EVIDENCE FOR THE INVOLVEMENT OF PURINERGIC SIGNALLING IN THE CONTROL OF RESPIRATION T. THOMAS, b; 1 * V. RALEVIC, c ; 1 M. BARDINI, a G. BURNSTOCK a and K. M. SPYER a a Autonomic Neuroscience Institute, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK b Department of Physiology, Division of Medical Sciences, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK c School of Biomedical Sciences, The University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK AbstractöThe ventrolateral medulla has a critical role in the generation and patterning of respiration via an extensive network of respiratory neurones. We have investigated the e¡ects of activating purinergic P2 receptors within the ventro- lateral medulla of the anaesthetised rat on the overall pattern of respiratory activity. In addition, using immunohisto- chemical techniques, we have identi¢ed the subtypes of P2X receptors in the ventrolateral medulla. Unilateral microinjection of ATP into the ventrolateral medulla reduced in a dose-dependent manner, or abolished, resting phrenic nerve discharge recorded as an indication of central inspiratory drive. ATP also elicited increases in blood pressure and variable changes in heart rate. These e¡ects were mimicked by microinjection of the P2X receptor agonist K,L-methylene ATP into the ventrolateral medulla. Whilst microinjection of suramin, a P2 receptor antagonist, had no e¡ect on resting cardiorespiratory variables it blocked the respiratory and cardiovascular e¡ects of ATP microinjected into the ventro- lateral medulla. Immunohistochemical staining using IgG antibodies showed that P2X 1 , P2X 2 , P2X 5 and P2X 6 , but not P2X 3 , P2X 4 or receptor subunits were localised in the rostral ventrolateral medulla. Our results indicate that several P2X receptor subtypes are localised within areas of the ventrolateral medulla that are important for cardiorespiratory control (including the pre-Bo « tzinger and Bo « tzinger complexes), and that activation of these receptors can have profound e¡ects on both the cardiovascular and the respiratory networks. Our pharmacological data suggest that di¡erent P2X subunits in this region may co-assemble to form hetero-oligomeric assemblies as well as homomultimers within this region. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: ATP, P2 receptors, immunohistochemistry, ventrolateral medulla, brainstem, breathing. ATP, acting via P2X ion channel receptors, is a fast neurotransmitter within the CNS (Jahr and Jessel, 1983; Edwards et al., 1992; Day et al., 1993; Torres et al., 1999). In recent years, there has been a growing body of evidence that ATP acting at P2X receptors within the medulla oblongata can modulate cardiovascular activity (Phillis et al., 1997; Horiuchi et al., 1999; Ralevic et al., 1999). In particular, we have shown that ATP applied iontophoretically to presumptive vasomotor neurones within the ventrolateral medulla (VLM) evokes a potent excitatory e¡ect (Ralevic et al., 1999). This may explain why activation of P2X receptors in VLM evokes a large increase in arterial pressure, accompanied by a signi¢cant increase in sympathetic nerve activity (Sun et al., 1992; Horiuchi et al., 1999). However, this area of the VLM also contains an exten- sive respiratory network, including the pre-Bo « tzinger and Bo « tzinger complexes and the nucleus ambiguus (Richter, 1996). This `ventral respiratory group' has a fundamental role in controlling the activity of the respiratory muscles. Whilst it is clear that ATP can act on the cardiovascular neurones in this area, the possibility that ATP might also modulate respiratory activity within the VLM has not been fully addressed. However, this prospect seems likely since we have recently illustrated that P2X receptors within the VLM are an important component in carbon dioxide-mediated changes in respiration (Thomas and Spyer, 2000). In this study we have addressed the question of whether ATP receptors within the VLM are involved in respiratory control. First, we have investigated the e¡ects of microinjection of ATP and its stable analogue K,L-methylene ATP (K,L-meATP) into the pre-Bo « tzinger and Bo « tzinger complexes on both cardiovascular and respiratory activity simultaneously. In addition, we have employed immunohistochemical techniques in order to visualise, and localise, the P2X receptor sub- types present within the VLM. Some of these results have been reported previously in preliminary abstracts (Ralevic et al., 1998; Thomas et al., 2000). 481 1 Joint senior author. *Corresponding author. Tel. : +44-121-414-6938 ; fax : +44-121-414- 6919. E-mail address : t.j.thomas@bham.ac.uk (T. Thomas). Abbreviations : K,L-meATP, K,L-methylene ATP; ABP, arterial blood pressure; HR, heart rate; NHS, normal horse serum; PBS, phosphate-bu¡ered saline ; VLM, ventrolateral medulla. NSC 5204 5-11-01 www.neuroscience-ibro.com Neuroscience Vol. 107, No. 3, pp. 481^490, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII:S0306-4522(01)00363-3 0306-4522 / 01 $20.00+0.00