USE OF ENHANCED SILVER STAINING COMBINED WITH ELECTRON-MICROSCOPIC IMMUNOLABELLING TO DEMONSTRATE THE COLOCALIZATION OF NEUROPEPTIDE-Y AND VASOACTIVE INTESTINAL POLYPEPTIDE IN CEREBROVASCULAR NERVES

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin-streptavidin-diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.