Involvement of noradrenergic transmission in the PVN on CREB activation, TORC1 levels, and pituitary-adrenal axis activity during morphine withdrawal

Abstract

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 α(1)- 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 (α(1)-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 We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations.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.

Figure 1. Effects of naloxone-induced morphine withdrawal on NA and MHPG levels at the PVN and on NA turnover (as estimated by the MHPG/NA ratio.

Morphine withdrawal increased MHPG production and NA turnover. Data represent the mean ± SEM 60 min after naloxone injection to control pellets- or morphine-treated rats. **p<0.01; ***p<0.001 vs. control pellets (placebo)+naloxone; ⁺p<0.05, ⁺⁺⁺p<0.001 vs. morphine-treated rats+saline; ^## p<0.01 vs. placebo-treated rats+saline.

Figure 2. Morphine withdrawal-induced CREB activation in the PVN is dependent on α₁-adrenoceptor stimulation.

Quantitative analysis and representative immunoblots (A) of pCREB in the PVN tissue isolated from placebo or morphine-dependent rats pretreated with prazosin before saline or naloxone injection to control and to morphine-dependent rats. Post hoc analysis revealed that the increase in CREB phosphorylation during morphine withdrawal was blocked by prazosin (1 mg/kg i.p.). Each bar represents mean ± SEM (% of controls); p: placebo pellets; m: morphine pellets; veh: vehicle; n: naloxone; praz: prazosin. **p<0.01 vs control pellets (placebo)+vehicle+naloxone; ⁺⁺⁺p<0.001 vs. morphine-treated rats+ to control and to morphine-dependent rats. vehicle+naloxone. PVN was also processed for pCREB immunohistochemistry. (B, C) represents immunohistochemical detection of pCREB in the PVN from morphine-treated rats receiving vehicle and naloxone (B) or prazosin plus naloxone (C). 3V: third ventricle. Scale bar: 100 µm. D: quantitative analysis of pCREB immunoreactivity the PVN. Data correspond to mean ± SEM. Post hoc analysis revealed a significant decrease in pCREB immunoreactivity in prazosin-pretreated rats. *p<0.05 versus morphine+vehicle+naloxone.

Figure 3. Morphine withdrawal-induced CREB activation in the PVN is not dependent on β-adrenoceptor stimulation.

Quantitative analysis and representative immunoblots (A) of pCREB in the PVN tissue isolated from placebo or morphine-dependent rats pretreated with propranolol before saline or naloxone injection to control and to morphine-dependent rats. Post hoc analysis revealed that the increase in CREB phosphorylation during morphine withdrawal was not modified by propranolol (3 mg/kg i.p.). Each bar represents mean ± SEM (% of controls); p: placebo pellets; m: morphine pellets; veh: vehicle; n: naloxone; prop: propranolol. **p<0.01 vs. control pellets (placebo)+naloxone; ⁺⁺p<0.01 vs. placebo-treated rats+propranolol+naloxone. PVN was also processed for pCREB immunohistochemistry. (B, C) represents immunohistochemical detection of pCREB in the PVN from morphine-treated rats receiving vehicle and naloxone (B) or propranolol plus naloxone (C). 3V: third ventricle. Scale bar: 100 µm. D: quantitative analysis of pCREB immunoreactivity the PVN. Data correspond to mean ± SEM. Post hoc analysis revealed no significant effects of propranolol pretreatment on pCREB immunoreactivity.

Figure 4. Increased pCREB into CRF neurons after naloxone-induced morphine withdrawal is α-1 adrenoceptor dependent.

PVN tissue isolated from placebo or morphine-dependent rats pretreated with vehicle or prazosin before naloxone injection was processed for pCREB and CRF double-label immunohistochemistry. Top panels (A–C) represent immunohistochemical detection of pCREB into CRF neurons after the different treatments. Low and high magnifications images show pCREB-positive (blue-black)/CRF-positive (brown) neurons in the PVN. Scale bar: 100 µm (low magnification); 20 µm (high magnification). 3V, third ventricle. Bottom panels (D) show quantitative analysis of pCREB-positive/CRF-positive and total CRF-positive (with or without pCREB) neurons in the PVN. Data shown are means ± SEM. Post hoc test revealed a higher number of pCREB-positive nuclei in CRF immunoreactive neurons after naloxone-induced morphine withdrawal. This increase was antagonized in prazosin-pretreated rats. The increase in number of CRF-positive neurons during morphine withdrawal was also blocked by prazosin. ***p<0.001 versus placebo (pla)+vehicle (veh)+naloxone (nx); ⁺⁺p<0.01 versus mor+veh+nx.

Figure 5. Noradrenergic activity is required for morphine withdrawal-induced TORC1 activation in the hypothalamic PVN.

Quantitative analysis and representative immunoblots of phosphorylated TORC 1 in the PVN tissue isolated from placebo or morphine-dependent rats pretreated with vehicle or prazosin before saline or naloxone injection to control and to morphine-dependent rats. Post hoc analysis revealed that the decrease in TORC phosphorylation induced by morphine withdrawal was reversed by prazosin. Each bar represents mean ± SEM (% of controls); p: placebo pellets; m: morphine pellets; veh: vehicle; n: naloxone; praz: prazosin. **p<0.01 vs. control pellets (placebo)+vehicle+naloxone; ⁺⁺⁺p<0.001 vs. morphine-treated rats+vehicle+naloxone.

Figure 6. Hypothalamus-pituitary-adrenal (HPA) axis activation during morphine withdrawal is attenuated by α₁- but not β-adrenoceptor blockade.

Placebo and morphine-dependent rats were pretreated with prazosin or propranolol and plasma levels of corticosterone (a marker of HPA axis activity) were determined 60 min after naloxone injection. Praz: prazosin; prop; propranolol; sal: saline; nx: naloxone. Each bar represents mean ± SEM. Post hoc analysis revealed a significant increase in plasma corticosterone concentration after naloxone-induced morphine withdrawal, which was attenuated in prazosin- but not in propranolol-pretreated rats. ***p<0.001 versus placebo+naloxone; ⁺⁺⁺p<0.001 versus morphine+saline; ^&&&p<0.001 versus placebo+prazosin+naloxone; ^$$$p<0.001 versus morphine+prazosin+saline; ^##p<0.01 versus morphine+naloxone; @@@p<0.001 versus placebo+propranolol+naloxone; ^%%%p<0.001 versus morphine+propranolol+saline.