Localization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization

doi:10.1016/S0006-8993(96)01070-0

Brain Research

Volume 744, Issue 1, 2 January 1997, Pages 47-56

https://doi.org/10.1016/S0006-8993(96)01070-0 Get rights and content

Abstract

The capacity to sense changes in the concentrations of extracellular ions is an important function in several cell types. For example, hormone secretion by parathyroid cells and thyroid C-cells is primarily regulated by the level of extracellular ionized calcium (Ca²⁺). The G-protein-coupled receptor that mediates the parathyroid cell response to Ca²⁺ has been cloned and we have used in situ hybridization to map calcium receptor (CaR) mRNA expression in the adult rat brain. Cells expressing CaR mRNA were present in many areas of the brain suggesting that a variety of cell types express the CaR. Particularly high numbers of CaR expressing cells were found in regions associated with the regulation of fluid and mineral homeostasis, most notably the subfornical organ. These data suggest that the capacity to detect changes in extracellular Ca²⁺ concentrations may have important functional consequences in several neural systems.

Introduction

The maintenance of bodily fluid and ionic homeostasis requires a complex interaction between several organ systems including the kidney, gut and brain. Each of these systems has specialized cells that respond to changes in extracellular ionic concentrations yet the cellular mechanisms by which such changes are detected are not well understood. The concentration of ionized calcium (Ca²⁺) in the plasma and extracellular fluids is the primary regulator of parathyroid hormone (PTH) secretion from the parathyroid gland and calcitonin (CT) secretion from thyroid C-cells. In this endocrine system, Ca²⁺ functions as a circulating hormone in a classical endocrine feedback loop to regulate PTH and CT secretion and thus maintain bodily calcium homeostasis. A large body of physiological and biochemical data led to the hypothesis that parathyroid cells, C-cells, and kidney cells express a cell surface receptor that enables them to detect and respond to small changes in the concentration of extracellular Ca²⁺3, 23. This hypothesis was recently confirmed by the cloning of calcium receptor (CaR) cDNAs from bovine and human parathyroid glands 4, 9, rat kidney [28]and rat C-cells [10]. More recently, a cDNA with a putative amino acid sequence identical to that cloned from rat kidney has been isolated from rat striatal cDNA library [33].

The parathyroid, kidney, and brain CaRs are predicted to be glycosylated proteins of approximately 140 kDa with an unusually large extracellular domain and the seven membrane spanning region characteristic of other G-protein-coupled receptors 4, 9, 28. CaRs share limited homology with only one other subfamily of G-protein-coupled receptors, the metabotropic glutamate receptors (mGluRs). The bovine parathyroid and rat kidney CaR clones are 92% identical at the amino acid level [28]. The bovine receptor couples to phosphatidylinositol turnover and the mobilization of intracellular calcium in parathyroid cells [3]and the bovine, human and rat CaRs all stimulate the mobilization of intracellular calcium when expressed in the human embryonic kidney 293 cell line (HEK 293 cells) [30].

Northern blot and RT-PCR analyses have indicated that CaR mRNA is present in a variety of tissues in addition to the parathyroid and thyroid glands including kidney, the gastrointestinal tract, and the brain [4]. The role of the CaR in these organs is uncertain; however, the identification of specific cell types that express this receptor could help to elucidate the functional significance of the CaR in these locations. In the present study, we have utilized in situ hybridization with probes complementary to the cloned rat CaR to perform more detailed localization of transcripts for the receptor in adult rat brain. Cells expressing the CaR mRNA are scattered widely throughout the brain suggesting the possibility of a key role for extracellular Ca²⁺-sensing in brain function. Most notably, a high density of CaR-expressing cells in the subfornical organ (SFO) and associated regions suggests that circulating Ca²⁺ may act at the CNS level, as well as at the parathyroid gland, in order to maintain bodily calcium homeostasis. In addition, CaR expression in other CNS regions may explain previously poorly understood actions of extracellular Ca²⁺ and further study of the role of CaR these areas may propose previously unknown roles for extracellular Ca²⁺-sensing in CNS function.

Section snippets

Animals and tissue preparation

Four adult male Sprague-Dawley rats, 150–200 g (Harlan Sprague Dawley, Madison, WI) were housed in hanging wire cages under incandescent lighting, with a 12:12 h light/dark cycle. They were provided with unlimited access to rat chow (Purina 5001) and tap water. The animals were anesthetized by an intramuscular injection of 23 mg ketamine HCl, 2 mg xylazine, decapitated and the brains were quickly removed and frozen in liquid nitrogen. Serial 16-μm sections were cut on a cryostat and thaw

Results

Cells expressing CaR mRNA are widely scattered throughout the CNS; however, the relative density of cells within a given region varies considerably. CNS regions that contain CaR-expressing cells are listed in Table 1. The relative density of CaR-expressing cells within a region were ranked on a scale of one to four, with one representing widely scattered cells and four representing the highest density of cells. In most areas CaR-expressing cells appeared as an intense cluster of silver grains

Discussion

We have used in situ hybridization with a 1.2 kb cRNA probe to the rat kidney CaR to map the specific anatomical localization of CaR expression within the rat brain. We have found CaR-expressing cells throughout the CNS; however, the density of these cells within specific regions varies considerably. Many areas contain isolated, widely scattered CaR-expressing cells whereas a limited number of nuclei contain a very high density of CaR-expressing cells.

Structurally, the CaR resembles the

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Research reportLocalization of calcium receptor mRNA in the adult rat central nervous system by in situ hybridization