The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons

Abstract

Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract.

Keywords: Cav; P7C3; P7C3A20; anxiety; neurogenesis; neuroprotection.

Graphical abstract

Figure 1.

Ca_v1.2 supports adult hippocampal neurogenesis. A, Left, Graphical representation of the dorsal, medial, and ventral dentate gyrus (DG) in which BrdU⁺ staining was quantified. Right, Representative images of BrdU- and hematoxylin-stained DG from forebrain-Ca_v1.2 cKO and WT littermate mice. B, Forebrain-Ca_v1.2 cKO mice show significantly lower BrdU⁺ cells in the DG compared with WT animals (B; WT, n= 4; KO, n=4; **p = 0.004, independent samples t test). C, D, Forebrain-Ca_v1.2 cKO mice also show lower DCX protein (C; WT, n=3; KO, n=3) and mRNA levels (D; WT, n=6; KO, n=6; ***p < 0.001, independent samples t test) compared with WT animals. All graphs are represented as mean ± SEM.

Figure 2.

Selective elimination of Ca_v1.2 in adult dentate gyrus results in lower neurogenesis. A, Top, Representative image of GFP-labeled cells within the dentate gyrus of Ca_v1.2^fl/fl mice injected with AAV2/2-Cre-GFP. Bottom, AAV2/2-Cre-GFP significantly decreased Ca_v1.2 mRNA compared with control AAV2/2-GFP injected mice (A; AAV2/2-GFP, n=5; AAV2/2-Cre, n= 5; ***p < 0.001, independent samples t test). B, Adult focal hippocampal knockout of Ca_v1.2 results in significantly lower BrdU⁺ cells in the dentate gyrus (AAV2/2-GFP, n=6; AAV2/2-Cre, n= 9; ****p < 0.0001, independent samples t test). All graphs are represented as mean ± SEM.

Figure 3.

Ca_v1.2 controls survival of young hippocampal neurons, associated with lower BDNF levels in the absence of differences in corticosterone levels or hippocampus volume. A, C, Graphical representation of BrdU pulse chase experiments to determine proliferation (A) versus survival (C). IHC, Immunohistochemistry. B, D, Forebrain-Ca_v1.2 cKO mice display normal proliferation as compared with WT animals, with no difference in BrdU+ cells 1 h after BrdU administration (B; WT, n=4; KO=3; p = 0.935, independent samples t test). Forebrain-Ca_v1.2 cKO mice do, however, show a deficit in survival of young hippocampal neurons, as indicated by significantly lower BrdU+ cells in the dentate gyrus 30 d after BrdU injection (D; WT n=7; KO, n=5; **p = 0.002, independent samples t test). Arrows point to BrdU-positive cells. E, BDNF protein levels are significantly lower in forebrain-Ca_v1.2 cKO mice compared with WT animals (WT, n=6; KO, n=10; *p = 0.005, independent samples t test). F, Corticosterone levels are not different between forebrain-Ca_v1.2 cKO mice and WT animals (Basal: WT, n=14; KO, n=15; Stressed: WT, n=15; KO, n=7; main effect of basal versus stressed ****p < 0.0001; main effect of genotype p = 0.4232, two-way ANOVA). G, Nissl staining showed no differences between forebrain-Ca_v1.2 cKO and WT thickness of the dentate gyrus (DG; p = 0.986, independent samples t test), CA1 (p = 0.518, independent samples t test) and CA3 (p = 0.898, independent samples Mann–Whitney U test) layers of the hippocampus (WT, n= 5; KO, n= 9). All graphs are represented as mean ± SEM.

Figure 4.

Treatment with P7C3-A20 restores hippocampal neurogenesis in forebrain-Ca_v1.2 cKO mice without affecting BDNF levels. A–C, Treatment with the neuroprotective compound P7C3-A20 significantly increased the levels of BrdU⁺ cells in the dentate gyrus (A, B; Veh vs P7C3-A20; ****p < 0.0001; WT-Veh, n=3; KO-Veh, n=3; WT-A20, n=3; KO-A20, n=3), DCX protein levels using immunohistochemistry (C), and mRNA levels (D; Veh: WT vs KO; *p = 0.029; WT: Veh vs P7C3-A20, **p = 0.005; KO: Veh vs P7C3-A20; ****p < 0.0001; WT-Veh, n=8; KO-Veh, n=7; WT-A20, n=9; KO-A20, n=8) of both WT and forebrain-Ca_v1.2 cKO adult animals compared to vehicle-treated groups. E, P7C3-A20 had no effect on BDNF protein levels in either group (WT: Veh vs P7C3-A20, p = 0.9996; KO: Veh vs P7C3-A20, p > 0.999; WT-VEH, n=8; KO-VEH, n=5; WT-A20, n=8; KO-A20, n=6). All graphs are represented as mean ± SEM.