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, 22 (5), 733-744

Loss of Hypothalamic Corticotropin-Releasing Hormone Markedly Reduces Anxiety Behaviors in Mice

Affiliations

Loss of Hypothalamic Corticotropin-Releasing Hormone Markedly Reduces Anxiety Behaviors in Mice

R Zhang et al. Mol Psychiatry.

Abstract

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, whereas extra-hypothalamic CRH has a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma adrenocorticotropic hormone, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open-field, elevated plus maze, holeboard, light-dark box and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict interest.

Figures

Figure 1
Figure 1
Generation of Crhflox mouse and Crh null mouse (a). Schematic diagram of mouse Crh gene targeting. Wild-type (WT) Crh allele, targeting vector, Neo allele, flox allele, null allele are drawn. Broken lines indicate corresponding locations on each allele or the vector. The targeting vector has two artificial insertions, of which the upstream insertion contains one loxP sequence between exon1 (e1) and exon2 (e2), while the downstream insertion located downstream of exon2 contains the neomycin resistant gene (Neo) flanked by two frt sequences accompanied by an adjacent downstream loxP sequence. The location of two internal BamHI sites and one artificial BamHI site are indicated. Thymidine kinase (TK) is a negative selection marker. Characterization of Crhflox (Crhfl/fl) and CrhKO (Crhdl/dl) mice (b–q). (b–g) Crh-immuno-reactive neurons and fibers were observed in the paraventricular nucleus of hypothalamus (PVH) and central amygdala (CeA) in CrhWT (b, c) and Crhflox (d, e) but not in CrhKO mice (f, g) (scale bar, 100um); (h–k) Adrenal hypoplasia in CrhKO mice. Adrenals from adult male Crhflox (h, i) and CrhKO (j, k) were haematoxylin-eosin stained after paraformaldehyde fixation. CrhKO mice revealed thinner zona fasciculata. Left panel, low magnification (scale bar, 100um); right panel, high magnification (scale bar, 100um); (l–q) Confirmation of fetal lung dysplasia in CrhKO which was rescued by in utero corticosterone replacement. Crhflox fetus revealed normal lung development (l, m) while deletion of Crh cause hypercellular lungs with thick alveolar septae and a paucity of air spaces (n, o). The deficiency of lung development was rescued by corticosterone administration (30ug/ml in drinking water) to female pregnant CrhKO mice (p, q). Left panel, low magnification (scale bar, 100um); right panel, high magnification (scale bar, 100um);
Figure 2
Figure 2
Characterization of Sim1CrhKO mice. Crhflox mice were crossed with Sim1Cre mice to delete the floxed Crh gene in Sim1-expressing tissues; (a–f) Representative images for Crh mRNA expression in the PVH by in situ hybridization (Bregma −0.70mm to Bregma −1.06mm). Top row, Crhflox (control); middle row, Sim1CrhKO; bottom row, CrhKO. Arrows point to PVH; (g) Semi-quantitative densitometry of film images showed that Crh was deleted in the PVH area of Sim1CrhKO mice, (Sim1CrhKO versus Crhflox, n=8 versus 8); (h) Q RT-PCR analysis revealed that Crh mRNA expression decreased in the PVH but not in the brainstem in Sim1CrhKO mice compared to the control (Sim1CrhKO versus Crhflox, n=5 versus 4; Control, filled bars; Sim1CrhKO, open bars); (i–l) Crh immunohistochemistry staining in the PVH and CeA. Sim1CrhKO showed less Crh-ir positive neurons and fibers in the PVH but comparable amount of Crh-ir positive staining in CeA area. Arrows point to PVH and CeA, scale bar: 100um; PVH, paraventricular nucleus of the hypothalamus; CeA, central nucleus of the amygdala; (m–r) Physiological characterization of Sim1CrhKO mice; (m–p) Adrenal hypoplasia in Sim1CrhKO versus Crhflox control mice: Whole adrenals and haematoxylin-eosin stained adrenal sections from the widest diameter, respectively, of Crhflox (m, o) and Sim1CrhKO (n, p) male mice; (q) Plasma ACTH was lower in male Sim1CrhKO (7) versus Crhflox (10) mice; (r) Plasma corticosterone was lower in Sim1CrhKO versus Crhflox mice at diurnal morning (9–10am) and evening (5–6pm) in males (n = 7 versus 10); *p<0.05 versus control.
Figure 3
Figure 3
Analysis of anxiety-like behaviors by the open field test and holeboard test. (a, b) Compared to Crhflox control mice, Sim1CrhKO mice spent more time in the center area and less time in wall areas while both groups spent equivalent amounts of time in neutral areas; c) Sim1CrhKO mice revealed decreased latency of first entry into center and neutral areas compared with control mice, whereas there were no differences for velocity; d) Sim1CrhKO mice entered into center more frequently compared to control animals; e) Holeboard analysis revealed Sim1CrhKO mice had significantly increased entrance frequency into the center compared to Crhflox controls, and that this occurred during the first and second 5 minute periods; f) Fine movement was significantly increased in Sim1CrhKO compared to Crhflox mice. *P<0.05, Sim1CrhKO versus Crhflox control mice (n = 7 versus 10).
Figure 4
Figure 4
Analysis of anxiety-like behaviors by the elevated plus maze (EPM), light-dark box and novel object recognition test (NORT). a, b) EPM analysis revealed that deletion of Crh in Sim1Cre neurons significantly decreased the entrance frequency into the closed arms compared to Crhflox control mice; c) Sim1CrhKO mice spent significantly more time in the open arms in term of duration and percentage compared to control mice; d) Sim1CrhKO compared with Crhflox mice had a shorter latency to emerge from darkness into the light box. However, there were no differences in other measured anxiety parameters (time spent in the light part, frequency into light box. d) There were no significant differences for duration in center and closed arms between control and Sim1CrhKO mice; e) Deletion of Crh from Sim1 neurons significantly decreased latency to novel objects while increased latency to familiar objects; f) Sim1CrhKO mice exhibited significantly increased touching frequency for novel objects while decreased exploring behaviors for familiar objects; *P<0.05, Sim1CrhKO versus Crhflox control mice (n = 7 versus 10).
Figure 5
Figure 5
Corticosterone supplementation did not reverse anxiolytic behaviors in Sim1CrhKO mice. a) Morning (7–8am) plasma corticosterone increased in CrhfloxCort and Sim1CrhKOCort animals during administration of corticosterone (5ug/ml) in drinking water compared to CrhfloxCon mice; b) Sim1CrhKOCort mice spent more time in the center of open field test compared to CrhfloxCon and CrhfloxCort mice; c) During EPM testing, Sim1CrhKO mice with corticosterone supplementation (Sim1CrhKOCort), had significantly increased cumulative duration and the entrance frequency into the open arms compared to Crhflox control mice, while CrhfloxCort animals had decreased time in the open arms; d) During lightbox testing, Sim1CrhKOCort mice showed a non-significant trend of more time in the light area; e) During holeboard testing, Sim1CrhKOCort mice showed increased fine movement compared to CrhfloxCon and CrhfloxCort animals; f) During novel object testing, Sim1CrhKOCort mice showed a non-significant trend towards increased cumulative duration for novel objects; *P<0.05, Sim1CrhKOCort and CrhfloxCort versus CrhfloxCon; #P<0.05, CrhfloxCort versus CrhfloxCon. For all tests, Sim1CrhKOCort (n = 4), CrhfloxCon (n = 5) and CrhfloxCort (n = 4).

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