Assessing behavioural effects of chronic HPA axis activation using conditional CRH-overexpressing mice

Abstract

The corticotropin-releasing hormone (CRH) and its cognate receptors have been implicated in the pathophysiology of stress-related disorders. Hypersecretion of central CRH and elevated glucocorticoid levels, as a consequence of impaired feedback control, have been shown to accompany mood and anxiety disorders. However, a clear discrimination of direct effects of centrally hypersecreted CRH from those resulting from HPA axis activation has been difficult. Applying a conditional strategy, we have generated two conditional CRH-overexpressing mouse lines: CRH-COE ( Del ) mice overexpress CRH throughout the body, while CRH-COE ( APit ) mice selectively overexpress CRH in the anterior and intermediate lobe of the pituitary. Both mouse lines show increased basal plasma corticosterone levels and consequently develop signs of Cushing's syndrome. However, while mice ubiquitously overexpressing CRH exhibited increased anxiety-related behaviour, overexpression of CRH in the pituitary did not produce alterations in emotional behaviour. These results suggest that chronic hypercorticosteroidism alone is not sufficient to alter anxiety-related behaviour but rather that central CRH hyperdrive on its own or in combination with elevated glucocorticoids is responsible for the increase in anxiety-related behaviour. In conclusion, the generated mouse lines represent valuable animal models to study the consequences of chronic CRH overproduction and HPA axis activation.

Fig. 1

Strategy for conditional overexpression of CRH. a Schematic representation of the ROSA26 (R26) locus, which was engineered to harbour a Cre-inducible Crh-LacZ expression unit (R26 ^flopCRH, flop: floxed stop). b Breeding to deleter-Cre or Pomc-Cre mice to remove the transcriptional terminator sequence (Cre recombinase expression pattern depicted in green). c Cre recombinase induced expression of CRH (depicted in orange) and β-galactosidase throughout the body (CRH-COE ^Del) or within the anterior pituitary (CRH-COE ^APit). R26 exons are indicated as black boxes, the transcriptional terminator as a STOP sign and loxP sites as green arrowheads. SA splice acceptor, IRES internal ribosomal entry side, pA polyA signal (Color figure online)

Fig. 2

Physiological and neuroendocrine alterations in male and female CRH-COE ^Del mice. a Body weight and b relative adrenal gland weights were significantly increased in male and female CRH–COE ^Del mice (black bars) compared to control littermates (white bars). c In addition, a decrease in relative thymus weight was observed in male CRH-COE ^Del mice. d Corticosterone levels measured in the morning (a.m.) and evening (p.m.) were significantly elevated in male and female COE mice, which also showed an attenuated stress response following 10-min of restraint stress compared to control animals. *Significantly different from control mice (P < 0.05), t trend (P ≤ 0.1), n.d. not determined, COE conditional overexpressing mice, Ctrl control littermates

Fig. 3

Behavioural characterization of male CRH-COE ^Del mice. a Locomotor activity in the open-field test was not altered in CRH-COE ^Del mice (black bars) compared to control littermates (white bars). Anxiety-related behaviour, as assessed in the b elevated plus-maze and c dark–light box test, was significantly increased in CRH-COE ^Del mice. d A mild increase in active stress-coping behaviour was observed in CRH-COE ^Del mice compared to control animals. *Significantly different from control mice (P < 0.05), t trend (P ≤ 0.1), COE conditional overexpressing mice, Ctrl control littermates

Fig. 4

Physiological and neuroendocrine alterations in male and female CRH-COE ^APit mice. a Body weight in homozygous male and female CRH-COE ^APit mice (black bars) was significantly decreased compared to control littermates (white bars). b A significant increase in relative adrenal gland weight was observed in male heterozygous (grey bars) and homozygous CRH-COE ^APit mice compared to control littermates. Relative adrenal gland weights were also significantly increased in homozygous female CRH-COE ^APit mice compared to their respective controls. c Relative thymus weight, assessed in males only, was significantly decreased in homozygous CRH-COE ^APit mice compared to control littermates. d A dosage-dependent increase in morning corticosterone levels was observed in heterozygous and homozygous male mice compared to control animals. Elevated coticosterone levels in the morning (a.m.), but not in the evening (p.m.) or in response to 10-min restraint stress, were also observed in female homozygous CRH-COE ^APit mice compared to control animals. *Significantly different from control mice (P < 0.05), not determined (n.d.). COE _het heterozygous conditional overexpressing mice, COE _hom homozygous conditional overexpressing mice, Ctrl control littermates

Fig. 5

Behavioural characterization of male CRH-COE ^APit mice. a Locomotor activity, measured in the open-field test, and anxiety-related behaviour, as assessed in the b elevated plus-maze and c dark–light box test, were not altered in CRH-COE ^APit (black bars) mice compared to control littermates (white bars). d An increase in swimming time was observed in homozygous CRH-COE ^APit mice in the FST. *Significantly different from control mice (P < 0.05), t = trend (P ≤ 0.1). COE _hom homozygous conditional overexpressing mice, Ctrl control littermates

Fig. 6

Baseline non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) of male CRH-COE ^APit mice. NREMS was significantly increased in CRH-COE ^APit mice (filled circles) during the second half of the dark period compared to control littermates (open circles). No differences in REMS could be detected between the two genotypes. White and black bars on the x-axis indicate the light and dark period, respectively. Vigilance states are indicated as percentages of 2 h means (±SEM). *Significantly different from control mice (P < 0.05). COE _hom homozygous conditional overexpressing mice, Ctrl control littermates