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

Stephen P. Arnerić, Marc A. Honig, Teresa A. Milner, Sandra Greco, Costantino Iadecola, Donald J. Reis

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)11-30
Number of pages20
JournalBrain research
Issue number1-2
StatePublished - Jun 28 1988
Externally publishedYes


  • Acetylcholine
  • Choline acetyltransferase
  • Cortical microvessel
  • Electron microscopy
  • High-performance liquid chromatography
  • Immunocytochemistry
  • Neurotransmitter release
  • γ-Aminobutyric acid

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology


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