Stabilization of Silver Chromate Golgi Impregnation in Rat Central Nervous System Neurons Using Photographic Developers. II. Electron Microscopy

The silver chromate precipitate present in neurons impregnated according to the Golgi-rapid and Golgi-Kopsch procedures can be stabilized by treatment with a photographic developer. In a complementary light microscopic study the stabilizing properties of various photographic developers were tested. Kodalith, Elon-ascorbic acid, HC-110, D-19 and Neutol proved to be the most successful. In the present electron microscopic study, we studied the distribution, shape and size of the particles found in Golgi-rapid and Golgi-Kopsch-impregnated neurons by treatment with each of these developers and, simultaneously, the effect of the developer on the preservation of the ultrastructural details. The reaction product after developer-treatment of Golgi-rapid material is sufficiently stable to withstand embedding and thin sectioning, whereas in Golgi-Kopsch material additional gold chloride “Honing” is necessary. In Golgi-impregnated, Kodalith-, Elon-ascorbic acid-, or HC-110-treated material the formed particles are small and located in the cytoplasm, limited by the plasma membranes of the impregnated profiles. In Golgi-impregnated, D-19 treated neurons, the formed particles are relatively coarse. The majority of these particles are within cytoplasm, but particles may also lie either across or entirely outside the plasma membranes of the impregnated profiles. A large number of the small particles in Golgi impregnated, Neutol-stabilized neurons can be seen partly or entirely outside the plasma membranes of the impregnated profiles. Good original ultrastructural preservation seems to be unaffected by developer treatment. Treatment of Golgi material with sodium bromide before stabilization (bromide substitution) results in the formation of small silver particles both inside and outside the impregnated profiles. The sodium bromide step of this procedure has an adverse effect on the preservation of ultrastructural detail.     

Chemical Stabilization of Golgi Silver Chromate Impregnations

Blocks of neural tissue were processed by a modified Golgi-Kopsch procedure and by the rapid Golgi method. Following the impregnation, the blocks were embedded in celloidin, sectioned at 100μm, and collected in 70% alcohol. The sections were then processed as follows: 1) rinsed in distilled water; 2) substituted with 0.4M sodium bromide for five minutes; 3) reduced in Kodak D-19 developer; and 4) treated in 0.5M sodium thiosulfate. The silver chromate deposits within the impregnated cells are converted successively to silver bromide and to reduced silver by this procedure. Sections so treated resist decomposition of the Golgi impregnation, and they may be counterstained with conventional aqueous cresyl violet to demonstrate the cytoarchitecture of the Golgi-impregnated tissue.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Silver Impregnation of Degenerating Axons in the Central Nervous System: A Modified Technic

Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr., soaked in 0.5% phosphomolybdic acid for 0.25-1.0 hr., and subsequently treated with 0.05% potassium permanganate for 4-10 min. (The duration of the latter treatment is critical and individually variable). Subsequent procedure is as follows: decolorize in a mixture of equal parts of 1% hydroquinone and 1% oxalic acid; wash thoroughly and soak sections in 1.5% silver nitrate for 20-30 min.; ammoniacal silver nitrate (silver nitrate 0.9 g., distilled water 20 ml., pure ethanol 10 ml., strong ammonia 1.8 ml., 2.5% sodium hydroxide 1.5 ml.) 0.5-1.0 min.; reduce in acidified formalin (distilled water 400 ml., pure ethanol 45 ml., 1% citric acid 13.5 ml., 10% formalin 13.5 ml.) 1 min.; wash, and pass section through 1 % sodium thiosulf ate (0.5-1.0 min.); wash thoroughly and pass sections through graded alcohols and xylene (3 changes); cover in neutral synthetic resin.     

A Silver Impregnation Technique for the Study of Steroid Receptors in Central Nervous System Tissue Prepared for Autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular stains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

Circuit-Specific Coinfection of Neurons in the Rat Central Nervous System with Two Pseudorabies Virus Recombinants

