Involvement of Noradrenergic Transmission in the PVN on CREB Activation, TORC1 Levels, and Pituitary-Adrenal Axis Activity during Morphine Withdrawal

Experimental and clinical findings have shown that administration of adrenoceptor antagonists alleviated different aspects of drug withdrawal and dependence. The present study tested the hypothesis that changes in CREB activation and phosphorylated TORC1 levels in the hypothalamic paraventricular nucleus (PVN) after naloxone-precipitated morphine withdrawal as well as the HPA axis activity arises from α1- and/or β-adrenoceptor activation. The effects of morphine dependence and withdrawal on CREB phosphorylation (pCREB), phosphorylated TORC1 (pTORC1), and HPA axis response were measured by Western-blot, immunohistochemistry and radioimmunoassay in rats pretreated with prazosin (α1-adrenoceptor antagonist) or propranolol (β-adrenoceptor antagonist). In addition, the effects of morphine withdrawal on MHPG (the main NA metabolite at the central nervous system) and NA content and turnover were evaluated by HPLC. We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations. Levels of the inactive form of TORC1 (pTORC1) were decreased during withdrawal. Prazosin but not propranolol blocked the rise in pCREB level and the decrease in pTORC1 immunoreactivity. In addition, the HPA axis response to morphine withdrawal was attenuated in prazosin-pretreated rats. Present results suggest that, during acute morphine withdrawal, NA may control the HPA axis activity through CREB activation at the PVN level. We concluded that the combined increase in CREB phosphorylation and decrease in pTORC1 levels might represent, in part, two of the mechanisms of CREB activation at the PVN during morphine withdrawal

Influencia del estrés prenatal en la ontogenia de receptores y péptidos opioides en el sistema nervioso central de la rata / M. Dolores Sánchez Carretero ; directoras M. Luisa Laorden Carrasco, M. Victoria Milanés Maquilón.

Tesis-Universidad de Murcia.MEDICINA ESPINARDO. DEPOSITO. MU-Tesis 494.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-1478

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

<div><p>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.</p> </div

Morphine-withdrawal aversive memories and their extinction modulate H4K5 acetylation and Brd4 activation in the rat hippocampus and basolateral amygdala

Chromatin modification is a crucial mechanism in several important phenomena in the brain, including drug addiction. Persistence of drug craving and risk of relapse could be attributed to drug-induced epigenetic mechanisms that seem to be candidates explaining long-lasting drug-induced behaviour and molecular alterations. Histone acetylation has been proposed to regulate drug-seeking behaviours and the extinction of rewarding memory of drug taking. In this work, we studied the epigenetic regulation during conditioned place aversion and after extinction of aversive memory of opiate withdrawal. Through immunofluorescence assays, we assessed some epigenetic marks (H4K5ac and p-Brd4) in crucial areas related to memory retrieval -basolateral amygdala (BLA) and hippocampus-. Additionally, to test the degree of transcriptional activation, we evaluated the immediate early genes (IEGs) response (Arc, Bdnf, Creb, Egr-1, Fos and Nfkb) and Smarcc1 (chromatin remodeler) through RT-qPCR in these nuclei. Our results showed increased p-Brd4 and H4K5ac levels during aversive memory retrieval, suggesting a more open chromatin state. However, transcriptional activation of these IEGs was not found, therefore suggesting that other secondary response may already be happening. Additionally, Smarcc1 levels were reduced due to morphine chronic administration in BLA and dentate gyrus. The activation markers returned to control levels after the retrieval of aversive memories, revealing a more repressed chromatin state. Taken together, our results show a major role of the tandem H4K5ac/p-Brd4 during the retrieval of aversive memories. These results might be useful to elucidate new molecular targets to improve and develop pharmacological treatments to address addiction and to avoid drug relapse

Effects of adrenalectomy on FosB/ΔFosB protein expression in the NAc(shell) pro-DYN-positive neurons and in the NTS TH-positive neurons from morphine dependent rats.

<p>ADX and non-operated (sham) rats were made dependent on morphine (mor) for 10 days. Controls received placebo pellets. Animals were perfused and the NAc and NTS were processed for double-labelled (FosB/ΔFosB and pro-DYN and FosB/ΔFosB and TH, respectively) immunohistochemistry. Panels A-B show immunohistochemical detection of FosB/ΔFosB into pro-DYN-positive neurons in sham- and ADX-morphine-dependent rats. Low and high magnifications images show FosB/ΔFosB-positive (blue-black)/pro-DYN-positive (brown) neurons (black arrow) and FosB/ΔFosB-negative/pro-DYN-positive (white arrow) immunoreactivity. Panels D-E show immunohistochemical detection of FosB/ΔFosB within TH-positive neurons in sham- and ADX-morphine-dependent rats. Low and high magnifications images show FosB/ΔFosB-positive (blue-black)/TH-positive (brown) neurons (black arrow) and FosB/ΔFosB-negative/TH-positive (white arrow) immunoreactivity. Scale bars: 100 µm (70X, low magnification); 20 µm (300X, high magnification). C: quantitative analysis of FosB/ΔFosB-positive/pro-DYN-positive neurons in the NAc(shell). F: quantitative analysis of FosB/ΔFosB-positive/TH-positive neurons in the NTS. Data correspond to mean ± SEM. ⁺p<0.05 versus sham+morphine. ac, anterior comisure, AP, area postrema; cc, canal central. Data correspond to mean ± SEM. ADX significantly attenuated the increase of FosB/ΔFosB expression in pro-DYN-positive neuron in the NAc and in TH-positive neurons in the NTS from morphine-dependent animals (***p<0.001 versus sham+placebo; ⁺p<0.05,⁺⁺⁺p<0.001 versus sham+morphine).</p

Table <b>1.</b> Effects of adrenalectomy on the number of CRH-positive neurons in the BNST, PVN, and CeA, pro-DYN-positive neurons in the NAc and TH-positive neurons in the NTS, in placebo- or morphine-pelleted animals.

<p>Each value represents the mean ± SEM. n = 6 per group. In ADX rats, Newman–Keuls’ post hoc comparison test revealed a significant decrease in the number of CRH⁺ neurons in the BNST and CeA (*p<0.05; ***p<0.001, versus sham+placebo; ⁺⁺⁺p<0.01, versus sham+morphine). CRH, corticotropin-releasing factor; PVN, hypothalamic paraventricular nucleus; BNST, bed nucleus of the stria terminalis; CeA, central nucleus amygdala; NAc, nucleus accumbens; NTS-A₂ nucleus of the solitary tract-A₂ noradrenergic cell group.</p