Forebrain CRF₁ modulates early-life stress-programmed cognitive deficits

Abstract

Childhood traumatic events hamper the development of the hippocampus and impair declarative memory in susceptible individuals. Persistent elevations of hippocampal corticotropin-releasing factor (CRF), acting through CRF receptor 1 (CRF₁), in experimental models of early-life stress have suggested a role for this endogenous stress hormone in the resulting structural modifications and cognitive dysfunction. However, direct testing of this possibility has been difficult. In the current study, we subjected conditional forebrain CRF₁ knock-out (CRF₁-CKO) mice to an impoverished postnatal environment and examined the role of forebrain CRF₁ in the long-lasting effects of early-life stress on learning and memory. Early-life stress impaired spatial learning and memory in wild-type mice, and postnatal forebrain CRF overexpression reproduced these deleterious effects. Cognitive deficits in stressed wild-type mice were associated with disrupted long-term potentiation (LTP) and a reduced number of dendritic spines in area CA3 but not in CA1. Forebrain CRF₁ deficiency restored cognitive function, LTP and spine density in area CA3, and augmented CA1 LTP and spine density in stressed mice. In addition, early-life stress differentially regulated the amount of hippocampal excitatory and inhibitory synapses in wild-type and CRF₁-CKO mice, accompanied by alterations in the neurexin-neuroligin complex. These data suggest that the functional, structural and molecular changes evoked by early-life stress are at least partly dependent on persistent forebrain CRF₁ signaling, providing a molecular target for the prevention of cognitive deficits in adults with a history of early-life adversity.

Figure 1.

A, B, Spatial learning and memory in adult wild-type and CRF₁-CKO mice that were exposed to either control condition or early-life stress. A, In the Y-maze test, all groups of animals spent more time exploring the novel arm versus the known ones. However, stressed wild-type mice performed significantly worse than wild-type controls, while stressed CRF₁-CKO mice displayed performance comparable to that of the controls. B, In the Morris water maze test, stressed wild-type mice took significantly longer to locate the hidden platform in the first spatial training day, indicating impaired acquisition of spatial information. In contrast, stressed CRF₁-CKO mice spent similar time to reach the platform compared with the controls. C, D, Spatial learning and memory in adult wild-type and CRF-COE mice. C, In the Y-maze test, wild-type mice showed preference to the novel arm, whereas CRF-COE mice visited the novel and familiar arms similarly. D, In the Morris water maze test, CRF-COE mice exhibited profound spatial learning impairments in the first two spatial training days and reduced cognitive flexibility in the reversal learning session. CT, Control; ES, early-life stress; KO, CRF₁-CKO; OE, CRF-COE. *p < 0.05, **p < 0.01 versus control wild-type group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus respective novel arm. n = 10–20 mice per group.

Figure 2.

A, B, Effects of early-life stress on mossy fiber-CA3 LTP (A) and SCCP-CA1 LTP (B) in adult wild-type and CRF₁-CKO mice. Representative traces for control and LTP are shown. A, After a HFS was delivered, mossy fiber-CA3 LTP was absent in stressed wild-type mice as indicated by significantly reduced amplitude of fEPSP in the last 10 min compared with wild-type controls. In contrast, CRF₁-CKO mice showed intermediate LTP. B, In the CA1 region, no difference in fEPSP potentiation was noticed between control and stressed wild-type mice, whereas LTP was surprisingly enhanced in the slices of stressed CRF₁-CKO mice. CT, Control; ES, early-life stress. *p < 0.05 versus the control group. ^###p < 0.001 versus stressed wild-type group. n = 4–6 mice per group.

Figure 3.

Effects of early-life stress on apical dendritic spine density in the stratum radiatum of CA3 and CA1 in adult wild-type and CRF₁-CKO mice. A, In the CA3 region, spine density was reduced in stressed wild-type and control CRF₁-CKO mice, whereas stressed CRF₁-CKO mice had more spines than control and stressed counterparts. B, Representative photomicrographs showing the apical dendrites and spines of Golgi-impregnated CA3 pyramidal neurons. C, In the CA1 region, stressed CRF₁-CKO mice had more exuberant spines than the control CRF₁-CKO and stressed wild-type groups. D, Representative photomicrographs showing the apical dendrites and spines of Golgi-impregnated CA1 pyramidal neurons. CT, Control; ES, early-life stress; KO, CRF₁-CKO. Scale bars, 2 μm. **p < 0.01, ***p < 0.001 versus the control group. ^#p < 0.05, ^##p < 0.01 versus the group under the same condition. n = 4 mice per group.

Figure 4.

Effects of early-life stress on excitatory and inhibitory synaptic density in area CA3 and CA1 in adult wild-type and CRF₁-CKO mice. A, The number of VGLUT1-positive synaptic puncta in CA3 stratum radiatum was significantly reduced in stressed wild-type but not stressed CRF₁-CKO mice. B, VGAT puncta density in area CA3 remained unchanged among groups. C, Representative confocal images taken from the stratum radiatum of CA3a (alongside stratum lucidum) showing VGLUT1- and VGAT-immunoreactive synaptic puncta. D, The number of VGLUT1-positive puncta in CA1 stratum radiatum was also decreased in stressed wild-type but not stressed CRF₁-CKO mice. E, VGAT puncta density in area CA1 was reduced by early-life stress in both wild-type and CRF₁-CKO mice. F, Representative confocal images taken from the stratum radiatum of CA1b (alongside stratum pyramidale) showing VGLUT1- and VGAT-immunoreactive synaptic puncta. CT, Control; ES, early-life stress; KO, CRF₁-CKO. Scale bars, 5 μm. *p < 0.05, **p < 0.01 versus the control group. n = 3–4 mice per group.

Figure 5.

Effects of early-life stress on hippocampal neurexin-1 and neuroligin-3 gene expression in adult wild-type and CRF₁-CKO mice. A, Neurexin-1 mRNA expression in area CA3 was reduced by early-life stress in wild-type but not CRF₁-CKO mice. B, Representative in situ hybridization images showing neurexin-1 mRNA expression in the hippocampus. C, Neuroligin-3 mRNA levels in area CA1 were reduced by early-life stress in both wild-type and CRF₁-CKO mice. D, Representative in situ hybridization images showing neuroligin-3 mRNA expression in the hippocampus. E, CRF₁ and neurexins partially colocalized in the perisomatic terminals and dendrites of CA1 pyramidal neurons. Arrows indicate CRF₁-EGFP and neurexins colocalized puncta. CT, Control; ES, early-life stress; KO, CRF₁-CKO. Scale bars: Left and middle two panels, 10 μm; right panel, 2 μm. *p < 0.05 versus the control group. n = 6–11 mice per group.