1 Dierent 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 aerent innervating the PVN and supraoptic nucleus (SON) during chronic opioid exposure and upon morphine withdrawal. 2 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 ± A 2 ) and the ventrolateral medulla (VLM ± A 1 ) and combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identi®cation 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. 3 Acute morphine administration produced an increase in Fos expression at hypothalamic nuclei and in the brainstem regions, and tolerance developed towards this eect. 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 signi®cant increase in Fos expression in response to morphine withdrawal. 4 These ®ndings 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.

We previously demonstrated that morphine withdrawal induced hyperactivity of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN) in rats, in parallel with an increase in the neurosecretory activity of the hypothalamus-pituitary-adrenocortical (HPA) axis, as evaluated by corticosterone release. These neuroendocrine effects were dependent on stimulation of a-adrenoceptors. In the present study, Fos immunostaining was used as a reflection of neuronal activity and combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurones during morphine withdrawal. Dependence on morphine was induced by 7-day chronic subcutaneous implantation of six morphine pellets (75 mg). Morphine withdrawal was precipitated by administration of naloxone (5 mg/kg subcutaneously) on day 8. Fos immunoreactivity in the PVN and also in the nucleus tractus solitarius (NTS)-A 2 and ventrolateral medulla (VLM)-A 1 cell groups, which project to the PVN, increased during morphine withdrawal. Following withdrawal, Fos immunoreactivity was present in most of the TH-positive neurones of the A 2 and A 1 neurones. In a second study, the effects of administration of adrenoceptor antagonists on withdrawal-induced Fos expression in the PVN were studied. Pre-treatment with a 1 -or a 2 -adrenoceptor antagonists, prazosin (1 mg/kg intraperitoneally) and yohimbine (1 mg/kg intraperitoneally), respectively, 20 min before naloxone administration to morphine-dependent rats markedly reduced Fos expression in the PVN. Similarly, pre-treatment with the b antagonist, propranolol (3 mg/kg intraperitoneally), significantly prevented withdrawal-induced Fos expression. Collectively, these results suggest the hypothesis that noradrenergic neurones in the brainstem innervating the PVN are active during morphine withdrawal, and that activation of transcriptional responses mediated by Fos in the HPA axis following withdrawal are dependent upon hypothalamic a-and b-adrenoceptors. The physiological response to morphine dependence and withdrawal invokes a number of complex neural and hormonal processes. Several lines of evidence provide support for the participation of noradrenergic pathways in the expression of morphine dependence (Maldonado 1997; Nestler and Aghajanian 1997), most of which have been derived from studies of the locus coeruleus. One of the prominent components of the morphine-withdrawal response is the central activation of the hypothalamus-pituitaryadrenocortical (HPA) axis (Pechnick 1993;Vargas et al. 1997;Fuertes et al. 2000a). Parvocellular neuroendocrine cells in the hypothalamic paraventricular nucleus (PVN) project to the median eminence and release corticotropinreleasing factor (CRF) and vasopressin (AVP) into the portal circulation when activated by a variety of stressors, resulting in the release of adrenocorticotropic hormone (ACTH) from anterior pituitary and the subsequent secretion of glucocorticoids from the adrenals. Previous anatomical and physiological dat...

Previous research has shown an increase in hypothalamo-pituitary-adrenal axis activity following naloxone administration to morphine-dependent rats. In the present study, we investigated the adaptive changes in the noradrenaline (NA) and dopamine (DA) systems in the hypothalamic paraventricular nucleus (PVN) during morphine dependence and withdrawal. Additionally, we examined the possible change in 3′,5′-cyclic adenosine monophosphate (cAMP) levels in that nucleus under the same conditions. Rats were made dependent on morphine by morphine or placebo (naïve) pellet implantation for 7 days. On day 8, rat groups received an acute injection of saline or naloxone (1 mg/kg subcutaneously) and were decapitated 30 min later. NA and DA content as well as their metabolite production in the PVN were estimated by HPLC/ED. Both plasma corticosterone levels and cAMP concentration in the PVN were measured by RIA. Naloxone administration to morphine-dependent rats (withdrawal) induced a pronounced increase in the production of both the NA metabolite MHPG and the DA metabolite DOPAC and an enhanced NA and DA turnover. Furthermore, an increase in corticosterone secretion was observed in parallel to the changes in catecholamine turnover. However, no alterations in cAMP levels were seen during morphine withdrawal. These results raise the possibility that catecholaminergic afferents to the PVN could play a significant role in the alterations of PVN functions and consequently in the pituitary-adrenal response during morphine abstinence syndrome. These data provide further support for the idea of adaptive changes in catecholaminergic neurons projecting to the PVN during chronic morphine exposure.

