Autoradiographic localization of muscarinic receptors on cultured, peptide-containing neurones from newborn rat superior cervical ganglion

Abstract

In order to identify subpopulations of cultured rat superior cervical ganglion (SCG) neurones which express muscarinic receptors, a combination of immunocytochemistry and autoradiography was performed on these cultures. Antibodies to vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were used to immunostain cultures that had previously been labelled with the irreversible muscarinic antagonist, [ 3H]propylbenzylylcholine mustard (PrBCM). Binding sites for [ 3H]PrBCM were observed on a large subpopulation of 65–85% of the ganglionic neuronal cell bodies. Specific labelling was not associated with non-neuronal cells found in these cultures. Approximately 60% of the SCG neurones were NPY-like immunoreactive (NPY-LI), a high proportion of which expressed muscarinic receptors. Five to 10% of the SCG neurones were VIP-LI, a small subpopulation of which displayed [ 3H]PrBCM binding sites. Receptor distribution on cell bodies was usually uniform, but occasionally, regions of high receptor density were seen. Dense networks of both varicose and non-varicose NPY-LI fibres were seen throughout the culture, a subpopulation of which expressed muscarinic receptors. Occasional VIP-LI fibres were also labelled with silver grains for [ 3H]PrBCM, but in less abundance than those for NPY-LI fibres. Conversely, neurones expressing muscarinic receptors were often immunonegative for either VIP or NPY: therefore, the identity of some of the neurones which express muscarinic receptors remains to be determined.