ATP acting as a neurotransmitter can cause fast responses via action on ligand-gated P2X receptors (P2X1-7). P2X2 and P2X3 receptor subunits have been localised immunohistochemically within the nodose ganglion, the location of vagal afferent neuronal somata (Vulchanova et al. 1997). We have also shown that the P2X2 receptor subunit is transported to vagal afferent fibre terminals in the nucleus tractus solitarii (NTS; Atkinson et al. 2000). Here we have studied the expression of P2X1, –2, –4 and –7 in the nodose ganglion and vagal afferent fibre terminals.
Male rats (100-150 g) were terminally anaesthetised with Sagatal (60 mg kg-1 I.P.), and perfused transcardially with artificial cerebrospinal fluid in which NaCl was replaced with sucrose (217 mM). Total RNA was isolated from pairs of nodose ganglia and reverse transcribed followed by 35 cycles of PCR using RT-PCR using P2X subunit specific primers. Subunit specific antisera (1:1000, Alomone Labs, Israel) were used to detect protein in 50 µm sections of nodose ganglion or medulla oblongata isolated from terminally anaesthetised rats (Sagatal, 60 mg kg-1 I.P.) perfused with 4 % paraformaldehyde/ 0-0.1 % glutaraldehyde. Primary antibodies were detected with donkey anti-rabbit Cy3 (1:1000, Jackson Immunochemicals) for fluorescence microscopy or for electron microscopy by 1 nm gold conjugated secondary antibodies (1:100, Amersham) which were then silver enhanced. In some rats vagal afferent fibres were anterogradely labelled by injection of biotinylated dextran amine (BDA) into nodose ganglion under halothane anaesthesia. After 3-7 days survival, the rats were perfused with fixative and sections cut as above, BDA detected by incubation in extra-avidin peroxidase (EAP, 1:1500, Sigma), followed by reaction in diaminobenzidine (DAB). P2X receptors were then detected using immunogold as above.
RT-PCR shows the presence of the transcripts for P2X1, –2, –4 and –7 receptor subunits in nodose ganglion. Immunohistochemistry revealed the presence of protein for all subunits. Qualitative estimates indicate that P2X2 receptor immunoreactivity (R-IR) was present in almost all cell bodies while P2X1 and P2X4 was in smaller numbers; all of these receptor subtypes exhibited cytoplasmic labelling. In contrast P2X7R-IR surrounded the nucleus in the majority of neurones. In anterogradely labelled vagal afferent terminals immunoreactivity for P2X2 and P2X7 could be detected. P2X1 and P2X4 R-IR have not been observed in vagal afferent fibres to date. However, these receptor subunits are present in a small number of nodose neurones and may therefore be present in a small number of vagal afferent terminals yet to be identified.This work was supported by The Wellcome Trust.
- Atkinson, L., Batten, T.F. & Deuchars, J. (2000). Neuroscience 99, 683-696.
Vulchanova, L., Riedl, M.S., Shuster, S.J., Buell, G., Surprenant, A., North, R.A. & Elde, R. (1997). Neuropharm. 36, 1229-1242.