Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S010


Colocalization of nitric oxide synthase and P2X2 receptor immunoreactivities in neurones of the rat hypothalamus and medulla

A.V. Gourine*, S.T. Yao†, A.J. Lawrence† and K.M. Spyer*

*Department of Physiology, Royal Free and University College London Medical School, Royal Free Campus, London NW3 2PF, UK and †Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia

There is evidence that both extracellular ATP, acting at P2X receptors, and nitric oxide (NO) are involved in the brainstem and hypothalamic mechanisms of cardiovascular and respiratory control (Lawrence, 1997; Phillis et al. 1997; Ralevich, 2000; Spyer & Thomas, 2000). It is unclear, however, whether ATP, acting at P2X receptors, and NO interact functionally within brainstem and hypothalamic structures involved in regulation of autonomic function. The present study investigated the extent of colocalization between neuronal NO synthase (NOS) and P2X2 receptor in neurones of the rat hypothalamus and medulla oblongata.

Experiments were carried out in accordance with UK legislation. Seven male Wistar-Kyoto rats weighing 250-300 g were deeply anaesthetised with pentobarbitone sodium (100 mg kg-1, I.P.) and then perfused transcardially with phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde in 0.1 M PBS. The brain was removed, postfixed and cryoprotected and 50 µm coronal slices of medulla and hypothalamus were cut on a freezing microtome. NOS- and P2X2 receptor-immunoreactive neurones were identified using antibodies against rat brain NOS (1:2000; Chemicon) and the rat P2X2 receptor (1:1000; Yao et al. 2000), respectively. Using immunofluorescence and confocal laser scanning microscopy it was found that a number of NOS-positive neurones in the brainstem and hypothalamus also exhibit P2X2 receptor immunoreactivity. Omission of either of the primary antibodies resulted in appropriate staining. P2X2 receptor-positive neurones constitute almost 50 % of the NOS-containing population in subpostremal nucleus of the solitary tract (NTS) and more than 60 % of the NOS-containing population in the commissural NTS. P2X2 receptor-positive neurones also constitute 95-100 % of NOS-immunoreactive neurones in both rostral and caudal ventral medulla. Substantial numbers of NOS-positive neurones in the hypothalamic supraoptic and paraventricular nuclei are also P2X2 receptor immunoreactive.

This study provides the first immunocytochemical evidence for the widespread colocalization of NO synthase and P2X2 receptors in neurones of brainstem and hypothalamic structures. Further physiological studies are needed to demonstrate whether functional interactions occur between purines and NO in central mechanisms of cardiovascular and respiratory control.

This work was supported by the BBSRC, The Wellcome Trust and the NHMRC (Australia).

Where applicable, experiments conform with Society ethical requirements