The changes in the content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in discrete brain nuclei during chronic opioids administration have not been well established. We evaluated the effects of acute and chronic morphine administration on the content of CRF and AVP in different hypothalamic and extrahypothalamic (bed nucleus of the stria terminalis, BNST) nuclei in rats. Concomitantly, changes in hypothalamic noradrenaline (NA) turnover [estimated by the 3-methoxy-4-hydroxyphenylethyleneglycol MHPG/NA ratio] and in plasma corticosterone release (as a marker of the activity of the hypothalamus-pituitary-adrenal axis) were determined. 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. Acute morphine injection to naïve rats increased both the release of corticosterone and the hypothalamic NA turnover. CRF and AVP showed no modifications in the paraventricular nucleus (PVN) or in the median eminence (ME). CRF content decreased in the ventromedian nucleus (VMN) and increased in the BNST, but did not change in the arcuate nucleus (AN). AVP was elevated in the supraoptic nucleus (SON) but not changed in the suprachiasmatic nucleus (SCN). In chronic morphine-treated rats, there was a pronounced decrease in the NA turnover and in the release of corticosterone, which indicates that tolerance develops to the acute effects of morphine. Correspondingly, CRF and AVP were enhanced in the PVN and decreased in the ME, when compared with naïve rats injected with morphine. CRF content was decreased in the AN and in the BNST, but increased in the VMN. The AVP content was decreased in the SON, and no modifications were seen in the SCN. The present study shows that, in addition to the modifications in corticosterone secretion and in hypothalamic NA turnover, chronic morphine administration produces a complex response in the CRF and AVP systems. These modifications might contribute to the behavioral, emotional and neuroendocrine alterations produced during opioid tolerance.

Chronic opiate exposure induces neurochemical adaptations in the noradrenergic system. Enhanced responsiveness of the hypothalamo-pituitary-adrenal axis after morphine withdrawal has been associated with hyperactivity of ascending noradrenergic input from the nucleus of the solitary tract (NTS-A(2)) cell group to the hypothalamic paraventricular nucleus (PVN). This study addressed the role of morphine withdrawal-induced corticosterone (CORT) release in regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis in adrenalectomized (ADX) rats supplemented with low CORT pellet (ADX plus CORT). Present results show that in sham-ADX rats, noradrenergic neurons in the NTS-A(2) became activated during morphine withdrawal, as indicated by increased TH mRNA expression. However, this induction of TH expression is not detected in ADX plus CORT rats that are unable to mount CORT secretory response to morphine withdrawal. Total TH protein levels were elevated in the NTS-A(2) from sham-operated rats during morphine dependence and withdrawal, whereas we did not find any alteration in ADX plus CORT animals. Furthermore, high levels of TH phosphorylated (activated) at Ser31 (but not at Ser40) were found in the A(2) area from sham-morphine withdrawn rats. Consistent with these effects, we observed an increase in the enzyme activity of TH in the PVN. However, induction of morphine withdrawal to ADX plus CORT animals did not alter the phosphorylation (activation) of TH in NTS-A(2) and decreased TH activity in the PVN. These results suggest the existence of a positive reverberating circle in which elevated glucocorticoids during morphine abstinence play a permissive role in morphine withdrawal-induced activation of noradrenergic pathway innervating the PVN.