Colocalization of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and hypothalamus
Introduction
Adrenal cortex secretes corticosteroid hormones in response to adrenocorticotropin (ACTH) and corticosteroids enter the brain where these steroids have a wide range of regulatory effects on the brain function, such as the regulation of neurotransmitters, neuronal excitability, growth and cell survival as well as neuroendocrine function (Meyer, 1985, Reul and de Kloet, 1985, McEwen et al., 1986). The effects of corticosteroids on the brain are mediated by two types of corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) (McEwen et al., 1986). It has been demonstrated that MR and GR had a different character in their affinity for ligands: MR has a higher affinity for corticosterone and aldosterone than GR (Beaumont and Fanestil, 1983, Reul and de Kloet, 1985). MR or GR mediates the gene transcription by binding to its hormone responsive element (HRE) in the DNA as a homodimer or heterodimer in a ligand-dependent manner (Evans, 1988). MR and GR can bind to the same HRE (Bas et al., 1996). MR and GR are closely related in the primary structure of an amino acid sequence, particularly in the DNA binding and ligand binding domains.
Adrenal corticosteroids regulate their own secretion via a negative feedback system at the level of the hypothalamus and the pituitary which inhibit the synthesis and/or release of corticotropin releasing hormone (CRH) and ACTH, establishing the hypothalamo–pituitary–adrenal (HPA) axis. The negative feedback of corticosteroids is mediated by GR at the hypothalamus and pituitary levels (de Kloet et al., 1998). Besides these regulatory systems, recent studies have demonstrated that the tonic inhibitory influence of corticosteroid on HPA activity was exerted via MR in the suprahypothalamic structure which includes the hippocampus (Meaney et al., 1996). The limbic structure restrains HPA activity via the inhibitory GABAergic system (Herman and Cullinan, 1997). At the lower level of corticosterone during the circadian trough MR is occupied and operated in a pro-active fashion in the maintenance of homeostasis, whereas at the higher level of corticosterone as seen at the circadian peak or after stress GR is activated in reactive mode (de Kloet, 2000).
Previous studies have been conducted with respect to the distribution of MR and/or GR in the brain by receptor autoradiography (Lombes et al., 1994), in situ hybridization (Herman et al., 1989) and immunohistochemistry (Ahima et al., 1991, Cintra et al., 1994, Bas et al., 1996). These studies have shown that MR and GR were distributed in each discrete region of the brain and partial colocalization of these two receptors in the hippocampus was noted.
We have been successful in raising specific antibodies against rat MR (Ito et al., 2000) and GR (Morimoto et al., 1996). Our previous studies along with in situ hybridization and green fluorescent protein imaging clearly demonstrated the cellular and subcellular distributions of MR and GR in the hippocampus in vivo (Ito et al., 2000) and in the primary cultured neurons in vitro (Nishi et al., 1999, Nishi et al., 2001).
In the present study, we focused on the colocalization and subcellular distribution pattern of MR and GR in the hippocampus as well as the hypothalamic paraventricular nucleus (PVN) and the arcuate nucleus (ARC) with the use of double immunohistochemistry and examined changes of these two receptors by adrenalectomy (ADX) and corticosterone replacement.
Section snippets
Animals
Wistar male rats weighing 220–300 g maintained in air-conditioned rooms (22 ± 1 °C) on a 12 h/12 h light/dark cycle under the conditions of free access to food and drinking water (for ADX-rats, 0.9% NaCl in drinking water was given throughout the experimental course). The rats were divided into three experimental groups: (1) sham-operated rats (n = 4), (2) 7-day ADX rats (n = 4), (3) 7-day orally corticosterone (10 mg/ml) administrated rats after ADX (n = 4).
All of the rats used in this study were treated
Immunoblotting
The affinity-purified anti-MR and anti-GR antibodies were subjected to immunoblotting experiments to examine their specificities. As shown in lane 1 of Fig. 1A, a single band with a molecular mass of 106 kDa was labeled by the MR antibody in the MR-transfected COS-1 cells, whereas no band with a molecular mass of 87 kDa for GR was detected with MR antibody (Fig. 1A, lane 2). On the other hand, the GR antibody recognized a major band with a molecular mass of 87 kDa only in the GR-transfected cells (
Specificity of the MR- and GR-antibodies
Previous immunocytochemical and green fluorescent protein (GFP)-labeled GR imaging studies have shown that GR was localized in the cytoplasmic region in the absence of ligand, and in the nucleus in the presence of ligand (Picard and Yamamoto, 1987, LaFond et al., 1988, Nishi et al., 2001). On the other hand, the result of immunocytochemical studies for MR has been controversial for subcellular distribution (McEwen et al., 1986, Funder and Sheppard, 1987, Herman et al., 1989, Ahima and Harlan,
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