Modulation of brain stem monoamines and c-aminobutyric acid by NK1 receptors in rats Qing-Ping Ma CA and Catherine Bleasdale DepartmentofPharmacology,MerckSharpandDohmeResearchLaboratories,NeuroscienceResearchCentre,TerlingsPark, HarlowCM202QR,UK CA CorrespondingAuthor:qingping__ma@merck.com Received13February2002; accepted15May2002 To understand the role of substance P in stress, anxiety and depression, we have investigated in rats the relationship between NK1 receptors and monoamines or GABA, and between substance P and serotonin (5-HT) in brain stem neurons by immunohistochemical double-staining techniques. In the periaqueductal gray and dorsal raphe nucleus, there was no colocalization between NK1and 5-HTor between NK1 and tyrosine hydroxylase (a marker for adrenaline and dopamine neurons). However, many GABA-positive neurons (4 50%) were NK1 positive, and some substance P-positive neurons were 5-HT positive as well. Almost all locus coeruleus noradrenaline neurons were NK1positive.Therefore, substance P may promote stress by activating noradrenaline neurons directly and inhibiting 5-HT neurons indirectly via GABA neurons. NeuroReport 13:1809^1812  c 2002LippincottWilliams& Wilkins. Key words:Dorsalraphenucleus;GABA;NK1receptor;Serotonin(5-HT);SubstanceP;Tyrosinehydroxylase INTRODUCTION Classical antidepressants and anxiolytics are mainly agents modulating monoamine and GABA functions [1–4]. In recent years, substance P (SP) has been implicated in mediating stress, anxiety and depression. One SP (NK1) receptor antagonist MK869 was shown to reduce vocaliza- tion of guinea pig puppies evoked by maternal separation, and to improve symptoms in depressed patients with a high or medium level of anxiety [5]. The relationship between SP and those classical neurotransmitters in mediating stress, anxiety and depression is still not clear. Monoamine and GABA neurons in the brain stem play an important role in stress, anxiety and depression. The selective serotonin reuptake inhibitors (SSRIs), which may act by enhancing the function of 5-HT system, have shown a good clinical efficacy in treating depression [2]. The dorsal raphe nucleus is the most important source of forebrain 5-HT, and its 5-HT neurons project extensively to most areas in the forebrain [6]. Noradrenaline has also been implicated in the action of classical antidepressants [1]. Locus coeruleus is the most important source of forebrain noradrenaline, which has been shown to play an important role in stress [3,7]. GABA neurons have an extensive distribution in the brain and many anxiolytics modulate GABA receptor function [4,8]. SP and NK1 receptors are also found in brain stem [9,10], which may interact with monoamine and GABA neurons in their action. Therefore, the aim of the present study is to investigate whether monoamine and GABA neurons in the brain stem, especially dorsal raphe nucleus and locus coeruleus, express NK1 receptors. MATERIALS AND METHODS Adult Sprague–Dawley rats (200–300 g) were terminally anesthetized and perfused through ascending aorta with 300 ml normal saline, followed by 500 ml 4% paraformalde- hyde and 0.1% glutaraldehyde (n ¼ 3), or by 500 ml 4% paraformaldehyde (n ¼ 3) in 0.1 M phosphate buffer (pH 7.4). Two animals were injected intracerebroventricularly (i.c.v.) with 100 mg colchicine 24 h before the perfusion. The brain was removed, post-fixed in 4% paraformaldehyde, and cryoprotected in 20% sucrose in 0.01 M phosphate buffered saline (PBS, pH 7.4). The tissue was cut at 30 mm and the sections collected in 0.01 M PBS. The sections were treated with 0.25% Triton X-100 for 15min, and then 1% bovine serum albumin for 15 min. All aspects of animal care and procedures were conducted in accordance with the Animals (Scientific Procedures) Act of UK and its associated guidelines. All efforts were made to minimize animal suffering and to reduce the number of animals used. For the double immunofluorescent staining, two primary antibodies raised in different species were diluted with 0.01 M PBS and mixed. After incubation in such an antibody mixture for 48 h at 41C, the sections were washed twice with 0959-4965  c LippincottWilliams&Wilkins Vol13 No 14 7 October 2002 1809 MOTIVATION,EMOTION,FEEDING,DRINKING NEUROREPORT Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.