Expression of alpha(1b) adrenoceptor mRNA in corticotropin-releasing hormone-containing cells of the rat hypothalamus and its regulation by corticosterone

Abstract

Considerable evidence supports a role for brainstem adrenergic and noradrenergic inputs to corticotropin-releasing hormone (CRH) cells of the hypothalamic paraventricular nucleus (PVN), in the control of hypothalamic-pituitary-adrenocortical (HPA) axis function. However, little is known about specific adrenoceptor (ADR) subtypes in CRH-containing cells of the PVN. Here we demonstrate, using dual in situ hybridization, that mRNA encoding alpha(1b) ADR is colocalized with CRH in the rat PVN. Furthermore, we confirm that these alpha(1b) ADR mRNA-containing cells are stress-responsive, by colocalization with c-fos mRNA after restraint, swim, or immune stress. To determine whether expression of alpha(1b) ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7, or 14 d. In situ hybridization revealed levels of alpha(1b) ADR mRNA were increased in the PVN 7 and 14 d after ADX, but were not altered in the hippocampus, amygdala, or dorsal raphe. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50 mg) or placebo for 7 d. Corticosterone replacement (10 mg) reduced the ADX-induced increase in PVN alpha(1b) ADR mRNA to control levels, whereas 50 mg of corticosterone replacement resulted in a decrease in PVN alpha(1b) ADR mRNA as compared with all other groups. Furthermore, levels of plasma corticosterone were significantly correlated (inverse relationship) with alpha(1b) ADR mRNA in the PVN. We conclude that alpha(1b) ADR mRNA is expressed in CRH-containing, stress-responsive cells of the PVN and is highly sensitive to circulating levels of corticosterone. Because activation of the alpha(1B) adrenoceptor is predominantly excitatory within the brain, we predict that this receptor plays an important role in facilitation of the HPA axis response.

Fig. 1.

Dual in situ hybridization to show coexpression of α_1b ADR mRNA in CRH mRNA-containing cells of the PVN. A, C, CRH mRNA in the PVN, labeled with a nonradioactive (digoxigenin) probe, viewed under bright-field illumination. B, D, α_1b ADR mRNA in the PVN, labeled with a radioactive (³⁵S) probe, viewed under dark-field illumination. Thebox in B represents the approximate area over which cells were counted on each side of the PVN.Arrows in C and D indicate examples of double-labeled cells. Scale bars: A, B, 400 μm; C, D, 50 μm. Subdivisions of the PVN:dp, dorsal parvocellular part; mpd,medial parvocellular part, dorsal zone; mpv, medial parvocellular part, ventral zone; pml, posterior magnocellular part, lateral zone.

Fig. 2.

Dual in situ hybridization to show coexpression of α_1b ADR mRNA in c-fos mRNA-containing cells of the PVN 30 min after IL-1β (5 μg/kg, i.p.). A, C, c-fos mRNA in the PVN, labeled with a nonradioactive (digoxigenin) probe, viewed under bright-field illumination. B, D, α_1b ADR mRNA in the PVN, labeled with a radioactive (³⁵S) probe, viewed under dark-field illumination. The box in B represents the approximate area over which cells were counted on each side of the PVN.Arrows in C and D indicate examples of double-labeled cells. Scale bars: A, B, 400 μm; C, D, 50 μm.

Fig. 3.

Expression of α_1b ADR mRNA in the PVN 1, 3, 7, or 14 d after bilateral adrenalectomy (ADX) or sham surgery. Data are expressed as percentage of integrated density with respect to (w.r.t.) sham. *p < 0.05; compared with appropriate sham group, Tukey post hoc comparison test.

Fig. 4.

In situ hybridization to show expression of α_1b ADR mRNA after bilateral ADX with low (10 mg) or high (50 mg) corticosterone replacement. A,Coronal brain section from a naive rat at the level of the PVN to show general distribution of α_1b ADR mRNA.B–F, Expression of α_1b ADR mRNA in the PVN of naive (B), SHAM (C), ADX (D), ADX plus 10 mg of corticosterone (E), or ADX plus 50 mg of corticosterone (F). Scale bar, 500 μm.

Fig. 5.

a, Expression of α_1bADR mRNA in PVN after bilateral adrenalectomy (ADX) with low (10 mg) or high (50 mg) corticosterone replacement. Data are expressed as integrated optical density (arbitrary units). *p < 0.05, **p < 0.01 compared with sham group; †p < 0.05, †††p < 0.001 compared with ADX group; ##p < 0.01 compared with ADX plus 10 group; Student–Newman–Keuls post hocmultiple comparisons test. b, Correlation analysis (Pearson; all groups, r² = 0.638; p < 0.001; ADX plus 10 mg group,r² = 0.717;p < 0.05) between levels of α_1b ADR mRNA in PVN and plasma corticosterone at time of killing, after bilateral adrenalectomy with corticosterone replacement (0, 10, or 50 mg, s.c.) for 7 d.