Elsevier

Brain Research

Volume 942, Issues 1–2, 28 June 2002, Pages 71-78
Brain Research

Research report
Decreased marble burying behavior in female mice lacking neuromedin-B receptor (NMB-R) implies the involvement of NMB/NMB-R in 5-HT neuron function

https://doi.org/10.1016/S0006-8993(02)02696-3Get rights and content

Abstract

Neuromedin B (NMB) is a mammalian bombesin-like peptide distributed widely in the central nervous system. This peptide exerts its function via the NMB receptor (NMB-R). Female NMB-R-deficient mice were used to study the role that NMB/NMB-R may play in 5-HT neuron function since this relationship was suggested in previous in vitro studies. As 5-HT neurons are thought to modulate marble burying behavior, a role for NMB-R in this behavior was assessed. Relative to wild-type mice, NMB-R-deficient mice showed decreased marble burying behavior. However, depletion of 5-HT by treatment with p-chlorophenylalanine (p-CPA) increased burying behavior in NMB-R-deficient mice suggesting that increased levels of 5-HT in the brain cause a decrease in burying behavior in NMB-R-deficient mice. While HPLC analysis showed that 5-HT content in the whole brain does not differ between NMB-R-deficient and wild-type mice, an immunohistochemical analysis of brain sections showed that 5-HT expression in the dorsal raphe (DR) nucleus is elevated in NMB-R-deficient mice. Furthermore, a quantitative RT-PCR analysis revealed that 5-HT1A-receptor gene expression is downregulated in NMB-R-deficient mice at the whole brain level. These behavioral and biological results suggest that NMB/NMB-R may modulate 5-HT neuronal activity by affecting DR function.

Introduction

Neuromedin B (NMB) is a mammalian bombesin (BN)-like peptide originally purified from porcine spinal cord [16]. NMB is widely distributed in the central nervous system [30] and its effects are mediated through the G-protein coupled neuromedin B receptor (NMB-R) [31]. Psychopharmacological studies show that NMB contributes to a wide variety of physiological and behavioral functions such as thermoregulation [11], spontaneous activity [10] and feeding behavior [12], [13], [25], [29], [33]. Despite this extensive body of research, the function(s) of endogenous NMB remains unclear. As NMB-R is distributed widely throughout the rat and mouse brain [18], [31], [32], NMB/NMB-R is predicted to play a role in many neurotransmission systems.

Several studies suggest that the NMB/NMB-R system interacts with 5-HT neurons. NMB-R is expressed in the dorsal raphe nucleus (DR) [31], a major source of 5-HT in the brain. Also, certain neurons in the rat DR are sensitive to BN-like peptides that elicit an excitatory response in the DR [22]. However, these neurons are insensitive to antagonists for another BN-like peptide receptor, gastrin-releasing peptide receptor (GRP-R) [23]. In the present study, we employed mice lacking NMB-R [19] to study the role for NMB/NMB-R in 5-HT neurons. We recently reported that male NMB-R-deficient mice exhibit altered emotional and/or anxiety-related behavior (risk assessment behavior) [34]. Risk assessment behavior is closely related to defensive behavior in mice [5], and may be controlled, at least in part, by 5-HT neurons [4].

On the basis of previous reports as well as our results, we chose a marble burying test paradigm to evaluate the relationship between the NMB/NMB-R system and 5-HT neurons with respect to behavior. Marble burying represents one type of defensive behavior in rodents. In response to aversive stimuli, mice and rats bury glass marbles [2], unpalatable food [24] or a shock prod [27]. Such behavior is clearly defensive and reflects certain types of anxiety [2] that are distinct from those measured by the light–dark box or elevated plus maze (in which no obvious threat is presented). Furthermore, several studies link defensive behavior (e.g. marble burying) to 5-HT function [9], [15] suggesting that elevated 5-HT activity in the synaptic cleft decreases burying behavior [1], [17]. It therefore follows that altered marble burying behavior in NMB-R-deficient mice may entail some dysfunction in 5-HT neurons.

We examined marble burying behavior in NMB-R-deficient mice. Subsequently, 5-HT content was measured by HPLC analysis at whole brain level, and 5-HT activity in the DR was determined immunohistochemically. Further, 5-HT1A-receptor gene expression was quantified in the brain. These behavioral and biological analyses indicate that NMB/NMB-R system may modulate 5-HT neuronal function by affecting the DR function.

Section snippets

Animals

Female mice were used as subjects because previous reports indicate that the marble burying behavior is more easily observed in female mice [17]. Female NMB-R-deficient mice and wild-type littermates were produced in our laboratory by mating NMB-R heterozygous males and females [34]. Groups, sample sizes, age, body weight, and generation of heterozygous male/female mice as well as treatments of each experiment are summarized in Table 1. Animal housing and all experiments were conducted in a

Marble burying test

Rearing and/or housing conditions are some of the critical determinants of behavior in laboratory animals [7], [14]. As such, we first examined marble burying behavior in wild-type and NMB-R-deficient mice that were housed either in a group or in isolation. Relative to wild-type mice, NMB-R-deficient mice exhibited decreased marble burying behavior under both housing conditions (Fig. 1A; F(22,3)=14.72, P<0.0001, one-way ANOVA; t=7.11, P<0.01, for group-housed groups; t=5.83, P<0.01, for

Discussion

We examined the role that NMB/NMB-R may play in 5-HT neuron function using female NMB-R-deficient mice. We assessed the defensive behavior of the mutant mice using a marble burying test and determined the changes in 5-HT in NMB-R-deficient mice using immunohistochemical and molecular biological assays.

Marble burying behavior is a characteristic defensive behavior of rodents that may be regulated by 5-HT [1], [2], [9], [17]. Here, we demonstrated that female NMB-R-deficient mice exhibit

Acknowledgements

We thank Dr. Ohki-Hamazaki for constructive discussions. This work was supported in part by research grants from The Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labour and Welfare, and Japan Science and Technology Corporation.

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These two authors contributed equally to this work.

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