Electron microscopic localization of acetylcholinesterase activity in the central nervous system: chemical basis of a catalytic activity of Hatchett's brown (cupric ferrocyanide) precipitate revealed by 3,3'-diaminobenzidine.

The chemical basis of the histochemical method for acetylcholinesterase described by Tago et al. was studied. The primary histochemical reaction of Tago's method is performed with diluted Karnovsky's medium, resulting in the in situ formation of Karnovsky's precipitate in minute quantities. The visualization of acetylcholinesterase activity is made in a secondary reaction where diaminobenzidine (DAB) is oxidized in the presence of hydrogen peroxide by the peroxidase-like activity of the histochemical precipitate. Since Karnovsky's precipitate is already known to be a mixture of Cu+3Fe (CN)6 and Cu++2Fe++(CN)6, the catalytic activity of these two complexes was studied in vitro comparatively to cupric ferricyanide (non-histochemical precipitate) and to copper and iron ions participating in the complexes. We showed that only the first two complexes intensely oxidized DAB with or without hydrogen peroxide. Thus, Tago's method is based on the same catalytic activity as Hanker's method (oxidation of DAB using non-diluted Karnovsky's medium without hydrogen peroxide). Since both complexes are able to oxidize DAB without hydrogen peroxide, we propose to describe this catalytic activity as oxidoreductase-like activity. Tago's method was modified for ultrastructural observations by avoiding metal intensification of the DAB precipitate. We obtained fine localization of DAB precipitate in the lumen of endoplasmic reticulum and Golgi apparatus of the neurons of substantia nigra.