N~~r~~e~enc~ Vol. 39, No, 3, pp. 775-785, 1990 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE Printe d in G re a t Brita in 030~~22/90 $3.00 + 0.00 Pergamon Press plc Q 1990 IBRO USE OF ENHANCED SILVER STAINING COMBINED WITH ELECTRON MICROSCOPICAL IMMUNOLABELLING TO DEMONSTRATE THE COLOCALIZATION OF NEUROPEPTIDE Y AND VASOACTIVE INTESTINAL POLYPEPTIDE IN CEREBROVASCULAR NERVES J. F. R. CAVANAGH, M. C. MIONE and G. BURNSTOCK* Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WClE 6BT, U.K. Abstract-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. T&is 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. Immunolabe4ling of the antigens for these peptides was performed sequentially with the biotin-streptavidindiaminobenzidine 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 s~~thectorny with guane~i~ne was localized in vasoactive intestinal ~ly~ptide-convening nerves. Furthe~ore, an immunohistochemical analysis of the pa~s~pathetic pterygopalatine ganglia in guanethidine-tr~ted 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 sympathec- tomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohisfochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunore~tivity to both these peptides in retrogradely Iabelied neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in p&vascular 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. Recent immunohist~hemical studies have revealed the existence of a population of neuropeptide Y (NPY)-immunoreactive cerebrovascular nerves that survive sympathectomy achieved either by bilateral removal of the superior cervical ganglia3 or ~anethidine treatment.“,” Moreover, an increase of NPY-like immunoreactivity (LI) in vasoactive intes- tinal polypeptide (VIP)-containing cerebrovascular nerves following superior cervical ganglionectomy has been demonstrated in the guinea-pig,j even though surgical sympathectomy leads to a decrease of NPY-immunoreactive cerebrovascular nerves. Using long-term ~anethidine treatment in developing rats, the compensatory expression of NPY in non- sympathetic nerves was shown to maintain the pat- tern and density of NPY immunoreactivity in cerebrovascular nerves.” *To whom correspondence should be addressed. Abbreoiurions: DAB, diaminobenzidine; FITC, ffuoreseeine isothiocyanate; LI, -like immunoreactivity; NDS, normal donkey serum; NPY, neuropeptide Y; PBS, phosphate-buffered saline; VIP, vasoactive intestinal polypeptide. The present study was undertaken to localize at the ultrastructural level the immunoreactivity for NPY present in rat cerebral vessels after long-term sym- pathectomy with guanethidine. The origin of these non-sympathetic NPY-immunoreactive cerebro- vascular nerves was also studied. The pte~gopalatine ganglion, a paras~pathetic ganglion, known to be the origin of VIP-L1 supply to rat cerebral vessels’* and also to contain NPY-immunoreactive neuronq5s6 was studied immunohistochemically in control and guanethidine-treated animals. Finally, the pathway of NPY/VIP-immunoreactive axons from the middle cerebral artery to this ganglion was studied in sympa- thectomized rats by use of a fluorescent neuronal tracer. Part of this work has been presented at the XIV International Symposium on Cerebral Blood Flow and Metabolism, Brain ‘89, and published in abstract form. ’ EXPERIMENTAL PROCEDURES Seven-day-old Wistar rats of both sexes were injected daily with guanethidine (Ismelin, CIBA), 60 mg/kg, s.c., for six weeks. Control rats were iniected with saline over the same period. Animals were perf&ion-fixed on the same day 775