A QUANTITATIVE MAPPING OF ACID PHOSPHATASE IN THE BRAIN OF THE RHESUS MONKEY.

THE distribution of acid phosphatase in the central nervous system as demonstrated by histochemical methods has been investigated by LANDOW, KABAT and NEWMAN (1942), WISLOCKI and DEMPSEY (1948), and by SHIMIZU (1950) in comparative studies that included mice, rats, guinea pigs, rabbits, cats and chickens. Numerous papers deal with the cytological distribution of acid phosphatase in selected regions of the brain of various species, but little attention has been given to gradations of enzyme activity among those regions. The present paper reports a mapping of acid phosphatase activity in the brain of the rhesus monkey. A quantitative histochemical method was used, based upon spectrophotometric measurements of dye extracts from standard size tissue discs taken from brain sections that had been incubated with a substrate for acid phosphatase. Previous mappings of oxidative enzymes (FRIEDE, 1961), and particularly a mapping of lactate dehydrogenase in the brain of the rhesus monkey (FRIEDE and FLEMING, 1963), facilitate comparison of enzyme patterns. MATERIAL AND METHODS Histochemical method. Preliminary testing and standardization of the quantitative method for acid phosphatase activity were done on cat and monkey brains; a detailed account of methodology will be given elsewhere. For the present experiment three adult, healthy rhesus monkeys were killed under pentothal anaesthesia; the brains were removed immediately and placed in 10 % formalin at 4". After 8 hr fixation, the material was cut into slices, 3-5 mm thick. Twenty-four hours after removal of each brain, 30 p frozen sections were cut, rinsed, and stored in chilled distilled water until all material from the brain had been prepared. All sections were transferred to a 4-litre Erlenmeyer flask containing a small portion of the buffer component of the incubation medium. The substrate and diazo salt were mixed with the remaining portion of the buffer and filtered as quickly as possible; the filtrate was added immediately to the sections and buffer in the Erlenmeyer flask and incubation was started. The incubation medium was a mixture of 1 mg of sodium-a-naphthyl phosphate (Signia Chemical Co. St Louis, Missouri) and 1 mg of the stable diazotate of o-amino azotoluene (Fast Garnet GBC) (Dajac Laboratories, Philadelphia) in 1 ml of 0.2 M-Walpole buffer of pH 5 (PEARSE, 1960). For each monkey brain approximately 2 I. of reaction mixture were used. The sections were incubated in an Eherbach water-bath shaker with constant agitation for 1 hr at 38". The reaction was stopped by transferring all sections into a large quantity of 10% formalin. The stained sections were mounted temporarily on a microslide covered with three layers of parafilm. Discs were cut from selected areas of the sections with stainless steel tubes, 1.1 mm and 2.4 mm in diameter. Each disc was placed in a stoppered test tube containing a drop of distilled water. Care was taken not to transfer particles of parafilm into the tubes since these would cause turbidity and false readings. The azo dye was extracted with 1 ml of chloroform-ethanol (1 :l), v/v and the colour density was determined within 10-30 min at 555 mp in microcells in a Beckman DU spectrophotometer. For the data collected in Table I an average of ten samples were taken from the regions indicated for each monkey, and means and standard deviations were calculated. The dependability of this method was tested in various experiments (unpublished data) the results of which are only briefly summarized: tissue fixed in formalin after incubation was used since it was impractical to cut discs from loose sections During the required 24-hr fixation period, some loss of