Neurotropic alphaherpesviruses have become popular tools for transynaptic analysis of neural circuitry. It has also been demonstrated that coinfection with two viruses expressing unique reporters can be used to define more complicated circuitry. However, the coinfection studies reported to date have employed nonisogenic strains that differ in their invasive properties. In the present investigation we used two antigenically distinct recombinants of the swine pathogen pseudorabies virus (PRV) in single and double infections of the rat central nervous system. Both viruses are derivatives of PRV-Bartha, a strain with reduced virulence that is widely used for circuit analysis. PRV-BaBlu expresses beta-galactosidase, and PRV-D expresses the PRV membrane protein gI, the gene for which is deleted in PRV-BaBlu. Antibodies to beta-galactosidase identify neurons infected with PRV-BaBlu, and antibodies monospecific for PRV gI identify neurons infected with PRV-D. The ability of these strains to establish coinfections in neurons was evaluated in visual and autonomic circuitry in which the parental virus has previously been characterized. The following conclusions can be drawn from these experiments. First, PRV-D is significantly more neuroinvasive than PRV-Bartha or PRV-BaBlu in the same circuitry. Second, PRV-D is more virulent than either PRV-Bartha or PRV-BaBlu, and PRV-BaBlu is less virulent than PRV-Bartha. Third, in every model examined, PRV-D and PRV-BaBlu coinfect some neurons, but single infections predominate. Fourth, prior infection with one virus renders neurons less permissive to infection by another virus. Fifth, prior infection by PRV-D is more effective than PRV-BaBlu in reducing invasion and spread of the second virus. Collectively, the data define important variables that must be considered in coinfection experiments and suggest that the most successful application of this approach would be accomplished by using isogenic strains of virus with equivalent virulence.     

The Differentiated Silver Impregnation of Certain Conducting Pathways in the Peripheral Nervous System

Feti of man, rabbit and mouse can be silver-impregnated in such a way that different kinds of nerve-fiber pathways in the peripheral nervous system are distinguishable from one another by different degrees of impregnation intensity. In the application of Bielschowsky's technic, it is important that the impregnation time in silver nitrate should be tested with respect to the species in question. This time is for man 6 weeks, for rabbit 3 weeks and for mouse 2–3 days.     

The Differentiated Silver Impregnation of Certain Conducting Pathways in the Peripheral Nervous System

Feti of man, rabbit and mouse can be silver-impregnated in such a way that different kinds of nerve-fiber pathways in the peripheral nervous system are distinguishable from one another by different degrees of impregnation intensity. In the application of Bielschowsky's technic, it is important that the impregnation time in silver nitrate should be tested with respect to the species in question. This time is for man 6 weeks, for rabbit 3 weeks and for mouse 2-3 days.     

A New Fixative Solution to Precede the Reduced Silver Impregnation of Arthropod Central Nervous Systems

Arthropod central nervous tissue is fixed for 1 hr at 20 C in 8% pure formic acid in 1:1 n-butanol/n-propanol prepared immediately before use (FBP), then washed for 15-30 min in 90% ethanol, and embedded in paraffin wax. Impregnation is by modified Ungewitter techniques in which the silver bath is preceded by mercury/cobalt mordanting, or by modified Holmes' methods following similar mordanting procedures. the methods yield high resolution of axons with minimal background staining, while the staining of neuronal somata is suppressed. They succeed with brains of crustacea and Odonata and other difficult materials. Tissues fixed in FBP are hard and require care in sectioning.     

Silver Impregnation of Degenerating Axon Terminals in the Central Nervous System: (1) Technic. (2) Chemical Notes

A new silver technic, tested on the brain of the rat, is described, especially suitable for demonstrating terminal degeneration within the central nervous system. It is a modification of the Glees method, designed to avoid use of tap water in preparing solutions. Some of the chemical principles underlying the process of reductive liberation of metallic silver from ammoniacal silver nitrate solutions are discussed.     

Tau Kinetics in Neurons and the Human Central Nervous System

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Expression of the Oligodendrocyte-Myelin Glycoprotein by Neurons in the Mouse Central Nervous System

Abstract: The oligodendrocyte-myelin glycoprotein (OMgp) is a 110-kDa glycosylphosphatidylinositol-linked protein that was initially identified as a myelin-specific protein but whose precise function remains unknown. In this study, immunohistochemistry, western blots, in situ hybridization, and northern blots were used to determine the distribution of OMgp in the mouse brain. OMgp is present in a concentration detectable on western blots in the brains of newborn mice, and its concentration gradually increases until day 24 of life. OMgp mRNA is also present in amounts detectable on northern blots in the brains of newborn mice, and its concentration gradually increases until day 21 of life, after which the concentration diminishes a little. Most of the OMgp in the mouse brain appears to be expressed in diverse groups of neurons, but it is particularly prominent in large projection neurons such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem, and anterior horn cells of the spinal cord. However, OMgp is not confined to these cells and is expressed in cells in the white matter as well. The OMgp gene is placed within an intron of the neurofibromatosis type I gene and on the opposite strand. This organization raises the possibility that there may be a relationship between the functions of the products of the two genes. In support of this possibility, we show that within the mouse CNS OMgp and neurofibromin are expressed in the same cell types.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Characterization and Biosynthesis of Cyclic-AMP-Binding Proteins in the Rat Central Nervous System

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Presence of Phospholipase D In Rat Central Nervous System Axolemma

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.     

A Silver Impregnation Technique for Normal Axons in the Human Central Nervous System for Celloidin and Epon Sections, with Substitutes for Soft Tap Water

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.