Elsevier

Neuroscience Research

Volume 30, Issue 1, January 1998, Pages 65-82
Neuroscience Research

Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody

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Abstract

The distribution of a metabotropic glutamate receptor mGluR2 in the central nervous system was immunohistochemically examined in the rat and mouse with a monoclonal antibody raised against an N-terminal sequence of rat mGluR2 (amino acid residues 87–134). Neuronal cell bodies with mGluR2-like immunoreactivity (mGluR2-LI) were clearly shown in the horizontal cells of Cajal in the cerebral cortex, neurons in the triangular septal nucleus and medial mammillary nucleus, Golgi cells and the unipolar brush cells in the cerebellar cortex, and Golgi-like and unipolar brush-like cells in the cochlear nucleus. Neuropil was intensely immunostained in the accessory olfactory bulb, bed nucleus of the accessory olfactory tract, neocortex, cingulate cortex, retrosplenial cortex, subicular and entorhinal cortices, stratum lacunosum-moleculare of CA1 and CA3, molecular layer of the dentate gyrus, periamygdaloid cortex, basolateral amygdaloid nucleus, bed nucleus of the anterior commissure, caudate-putamen, accumbens nucleus, thalamic reticular nucleus, anteroventral and paraventricular thalamic nuclei, granular layer of the cerebellar cortex, anterior and ventral tegmental nuclei, granular layer of the cochlear nucleus, and parvicellular part of the lateral reticular nucleus. Many axons in the white matter and fiber bundles were also immunostained. No glial cells with mGluR2-LI were found. No particular species differences were found in the distribution pattern of mGluR2-LI between the rat and mouse. The results indicate that mGluR2 is expressed not only in somato-dendritic domain, but also in axonal domain of excitatory and inhibitory neurons.

Introduction

The metabotropic glutamate receptors (mGluRs) are involved in many brain functions and pathology, and functional diversity of mGluRs is reflected in the presence of multiple receptor subtypes (for review, see Nakanishi and Masu, 1994, Pin and Duvoisin, 1995). Eight subtypes of mGluRs have so far been cloned, several of which exist in alternative splicing variants. They are classified into three groups according to their amino acid sequence similarities, transduction mechanism, and agonist selectivity: mGluR1 and mGluR5 (group I); mGluR2 and mGluR3 (group II); mGluR4, mGluR6, mGluR7 and mGluR8 (group III) (for review, Nakanishi and Masu, 1994, Pin and Duvoisin, 1995; also see Flor et al., 1997).

Group II mGluRs, mGluR2 and mGluR3, are about 70% identical to each other in amino acid sequence, negatively linked to adenylate cyclase activity via G protein, and selectively activated with (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (Tanabe et al., 1993, Hayashi et al., 1993). On the other hand, the in situ hybridization histochemistry in the rat has indicated the distinct patterns of distribution of mGluR2 and mGluR3 (Ohishi et al., 1993a, Ohishi et al., 1993b), and revealed the most prominent expression of mGluR2 mRNA in the accessory olfactory bulb and the hippocampal dentate gyrus. In the rat accessory olfactory bulb, it has been indicated that activation of mGluR2 on granule cell dendrites at the presynaptic sites of the dendrodendritic synapses between the granule cells and the mitral cells inhibits the GABAergic transmission from the granule cells to the mitral cells (Hayashi et al., 1993). In the mouse hippocampus, activation of mGluR2 on mossy fiber terminals from the granule cells of the dentate gyrus has been suggested to suppress glutamatergic synaptic transmission from the mossy fiber terminals to the hippocampal pyramidal neurons in CA3 (Yokoi et al., 1996).

The previous studies also reported the distribution of immunoreactivity for group II mGluRs in the rat brain by using polyclonal antibodies (Ohishi et al., 1994, Petralia et al., 1996). The antibodies used in these studies, however, recognize both mGluR2 and mGluR3. Thus, we raised a monoclonal antibody against an N-terminal sequence of rat mGluR2 (MAb mG2Na-5; Neki et al., 1996a) and obtained immunohistochemical evidence indicating that mGluR2 is located not only presynaptically but also postsynaptically (Neki et al., 1996a, Neki et al., 1996b). In the present study, the distribution of immunoreactivity for mGluR2 was examined systematically throughout the brain and spinal cord in the adult rat and mouse. The brains of the mGluR2 gene-lacking mouse (Yokoi et al., 1996) were also immunostained with the monoclonal antibody to confirm the specificity of mGluR2-like immunoreactivity. The distribution pattern of mGluR2-like immunoreactivity (mGluR2-LI) as revealed with the monoclonal antibody for mGluR2 was different from that as revealed with the polyclonal antibody for mGluR2/3.

Section snippets

Materials and methods

A total of eight adult male rats weighing between 200 and 300 g (Sprague–Dawley; Oriental Bioservice, Kyoto, Japan) were used to examine the distribution of mGluR2-like immunoreactivity (mGluR2-LI) in the brain and spinal cord by using the mouse monoclonal antibody, which was produced by using a glutathione S-transferase fusion protein containing an N-terminal sequence of rat mGluR2, putative extracellular amino acid residues 87–134 (MAb mG2Na-5; Neki et al., 1996a). Six male adult wild-type

Results

The both groups of the sections, the sections fixed by PA fixation and those fixed by non-PA fixation, were immunostained in each of the CNS regions in the rat and mouse. In the sections fixed by PA fixation, mGluR2-LI was observed as diffuse immunostaining of neuropil. In most of mGluR2-like immunoreactive neuropil, no profiles of immunostained neurons were detectable. On the other hand, in the sections fixed by non-PA fixation, neuronal cell bodies and proximal dendrites were immunostained,

Discussion

In the previous immunohistochemical studies so far reported, two kinds of polyclonal antibodies were used to examine the distribution of group II mGluRs. Ohishi et al. (1994)made a polyclonal antibody from a bacterial fusion protein, of which immunoreactivity was blocked with a synthetic 25-amino acid peptide corresponding to a C-terminus of rat mGluR2 (residues 848–872). This 25-amino acid sequence differed by five amino acids from that of rat mGluR3 C-terminus (residues 855–879), and the

Acknowledgements

The authors are grateful for photographic help of Mr Akira Uesugi. The authors also express their gratitudes for the support of Drs Satoru Fukuchi, Ritsu Hayashi, Sohzaburo Hayashi, Mizuho Katsurada, Hitoshi Kawai, Yutaka Kitani, Toshihiko Kuroda, Keiko Kumagai, Hiroshi Matsubara, Hiroshi Matsushima, Chisato Minakuchi, Gonpei Niwa, Hajime Oda, Masahiko Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Tamaki, Eizo Watanabe, Kazuo Yoshino, and Toshiaki Yoshino. This work was supported in

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