Use of enhanced silver staining combined with electron microscopical 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.
Cavanagh
J F
JF
Department of Anatomy, University College London, U.K.
Mione
M C
MC
Burnstock
G
G
D000818:Animals; D002536:Cerebral Arteries / Q000166:cytology / Q000294:innervation* / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D005726:Ganglia, Parasympathetic / Q000166:cytology* / Q000648:ultrastructure; D007124:Immunoenzyme Techniques; D008297:Male; D016253:Microscopy, Immunoelectron; D009474:Neurons / Q000166:cytology* / Q000648:ultrastructure; D009478:Neuropeptide Y / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D012834:Silver; D013194:Staining and Labeling; D014660:Vasoactive Intestinal Peptide / Q000032:analysis*
D009478:Neuropeptide Y; D014660:Vasoactive Intestinal Peptide; D012834:Silver