Genome-wide analysis of glucocorticoid-responsive transcripts in the hypothalamic paraventricular region of male rats

Purpose: we sought to search for genes that are transcribed or repressed in a glucocorticoid (GC)-dependent manner in a hypothalamic region containing the paraventricular nucleus of the hypothalamus (PVH), thereby unraveling mechanisms underlying GC-induced neuroendocrine and autonomic regulation. Methods: we employed unbiased quantification of the abundance of DNA transcripts provided by RNA-seq in the PVH region of male rats and identified genes whose expression is enhanced or suppressed by the 12-day exposure to a high dose of corticosterone (Cort) by Cort implant (12-day high Cort) or deprivation of corticoids for 7 days [7-day adrenalectomy (ADX)] (Experiment 1), as well as by acute Cort increase (Experiment 2). Sham-ADX animals served as controls in Experiment 1. In Experiment 2, rats underwent ADX and supplemented with Cort in drinking water. The animals were decapitated at 1 h [acute Cort (1) 1 h] or 3 h [acute Cort (1) 3 h] following injection of HBC Cort (1 mg), a dose shown to elicit plasma Cort surge comparable to that during acute stress. A group of rats without Cort injection served as controls (ADX + Cort suppl). Differential expression was calculated as fold-changes in gene expression [measured as fragments per kilobase of exon per million reads mapped (FPKM)]. The Benjamini & Hochberg method was applied to correct the unavoidable false positive data ensued from the multiple tests, and a q-value was calculated as the false discovery rate-adjusted p-value. The q-value < 0.05 was used as the cut-off criterion. Pearson’s correlation analysis was carried out for grouping genes that showed similar expression patterns. qRT–PCR validation was performed using Power SYBR Green PCR mix. Semi-quantitative in situ hybridization was carried out using 35S-labeled cRNA probes. Results: some canonical GC target genes, among others of a broad functional spectrum, were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in Avp and Aplnr expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, 6 immediate early genes, Bsx, Egr1, Fos, Fosb, Junb, and Nr4a3, were upregulated at 1 h after Cort injection, and the changes were confirmed by qRTPCR and semi-quantitative in situ hybridization. Conclusions: the present study uncovered a broad spectrum of Cort-responsive genes, potentially involved in the regulation of the HPA-axis, as well as other neuroendocrine and pre-autonomic functions of the PVH. Transcriptome profiles of the hypothalamic region containing the PVH were generated by deep sequencing, in duplicate, using Illumina HiSeq2000.