Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is known if the cells of origin are intra- or extracortical nor perivascular fibers colocalize ACh and VIP. Cortical cholinergic innervation arises primarily from the basal forebrain and to a small extent from intrinsic bipolar ACh neurons thought to be the sole source of cortical VIP. In order evaluate if intracortical perivascular ACh terminals could be distinguished from those of the basal forebrain by their colocalization with VIP, we performed a double immunofluorescence study and determined the percentage of colocalization of choline acetyltransferase (ChAT) and VIP in cortical neurons, as well as in terminal fields associated with intracortical blood vessels. From a total of 2103 cells examined in all cortical areas, VIP neurons accounted for the largest population (58.3%) followed by ChAT-positive cells (28.2%) with only 13.5% of cells being double-labelled for VIP and ChAT. Of the cortical ChAT-immunostained cells (n = 878), 32.3% colocalized VIP whereas only 18.8% of VIP neurons (n = 1509) also contained ChAT. In various cortical areas, ChAT cell bodies were seen to be contacted by VIP terminals which surrounded closely their cell soma and proximal dendrites. Perivascular fibers studied by double immunofluorescence and confocal microscopy were of three categories including cholinergic, VIPergic with a smaller population of fibers which costained for both ChAT and VIP. These results show hat cortical VIP neurons are much more numerous than those containing ChAT, and that a majority of VIP neurons do not colocalize with ChAT. This observation indicates that ACh and VIP are primarily located in distinct neuronal populations and that VIP cannot be used as a marker of intracortical ACh neurons and terminals. Our results further suggest that intracortical blood vessels are primarily under the influence of distinct ChAT and VIP perivascular fibers. Also, the presence of a subset of VIP and ChAT/VIP fibers in association with intracortical blood vessels strongly suggests a role for cortical bipolar neurons in local cerebrovascular regulation. The origin of the perivascular ChAT fibers which do not colocalize VIP, however, remains unknown.
Bibliographical noteFunding Information:
The authors are most grateful to Dr. D. Linville for the colchicine injections, Mr. C. Hodge for professional photographic work and Ms. L. Michel for preparation of the manuscript. We also want to thank Drs. B. Berger, C. Sotelo and L. Descarries for their helpful suggestions or comments throughout the realization of this study. This work was supported by a grant (MA-9967) and a Scholarship from the Medical Research Council (MRC) of Canada to E.H. and a grant (NS 12311) from the National Institute of Health (NIH) of the United States to B.K.H., Division of Neuroscience Research in Psychiatry, University of Minnesota. Contribution number 109. We also acknowledge the support from the 'Fonds de la Recherche en SantE du QuEbec (FRSQ) to A.C.
- Cerebral blood flow
- Cerebral circulation
- Cerebral cortex
- Double immunofluorescence