The ventrolateral medulla (VLM) functions as a primary central chemoreceptive area, responsible for sensitivity to increases in arterial levels of PCO2 and mediating the ventilatory response to hypercapnia (Loeschcke, 1982). ATP acting via P2X receptors may be involved in mediating changes in the activity of medullary respiratory neurones during hypercapnia, thus playing an important role in central chemoreception (Spyer & Thomas, 2000). The objective of the present study was to determine whether respiratory neurones in the VLM contain P2X2 and/or P2X1 receptor subunits.
Experiments were performed in male Sprague-Dawley rats (300-340 g) anaesthetised (pentobarbitone sodium 60 mg kg-1 I.P., then 10 mg kg-1 I.V. as required), injected with gallamine triethiodide (10 mg kg-1, I.V., then 2-4 mg kg-1 h-1 I.V.) and artificially ventilated. All studies were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986. Adequate anaesthesia was ensured by maintaining stable levels of blood pressure, and heart and central respiratory rate. Respiratory neurones located in the area of rostral VLM (stereotaxic co-ordinates: 2.0-2.5 mm rostral to the calamus scriptorius, 1.5-2.0 mm lateral to midline and 2.6-3.0 mm ventral from the dorsal surface of the medulla oblongata) were labelled with Neurobiotin using the juxtacellular method (Pinault, 1996). Animals were transcardially perfused with 4 % paraformaldehyde, the brains removed and 50 µm coronal sections of the medulla cut using a vibratome. The filled cells were visualised by incubating the sections in avidin FITC (1:1000, Vector Labs). The sections were then incubated in rabbit polyclonal antibodies raised against either the P2X2 (1:1000, Dr J.A. Barden, University of Sydney, Australia) or P2X1 (1:1000; Alomone Labs, Israel) receptor subunits. These were visualised by incubation in a Cy3 conjugated secondary antibody (1:1000, Jackson Immunoresearch) and the sections viewed under a fluorescence microscope.
It was found that (i) out of twelve labelled expiratory neurones, six (50 %) were strongly immunoreactive for the P2X2 receptor subunit; (ii) only two out of seven juxtacellularly labelled pre-inspiratory neurones were immunoreactive for the P2X2 receptor subunit; (iii) none of the labelled VLM inspiratory neurones (n = 4) was detectably immunoreactive for the P2X2 receptor subunit; (iv) none of the labelled VLM neurones with respiratory-related discharge (n = 15) were detectably immunoreactive for the P2X1 receptor subunit.
This suggests that in the VLM only a relatively small proportion of neurones that display rhythmic respiratory-related activity contain P2X2 receptor subunit and that it is unlikely that these neurones contain P2X1 receptor subunit either. P2X2 receptor subunit immunoreactivity is confined predominantly to the population of the VLM expiratory neurones indicating that extracellular ATP can be an important modulator of their activity.
This work was supported by the BBSRC.