Glucocorticoid regulation of stress-induced mineralocorticoid receptor gene transcription in vivo.

Abstract

The neuronal mineralocorticoid receptor (MR) binds endogenous corticosterone (B) with high affinity.' The ligand-activated MR is transported to the cell nucleus, where it is capable of affecting transcription of a wide variety of genes.2 Within the central nervous system, the MR is of highest abundance within the hippocampus, thus leading to its hypothetical association with hippocampally mediated negative feedback control of the hypothalamo-pituitary-adrenocortical (HPA) stress axis.3~~ Understanding regulation of the neuronal MR is critical to elucidating its true function within the nervous system. In the present experiments, we employed a semiquantitative in situ hybridization strategy to assess in vivo stress regulation of neuronal MR mRNA and gene expression. For characterization of the MR intron probe, brains of normal Sprague-Dawley rats were sectioned at 20 pm and sections through the hippocampus processed for in situ hybridization. Adjacent series were hybridized with probes complementary to 1) the 3' coding and proximal 3' untranslated region of the MR mRNA (exon probe) and 2) the 5' portion of a coding region intron lying between exons 2 and 3 (intron probe). The latter probe was designed to recognize unspliced or partially-spliced gene transcripts present in the cell nucleus as heteronuclear (hn) RNA. An additional series was preincubated with R N h e A to verify probe specificity. Sections were dipped in NTB2 liquid emulsion and counterstained with cresyl violet. For analysis of stress and glucocorticoid effects on MR gene and mRNA regulation, rats received either adrenalectomy (ADX) or sham operation (SHAM), and were subsequently implanted with pellets containing either cholesterol (ADX-C and SHAM-C rats) or 30% B/70% cholesterol (ADX-B rats). Acute restraint stress (plastic restraint cages, 60 m) was imposed 5 days following pellet implantation, and groups of animals were killed 60 and 120 m post-stress, with unstressed rats serving as controls. Blood samples from all rats were assayed for B. Localization of signal generated by MR exon and intron probes are shown in FIGURE 1.MR exon signal was located diffusely throughout the cell (FIG. 1A,B) and showed a characteristic distribution across hippocampal subfields and dentate gyrus (FIG. lC).%' Cytoplasmic localization was confirmed on Nissl-counterstained sections (data not shown). In contrast, the MR intron probe showed a punctate distribution consistent with nuclear localization (FIG. lD,E). MR intron signal (FIG. 1F) could be localized to all hippocampal subfields, and was significantly less intense than exon signal at equal exposure times. The localization data indicate 1) compart-