Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Nov;84(22):8145–8149. doi: 10.1073/pnas.84.22.8145

Spectrophotometry in vivo, a technique for local and direct enzymatic assays: application to brain acetylcholinesterase.

G Testylier 1, P Gourmelon 1
PMCID: PMC299495  PMID: 3479782

Abstract

In vivo enzymology is not widely studied due to the lack of a well-adapted technology. We have developed a system that allows local and long-term spectrophotometric assays in brain tissue of live animals. It utilizes a miniaturized optical probe consisting of a multibarrel micropipette for reagent injections and optical fibers for light absorption measurements. We have applied this system to the colorimetric determination of brain acetylcholinesterase activity in rats. The reproducibility of the assay was demonstrated by repetitive assays over 24 hr, its specificity was established through the use of a highly specific organophosphorus inhibitor, and the activities measured in different brain areas agreed with the known distribution of acetylcholinesterase. No electroencephalographic abnormalities and no change in vigilance level were observed in the experimental animals. This methodology should prove to be useful for the colorimetric measurement of different enzymes or metabolites in various organs.

Full text

PDF
8145

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abdel-Latif A. A. Reaction of catecholamines with hydroxylamine and its application to the assay of catechol O-methyl transferase. Anal Biochem. 1969 Jun;29(3):468–475. doi: 10.1016/0003-2697(69)90331-5. [DOI] [PubMed] [Google Scholar]
  2. Butcher L. L., Talbot K., Bilezikjian L. Acetylcholinesterase neurons in dopamine-containing regions of the brain. J Neural Transm. 1975;37(2):127–153. doi: 10.1007/BF01663629. [DOI] [PubMed] [Google Scholar]
  3. Chen J. C., Rhee K. K., Beaudry D. M., Ramirez V. D. In vivo output of dopamine and metabolites from the rat caudate nucleus as estimated with push-pull perfusion on-line with HPLC-EC in unrestrained, conscious rats. I. Chromatographic and biological validation. Neuroendocrinology. 1984 May;38(5):362–370. doi: 10.1159/000123918. [DOI] [PubMed] [Google Scholar]
  4. Couraud J. Y., Di Giamberardino L. Axonal transport of the molecular forms of acetylcholinesterase in chick sciatic nerve. J Neurochem. 1980 Nov;35(5):1053–1066. doi: 10.1111/j.1471-4159.1980.tb07859.x. [DOI] [PubMed] [Google Scholar]
  5. ELLMAN G. L., COURTNEY K. D., ANDRES V., Jr, FEATHER-STONE R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  6. Gonon F., Buda M., Cespuglio R., Jouvet M., Pujol J. F. In vivo electrochemical detection of catechols in the neostriatum of anaesthetized rats: dopamine or DOPAC? Nature. 1980 Aug 28;286(5776):902–904. doi: 10.1038/286902a0. [DOI] [PubMed] [Google Scholar]
  7. Goossens P., Viret J., Leterrier F. Rat brain acetylcholinesterase turnover in vivo: use of a radioactive methylphosphonothiate irreversible inhibitor. Biochem Biophys Res Commun. 1984 Aug 30;123(1):71–77. doi: 10.1016/0006-291x(84)90381-4. [DOI] [PubMed] [Google Scholar]
  8. Hoover D. B., Muth E. A., Jacobowitz D. M. A mapping of the distribution of acetycholine, choline acetyltransferase and acetylcholinesterase in discrete areas of rat brain. Brain Res. 1978 Sep 22;153(2):295–306. doi: 10.1016/0006-8993(78)90408-0. [DOI] [PubMed] [Google Scholar]
  9. Mesulam M. M., Van Hoesen G. W. Acetylcholinesterase-rich projections from the basal forebrain of the rhesus monkey to neocortex. Brain Res. 1976 Jun 4;109(1):152–157. doi: 10.1016/0006-8993(76)90385-1. [DOI] [PubMed] [Google Scholar]
  10. Sandberg M., Lindström S. Amino acids in the dorsal lateral geniculate nucleus of the cat--collection in vivo. J Neurosci Methods. 1983 Sep;9(1):65–74. doi: 10.1016/0165-0270(83)90110-3. [DOI] [PubMed] [Google Scholar]
  11. Shimizu T., Nomiyama S., Hirata F., Hayaishi O. Indoleamine 2,3-dioxygenase. Purification and some properties. J Biol Chem. 1978 Jul 10;253(13):4700–4706. [PubMed] [Google Scholar]
  12. Soubrié P., Reisine T. D., Glowinski J. Functional aspects of serotonin transmission in the basal ganglia: a review and an in vivo approach using the push-pull cannula technique. Neuroscience. 1984 Nov;13(3):605–625. doi: 10.1016/0306-4522(84)90082-4. [DOI] [PubMed] [Google Scholar]
  13. Svaasand L. O., Doiron D. R., Dougherty T. J. Temperature rise during photoradiation therapy of malignant tumors. Med Phys. 1983 Jan-Feb;10(1):10–17. doi: 10.1118/1.595373. [DOI] [PubMed] [Google Scholar]
  14. Vigny M., Bon S., Massoulié J., Leterrier F. Active-site catalytic efficiency of acetylcholinesterase molecular forms in Electrophorus, torpedo, rat and chicken. Eur J Biochem. 1978 Apr 17;85(2):317–323. doi: 10.1111/j.1432-1033.1978.tb12241.x. [DOI] [PubMed] [Google Scholar]
  15. WEISSBACH H., SMITH T. E., DALY J. W., WITKOP B., UDENFRIEND S. A rapid spectrophotometric assay of mono-amine oxidase based on the rate of disappearance of kynuramine. J Biol Chem. 1960 Apr;235:1160–1163. [PubMed] [Google Scholar]
  16. Woolf N. J., Butcher L. L. Cholinergic neurons in the caudate-putamen complex proper are intrinsically organized: a combined Evans blue and acetylcholinesterase analysis. Brain Res Bull. 1981 Nov;7(5):487–507. doi: 10.1016/0361-9230(81)90004-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES