Elsevier

Brain Research

Volume 1032, Issues 1–2, 25 January 2005, Pages 162-170
Brain Research

Research report
Immunohistochemical localization of gastrin-releasing peptide receptor in the mouse brain

https://doi.org/10.1016/j.brainres.2004.10.068Get rights and content

Abstract

Gastrin-releasing peptide (GRP) is a mammalian bombesin (BN)-like peptide that binds with high affinity to the GRP receptor (GRP-R). Previous behavioral studies using mice and rats showed that the GRP/GRP-R system mediates learning and memory by modulating neurotransmitter release in the local GABAergic network of the amygdala and the nucleus tractus solitarius (NTS). To date, the precise distribution of GRP-R in the brain has not been elucidated. We used a synthetic peptide derived from mouse GRP-R to generate affinity-purified antibodies to GRP-R and used immunohistochemistry to determine the distribution of GRP-R in the mouse brain. The specificity of anti-GRP-R antibody was confirmed in vitro using COS-7 cells transiently expressing GRP-R and in vivo using GRP-R-deficient and wild-type mouse brain sections. GRP-R immunoreactivity was widely distributed in the isocortex, hippocampal formation, piriform cortex, amygdala, hypothalamus, and brain stem. In particular, GRP-R immunoreactivity was observed in the lateral (LA), central, and basolateral amygdaloid (BLA) nuclei and NTS, which are important regions for memory performance. Double-labeling immunohistochemistry demonstrated that subpopulations of GRP-R are present in GABAergic neurons in the amygdala. Consequently, GRP-R immunoreactivity was observed in the GABAergic neurons of the limbic region. These anatomical results provide support for the idea that the GRP/GRP-R system mediates memory performance by modulating neurotransmitter release in the local GABAergic network.

Introduction

The mammalian bombesin (BN)-like peptides, gastrin-releasing peptide (GRP) [12] and neuromedin-B (NMB) [13], are widely distributed throughout the central nervous system and gastrointestinal tract. GRP is a homolog of amphibian BN, and NMB is the mammalian counterpart of another amphibian BN-like peptide, ranatensin [10]. Mammalian BN-like peptides have a wide spectrum of physiological effects, including regulation of exocrine and endocrine secretions, smooth muscle contraction, homeostasis, and behavior [10]. The mammalian BN-like peptides bind to G-protein-coupled BN receptors, GRP receptor (GRP-R) [2], [28], NMB receptor (NMB-R) [30], and an orphan receptor known as BN receptor subtype-3 (BRS-3) [3], [5]. To characterize the function of these endogenous BN-like peptides, we established and analyzed mouse lines deficient in GRP-R [32], NMB-R [17], or BRS-3 [16].

Several studies have shown that BN and GRP improve learning and memory retention in rats and mice. For example, peripheral administration of BN and/or GRP after training enhances memory retention of inhibitory avoidance tests [4], [19], [25]. The transport of peripherally administrated BN/GRP to the brain through the blood–brain barrier has not been reported. However, vagotomy inhibits the memory enhancing effects of both BN and GRP, suggesting that these peptides produce their effect by stimulating ascending vagal pathways [4]. The vagal fibers enter the midbrain at the level of the nucleus tractus solitarius (NTS), and NTS neurons project directly to the amygdala [23]. Immediate inactivation of the amygdala or NTS after inhibitory avoidance training attenuates the memory-enhancing effect of BN [19]. In addition, unilateral infusion of BN into the NTS enhances memory retention in inhibitory avoidance and radial arm maze tasks [33]. These results indicate that an intact amygdala and NTS may be required for memory retention mediated by the GRP/GRP-R system.

Intraperitoneal administration of a BN/GRP antagonist impairs memory retention in inhibitory avoidance tests [21], [26]. Memory retention in the inhibitory avoidance test is also disrupted by posttraining bilateral infusion of a BN/GRP receptor antagonist into the dorsal hippocampus and the basolateral amygdaloid nucleus (BLA) [20], [22]. In addition, unilateral injection of muscimol, a γ-aminobutyric acid type A (GABAA) receptor agonist, into the BLA blocks the memory-impairing effect of posttraining systemic administration of a BN/GRP-R antagonist [22]. Furthermore, Shumyatsky et al. [27] have shown that GRP-R-deficient mice have enhanced long-term potentiation in the lateral amygdaloid nucleus (LA) and persistent long-term fear memory. These authors also reported that GRP-R mRNA is expressed in GABAergic interneurons of the LA. Electrophysiological studies showed that the GRP/GRP-R system increases GABAergic inhibition in principal neurons [27]. These findings suggest that the GRP/GRP-R system modulates memory performance in the amygdala by regulating the GABAergic local network. However, the precise distribution of GRP-R in the brain has not been defined due to the lack of a suitable GRP-R-specific antibody. Therefore, to address this issue, we used GRP-R-specific antibody in immunohistological analyses to investigate the distribution of GRP-R in the limbic regions of the mouse brain, which are known to be important for memory performance [19], [20], [22], [27], [33].

Section snippets

Antibody production

A synthetic peptide was prepared based on the predicted amino acid sequence 179–190 (FSDLHPFHVKDT) of the second extracellular loop of mouse GRP-R [2]. An additional cysteine was included at the C terminus. A BLAST search was performed to confirm that the chosen peptide sequence was unique. The peptide was injected into rabbits to produce polyclonal antisera (IWAKI, Japan). GRP-R-specific polyclonal antisera were affinity-purified using the Sulfolink purification system (Pierce, USA). The

Specificity of anti-GRP-R antibody

We generated an affinity-purified polyclonal antibody against a mouse GRP-R-derived synthetic peptide. To confirm the specificity of this anti-GRP-R antibody, GRP-R cDNA subcloned into the pIRES2-EGFP vector (GRP-R/pIRES2-EGFP) was transfected into COS-7 cells, and the transfected cells were immunostained with anti-GRP-R antibody. Specific GRP-R immunoreactivity (in red) was detected in the cell body, and this signal merged with the EGFP signal (Fig. 1a). Immunocytochemistry was also performed

Discussion

Previous behavioral studies using mice and rats have shown that the GRP/GRP-R system mediates memory retention in the amygdala and NTS [19], [33]. However, the lack of availability of a GRP-R-specific antibody has precluded the elucidation of the precise distribution of GRP-R in the brain. Thus, in our present work, we used a synthetic peptide to generate affinity-purified antibodies against mouse GRP-R. The amino acid sequence of mouse GRP-R is highly homologous to mouse NMB-R (54% identity)

Acknowledgments

We thank Dr. A. Furuta and Dr. K. Ayukawa for helpful suggestions and discussions on immunohistochemical analyses. We are also grateful to S. Kikuchi for technical assistance and E. Sato for breeding the mice used in this study. This work was supported by research grants from the Ministry of Health, Labour and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Pharmaceuticals and Medical Devices Agency of Japan.

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