Abstract
The predicted C-terminal dodecapeptide of the human vesicular acetylcholine transporter (VAChT), deduced from the unique open reading frame of the recently cloned human VAChT cDNA, was conjugated through an N-terminal cysteine to keyhole limpet hemocyanin and used as an immunogen to generate polyclonal antihuman VAChT antibodies in rabbits. The distribution of the VAChT antigen in representative regions of the cholinergic nervous system was examined and compared to that of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), a specific marker for cholinergic neurons.
VAChT immunoreactivity was localized in cell bodies of neurons in the basal forebrain and ventral horn of the spinal cord, regions in which major cholinergic projection systems to the cerebral cortex and to skeletal muscle, respectively, originate. The primate caudate nucleus contained numerous VAChT-positive interneurons. VAChT immunoreactivity was visualized in both cell bodies and extensive terminals in striatal interneurons, in contrast to formalin-fixed, deparaffinized sections stained for ChAT, in which cell bodies and fibers were stained but nerve terminals were less well visualized than with the VAChT antiserum. VAChT-positive nerve fibers were visualized in routinely immersion-fixed, paraffin-embedded human cerebral cortex, comparable to the density of fibers observed in perfusion-fixed Bouin’s-postfixed monkey cerebral cortex. Extensive investment of virtually all principal ganglion cells of thoracic sympathetic ganglia of monkey and human with VAChT-positive nerve terminals was observed. VAChT-positive cell bodies, presumably corresponding to cholinergic sympathetic sudomotor neurons, were a significant fraction of the total principal cell population in monkey and human thoracic sympathetic ganglia.
VAChT is a specific marker for cholinergic neurons in human and rhesus monkey, visualizing especially nerve terminals more extensively than antibodies against the cholinergic biosynthetic enzyme ChAT, in routinely fixed tissue. VAChT immunoreactivity in cholinergic nerve terminals of the central and peripheral nervous systems ought to prove useful for visualizing cholinergic synapses and neuroeffector junctions, and their functional status during development and in neurodegenerative and autonomic disease.
Similar content being viewed by others
References
Alfonso A., Grundahl K., Duerr J. S., Han H.-P., and Rand J. B. (1993) TheCaenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter.Science 261, 617–619.
Bejanin S., Cervini R., Mallet J., and Berrard S. (1994) A unique gene organization for two cholinergic markers, choline acetyltransferase and a putative vesicular transporter of acetylcholine.J. Biol. Chem. 269, 21,944–21,947.
Bruce G., Wainer B. H., and Hersh L. B. (1985) Immunoaffinity purification of human choline acetyltransferase: comparison of the brain and placental enzymes.J. Neurochem. 45, 611–620.
Butcher L. L., Oh J. D., Woolf N. J., Edwards R. H., and Roghani A. (1992) Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.Neurochem. Int. 21, 429–445.
Coyle J. T., Price D. L., and DeLong M. R. (1983) Alzheimer’s disease: a disorder of cortical cholinergic innervation.Science 219, 1184–1190.
Dun N. J. and Perlman R. L., eds. (1985)Neurobiology of Acetylcholine. Plenum, New York.
Eckenstein F. P., Baughman R. W., and Quinn J. (1988) An anatomical study of cholinergic innervation in rat cerebral cortex.Neuroscience 25, 457–474.
Engel A. G. (1984) Myasthenia gravis and myasthenic syndromes.Ann. Neurol. 16, 519–534.
Erickson J. D., Varoqui H., Schäfer M., Diebler M.-F., Weihe E., Modi W., Rand J., Eiden L. E., Bonner T. I., and Usdin T. (1994) Functional characterization of the mammalian vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus.J. Biol. Chem. 269, 21,929–21,932.
Goldberg A. M. and Hanin I., eds. (1976)Biology of Cholinergic Function. Raven, New York.
Ibanez C., Pelto F., Huikko M., Soder O., Ritzen E. M., and Hersh L. B. (1991) Expression of choline acetyltransferase mRNA in spermatogenic cells results in an accumulation of the enzyme in the postacrosomal region of mature spermatozoa.Proc. Natl. Acad. Sci. USA 88, 3676–3680.
Liberini P. and Cuello A. C. (1995) Primate models of cholinergic dysfunction.Funct. Neurol. 10, 45–54.
Mesulam M.-M., Mash D., Hersh L., Bothwell M., and Geula C. (1992) Cholinergic innervation of the human striatum, globus pallidus, subthalamic nucleus, substantia nigra and red nucleus.J. Comp. Neurol. 323, 252–268.
Mesulam M. M., Mufson E. J., Levey A. I., and Wainer B. H. (1984) Atlas of cholinergic neurons in the forebrain and upper brainstem of the macaque based on monoclonal choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry.Neuroscience 12, 669–686.
Paxinos G. and Butcher L. L. (1985) Organizational principles of the brain as revealed by choline acetyltransferase and acetylcholinesterase distribution and projections, inThe Rat Nervous System, vol. 1 (Paxinos G., ed.), Academic, New York, pp. 487–521.
