Proceedings of The Physiological Society

University of Leeds (2002) J Physiol 544P, S012


Light and electron microscopic localisation of P2X4 receptor subunit immunoreactivity in the dorsal vagal complex of the rat

Fathia Ashur and Jim Deuchars

School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK

Ionotropic ATP-gated receptors comprise three (or multiples of three) of the seven subunits cloned so far, P2X1-7. P2X4 receptor subunits (P2X4RS) are expressed throughout the CNS. In the nucleus tractus solitarii (NTS) an antibody against an extracellular portion of the P2X4RS revealed sparsely distributed P2X4RS-positive neurones, mainly in the intermediate portion of the commissural NTS, and along the postremal border (Yao et al. 2001). Here we compare this pattern with that obtained using an antibody against a peptide sequence corresponding to an intracellular portion of the subunit (C-terminus, amino acids 370-388, Alomone Labs), as well as examine the localisation ultrastructurally.

Male Wistar rats (150-200 g) were humanely killed by anaesthesia with sodium pentobarbital (Sagatal, 60 mg kg-1 I.P.) and transcardial perfusion with fixative containing 0.1 M phosphate buffer, 4 % paraformaldehyde and 0-0.5 % glutaraldehyde. Transverse sections (50 mm) of medulla oblongata were cut on a vibrating microtome and incubated in rabbit anti-P2X4RS (1:500) for 12-24 h. Sections were washed and placed in either donkey anti-rabbit IgG conjugated to Cy3 (1:1000, Jackson) or to a biotinylated anti-rabbit IgG (1:500; Jackson Immunoresearch) followed by peroxidase conjugated extravadin (1:1500; Sigma), visualised with diaminobenzidine. Sections were subsequently prepared for fluoroscence or light and electron microscopy.

Using fluorescence microscopy neuronal somata in the dorsal vagal nucleus contained moderate levels of P2X4RS immunoreactivity, while somata in the NTS were very lightly labelled. A dense band of immunoreactivity was observed in the sub-postremal NTS. The area postrema was densely labelled. In the NTS conventional light microscopy revealed fine punctate staining of the neuropil. In addition, some stained elements appeared to be glial in nature, although glial somata were not evident. Electron microscopy revealed that immunoreactivity was present both pre- and postynaptically in neurones. Post-synaptically the peroxidase reaction product could be located at the synaptic specialisation. However, presynaptically the reaction product was rarely located at the synaptic grid. In addition, glial cell processes surrounding presynaptic terminals were also labelled.

These data indicate diverse subcellular localisation of the P2X4RS in the NTS. We are currently investigating the origin and chemical nature of the presynaptic terminals bearing the P2X4 receptors, as well as those that are adjacent to P2X4RS containing glial processes.

All procedures accord with current UK legislation.

Where applicable, experiments conform with Society ethical requirements