Dorsal raphé nucleus glucocorticoid receptors inhibit tph2 gene expression in male C57BL/6J mice

Abstract

The serotonergic dorsal raphé nucleus (DRN) expresses glucocorticoid receptors (GR), and systemic glucocorticoids have been shown to regulate expression and activity of tryptophan hydroxylase isoform 2, the rate-limiting enzyme for serotonin synthesis in brain. We have used intra-DRN injection of pseudotyped adeno-associated virus AAV2/9 transducing either green fluorescent protein (GFP control) or Cre recombinase (DRN GR deletion) in floxed GR mice to determine if DRN GR directly regulate DRN mRNA levels of tryptophan hydroxylase 2 (tph2). In a separate set of similarly-treated floxed GR mice, we also measured limbic forebrain region concentrations of serotonin (5-hydroxytryptamine; 5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA). DRN GR deletion increased tph2 mRNA levels in the dorsal, lateral wing, and caudal parts of the DRN without altering tissue concentrations of 5-HT, 5-HIAA, or the 5-HIAA/5-HT ratio in limbic forebrain regions. We conclude that DRN GR inhibit DRN tph2 gene expression in mice without marked effects on serotonin metabolism, at least under basal conditions at the circadian nadir. These data provide the first evidence of localized control of DRN tph2 mRNA expression by DRN GR in mice.

Keywords: 5-hydroxyindoleacetic acid (5-HIAA); Dorsal raphé nucleus; Glucocorticoid; Serotonin; Tryptophan hydroxylase.

Figure 1

Representative images of glucocorticoid receptor (GR) expression in the dorsal raphé nucleus (DRN, bregma −4.36 mm to −4.96 mm) and locus coeruleus (bregma −5.30 mm) in floxed GR mice after DRN injections of AAV2/9 virus transducing green fluorescent protein (GFP control) or Cre recombinase (GR deletion). Tryptophan hydroxylase isoform 2 immunoreactivity (Tph2; green pseudocolor) indicates the boundaries of the DRN, while GR immunoreactive nuclei are shown in red pseudocolor. Yellow dashed lines indicate the cerebral aqueduct; the fourth ventricle is above and medial to locus coeruleus images. Numbers on left indicate distance from bregma [29].

Figure 2

A, Representative examples of tryptophan hydroxylase isoform 2 (tph2) in situ hybridization histochemistry in the DRN of floxed GR mice after DRN injections of AAV2/9 virus transducing green fluorescent protein (GFP control) or Cre recombinase (GR deletion). White outlines show the dorsal DRN (DRD), the lateral wings (LW), the caudal DRN (DRC), and the interfascicular DRN (DRI), as defined by Donner et al. [8]. B, Semi-quantitative analysis of tph2 in situ hybridization histochemistry in GFP control (white bars) and DRN GR deletion groups (black bars). Numbers on the left indicate distance from bregma [29]. Mice were euthanized 3 weeks after DRN injection under basal conditions within 2h of lights-on. *, P < 0.05 vs. GFP control; n = 5 per group.