Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential roles of CRF receptors 1 and 2

Abstract

The differential modulation of learning and anxiety by corticotropin-releasing factor (CRF) through CRF receptor subtypes 1 (CRFR1) and 2 (CRFR2) is demonstrated. As learning paradigm, context- and tone-dependent fear conditioning of the mouse was used. Injection of CRF into the dorsal hippocampus before training enhanced learning through CRFR1 as demonstrated by the finding that this effect was prevented by the local injection of the unselective CRFR antagonist astressin, but not by the CRFR2-specific antagonist antisauvagine-30 (anti-Svg-30). In contrast, injection of CRF into the lateral intermediate septum impaired learning through CRFR2, as demonstrated by the ability of antisauvagine-30 to block this effect. When antisauvagine-30 was injected alone into the lateral intermediate septum, learning was enhanced. Such tonic control of learning was not observed when astressin or antisauvagine-30 was injected into the dorsal hippocampus. Injection of CRF after the training into the dorsal hippocampus and the lateral intermediate septum also enhanced and impaired learning, respectively. Thus, it was indicated that CRF acted on memory consolidation. It was concluded that the observed effects reflected changes of associative learning and not arousal, attention, or motivation. Although a dose of 20 pmol human/rat CRF was sufficient to affect learning significantly, a fivefold higher dose was required to induce anxiety by injection into the septum. Immobilization for 1 hr generated a stress response that included the induction of anxiety through septal CRFR2 and the subsequent enhancement of learning through hippocampal CRFR1. The involvement of either receptor subtype was demonstrated by region-specific injections of astressin and antisauvagine-30.

Fig. 1.

Anatomical localization of the injection sites for CRF receptor agonists and antagonists. Native brain sections of mice injected with methylene blue (a) and sections counterstained with nuclear fast red (b). Scale bar, 400 μm. i.c.v., Intracerebroventricular; DG, dentate gyrus; CA1, hippocampal subfield; LS, lateral septum; LV, lateral ventricle; MS, medial septum; TS, trigonal septal nucleus.

Fig. 2.

Dose-dependent effects of CRF-like peptides. Mice were injected intracerebroventricularly with h/rCRF, h/r CRF(6–33), rUcn, and oCRF 5 min before training. Context-dependent (a) and tone-dependent (b) fear conditioning were determined 24 hr later. Statistically significant differences: *p < 0.01 versus aCSF.

Fig. 3.

Region-specific effects of CRF-like peptides. Context- and tone-dependent fear conditioning after i.h. (a), i.s. (b), intracortical (c), or intracaudate (e) injection of 20 pmol peptide per mouse. Additional mice subjected to i.h. treatment but without receiving a shock (no shock) were used to test whether i.h. injection of h/rCRF produced immobility or place aversion (a). Statistically significant differences: *p < 0.01 versus aCSF; **p < 0.001 versus aCSF.

Fig. 4.

Dose-dependent effect of h/rCRF injected i.h. or i.s. Context- and tone-dependent fear conditioning of mice injected i.h. (a) or i.s. (b) 5 min before training was determined 24 hr later. Statistically significant differences: *p < 0.01 versus aCSF; **p < 0.001 versus aCSF.

Fig. 5.

Injections of h/rCRF before and after training. h/rCRF (20 pmol per mouse) was injected i.h. (a) and i.s. (b) before and after training as indicated. Freezing to context, novel context, and tone is presented. Statistically significant differences: *p < 0.001 versus aCSF.

Fig. 6.

Receptor-specificity of the effect of h/rCRF on fear conditioning. The CRFR antagonists astressin [300 ng (85 pmol) per mouse] or anti-Svg-30 [400 ng (100 pmol) per mouse] were injected 10 min before i.h. (a) or i.s. (b) administration of h/rCRF [100 ng (20 pmol) per mouse], which was applied 5 min before training. Statistically significant differences: *p < 0.001 versus aCSF.

Fig. 7.

Anxiogenic action of h/rCRF in the lateral intermediate septum. The time spent (a) and number of entries (b) on the open arms of an elevated plus-maze were determined 30 min after h/rCRF injection. The antagonists astressin (85 pmol per mouse) and anti-Svg-30 (100 pmol per mouse) were injected i.s. 10 min before the application of h/rCRF (500 ng per mouse), which was used 30 min before the plus-maze test. Statistically significant differences: *p < 0.01 versus aCSF.

Fig. 8.

Stress-induced enhancement of fear conditioning through hippocampal CRFR1. a, In mice subjected for 1 hr to immobilization and trained 3 hr later, fear conditioning to context and tone was significantly enhanced (p < 0.01). b, This effect was fully antagonized by astressin injected i.h. (85 pmol per mouse) either immediately before immobilization stress (astressin + stress) or 15 min before the training (stress + astressin). Statistically significant differences: *p < 0.001 versus nonstressed mice.

Fig. 9.

Stress-induced anxiety mediated through septal CRFR2. In mice subjected for 1 hr to immobilization, the time spent (a) and number of entries (b) on the open arms of an elevated plus-maze were significantly reduced after 30 min. CRFR antagonists astressin (85 pmol per mouse) and anti-Svg-30 (100 pmol per mouse) injected i.s. fully prevented the decrease of the time spent (c) and number of entries (d) on the open arms of the plus-maze observed 30 min after stress. Statistically significant differences: *p < 0.01 versus nonstressed mice.