Corticotropin (ACTH) acts directly on amygdala neurons to down-regulate corticotropin-releasing hormone gene expression

Abstract

The hormone corticotropin (ACTH) is employed as therapy for diverse neurological disorders, but the mechanisms for its efficacy remain unknown. ACTH promotes the release of adrenal steroids (glucocorticoids), and most ACTH effects on the central nervous system (CNS) have been attributed to activation of glucocorticoid receptors. However, in several human disorders, ACTH has therapeutic actions that differ qualitatively or quantitatively from those of steroids. This study tested the hypothesis that ACTH directly influences limbic neurons via the recently characterized melanocortin receptors and focused on the effects of ACTH on the expression of corticotropin-releasing hormone (CRH), a neuropeptide involved in neuroimmune functions and in certain developmental seizures. The results demonstrated that ACTH potently reduced CRH expression in amygdala neurons. This down-regulation was not abolished by experimental elimination of steroids or by blocking their receptors and was reproduced by a centrally administered ACTH fragment that does not promote steroid release. Importantly, selective blocking of melanocortin receptors prevented ACTH-induced down-regulation of CRH expression. Taken together, these data indicate that ACTH activates central melanocortin receptors to modulate CRH gene expression in amygdala, supporting the notion that direct, steroid-independent actions of ACTH may account for some of its established clinical effects on the CNS.

Fig 1

Differential regulation of CRH mRNA levels in ACe, hippocampus, and hypothalamic PVN by ACTH and adrenal steroids. Photomicrographs of coronal brain sections subjected to ISH for CRH mRNA. CRH mRNA expression in ACe (arrows, top row), PVN (arrows, second row), and hippocampus (third row) are shown. ACTH treatment decreased CRH mRNA signal over ACe. Adrenalectomy (ADX), by increasing endogenous ACTH levels, led to a similar reduction of CRH gene expression in ACe (top row). In contrast to the ACe, ADX resulted in up-regulation of CRH mRNA levels in PVN (middle row) and reduced CRH mRNA signal in hippocampus (third row) compared to control sections. The bottom row depicts representative darkfield photomicrographs of ACe (encircled region in left two panels) and PVN (right two panels). A marked reduction in the number of CRH-expressing cells is evident in ACTH-treated animals compared to controls. In PVN, increased CRH mRNA signal is visible in sections from an ADX animal. Scale bar = 1,500 μm in top two rows, 90 μm in third row, 200 μm in bottom row. Asterisks = basolateral nucleus; mpd, mpv = medial dorsal and medial ventral parvicellular cell groups of PVN, respectively.

Fig 2

Effects of ACTH on CRH mRNA levels in ACe. (A) Semiquantitative analysis of CRH mRNA expression in ACe of immature rats subjected to selective hormone level manipulations. Signal was analyzed over ACe following ISH as discussed in Materials and Methods. (B) CRH mRNA levels in ACe correlated inversely with plasma ACTH. Thus, ACTH administration (ACTH i.p.) or augmentation of endogenous ACTH (ADX) or both (ACTH i.p. + ADX) significantly reduced CRH mRNA levels in ACe compared to both the i.p. vehicle-injected and the sham-adrenalectomy, vehicle-injected control groups. No correlation between plasma corticosterone levels and ACe-CRH mRNA was observed (C). *Significant difference from control and sham ADX; **significant difference from all other groups (p < 0.05). Values depict means ± SEM. ADX = adrenalectomy.

Fig 3

CRH mRNA expression in ACe is down-regulated by centrally administered ACTH analog that is devoid of glucocorticoid secretion. Semiquantitative analysis (see Materials and Methods) of CRH mRNA signal in sections of animals receiving an i.c.v. infusion of ACTH_4–10, an analog binding melanocortin receptors but not stimulating corticosterone (Cort). Although infusion of ACTH_4–10 did not induce Cort secretion (hatched bars, scale on right), this analog reduced ACe-CRH mRNA levels significantly (solid bars, left scale). Values are means ± SEM; *p < 0.05.

Fig 4

Effects of ACTH on CRH mRNA expression in ACe require activation of MC4-Rs but not of glucocorticoid receptors. (A) High endogenous plasma ACTH levels were achieved by ADX, significantly reducing CRH mRNA levels (single asterisks). Administration of SHU9119, however, abolished ACTH-induced reduction of CRH mRNA. (B) In contrast, coadministered RU 38486 failed to block the effects of ACTH (i.p.) on CRH mRNA expression in ACe. In fact, CRH mRNA levels in ACTH+ RU 38486 treated rats were significantly lower from those given ACTH alone, as denoted by double asterisks. Values denote means ± SEM of values achieved using semiquantitative analysis (see Materials and Methods). i.c.v. Controls and i.p. controls received vehicle via the noted routes; ADX/icv were adrenalectomized and given i.c.v. vehicle. Diamonds over the i.p. control and ACTH groups indicate that they correspond to groups shown in Fig 2A.

Fig 5

Differential regulation of CRH mRNA expression in hippocampal regions of immature rats by glucocorticoids and ACTH. Semiquantitative analysis of signal over dentate gyrus (A), CA3 (B), and CA1 (C) was performed after ISH, as described in Materials and Methods. CRH mRNA levels in CA1 (the region rich in steroid receptors in the immature rat) were reduced by elimination of GCs (ADX), regardless of ACTH levels (C). CRH mRNA expression in other hippocampal regions was not appreciably altered by these hormonal manipulations.*Significant difference from i.p. vehicle-injected control and sham-ADX animals given i.p. vehicle (p < 0.05). Values are means ± SEM. ADX = adrenalectomy.