerant to morphine decreased hypothalamic MHPG concentration, and reduced hypothalamic NA turnover. Chronic infusion of nimodipine concurrently with morphine prevented the decrease in NA turnover during tolerance. After naloxone administration to tolerant rats we found a striking parallelism between an increased activity of the hypothalamic-pituitary-adrenal axis and an enhanced activity of noradrenergic neurons projecting to the hypothalamus. However, hypothalamic NA turnover and MHPG concentration, both elevated during withdrawal, returned to control levels in rats infused chronically with nimodipine, concomitantly with a reduction of the secretion of corticosterone. Taken together, these data indicate that increased noradrenergic neuronal activity in the hypothalamic nerve terminals is associated with the neuroendocrine morphine withdrawal syndrome and suggest that an up-regulated calcium system might contribute to the activation of these neurons.

Catecholaminergic mediation of morphine-induced activation of pituitary-adrenocortical axis in the rat: implication of alpha- and beta-adrenoceptors.

The present study investigates the role of hypothalamic catecholamines in the effects of morphine on hypothalamo-pituitary-adrenocortical (HPA) axis. Acutely administered morphine (30 mg/kg i.p) increased plasma corticosterone and reduced the hypothalamic noradrenaline (NA) content but it did not change either the dopamine (DA) concentration or the ratio DOPAC/DA. After reserpine administration the hypothalamic contents of NA and DA were drastically reduced without changing plasma corticosterone concentrations. The increase in plasma corticosterone induced by morphine was significantly reduced by the pretreatment with reserpine. The alpha 1- and alpha 2-antagonists prazosin and yohimbine, respectively, significantly antagonized the effect of morphine on plasma corticosterone. The beta-antagonist propranolol also significantly attenuated the increase of corticosterone secretion induced by morphine. The results suggest that the action of the opiate on HPA axis activity may be dependent on stimulatory catecholaminergic systems which utilize alpha 1-, alpha 2- and beta-adrenoceptors.

Effects of intracerebroventricular clonidine on the hypothalamic noradrenaline and plasma corticosterone levels of opiate naive rats and after naloxone-induced withdrawal.

The aim of this study was to determine whether hypothalamic noradrenergic neuronal activity contributes to the abstinence-induced hypersecretion of corticosterone during naloxone-induced withdrawal. With this purpose the effects of intracerebroventricular (i.c.v.) clonidine on hypothalamic noradrenaline (NA) and plasma corticosterone were studied in chronically placebo-treated rats (controls) and during naloxone-induced morphine withdrawal. In control rats, clonidine (10 micrograms) significantly increased plasma levels of corticosterone without changing the hypothalamic content of NA. Naloxone (1 mg/kg, s.c.) also increased plasma corticosterone levels and clonidine administered prior to naloxone, antagonized the effect of naloxone on plasma corticosterone. In chronically morphine-treated rats, naloxone treatment induced an increase in plasma corticosterone and reduced the hypothalamic NA content. Clonidine significantly prevented the reduction in the hypothalamic NA, without modifying plasma levels of corticosterone. The results show an interaction between opioid-receptors and alpha 2-adrenoceptors in the hypothalamus, and suggest that mechanisms other than hyperactivity of NA neurons contribute to the hypothalamus-pituitary-adrenocortical (HPA) axis hyperactivity during the opiate withdrawal.

Modulation by catecholamine of hypothalamus-pituitary-adrenocortical (HPA) axis activity in morphine-tolerance and withdrawal.

1. Hypothalamic noradrenaline (NA), dopamine (DA) and plasma corticosterone concentrations were determined after acute morphine administration to both naive and morphine-tolerant rats and during naloxone-induced withdrawal. 2. Acutely administered morphine (30 mg/kg) significantly increased the plasma level of corticosterone and reduced the NA and DA content in the hypothalamus. Naloxone (1 mg/kg), administered before morphine, blocked the effect of the opiate on both plasma corticosterone and hypothalamic NA concentration. 3. In chronically morphine-treated rats, a challenge dose of morphine (30 mg/kg) neither modified the plasma corticosterone level nor the NA concentration, while DA content was significantly enhanced. 4. After naloxone-induced withdrawal, the hypothalamic content of NA was significantly reduced, simultaneously with an increase in plasma corticosterone, while DA content remained unchanged. 5. These results suggest that the hypothalamic noradrenergic neurons are mainly mainly implicated in the effect of acute morphine on the hypothalamus-pituitary-adrenocortical (HPA) axis and in the tolerance development to this effect. The results also suggest that a hyperactivity of noradrenergic pathways in the hypothalamus would be one of the physiologically relevant mechanisms mediating the neuroendocrine opiate withdrawal at the HPA level.

L-type Ca2+ channel ligands modulate morphine effects on the hypothalamus-pituitary-adrenocortical axis in rats.

The role of the L-type Ca2+ channel in the acute effects of morphine on the hypothalamo-pituitary-adrenocortical (HPA) system was studied by administration of the Ca2+ channel agonist, BAY K 8644, and the antagonists, verapamil and nimodipine, to rats. Morphine (30 mg/kg i.p.) induced an increase in corticosterone secretion 30 min after injection, which was correlated with a simultaneous change in hypothalamic noradrenaline (NA) and dopamine (DA) contents. Pretreatment with verapamil (10 or 20 mg/kg i.p.) or nimodipine (5 mg/kg i.p.) antagonized the HPA activation induced by morphine, blocking both the decrease in hypothalamic NA levels and the elevation in plasma corticosterone induced by the opioid. BAY K 8644 (2 mg/kg i.p.) potentiated the effects of morphine, decreasing the hypothalamic NA content and increasing the release of corticosterone. The Ca2+ channel antagonist, nimodipine, given alone induced a slight reduction in hypothalamic NA content but did not modify plasma corticosterone levels. Verapamil given alone did not alter HPA activity. Instead, the Ca2+ agonist decreased the hypothalamic catecholamine content and increased plasma corticosterone levels. These results indicate that Ca2+ influx is necessary for the expression of opioid actions on the HPA system, and suggest that the Ca2+ flux in hypothalamic neurons is functionally linked to activation of opioid receptors.