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

Abstract

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).

Experimental approach: The contribution of CRF₂ signalling in opiate withdrawal was assessed by i.c.v. infusion of the selective CRF₂ antagonist, antisauvagine-30 (AS-30). Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with AS-30 or saline 10 min before naloxone and the physical signs of abstinence, the HPA axis activity, NA turnover, TH activation and CRF₂ expression were measured using immunoblotting, RIA, HPLC and immunohistochemistry.

Key results: Rats pretreated with AS-30 showed decreased levels of somatic signs of naloxone-induced opiate withdrawal, but the corticosterone response was not modified. AS-30 attenuated the increased production of the NA metabolite, 3-methoxy-4-hydroxyphenylglycol, as well as the enhanced NA turnover observed in morphine-withdrawn rats. Finally, AS-30 antagonized the TH phosphorylation at Serine40 induced by morphine withdrawal.

Conclusions and implications: These results suggest that physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by CRF₂ signalling. Furthermore, they indicate a marginal role for the HPA axis in CRF₂-mediation of opiate withdrawal.

Figure 1

Attenuation of the severity of somatic signs of naloxone-precipitated morphine withdrawal by antisauvagine-30 microinfusion. The following variables were counted: (A) wet dog shakes; (C) body weight loss; (D) paw tremour and checked: (B) piloerection; (E) mastication; (F) teeth chattering. Somatic signs of withdrawal were observed during 30 min immediately after naloxone injection (1 mg·kg⁻¹ s.c.). A global withdrawal score (G) was calculated for each animal as described in the Methods. Data are expressed as mean ± SEM. ⋆P < 0.050; ⋆⋆⋆P < 0.001, versus morphine + vehicle (VEH) + naloxone; ⋆⋆P < 0.01; ⋆⋆⋆P < 0.001 versus similar groups receiving chronic placebo.

Figure 2

Antisauvagine-30 (AS-30) attenuated the plasma ACTH (A) but not the corticosterone (B) response to naloxone-induced morphine withdrawal. Data represent the mean ± SEM of plasma ACTH and corticosterone concentration 30 min after naloxone injection to control pellets- or morphine-treated rats administered vehicle, or AS-30 10 min before naloxone administration. ***P < 0.001 versus control pellets + vehicle + naloxone; ⁺⁺⁺P < 0.001 versus placebo + AS-30 + naloxone; #P < 0.05 versus morphine + vehicle + naloxone. ACTH, adrenocorticotropic hormone.

Figure 3

Effects of CRF₂ 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. AS-30 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 AS-30 10 min before naloxone (nx) administration. *P < 0.05; **P < 0.01 versus control pellets (placebo) + saline + naloxone; ⁺P < 0.05 versus control pellets + AS-30 + nx; #P < 0.05; ## P < 0.01 versus morphine-treated rats + saline + naloxone. CRF₂, corticotropin-releasing factor type-2 receptor; NA, noradrenaline; MHPG, 3-methoxy-4-hydroxyphenylethylen glycol; PVN, paraventricular nucleus; AS-30, antisauvagine-30; veh, vehicle.

Figure 4

Morphine withdrawal-induced TH phosphorylation at Ser40 is dependent on the activation of CRF₂ 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 the administration of naloxone in the absence or presence of an i.c.v. infusion of AS-30 (20 µg·2 µL⁻¹) 10 min before naloxone administration. Data correspond to mean ± SEM. In morphine-dependent rats, a post hoc comparison test revealed a significant increase in TH phosphorylation at Ser31 during morphine withdrawal, which was not antagonized by AS-30. By contrast, the increase in TH phosphorylated at Ser40 after naloxone-precipitated morphine withdrawal was attenuated in rats pretreated with AS-30. *P < 0.05; **P < 0.01 versus control pellets (placebo) + saline + naloxone; ⁺P < 0.05 versus control pellets + AS-30 + naloxone; #P < 0.05 morphine-treated rats + saline + naloxone. TH, tyrosine hydroxylase; CRF₂, corticotropin-releasing factor type-2 receptor; NTS-A_2, nucleus of the solitary tract-A2 noradrenergic cell group; AS-30, antisauvagine-30; veh, vehicle; nx, naloxone.

Figure 5

Representative immunoblots of CRF₂ 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 loading control. Data represent the optical density of immunoreactive bands expressed as a percentage (%) of the mean ± SEM of placebo control band. ***P < 0.001 versus control group. CRF₂, corticotropin-releasing factor type-2 receptor; NTS, nucleus of the solitary tract; veh, vehicle; nx, naloxone; pla, placebo; mor, morphine.