Electron microscopic localisation of P2X 4 receptor subunit immunoreactivity to pre- and post-synaptic neuronal elements and glial processes in the dorsal vagal complex of the rat

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P2X receptors are ligand gated ion channels activated by extracellular ATP. There are seven P2X subunits, P2X(1-7), and all are expressed in the CNS. The P2X(4) receptor subunit (P2X(4)R) is likely to be important in the CNS as it has been reported to be expressed throughout the brain and spinal cord. However, P2X(4)Rs have been identified as restricted to neurones, only in glia or expressed in both neurones and glia with no discernible relationship to CNS region or epitope target of antibodies used for staining. In addition, although there are particularly high levels of mRNA encoding P2X(4)R in the brainstem, previous immunohistochemical studies have revealed only indistinct staining. We therefore examined the distribution of P2X(4)R in the dorsal vagal complex (DVC) of the brainstem using immunohistochemistry in sections obtained from adult Wistar rats transcardially perfused with aldehyde fixatives. When this revealed staining identifiable only as small puncta at the light microscope level, we examined the area with electron microscopy. This ultrastructural study revealed that P2X(4)R immunoreactivity (IR) was present in neurones at both pre- and post-synaptic sites as well as in glial cell processes and somata. This P2X(4)R-IR was localised adjacent to plasma membranes, as well as internally in membrane bound structures resembling endosomes. Immunoreactivity in endosomes was more prominent following antigen retrieval protocols. Localisation of P2X(4)R-IR in astrocytes, identified by the presence of glial fibrillary acidic protein (GFAP), was confirmed using immunofluorescence. The presence of P2X(4)Rs in the dorsal vagal complex is consistent with expression studies, but some reasons for a lack of correlation with pharmacological studies are discussed. The P2X(4)R is therefore expressed by neurones and glia in the dorsal vagal complex and may play a role in mediating extracellular signalling by ATP in this region.