Elsevier

Brain Research

Volume 1030, Issue 2, 31 December 2004, Pages 221-233
Brain Research

Research report
Immunohistochemical mapping of calcitonin receptors in the adult rat brain

https://doi.org/10.1016/j.brainres.2004.10.012Get rights and content

Abstract

Calcitonin receptors (CTR) have previously been identified in specific regions of the rat central nervous system using in situ hybridization or autoradiography with iodinated ligands. In this study, the results of immunohistochemical mapping of CTR in the adult rat brain are reported, using a potent and recently developed antibody that recognizes an intracellular epitope of the rat CTR, and high-resolution immunofluorescence techniques. Abundant expression was found in the brain, with highest densities in the nucleus accumbens, lateral arcuate nucleus, lateral substantia nigra, bed nucleus of the stria terminalis, locus coeruleus, area postrema, nucleus of the solitary tract, and some of the nuclei of the reticular formation. These results are in close correspondence with previous mapping studies. However, we detected CTR immunoreactivity in several additional brain areas, as the ventromedial, lateral and posterior hypothalamus, where CT binding has not yet been described. Our detailed mapping of the CTR in the rat brain has identified CTR-positive cells that will be important for subsequent characterization of behavioral functions associated with the actions of CT-related peptides.

Introduction

Calcitonins (CT) are a family of peptides, which have a wide variety of physiological actions affecting calcium homeostasis, food intake, gastric acid secretion, the release of pituitary hormones and pain perception [44]. Some of these effects are supposed to be exerted through direct action on the central nervous system (CNS) via specific binding to CT receptors (CTRs).

These CTRs belong to the superfamily of seven-transmembrane domain G-protein-coupled receptors [14]. According to the molecular structure, tissue distribution and ligand binding affinity, two different isoforms of the rat CTR have been identified, namely, C1a and C1b [2], [34].

The C1a isoform is abundant in peripheral tissues, for instance, in kidney [2], [39] and in the developing CNS [41] and has a high affinity for salmon calcitonin (sCT, Kd=8 pM [2]), but less for rat and human CT [13], [34]. The C1b isoform, which has an additional 37-amino-acid sequence, was also found expressed in kidney [39]. This isoform has a lower affinity for sCT (48 pM [2]) than C1a and negligible interaction with human CT in transfected COS-1 cells [13], [34]. The difference in the binding affinities suggested that the two isoforms have different physiological ligands and thus different functional roles. However, these features, especially in the case of the C1b isoform, have not yet been well characterized.

The pancreatic polypeptide amylin (37 amino acids), a member of the CT family, shares certain biological actions with sCT, especially an inhibitory effect on food intake [15], [18], [19]. Both isoforms of the CTR, when co-expressed with receptor activity modifying protein (RAMP) 1, 2 or 3 on the surface of different cell lines, may function as an amylin receptor [6], [22], [27], [40], [46]. Such RAMP-coupled CTRs also exhibit high affinities for sCT. The anorectic actions of both amylin and sCT are thought to be exerted through RAMP-coupled CTRs expressed on neurons of the area postrema [18], [19] where the expression of RAMP2 and 3 mRNA has already been reported [4], [38].

The distribution of CTR in the adult rat CNS has only been mapped using in vitro autoradiography [10], [11], [12], [24], [37] and in situ mRNA hybridization [36] so far. However, a recently developed and potent CTR antibody now enables the application of immunohistochemistry (IHC) and has already been used in mapping studies of the fetal CNS [41] and the developing rat kidney [39]. This novel antibody directly detects the CTR protein by recognizing an epitope common to both C1a and C1b isoforms. While in situ hybridization locates only the mRNA of the CTR gene and in vitro autoradiography detects the pharmacological binding of a ligand, IHC enables identification of the receptor at the protein level. Thus IHC, besides confirming some of the previous results, will also greatly extend our knowledge about the distribution of CTR protein itself.

There are conflicting data derived from functional mapping by central microinjections of sCT in the CNS and mapping studies of CTR. The actions of sCT were evident in regions of the brain where previously no CTR has been detected, or on the contrary, no effect was observed where high densities of CTR were reported [5]. The CTR mapping data presented in this study were correlated with the functional studies as reported to clarify some of these apparent incongruities.

In our current paper, we present the results of an immunohistochemical mapping of the CTR C1(a+b) in the adult rat brain, using the recently developed CTR antibody. The findings as outlined above are compared with previous data derived using in vitro autoradiography or in situ hybridization, as well as with functional mapping studies.

Section snippets

Materials and methods

Adult male Wistar rats (n=3) weighing 240 g were anesthetized with an intraperitoneal pentobarbital injection (Nembutal; Abbott Laboratories, USA; 80 mg/kg) and perfused transcardially with ice-cold phosphate buffer (PhB 0.1M), followed by 4% paraformaldehyde (in 0.1M PhB). The brains were removed and kept in 4% paraformaldehyde for additional 2 h to achieve proper tissue fixation. After 48 h of incubation in 20% sucrose solution at 4 °C (in 0.1M PhB) for cryoprotection, the samples were

Results

The rabbit polyclonal anti-rodent CTR antibody used in this study has been described and characterized in previous studies [39], [41]. Furthermore, immunofluorescence studies in transfected COS cells (Dr Roman Muff, unpublished data) demonstrated that the antibody resulted in a strong signal with rat CTR, but did not cross-react with rat calcitonin receptor-like receptor (V5-tagged) and poorly with human CTR (c-myc tagged).

The anatomical mapping of the CTR antibody that recognizes both C1a and

Discussion

The anatomical distribution of CTR protein (including both isoforms C1a and C1b) in the adult rat brain, as investigated by immunohistochemistry, is reported here for the first time. We present a detailed topographical localization and a scored analysis of the CTR proteins throughout the CNS (Table 1).

In the present study, the nuclei ArcL, the SNL, the NTS and the AP showed the highest density of CTR expressing neurons. With respect to the fibers, the most dense immunofluorescence labeling was

Acknowledgements

We wish to sincerely thank Dr Roman Muff (Zurich) for generously providing the additional data useful in the further characterization of the anti-CTR antibody and described in the Results. This project has been supported by the Swiss National Research Foundation (#3100-066694) and the Olga Mayenfisch Foundation.

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