Enzyme histochemical methods applied in the brain

Catalytic enzyme histochemistry offers the possibility to demonstrate enzymes qualitatively and their activities quantitatively in brain sections at those sites where they are localized. To get an appropriate histochemical demonstration of enzymes, requirements are to be fulfilled with respect to the preparation of brain tissue, the detection methods, and the incubation conditions. For enzyme demonstration at the light microscopic level, brain tissue should be frozen as quickly as possible and for those at the electron microscopic level perfusion fixation using low concentrations of aldehydes seems to be best suited. The detection of enzymes in brain sections is preferentially performed by the so-called precipitation reactions with metallic ions, the tetrazolium and the diaminobenzidine methods. The application of these methods was shown in the example of aspartate aminotransferase, glutamate dehydrogenase, and cytochrome c oxidase. In the detection of enzymes incubation conditions should be chosen so that soluble enzymes cannot diffuse out of the sections into the incubation media and that the activities of enzymes are completely demonstrated. On the whole, all the precipitation reactions result in a water-insoluble reaction product which is precipitated at the enzymatic sites in brain sections. Finally, it is shown that scanning microphotometry is a valuable tool for the quantification of enzyme activities in brain sections. It is concluded that catalytic enzyme histochemistry using improved detection methods could be a source of results complementary to those provided by immunocytochemistry and microchemistry.     

A quantitative histochemistry technique for measuring regional distribution of acetylcholinesterase in the brain using digital scanning densitometry

Studies of brain acetylcholinesterase (AChE) are traditionally based on biochemical assays, immunoreactivity, and histochemistry. Conventional histochemistry yields rich morphological data from tissue sections but yields quantitative results only with great difficulty. Several histochemical methods developed in recent years, including microdensitometry, microphotometry, and video-based histochemistry, are effective in quantitative and detailed study of AChE in tissue sections. However, they are usually time-consuming. As we report here, we adapted digital scanning densitometry to quantitate AChE histochemical staining in brain sections. The AChE and butyrylcholinesterase (BuChE), as measured by the method, were heterogeneously distributed throughout the brain, results that are consistent with those obtained by biochemical methods. The staining intensity is dependent on section thickness, substrate concentration, and reaction time. The cholinesterase inhibitor methyl paraoxon significantly decreased AChE staining intensity. Furthermore, data acquired from densitometry are similar to those obtained by video-based microscopy or by spectrophotometry. The advantage of the densitometric measurements compared to other quantitative histochemical methods is that it is very rapid while collecting data that are equivalent in quality. Because the digital scanning densitometers provide high quality and sensitive imaging, wide dynamic ranges, and convenient image analysis software, they are very useful tools in quantitative histochemistry.     

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.     

Quantitative enzyme histochemistry in the brain

Two main groups of quantitative methods are used in the brain to relate enzymatic processes to cellular structures, i.e. the methods of microchemistry and microscopic histochemistry. Microchemistry tries to quantify enzyme activities in very small brain regions by miniaturizing biochemical methods, whereas microscopic histochemistry applies staining procedures to tissue sections, preserving the structural relationship that is present in situ and giving topological information on the distribution of enzymes which is indispensable in structural heterogeneous tissue as is the brain. The present review deals preferentially with microscopic methods and, in particular, with scanning microphotometry (image plane scanning). Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. end-point and kinetic (continuous monitoring) measurements which are described in detail. Methods for the microphotometric demonstration of certain important dehydrogenases (isocitrate dehydrogenases, succinate dehydrogenase, NAD-linked malate dehydrogenase, glutamate dehydrogenase and glycerol 3-phosphate dehydrogenase), of cytochrome c oxidase, hexokinase and acetylcholinesterase are presented. These methods were adapted for giving optimal demonstration of enzyme activities in the rat hippocampus. The examples are given to illustrate the aptitude and possibilities of this technique in the quantification of enzymes in the complex matrix of the brain.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Histochemistry of carbohydrates as performed by physical development procedures

Summary The histochemistry of carbohydrates demonstrated by means of physical development procedures has been reviewed in terms of the use and reliability of the procedures, physical developers, practice of the procedures, a fundamental series of light and electron microscopic methods and certain other promising aspects of this area of histochemistry. A line of fundamental light- and electron-microscopic histochemical methods for carbohydrates using physical development procedures such as periodic acid thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD), high- or low-iron diamine (HID or LID)-TCH-SP-PD and lectin-gold (LT-G)-PD and related methods has been found to be more efficient, compared with those without physical development procedures. Since a series of other promising histochemical methods for carbohydrates using physical development procedures have been derived or are now being introduced, these procedures could be regarded as an unusually potent vehicle for effectively advancing carbohydrate histochemistry in both light and electron microscopy.     

Innervation of the renal cortex.