Perry E. K., Perry R. H., Blessed G., and Tomlinson B. E. (1977) Necropsy evidence of central cholinergic deficits in senile dementia.Lancet 1, 189.
Perry E. K., Tomlinson B. E., Blessed G., Bergman K., Gibson P. H., and Perry R. H. (1978) Correlation of cholinergic abnormalities with senile plaques and mental test-scores in senile dementia.Br. Med. J. 2, 1457–1459.
Roghani A., Feldman J., Kohan S. A., Shirzadi A., Gundersen C. B., Brecha N., and Edwards R. H. (1994) Molecular cloning of a putative vesicular transporter for acetylcholine.Proc. Natl. Acad. Sci. USA 91, 10,620–10,624.
Saper C. B. (1990) Cholinergic system, inThe Human Nervous System (Paxinos G., ed.), Academic, New York, pp. 1095–1113.
Schäfer M. K.-H., Nohr D., Romeo H., Eiden L. E., and Weihe E. (1994a) Pan-neuronal expression of chromogranin A in rat nervous system.Peptides 15, 263–279.
Schäfer M. K.-H., Weihe E., Varoqui H., Eiden L. E., and Erickson J. D. (1994b) Distribution of the vesicular acetylcholine transporter (VAChT) in the central and peripheral nervous systems of the rat.J. Mol. Neurosci. 5, 1–18.
Schäfer M. K.-H., Tao-Cheng J.-H., Erickson J. D., Weihe E., and Eiden L. E. (1995) Anatomical and subcellular distribution of the endocrine and neuronal vesicular monoamine transporters (VMAT1 and VMAT2) and the vesicular acetylcholine transporter (VAChT).Soc. Neurosci. Abstr. 21, 783.
Schemann M., Sann H., Schaaf C., and Mader M. (1993) Identification of cholinergic neurons in enteric nervous system by antibodies against choline acetyltransferase.Am. J. Physiol. 265, 1005–1009.
Selden N., Geula C., Hersh L., and Mesulam M.-M. (1994) Human striatum: chemoarchitecture of the caudate nucleus, putamen and ventral striatum in health and Alzheimer’s disease.Neuroscience 60, 621–636.
Semba K. and Fibiger H. C. (1989) Organization of central cholinergic systems, inProgress in Brain Research, vol. 79 (Nordberg A., Fuxe K., Holmstedt B., and Sundwall A., eds.), Elsevier, Amsterdam, pp. 37–63.
Sofroniew M. V., Campbell P. E., Cuello A. C., and Eckenstein F. (1985) Central cholinergic neurons visualized by immunohistochemical detection of choline acetyltransferase, inThe Rat Nervous System (Paxinos G., ed.), Academic, New York, pp. 471–485.
Steriade M. and Buzsaki G. (1990) Parallel activation of thalamic and cortical neurons by brainstem and basal forebrain cholinergic systems, inBrain Cholinergic Systems (Steriade M. and Biesold D., eds.), Oxford University Press, Oxford, pp. 3–62.
Usdin T., Eiden L. E., Bonner T. I., and Erickson J. D. (1995) Molecular biology of vesicular acetylcholine transporters (VAChTs).TINS 18, 218–224.
Varoqui H., Diebler M.-F., Meunier F.-M., Rand J. B., Usdin T. B., Bonner T. I., Eiden L. E., and Erickson J. D. (1994) Cloning and expression of the vesamicol binding protein from the marine ray Torpedo. Homology with the putative vesicular acetylcholine transporter UNC-17 fromCaenorhabditis elegans.FEBS Lett. 342, 97–102.
Wainer B. H. and Mesulam M.-M. (1990) Ascending cholinergic pathways in the rat brain, inBrain Cholinergic Systems (Steriade M. and Biesold D., eds.), Oxford University Press, Oxford, pp. 65–119.
Weihe E., Nohr D., Sharer L., Murray E., Rausch D., and Eiden L. (1993) Cortical astrocytosis in juvenile rhesus monkeys infected with simian immunodeficiency virus.NeuroReport 4, 263–266.
Weihe E., Schäfer M. K.-H., Erickson J. D., and Eiden L. E. (1994) Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat.J. Mol. Neurosci. 5, 149–164.
Weiner N. and Taylor P. (1985) Neurohumoral transmission: the autonomic and somatic motor nervous systems, inThe Pharmacological Basis of Therapeutics (Gilman A. G., Goodman L. S., Rall T. W., and Murad F., eds.), MacMillan, New York, pp. 66–99.
Wu D. and Hersh L. B. (1994) Choline acetyltransferase: celebrating its fiftieth year.J. Neurochem. 62, 1653–1663.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schäfer, M.K.H., Weihe, E., Erickson, J.D. et al. Human and monkey cholinergic neurons visualized in paraffin-embedded tissues by immunoreactivity for VAChT, the vesicular acetylcholine transporter. J Mol Neurosci 6, 225–235 (1995). https://doi.org/10.1007/BF02736782
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02736782