A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue.

We introduce a one-step histochemical method with cobalt as the precipitating agent for ferrocyanide for the light microscopic demonstration of acetylcholinesterase activity. This method was used to demonstrate acetylcholinesterase in normal cortical fibers and neurons, as well as pathological elements such as plaques and tangles. This procedure can also be easily combined with immunohistochemical methods that use diaminobenzidine as a chromogen.

Cytochemical Localization of Transferase Activities: Carnitine Acetyltransferase

Two methods for the cytochemical detection of free CoA and their utilization in the fine-structural localization of carnitine acetyltransferase in rat heart are described. The first utilizes the reducing property of the SH group of CoA to reduce potassium ferricyanide to potassium ferrocyanide, which in the presence of uranyl ions forms an electron-dense precipitate of uranyl ferrocyanide. The second utilizes the mercaptide-forming property of the free SH group of CoA, which forms a precipitate with cadmium ions. Using the uranyl-ferrocyanide method, reaction product due to endogenous enzymic activity was found on and between the cristae and between the inner and outer membranes of the mitochondria in fresh heart muscle. In aldehyde-fixed tissue activity was recorded only between the inner and outer membranes. Endogenous activity was removed by preincubation of the tissue in a solution of ferricyanide. On addition of acetyl CoA and carnitine to the incubation medium, fresh tissue, which had been preincubated in ferricyanide, showed reaction product between and on the cristae and between the inner and outer membranes of the mitochondria, while fixed tissue showed reaction product in the latter position only. In both cases the activity between the outer and inner mitochondrial membranes was dependent on both acetyl CoA and carnitine, while the cristae reaction occurred in the absence of carnitine, but required acetyl CoA. All activity was inhibited by mercuric chloride. Acetyl carnitine reduced the activity in the fixed tissue and had severe effects on the structure of fresh mitochondria. These results suggest the presence of carnitine acetyltransferase, which survives aldehyde fixation, on the inner surface of the outer mitochondrial membrane and/or the outer surface of the inner mitochondrial membrane. A second enzyme which released CoA from acetyl CoA occurred in relation to the cristae of unfixed mitochondria. The cadmium method was less satisfactory than the uranyl-ferrocyanide method but with fixed tissue gave confirmatory results.

THE SITE OF FERRICYANIDE REDUCTION BY REDUCTASES WITHIN MITOCHONDRIA AS STUDIED BY ELECTRON MICROSCOPY

In the present investigation the ultrastructural site of ferricyanide reduction by reductases in mitochondria in tissues (heart and kidney) of the normal adult rat was studied by the copper ferrocyanide method developed recently in our laboratory. Substrates used were sodium succinate, cytochrome c, dihydronicotinamide-adenine dinucleotide (NADH2), sodium dl-β-hydroxybutyrate, phenazine methosulfate and ubiquinone. All reductases tested were positive in mitochondria and the activities of the succinate-ferricyanide reductase, the cytochrome c-ferricyanide reductase, the NADH2-ferricyanide reductase and β-hydroxybutyrate-ferricyanide reductase were most evident. An ambiguous trace of activity was observed when phenazine methosulfate and ubiquinone were used as substrate. It was also observed that not all of the mitochondria revealed the reductase activity and the existence of functional heterogeneity among mitochondria was postulated. In enzymatically positive mitochondria, the reaction product, copper ferrocyanide, was found both in the mitochondrial membranes and in spaces such as the intracristal space and the outer space. The functional significance of the positive reaction in spaces is briefly discussed.

A THIOCHOLINE-LEAD FERROCYANIDE METHOD FOR ACETYLCHOLINESTERASE

The principle employed in the thiocholine-copper ferrocyanide method of Karnovsky and Roots has been adopted for the development of a similar procedure in which lead, complexed with Tris-acetate buffer, is used as the trapping agent for the ferrocyanide ion, formed by the preferential reduction of ferricyanide by thiocholine released enzymatically from acetylthiocholine. The resulting colloidal, faintly yellowish white precipitate, Pb2Fe(CN)6, can be viewed directly by light or phase contrast microscopy, or more easily following its conversion to PbS. The method has been shown to produce extremely fine localization of acetylcholinesterase in the cat superior cervical and stellate ganglia and at the motor end plates of mouse intercostal muscle.