Transient early-life forebrain corticotropin-releasing hormone elevation causes long-lasting anxiogenic and despair-like changes in mice

Abstract

During development, early-life stress, such as abuse or trauma, induces long-lasting changes that are linked to adult anxiety and depressive behavior. It has been postulated that altered expression of corticotropin-releasing hormone (CRH) can at least partially account for the various effects of stress on behavior. In accord with this hypothesis, evidence from pharmacological and genetic studies has indicated the capacity of differing levels of CRH activity in different brain areas to produce behavioral changes. Furthermore, stress during early life or adulthood causes an increase in CRH release in a variety of neural sites. To evaluate the temporal and spatial specificity of the effect of early-life CRH exposure on adult behavior, the tetracycline-off system was used to produce mice with forebrain-restricted inducible expression of CRH. After transient elevation of CRH during development only, behavioral testing in adult mice revealed a persistent anxiogenic and despair-like phenotype. These behavioral changes were not associated with alterations in adult circadian or stress-induced corticosterone release but were associated with changes in CRH receptor type 1 expression. Furthermore, the despair-like changes were normalized with antidepressant treatment. Overall, these studies suggest that forebrain-restricted CRH signaling during development can permanently alter stress adaptation leading to increases in maladaptive behavior in adulthood.

Figure 1.

Expression of CRH in FBCRHOE and control mice on and off doxycycline (doxy). A, B, Representative sections showing CRH mRNA expression from FBCRHOE, Tetop-CRH, CaMKII-tTA, and wild-type (WT) mice off (A) or on (B) doxy chow. C, Quantitation of CRH mRNA expression in FBCRHOE^life (off doxy entire lives) and control^life mice (off doxy entire lives) in the thalamus, PVN, CeA, BLA, caudate/putamen, somatosensory cortex (frontal Ctx), cingulate cortex (cingulate Ctx), DG, CA1, and CA3. In situ signal is normalized with mean control value for each individual anatomical area. D, Quantitation of CRH mRNA expression in FBCRHOE^dev (on doxy P21–P56 when tested) and control^dev mice (on doxy P21–P56 when tested). E, Sections from FBCRHOE^life and Tetop-CRH mice off doxy showing CRH immunohistochemical staining. Left panel, 10× magnification of hippocampus in FBCRHOE section with inset magnification of granule cell axon staining. FBCRHOE^life, Double transgenic mice off doxy entire lives; control^life, control mice off doxy entire lives; FBCRHOE^dev, double transgenic mice off doxy E0–P21 and on doxy >P21; control^dev, control mice off doxy E0–P21 and on doxy >P21. Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001 FBCRHOE^life vs control^life.

Figure 2.

Expression of CRH in FBCRHOE^dev and control^dev mice during development. Representative sections showing CRH mRNA expression from FBCRHOE^dev (OE) and control^dev (Cntl) mice at E12, E15, P1, P3, P7 and P14. All mice were off doxycycline when analyzed. Overexpression of CRH likely occurs between E12 and E15.

Figure 3.

Examination of corticosterone in mice exposed to CRH during development only. A, Basal corticosterone is increased in P15 and P20 transient overexpressors (FBCRHOE^dev) mice (n = 4–5) compared with control^dev mice (n = 4–5). B, No differences are seen between adult FBCRHOE^dev (n = 16) and control^dev mice (n = 16) in circadian corticosterone. C, Adult FBCRHOE^dev mice (n = 9) show no alterations in plasma corticosterone 0 or 90 min following restraint stress compared with control^dev mice (n = 9). OE, FBCRHOE^dev; Cntl, control^dev. Student's t test; *p = 0.05 compared with control^dev at specified postnatal day.

Figure 4.

FBCRHOE^dev mice show increased anxiety-like behavior. A–D, In the open-field test, FBCRHOE^dev (n = 11) mice show increased latency to enter the center of the open field (A), decreased time in the center of the open field (B), decreased number of entries center of the open field (C), and decreased distance traveled in the center of the open field compared with control^dev mice (n = 11) (D). E, FBCRHOE^dev show equivalent total distance traveled in open-field testing compared with control^dev mice. F–H, In the L:D preference task, FBCRHOE^dev mice (n = 11) show increased latency to enter the light (F), decreased number of entries into the light (G), and decreased time in the light compared with control^dev mice (n = 11) (H). Student's t test; *p ≤ 0.05; **p ≤ 0.01 compared with control^dev mice.

Figure 5.

FBCRHOE^dev mice exhibit despair-like behavior in the TST and FST. A, In the TST, FBCRHOE^dev mice (n = 12) show reduced activity compared with control^dev mice (n = 12). B, C, In the FST, FBCRHOE^dev mice (n = 9) show reduced activity (B) and reduced latency to float (C) compared with control^dev mice (n = 11). Student's t test; *p ≤ 0.05; **p = 0.01 compared with control^dev mice.

Figure 6.

Effects of chronic imipramine [antidepressant (AD)] on FBCRHOE^dev despair-like behavior. A, FBCRHOE^dev mice (n = 6) treated with chronic imipramine show no difference in TST activity compared with control^dev mice (n = 6) treated with imipramine. B, FBCRHOE^dev mice (n = 6) treated with chronic imipramine show no difference in FST activity compared with control^dev mice (n = 6) treated with imipramine. Saline (vehicle)-treated FBCRHOE^dev continued to show despair-like behavior compared with control^dev saline-treated mice in the TST (A) and FST (B). Bonferroni test; *p < 0.05 versus saline-treated control^dev.

Figure 7.

FBCRHOE^dev mice show changes in CRHR1 mRNA expression that are reversed by imipramine treatment. A, Quantitation of GR mRNA from in situ hybridization of FBCRHOE^dev mice and control^dev mice in the PVN, CeA, BLA, DG, and CA1. FBCRHOE^dev mice (n = 9) show equivalent GR expression compared with control^dev mice (n = 9). Right, Representative sections showing GR in situ signal. B, Quantitation of basal CRHR1 mRNA in FBCRHOE^dev and control^dev mice. FBCRHOE^dev mice (n = 3–9) show increased CRHR1 expression in the cingulate cortex (Ctx), DG and CA1 compared with control^dev mice (n = 4–9). On right, representative sections showing CRHR1 in situ signal in brain and pituitary gland (Pit). C, Chronic imipramine reduces CRHR1 expression in the Ctx and CA1 in FBCRHOE^dev mice (n = 4 per treatment). No differences were observed between control^dev mice treated with saline (n = 4) or imipramine (n = 4) for either area. Student's t test; *p < 0.05; **p = 0.01 compared with control^dev (B) or imipramine FBCRHOE^dev (C).