Neuronal and endothelial sites of acetylcholine synthesis and release associated with microvessels in rat cerebral cortex: ultrastructural and neurochemical studies

We sought to establish what proportion of the cholinergic innervation of the cerebral cortex (CX) is associated with intraparenchymal blood vessels by using immunocytochemical and neurochemical techniques, and whether [³H]acetylcholine ([³H]ACh) is synthesized and released by elements associated with cortical microvessels (MV). MVs and, for comparison, tissue homogenates were prepared using sucrose gradient/differential ultracentrifugation methods. Efficacy of the separation technique was indicated by the activity of γ-glutamyltranspeptidase (up to 29.2-fold enrichment), an endothelial cell marker enzyme, in the MV fraction and microscopy. The size of isolated microvessels ranged from 5 to 40 μm (o.d.) with 67.7% of the vessels less than 10 μm and 32.2% between 11 and 40 μm (690 vessels measured from 4 animals). By electron microscopy immunoreactive choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, was localized to: (a) axons and axon terminals opposed to the basal laminae of capillaries and small arterioles, and (b) capillary endothelial cells. ChAT-labeled elements associated with MVs were most prominent in layers I, III and V of the CX consistent with the local pattern of cholinergic innervation. The absolute amount of ACh synthesized (pmol Ach/100 mg wet wt.) by elements associated with cortical MVs was relatively small (2.3% total cortical homogenate activity). Inhibition of MV ChAt activity to 5% of control by the specific ChAT inhibitor, 4-naphthylvinylpyridine, and HPLC analysis of the product, indicated that authentic ACh was measured. Other tissues similarly synthesized small amounts of ACh relative to the CX, caudate nucleus (CN, 2.4%), cerebellum (CRB, 1.4%) and liver (LIV, 3.9%). Consistent with the known extent of the cholinergic innervation of the tissues examined, the rank order of ChAT associated for both MVs and homogenate were: CN > CX > > CRB > LIV. However, based on the specific activities of ChAT, cortical MVs have the remarkable capacity to synthesize ACh at rates 95% greater than cortical (S₁ fraction) homogenate (59.0 ± 3.5 nmol/mg protein/40 min; n = 7), which is enriched in nerve terminals. Except for LV (+11%), other tissues also had remarkably high ChAT activity in MV (% above corresponding homogenate; P < 0.05, n = 5): CN (+269) and CRB (+313). Release of [³H]ACh from MVs and, for comparison, nerve terminals were graded to K⁺ depolarization stimulus (5-55 mM), maximal with 55 mM K⁺ and CA²⁺ dependent. The K⁺-evoked release of neurotransmitter amino acids aspartate and γ-aminobutyric acid (GABA), unlike [³H]ACh, was only observed from nerve terminals. This differential pattern of neurotransmitter release suggest a selective innervation of cholinergic neurons with the cortical microvasculature and that contamination of the MV fraction by non-vascularly related neurons in unlikely. We conclude that the synthesis and release of ACh, at the level of cortical MVs, is consistent with evidence for a potent mechanism for the neural control of the cerebral circulation by ACh.