Glucocorticoid receptors participate in the opiate withdrawal-induced stimulation of rats NTS noradrenergic activity and in the somatic signs of morphine withdrawal

Abstract

Background and purpose: Recent evidence suggests that glucocorticoid receptor (GR) is a major molecular substrate of addictive properties of drugs of abuse. Hence, we performed a series of experiments to further characterize the role of GR signalling in opiate withdrawal-induced physical signs of dependence, enhanced noradrenaline (NA) turnover in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) phosphorylation (activation) as well as GR expression in the nucleus of the solitary tract noradrenergic cell group (NTS-A₂).

Experimental approach: The role of GR signalling was assessed by i.p. pretreatment of the selective GR antagonist, mifepristone. Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with mifepristone or vehicle 30 min before naloxone and physical signs of abstinence, NA turnover, TH activation, GR expression and the hypothalamus-pituitary-adrenocortical axis activity were measured using HPLC, immunoblotting and RIA.

Key results: Mifepristone alleviated the somatic signs of naloxone-induced opiate withdrawal. Mifepristone attenuated the increase in the NA metabolite, 3-methoxy-4-hydroxyphenylethylen glycol (MHPG), in the PVN, and the enhanced NA turnover observed in morphine-withdrawn rats. Mifepristone antagonized the TH phosphorylation at Ser³¹ and the expression of c-Fos expression induced by morphine withdrawal. Finally, naloxone-precipitated morphine withdrawal induced up-regulation of GR in the NTS.

Conclusions and implications: These results suggest that the physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by GR signalling. Overall, the present data suggest that drugs targeting the GR may ameliorate stress and aversive effects associated with opiate withdrawal.

Figure 1

Attenuation of the severity of somatic signs of naloxone-precipitated morphine withdrawal by mifepristone. Counted (A: wet-dog shakes; G: body weight loss) and assessed (B: tremor; C: sniffing; D: ptosis; E: teeth chattering; F: rinorrhoea; H: piloerection; I: chromodacryorrhoea). Somatic signs of withdrawal were observed for 30 min immediately after naloxone injection (1 mg·kg⁻¹ s.c.). A global withdrawal score (J) was calculated for each animal as described in the Methods. Data are expressed as mean ± SEM. ^##P < 0.01, ^###P < 0.001, versus morphine + vehicle (veh) + naloxone (nx); **P < 0.01, ***P < 0.001, versus placebo + veh + nx; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, versus placebo + mifepristone (mife) + naloxone (nx).

Figure 2

Effects of GR blockade on NA (A) and MHPG (B) levels at the PVN and on the morphine withdrawal-induced increased NA turnover (as estimated by the MHPG/NA ratio; C) in control and in morphine-dependent rats after administration of naloxone. Mifepristone (50 mg·kg⁻¹) attenuated morphine withdrawal-induced increase in MHPG levels and NA turnover. Data represent the mean ± SEM 30 min after naloxone injection to control pellets- or morphine-treated rats receiving vehicle or mifepristone thirty min before naloxone administration. *P < 0.05, **P < 0.01, versus control pellets (placebo) + vehicle (veh) + naloxone (nx); ^#P < 0.05, ^##P < 0.01, versus morphine + veh + nx.

Figure 3

Effects of mifepristone on neuronal activation in the NTS in response to morphine withdrawal. Representative immunoblots of c-Fos in NTS tissues isolated from placebo or morphine-dependent rats 30 min after administration of naloxone in absence or presence of mifepristone (50 mg·kg⁻¹) 30 min before naloxone administration. β-Actin was used as a loading control. Data represent the optical density of immunoreactive bands expressed as a percentage (%) of the mean ± SEM of placebo control band. *P < 0.05, versus the corresponding control group receiving placebo + vehicle (veh) + naloxone (nx); ^#P < 0.05, versus morphine + veh + nx.

Figure 4

Effects of GR blockade on morphine withdrawal-induced TH phosphorylation at Ser³¹ and Ser⁴⁰ in the NTS-A2 noradrenergic cell group. Representative immunoblots of TH phosphorylated at Ser³¹ (A) and Ser⁴⁰ (B) in NTS tissues isolated from placebo or morphine-dependent rats 30 min after administration of naloxone in the absence or presence of mifepristone (50 mg·kg^-1) 30 min before naloxone administration. β-Actin was used as a loading control. Data represent the optical density of immunoreactive bands expressed as a percentage (%) of the mean ± SEM of placebo control band. In morphine-dependent rats, post hoc comparison test revealed a significant increase in TH phosphorylation at Ser³¹ during morphine withdrawal, which was attenuated by mifepristone. By contrast, the increase in TH phosphorylated at Ser⁴⁰ after naloxone-precipitated morphine withdrawal did not was attenuated in rats pretreated with mifepristone *P < 0.05, **P < 0.01, versus control pellets (placebo) + vehicle (veh) + naloxone (nx); ^#P < 0.05, versus morphine-treated rats + veh + nx.

Figure 5

Representative immunoblots of GR expression in NTS tissue isolated from control pellets-treated (placebo) or morphine-dependent rats 30 min after administration of naloxone. β-Actin was used as a loading control. Data represent the optical density of immunoreactive bands expressed as the percentage (%) of the mean ± SEM of placebo control band. **P < 0.01, versus control group.

Figure 6

GR blockade did not modify the plasma corticosterone response to naloxone-induced morphine withdrawal. Data represent the mean ± SEM of plasma corticosterone concentration 30 min after naloxone injection to control pellets- or morphine-treated rats receiving vehicle or mifepristone (50 mg.kg⁻¹) 30 min before naloxone administration. ***P < 0.001, versus control pellets + vehicle (veh) + naloxone; ⁺⁺⁺P < 0.001, versus placebo + mifepristone (mife) + naloxone.