The presence of cholinergic nerve fibers in the brain vasculature has been a matter of controversy, partly due to the lack of a reliable histochemical marker. Accordingly, no distinct information about the origin and pathways for such fibers has been available. In the present study on the rat pial vasculature, utilizing a choline acetyltransferase (ChAT) antibody, which is able to demonstrate this enzyme in peripheral nervous tissue, evidence was obtained for an innervation by cholinergic fibers of large pial arteries. Vasoactive intestinal polypeptide (VIP) was present in or in close association with these fibers. By the aid of the retrograde axonal tracer True Blue (TB) applied to the middle cerebral arterial wall, such fibers were shown to originate in a subgroup of ChAT-positive cells in the sphenopalatine, otic, and internal carotid ganglia, which, in addition, contained VIP. The ChAT-positive pial nerve fibers were few in relation to the VIP-immunoreactive fibers, as was also illustrated by the few TB-positive cells in the ganglia that were ChAT positive as compared with the number of cells that were VIP positive. Only a small population of ChAT-containing neurons in these ganglia appeared to project to the pial vessels. The pathway from the sphenopalatine ganglion is via a membranous structure on the medial orbital wall, through the ethmoidal foramen, and along the internal ethmoidal artery to reach the circle of Willis. The fibers from the internal carotid and otic ganglia probably bridge to the internal carotid artery in the carotid canal, those from the otic ganglion after an initial course in the lesser superficial petrosal nerve. In addition, a weak accumulation of ChAT immunoreactivity was observed along thick nerve bundles in the wall of large and smaller pial arteries. VIP fibers were running at some locations along these nerve bundles.
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