Abstract
Localization of cholinesterases in the tissues of the American cockroach was studied histochemically by thiocholine method which was developed by Koelle and his coworkers. For the quantitative estimation of the activities of cholinesterases, the manometric method was used, and the effects of the fixatives and the salt concentrations used in the histochemical procedures on the activities of cholinesterases were estimated.
The optimum pH for the maximum activity of cockroach cholinesterases was 7.2 to 8.0, and the optimum acetylcholine concentration of the substrate was 0.03 to 0.008M. Therefore, cholinesterases of the cockroach are very similar to the specific or true cholinesterases of mammals.
For detail observation of the sites of cholinesterases in the tissues of the cockroach, it is desirable to prepare the sections as thin as possible. On the other hand, to keep the activities of the enzymes as high as possible, it is better to prepare the sections from fresh tissues, but it is difficult to cut thin sections from fresh materials. To facilitate thin sectioning, tissues were fixed in 10 per cent neutral formaline for 48 hours at room temperature varied from 5 to 13°C. By this treatment, activities of cholinesterases decreased to 54 percent as compared with that of the fresh tissues. Fixation of the tissues in pure acetone for two hours also gave good result, but prolonged fixation destroyed the cholinesterase activity. Fixation with ethyl alcohol for more than two hours badly destroyed the enzymes, so it is not recommendable.
Media that contained 25 per cent of sodium sulphate were used in histochemical study for the purpose of eliminating artifacts which caused diffusion of cholinesterases, but sodium sulphate itself destroyed 42 per cent of cholinesterase activity. Incubation media used in histochemical method contained 25 per cent of sodium sulphate and some of cupper sulphate and glycine, and their pH were 6.0. In these media, activities of cholinesterase were adout 20 per cent as compared with that of optimum condition. Though these media were far from the optimum condition for the maximum activity of the enzyme, but were sufficient to detect sites of cholinesterases in the tissues.
Activities of cholinesterases were found in all nervous tissues such as brain, ganglions, nerve cords, and peripheral nerve fibres in muscles and both digestive organs and reproductive organs. No choliesterase activity was found in dorsal vessel, fat bodies and trachea. Low activity of cholinesterases was found in hemolymphs, but their localization in the hemocytes were not detectable.
In general cholinesterases in the American cockroach were of specific type and hydrolyzed acetylthiocholine more easily than butylthiocholine. But cholinesterases of the digestive and reproductive organs hydrolyzed much more butylthiocholine than that of other tissues, so they might be of both specific and non-specific types. Whether the activities of cholinesterases in the muscles, digestive organs and reproductive organs would be derived from the nervous system which ended in these tissues or from the cells of these tissues themselves were not determined.
Cholinesterases were found in cytoplasms, but not in nuclei. In nervous system, activities of cholinesterases were high on the nerve sheaths and surfaces of neurons, and we could detect the runnings of neurons by the distribution of the enzymes. A very high activity of cholinesterases was found in the brain, but their distribution in it was not uniform. In some parts of the brain little or no cholinesterase activity was found. This might indicate the different physiological functions in the different regions of the brain.
