Reduced Gene Dosage of the Psychiatric Risk Gene Cacna1c Is Associated with Impairments in Hypothalamic-Pituitary-Adrenal Axis Activity in Rats

Abstract

Common and rare variation in CACNA1C gene expression has been consistently associated with neuropsychiatric disorders such as schizophrenia, bipolar disorder, and major depression. However, the underlying biological pathways that cause this association have yet to be fully determined. In this study, we present evidence that rats with a reduced gene dosage of Cacna1c have increased basal corticosterone levels in the periphery and reduced the expression of Nr3c1 encoding the glucocorticoid receptor in the hippocampus and hypothalamus. These results are consistent, with an effect of Cacna1c dosage on hypothalamus-pituitary-adrenal (HPA) axis function. Heterozygous Cacna1c rats had lower levels of the histone markers H3K4me3 and H3K27acat exon 1₇ of the Nr3c1 gene. These histone modifications are typically linked to increased gene expression, but here were not associated with changes in the expression of exon 1₇ variants under non-stress conditions. Heterozygous Cacna1c rats additionally show increased anxiety behaviours. These results support an association of Cacna1c heterozygosity with the altered activity of the HPA axis and function in the resting state, and this may be a predisposing mechanism that contributes to the increased risk of psychiatric disorders with stress.

Keywords: Cacna1c; L-type voltage-gated calcium channel; anxiety; bipolar disorder; glucocorticoid; hippocampus; schizophrenia; stress.

Figure 1

(A). Cacna1c^+/− rats (HET) had reduced Nr3c1 gene expression in the hippocampus and the hypothalamus compared to wild types (WTs). (B). There were no differences in Nr3c2 expression in any brain region tested. Medians and quartiles are depicted on each plot (whiskers run to the smallest datapoint within 1.5 × IQR below Q1 and the largest datapoint within 1.5IQR above Q3). Measures from individual rats are shown as black dots. Results were considered significant if p < 0.05 (*). Although transformations were used in the analysis, for clarity, untransformed data are presented here (see Figure S1 for visualisation of transformed data). Nr3c1: hippocampus, HET n = 11, WT n = 15; PFC, HET n = 7, WT n = 11; Hypo HET n = 8, WT n = 7. Nr3c2: hippocampus, HET n = 10, WT n = 13; PFC, HET n = 6, WT n = 10; Hypo HET n = 6, WT n = 7.

Figure 2

(A) Top: The % of methylation at each targeted CpG site in the hippocampus of WT rats and Cacna1c^+/− rats is displayed (top left) with no significant differences between the genotypes, with the exception of CpG 14 (top right). The highest % methylation was within at the 5′ CpG site (CpG₁₆) within the NGFIA binding site. Bottom: Schematic showing the genetic sequence that indicates Exon 1₇ within the promoter region of Nr3c1 in the rat [49]. The red region indicates the sequence of Exon 1₇, with the NGFIA binding site contained within, highlighted in purple. The sequence analysed by this study is underlined, and the CpG dinucleotides investigated are in bold (corresponding to CpGs 10–17 [49]. n = 12 per group. (B–E) Cacna1c^+/− rats show reduced DNA interacting with histone modification markers of active transcription H3K4me3 and H3K27ac within the exon 1₇ region (B,C). No differences were seen in the region closer to the transcription start site (D,E). Medians and quartiles are depicted on each plot (whiskers run to the smallest datapoint within 1.5 × IQR below Q1 and the largest datapoint within 1.5IQR above Q3). n = 10 per group except c, where n = 11. Measures from individual rats are shown as black dots. Although transformations were used in the analysis, for clarity, untransformed data are presented here (see Figure S2 for visualisation of transformed data). Results were considered significant if p < 0.05 (*) or p < 0.05 (**).

Figure 3

(A) Male Cacna1c^+/− rats (HETs) had higher circulating corticosterone levels than WT males (n = 19 per group). (B) Male Cacna1c^+/− rats had similar CRH hormone levels to WT males (n = 12 per group). In a separate mixed-sex cohort (n: male WT = 13, male HET = 13, female WT = 11, and female HET = 12) (C), Cacna1c^+/− rats showed an increased peripheral corticosterone concentration compared to WT rats. (D) A profound sex difference in corticosterone levels was also observed, with females showing higher levels than males. (E) There was no difference in the ratio of peripheral corticosterone/CBG between genotypes (p = 0.248). (F) Corticosterone/CBG did not differ between sexes (p = 0.199). There were no sex-by-genotype interactions (p > 0.05, see main text for details), and main effects are presented only. Medians and quartiles are depicted on each plot (whiskers run to the smallest datapoint within 1.5 × IQR below Q1 and the largest datapoint within 1.5IQR above Q3). Measures from individual rats are shown as black dots. Although transformations were used in the analysis, for clarity, untransformed data are presented here (see Figure S4 for visualisation of transformed data). * p < 0.05, *** p < 0.001.

Figure 4

(A) There was a reduction in time spent in the inner 50% of an OF arena in Cacna1c^+/- rats (HET) compared to WT animals. (B) In the same cohort, there were no differences of genotype regarding the time spent in the open arms of the EPM. However, female rats spent more time in the open arms compared to males, which was associated with WT females spending more time in the open arms than WT males. Medians and quartiles are depicted on each plot (whiskers run to the smallest datapoint within 1.5 × IQR below Q1 and the largest datapoint within 1.5IQR above Q3). Although transformations were used in the analysis, for clarity, untransformed data are presented here (see Figure S6 for visualisation of transformed data). Measures from individual rats are shown as black dots. * p < 0.05. Male HET n = 17; male WT n = 11; female HET n = 17; female WT n = 11.