Proceedings of The Physiological Society
University of York (2002) J Physiol 539P, S006
Neuronal and endothelial localisation of the angiotensin type 1 receptor in the medulla oblongata of the rat
Zai Ahmad*, Julian F.R. Paton and Jim Deuchars*
*School of Biomedical Sciences, University of Leeds, Leeds and Department of Physiology, University of Bristol, Bristol, UK
The angiotensin II type 1 receptor (AT1R) has been implicated in a variety of autonomic pathways in the medulla oblongata. To investigate these autonomic pathways we have examined the localisation of the AT1R using immunohistochemistry at light and electron microscopic levels and determined some neuro-transmitters co-localised with the AT1R.
Male Wistar rats (100-200 g; n = 5) were humanely killed by terminal anaesthesia (Sagatal, 60 mg kg-1, I.P.) and transcardial perfusion with fixative containing 4 % paraformaldehyde/0-0.5 % glutaraldehyde in 0.1 M phosphate buffer. Transverse sections (50 µm) of medulla oblongata were cut on a vibrating microtome and incubated in rabbit anti-AT1R (1:400 Autogen Bioclear) 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) followed by peroxidase conjugated extravadin (1:1000; Sigma), visualised with diaminobenzidine (DAB). For double labelling using fluorescence, sections were incubated in either sheep anti-PNMT (1:2000, Chemicon) or mouse anti-TH (1:2000, Chemicon). These double labelling fluorescence procedures as well as anterograde labelling of vagal afferents with AT1R detection (n = 5) were conducted using the methods described previously (Atkinson et al. 2000).
At the LM level AT1R immunoreactive neurones were identified by intense labelling predominantly localised to the membrane of somata and dendrites, but also to a lesser extent within the cytoplasm. Labelled neurones were in the nucleus tractus solitarii (NTS), rostral ventrolateral medulla (RVLM), raphe obscurus and the intermedius nucleus of the medulla. In the NTS, neurones were mainly in the dorsomedial and commissural subdivisions. Diffuse punctate labelling was also observed throughout the NTS as well as in the nucleus ambiguus (NA). Fibre-like labelling was apparent in the spinal trigeminal nucleus. Ultrastructural analysis confirmed that the AT1R-IR was located on neuronal somata and dendrites in the NTS and RVLM, but also to synaptic terminals in the NTS, NA and the RVLM. In the NTS, some of the AT1R-IR terminals were identified as vagal afferents by prior anterograde labelling from the nodose ganglion. In addition to neurones, ultrastructural examination revealed immunoreactivity in endothelial cells lining small arterioles.
These data show that AT1 receptors are present in vagal afferent but not vagal efferent neurones, putative pre-sympathetic neurones in the RVLM neurones and presynaptic terminals in the RVLM and NA.
This work was supported by the BHF, BBSRC and The Wellcome Trust.
Where applicable, experiments conform with Society ethical requirements