Morphologic studies of renal innervation have utilized the methods of histochemistry and electron microscopy. Much information has been derived from examination of the renal cortex in monkey and rat. Fluorescence histochemistry shows a rich adrenergic innervation. Acetylcholinesterase can be demonstrated histochemically in the renal nerves by light and electron microscopy. Studies in the rat using 6-hydroxydopamine, a drug that selectively destroys adrenergic nerves, indicate that the glomerular arterioles and surrounding tubules are innervated by adrenergic nerves containing acetylcholinesterase. Distinct neurovascular and neurotubular junctions are observed the electron microscope. They are anatomically consistent with being the sites of synaptic transmission. Ultrastructural analysis of serial sections reveals that single individual axons contact multiple vascular cells and renal tubules. We now have a considerable body of information concerning the morphology of renal innervation are are beginning to appreciate the role of the renal nerves in kidney function.     

Acid phosphatase distribution in the liver in cirrhosis

The distribution of acid phosphatase in liver cirrhosis, as well as in its reverse development, was investigated in mice using histochemistry and electron histochemistry methods. Histochemistry demonstrated a sharp activity increase of acid phosphatase (as compared with the same in the material of partial hepatectomy) in liver cells (especially hepatocytes) during liver cirrhosis regression 10 days after a partial hepatectomy. Electron histochemistry has shown the enzyme withdraw out of hepatocytes and connective tissue cells of fibrotic stratum in the extra-cell medium. The reaction product localized on the neighbouring collagen fibres giving evidence that during reverse development of liver cirrhosis the lisosomal enzyme release from specified cells by means of exocytosis and they are involved in the lysis of collagen.     

Histochemistry for nanomedicine: Novelty in tradition

During the last two centuries, histochemistry has provided significant advancements in many fields of life sciences. After a period of neglect due to the great development of biomolecular techniques, the histochemical approach has been reappraised and is now widely applied in the field of nanomedicine. In fact, the novel nanoconstructs intended for biomedical purposes must be visualized to test their interaction with tissue and cell components. To this aim, several long-established staining methods have been re-discovered and re-interpreted in an unconventional way for unequivocal identification of nanoparticulates at both light and transmission electron microscopy.Key words: histochemistry, immunohistochemistry, nanoparticles, light microscopy, transmission electron microscopy     

Some observations on the fine structure of light and dark cells in the gall bladder epithelium of the mouse

Summary Electron microscopic observations have been made of the two epithelial cell types, light barrel-shaped and dark rod-shaped cells in the gall bladder of the mouse.The light cells have a voluminous cytoplasm of low electron opacity in which cell organelles such as mitochondria, elements of granular endoplasmic reticulum, and free ribosomes undergo more or less degenerative changes. However, there are a relatively abundant Golgi apparatus and numerous lysosomal dense bodies. The ultrastructural features of the light cells suggest that they are an aged, degenerative cell type with declining functional activity and a high degree of hydration.The dark cells are characterized by a high concentration of mitochondria and free ribosomes, more or less distinctive elements of granular endoplasmic reticulum, and well developed components of the Golgi apparatus. Such ultrastructural characteristics indicate that the dark cell type has a high synthetic activity.What has been observed in the present study can well be correlated with the results of previous studies on the same cells by methods of light microscopic histochemistry.     

Localisation of monoamines in nerves of the posterior salivary gland and salivary centre in the brain of Octopus

Summary Slices of the posterior salivary gland and of the superior buccal lobe of the brain of Octopus vulgaris take up ³H-5-hydroxytryptamine in vitro. By light and electron microscopical radioautography the uptake is localised in certain neuronal perikarya and axonal varicosities in the superior buccal lobe, and in nerves that end in the secretory tubules of the posterior salivary gland. These structures do not incorporate ³H-noradrenaline. After formaldehyde histochemistry, monoamine fluorescence is found in some neuronal perikarya of the superior buccal lobe, and in nerves entering the secretory tubules of the gland. The posterior salivary gland nerve, which originates in the superior buccal lobe and supplies the gland, shows a pronounced accumulation of fluorescence on the proximal side of a ligature applied in vivo. It is suggested that monoamines are transported from the brain to the gland by the posterior salivary gland nerves.J. B. would like to thank the Science Research Council, Great Britain, for financial support.     

The structure of the coeliac ganglion of a teleost fish Myoxocephalus scorpius

Summary In order to compare the structure of a teleost sympathetic ganglion with those of other vertebrates, light, fluorescence histochemical and electron microscopy were carried out on the coeliac ganglion of the scorpion fish, Myoxocephalus scorpius. In common with studies on other vertebrates, fluorescence histochemistry distinguished two cell types: a) principal neurones which exhibited low levels of specific catecholamine fluorescence and comprise the majority of neurones in the ganglia, and b) smaller intensely fluorescent cells, some of which had processes tens of micrometers long.With the electron microscope, the principal cells were seen to make axodendritic and axosomatic synapses with axons containing mainly 30 nm agranular vesicles at the synaptic site while in other vertebrates usually only one or other synaptic association is present.Both the somata and the processes of intensely fluorescent cells contain 300–600 nm diameter vesicles many of which have electron dense cores. These cells are also innervated by axons containing 30 nm agranular vesicles.     

