PROPERTIES OF CELL NUCLEI ISOLATED FROM VARIOUS REGIONS OF RAT BRAIN: DIVERGENT CHARACTERISTICS OF CEREBELLAR CELL NUCLEI

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 10⁸ per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of 'large' and 'small' cell nuclei to cell types in the brain is discussed.     

ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).     

Some cytochemical correlations between oxidase activity (cytochrome and peroxidase) and chemical structure of bis(phenylenediamines)

Summary A series of 17 bis(phenylenediamine) derivatives have been prepared and compared with 3,3-diaminobenzidine (DAB) with regard to their ability to demonstrate cytochrome oxidase activity, peroxisome activity, horseradish peroxidase activity, erthrocytic peroxidase activity in cytochemical preparations, and bovine catalase activity in in vitro experiments. The results are tabulated, some illustrative photomicrographs are included and interesting correlations are discussed.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U. S. Public Health Service.Acknowledgement for technical assistance is due Charles Hatton and William Brown.     

Preparation and structure-activity relationship of reagents for cytochrome oxidase activity: potential for light and electron microscopy

Summary The syntheses of a series of p-substituted aromatic diamines and some representative thioureido- and mercapto-naphthols are described. Their cytochemical behavior in the Nadi reaction using fresh frozen sections of rat heart was studied in an attempt to learn more about the structural requirements of the Nadi reaction for cytochrome oxidase.The lipid solubility of the dyes formed from either N,N-dimethyl-p-phenylenediamine or ADN (4-amino-1-N,N-dimethylnaphthylamine) with 1-naphthol can be decreased by substituting hydrophilic hydroxyethyl or dihydroxypropyl side chains for one or more of the methyl groups in these reagents. However, these dyes diffuse readily, fade rapidly and are too soluble in water as well as lipid, which renders them unsuitable for light and electron microscopy.Burstone's reagent PPD (N-phenyl-p-phenylenediamine) and ADN (4-amino-1-N,N-dimethylnaphthylamine) are capable of undergoing self condensation either alone or in the presence of an unreactive naphthol to give colored indamines which osmicate readily. Blocking either the 4-position of N-phenyl-p-phenylenediamine, the secondary amino group, or both of these positions, with a methyl group did not prevent indamine formation.N-Benzyl-, N-4-methylbenzyl- and N-4-methoxybenzyl-p-phenylenediamine are good reagents for the demonstration of cytochrome oxidase activity in the Nadi reaction with light microscopy. They produce blue indoaniline dyes with 1-naphthols and do not self condense to form highly colored pigments. These indoanilines are osmiophilic eliminating the necessity for introducing osmiophilic groups for electron microscopic studies. Their drawback in electron microscopy is in the droplet nature of the deposit.Incorporating a carbonyl or carboxyl function into the 2-position or a bulky substituent into the 5-position of 1-naphthol prevented indoaniline formation.A new method for the cytochemical demonstration of cytochrome oxidase is presented utilizing a new reagent N,N-bis(p-aminophenyl)-1,3 xylylenediamine (XIII) (BAXD). This reagent is polymerized to an insoluble osmiophilic polymer distributed in non-droplet form, thus providing a useful method for demonstrating cytochrome oxidase activity in light and electron microscopy.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland. Presented (in part) as a Presidential Address (AMS) at the Third International Congress of Histo- and Cytochemistry on August 19, 1968 in New York, N.Y. Acknowledgement is due Lionel, Katzoff for preparation of the thiolnaphthols XXXV and XXXVI, and to Norberto I. Schinitman for the osmium analysis.     

Ultrastructural cytochemistry of p-N,N-dimethylamino-beta-phenethylamine (DAPA) oxidation reactions.

The ultracytochemical localization of amine oxidase (AO) activity is demonstrated with a new substrate, p-N,N-dimethylamino-beta-phenethylamine (DAPA). DAPA was designed to yield a stronger reducing agent on oxidation by monoamine oxidase (MAO) than is obtained from the MAO substrate, tryptamine, upon oxidation. Thus MAO and possibly other oxidase(s) can be demonstrated with DAPA and the tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). The latter is a nonosmiophilic tetrazolium salt which is reduced to an osmiophilic formazan. In addition, DAPA itself demonstrates AO activity ultracytochemically with and without BSPT. We speculate that either oxidative polymerization of DAPA or Schiff's base formation with protein after aldehyde formation is responsible for the latter reaction, which is made permanent for ultracytochemical localization by osmication at a later step. DAPA oxidation reaction products are demonstrated in guinea pig kidney, specifically in the endoplasmic reticulum, nuclear envelope and mitochondrial outer compartments and cristae. Differences in reaction product characteristics and localization in relation to formaldehyde fixation and the localization of reaction product in mitochondrial cristae, as well as outer compartments, suggest that DAPA oxidation is mediated through one or more MAOs and possible other oxidases.