Cristina Núñez

Morphine withdrawal‐induced c‐fos expression in the hypothalamic paraventricular nucleus is dependent on the activation of catecholaminergic neurones

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 α‐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/kgsubcutaneously) on day 8. Fos immunoreactivity in the PVN and also in the nucleus tractus solitarius (NTS)‐A2 and ventrolateral medulla (VLM)‐A1 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 A2 and A1 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 α1‐ or α2‐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 β 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 α‐ and β‐adrenoceptors.

Activation of stress-related hypothalamic neuropeptide gene expression during morphine withdrawal.

Morphine withdrawal results in serious affective and somatic symptoms including activation of the hypothalamo–pituitary–adrenocortical (HPA) axis. To reveal secretory, activational and transcriptional changes in the hypothalamus of morphine‐dependent rats during naloxone precipitated opioid withdrawal, we measured corticosterone secretion, c‐Fos induction and heteronuclear (hn)RNA levels of corticotropin‐releasing hormone (CRH) and arginine vasopressin (AVP) in naïve and morphine dependent animals injected with saline or 5 mg/kg naloxone. Naloxone precipitated morphine withdrawal resulted in a significant increase in corticosterone secretion and induction of neuronal activation in the hypothalamic paraventricular nucleus (PVH) 2 h after challenge. Using probes complementary to intronic sequences of genes encoding neuropeptides in parvocellular neurosecretory neurons of the PVH, we found robust increases in CRH and AVP hnRNAs in morphine dependent rats during naloxone precipitated withdrawal. Naïve rats and animals that were implanted with morphine pellets for 8 days did not display significant up‐regulation of ongoing neuropeptide expression in the parvocellular compartment of the PVH. In addition to hypophyseotropic neurons, naloxone precipitated withdrawal resulted in a marked activation in autonomic‐related projection neurons in PVH and in the magnocellular neurons in the PVH and supraoptic nuclei. These activations however were not associated with induction of CRH or AVP hnRNAs.

Effects of Corticotropin-Releasing Factor Receptor-1 Antagonists on the Brain Stress System Responses to Morphine Withdrawal

The role of stress in drug addiction is well established. The negative affective states of withdrawal most probably involve recruitment of brain stress neurocircuitry [e.g., induction of hypothalamo-pituitary-adrenocortical (HPA) axis, noradrenergic activity, and corticotropin-releasing factor (CRF) activity]. The present study investigated t

Elevated Glucocorticoid Levels Are Responsible for Induction of Tyrosine Hydroxylase mRNA Expression, Phosphorylation, and Enzyme Activity in the Nucleus of the Solitary Tract during Morphine Withdrawal

he role of CRF receptor-1 subtype (CRF1R) on the response of brain stress system to morphine withdrawal. The effects of naloxone-precipitated morphine withdrawal on noradrenaline (NA) turnover in the paraventricular nucleus (PVN), HPA axis activity, signs of withdrawal, and c-Fos expression were measured in rats pretreated with vehicle, CP-154526 [N-butyl-N-ethyl-2,5-dimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[3,2-e]pyrimidin-4-amine], or antalarmin (selective CRF1R antagonists). Tyrosine hydroxylase-positive neurons expressing CRF1R were seen at the level of the nucleus tractus solitarius-A2 cell group in both control and morphine-withdrawn rats. CP-154526 and antalarmin attenuated the increases in body weight loss and irritability that were seen during naloxone-induced morphine withdrawal. Pretreatment with CRF1R antagonists resulted in no significant modification of the increased NA turnover at PVN, plasma corticosterone levels, or c-Fos expression that was seen during naloxone-induced morphine withdrawal. However, blockade of CRF1R significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin levels. These results suggest that the CRF1R subtype may be involved in the behavioral and somatic signs and in adrenocorticotropin release (partially) during morphine withdrawal. However, CRF1R activation may not contribute to the functional interaction between NA and CRF systems in mediating morphine withdrawal-activation of brain stress neurocircuitry.

Induction of FosB/ΔFosB in the brain stress system‐related structures during morphine dependence and withdrawal

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.

CRF2 mediates the increased noradrenergic activity in the hypothalamic paraventricular nucleus and the negative state of morphine withdrawal in rats

J. Neurochem. (2010) 114, 475–487.

Changes in metabolic-related variables during chronic morphine treatment

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).