Histochemical localization of acetylcholinesterase in isolated brain synaptosomes

Summary The distribution of acetylcholinesterase in isolated brain synaptosomes of the rat and calf was studied with electron microscopic histochemistry. The enzyme was observed mainly in the membrane of the postsynaptic neurone and to a much lesser extent in the axon membrane.     

Characteristics of the ultrastructural organization of the nuclei of somatic cell hybrids

A study was made of the ultrastructure of cell nuclei of two types of hybrid clones obtained from the fusion of Chinese hamster with human skin fibroblasts, and from that of mouse hepatoma cells with mink fibroblasts. In cell nuclei of the eight hybrid clones deep invaginations of the inner membrane, not characteristic of the parent cells, were revealed. Analysis of serial sections, and application of electron microscopic radioautography and histochemistry have suggested that these structures are associated with the nuclear envelope which is necessary for regulating the superfluous chromosome localization in the hybrid nucleus.     

Structuro-functional organization of the fibrillar component and ground substance of human rib cartilage

A complex morphological investigation (histology, histochemistry, scanning and transmissive electron microscopy, electron histochemistry) has been performed to study the intercellular substance of the costal hyalinous cartilage. It has been demonstrated that the fibrillar framework of the costal cartilage consists of branching collagenous fibrillae, chaotically scattering. The fibrillae are surrounded with the ground substance; one of its components is the reticular ruthenium-positive structure.     

Ultrastructural characteristics of the localization of the enzymes of cyclic nucleotide metabolism in the mammalian brain

The enzymes involved in cyclic nucleotide turnover (adenylate cyclase, guanylate cyclase, phosphodiesterase) were located in various regions of the brain, in synaptosomes and isolated synaptic membranes, using electron cytochemical methods. The interaction of the above enzymes in the processes of adrenergic and cholinergic cell reception in the CNS are discussed.     

Nitric oxide synthase is localized predominantly in the Golgi apparatus and cytoplasmic vesicles of vascular endothelial cells

The localization of nitric oxide synthase (NOS) in vascular endothelial cells of submucosal blood vessels from the guinea-pig ileum was examined using NADPH diaphorase histochemistry at the light microscopic level, and endothelial NOS immunohistochemistry at the light and electron microscopic level. The pattern of staining observed following NADPH diaphorase histochemistry and endothelial NOS immunohistochemistry was identical. Endothelial cells of the arterioles, capillaries and venules showed small patches of intense, perinuclear staining. Under the electron microscope, endothelial NOS immunoreactivity was found predominantly in association with the Golgi apparatus and with the membranes of some vesicles. Small regions of the plasma membrane and the rough endoplasmic reticulum also showed some immunoreactivity. The presence of NOS in the Golgi apparatus and in vesicles raises the possibility that NOS may be exteriorized by endothelial cells, and hence that nitric oxide is synthesized extracellularly.     

Brain glycogen during non-diapausing and diapausing development of Pieris brassicae larvae and pupae

Summary

Histological and cytological localization of glycogen was studied in the brain of Pieris brassicae larvae and pupae by histochemistry and electron microscopy. The major glycogen deposits were observed in glial cells located between the cortex and the neuropile and also in perineurial cells. The concentrations of brain glycogen were measured in the larvae and the pupae during non-diapausing and diapausing development. In addition, we demonstrated that starvation reduces the density of the brain glycogen deposits as well as the concentrations of glycogen.     

Fine structural localization of peroxidase in the rat thyroid

Summary The fine structural localization of a peroxidase activity in the rat thyroid follicular epithelial cell was studied by histochemistry at electron microscopic level. The reaction product is recognized chiefly in the cisternae of the elements of granular endoplasmic reticulum and of nuclear envelope. Golgi vesicles or apical small vesicles, mitochondria, and dense granules are sometimes positive for this reaction. The relationship between the fine structural localization of peroxidase and the site of the iodination of thyroglobulin is discussed.     

The nature of cnidarian desmocytes

The electron microscope reveals that the cnidarian desmocyte is an ectodermal cell which forms acidophil protein tonofibrillae intracellularly. One end of the cell is bound to mesogleal fibrils; the other becomes embedded in the thickening cuticle. The bundle of tonofibrillae later becomes rivetshaped and the cell dies, but still the mesoglea remains bound to the cuticle by means of the rivet. The histochemistry and formation of the rivet as well as the comparative cytology of cnidarian desmocytes are discussed.