Glucocorticoids Regulation of FosB/ΔFosB Expression Induced by Chronic Opiate Exposure in the Brain Stress System

To reveal neuroendocrine/neurochemical changes that are responsible for the robust metabolic alterations seen during chronic morphine treatment we followed hormonal-, transcriptional- and behavioral changes during chronic morphine administration in adult male Wistar rats. Animals were implanted with increasing amount of slow release morphine tablets for 8 days. Morphine treated animals gain significantly less weight than placebo implanted controls. This weight loss is due to the dramatic decrease in the food intake and caloric efficiency in the first days of drug administration and to the lasting disregulated feeding pattern. Changes in feeding behavior included increase of diurnal and decrease of nocturnal feeding frequency in morphine treated rats. Significantly less leptin and insulin plasma levels were detected in morphine implanted animals than in placebo implanted controls, while adiponectin and ACTH concentration remain unchanged. Morphine treated rats display an increase of FosB/Delta FosB immunoreactivity at brain sites that have been implicated regulation of food intake and energy expenditure, including hypothalamic arcuate, paraventricular and ventromedial nuclei and in the lateral hypothalamic area as well as in the caudal brainstem. However, morphine-induced long-term metabolic alterations were not accompanied with any significant changes in the expression of anorexigenic neuropeptides POMC and CART in the hypothalamus and in the brainstem. The disregulated feeding pattern was not reflected in changes of orexin transcription, however, a compensatory upregulation was revealed in hypothalamic NPY expression.

Morphine regulates Argonaute 2 and TH expression and activity but not miR‐133b in midbrain dopaminergic neurons

Chronic use of drugs of abuse profoundly alters stress-responsive system. Repeated exposure to morphine leads to accumulation of the transcription factor ΔFosB, particularly in brain areas associated with reward and stress. The persistent effects of ΔFosB on target genes may play an important role in the plasticity induced by drugs of abuse. Recent evidence suggests that stress-related hormones (e.g., glucocorticoids, GC) may induce adaptations in the brain stress system that is likely to involve alteration in gene expression and transcription factors. This study examined the role of GC in regulation of FosB/ΔFosB in both hypothalamic and extrahypothalamic brain stress systems during morphine dependence. For that, expression of FosB/ΔFosB was measured in control (sham-operated) and adrenalectomized (ADX) rats that were made opiate dependent after ten days of morphine treatment. In sham-operated rats, FosB/ΔFosB was induced after chronic morphine administration in all the brain stress areas investigated: nucleus accumbens(shell) (NAc), bed nucleus of the stria terminalis (BNST), central amygdala (CeA), hypothalamic paraventricular nucleus (PVN) and nucleus of the solitary tract noradrenergic cell group (NTS-A2). Adrenalectomy attenuated the increased production of FosB/ΔFosB observed after chronic morphine exposure in NAc, CeA, and NTS. Furthermore, ADX decreased expression of FosB/ΔFosB within CRH-positive neurons of the BNST, PVN and CeA. Similar results were obtained in NTS-A2 TH-positive neurons and NAc pro-dynorphin-positive neurons. These data suggest that neuroadaptation (estimated as accumulation of FosB/ΔFosB) to opiates in brain areas associated with stress is modulated by GC, supporting the evidence of a link between brain stress hormones and addiction.

Morphine administration modulates expression of Argonaute 2 and dopamine-related transcription factors involved in midbrain dopaminergic neurons function

Epigenetic changes such as microRNAs (miRs)/Ago2‐induced gene silencing represent complex molecular signature that regulate cellular plasticity. Recent studies showed involvement of miRs and Ago2 in drug addiction. In this study, we show that changes in gene expression induced by morphine and morphine withdrawal occur with concomitant epigenetic modifications in the mesolimbic dopaminergic (DA) pathway [ventral tegmental area (VTA)/nucleus accumbens (NAc) shell], which is critically involved in drug‐induced dependence. We found that acute or chronic morphine administration as well as morphine withdrawal did not modify miR‐133b messenger RNA (mRNA) expression in the VTA, whereas Ago2 protein levels were decreased and increased in morphine‐dependent rats and after morphine withdrawal, respectively. These changes were paralleled with enhanced and decreased NAc tyrosine hydroxylase (TH) protein (an early DA marker) in morphine‐dependent rats and after withdrawal, respectively. We also observed changes in TH mRNA expression in the VTA that could be related to Ago2‐induced translational repression of TH mRNA during morphine withdrawal. However, the VTA number of TH‐positive neurons suffered no alterations after the different treatment. Acute morphine administration produced a marked increase in TH activity and DA turnover in the NAc (shell). In contrast, precipitated morphine withdrawal decreased TH activation and did not change DA turnover. These findings provide new information into the possible correlation between Ago2/miRs complex regulation and DA neurons plasticity during opiate addiction.