Bodian's Silver Method Research Articles

The changing pattern of fibre bundles that pass through the optic chiasm of mice.

The organization of retinofugal fibres in the developing and adult mouse has been studied with transmission electron microscopy, autoradiography and the Bodian silver method. It has previously been shown that all retinal ganglion cell axons are in glial-wrapped bundles in the developing and adult optic nerve, but are not in similar bundles close to the chiasm. In the embryonic mouse this region shows a transition in glial morphology from an interfascicular to a radial type and here retinofugal fibres begin to form a new order related to their age. Growth cones become concentrated at the pial surface of the juxtachiasmatic nerve and older fibres are restricted to deeper regions. This same age-related order is also evident in the optic tract. However, the age-related order is lost within the chiasm, where growth cones, young and old fibres are again mingled in distinct bundles as they cross the mid-line. This study is particularly concerned with the structure of the mid-line bundles. These fibre bundles cross each other at right angles, and are recognizable in fetal and adult mice. In the adult, monocular injections of H3 proline followed by autoradiographic study show that the individual mid-line bundles are monocular and that they fuse again, losing the fascicular structure as they leave the chiasm and enter the tract. In the fetus and in the adult, the bundles generally lack a complete glial wrapping so that growth cones can lie in intimate contact with two crossing bundles, one coming from the left eye, the other from the right. The interesting question about the mechanisms that keep growth cones from entering the wrong bundles when they are in this position remains to be addressed.

Large amounts of neocortical βA4 deposits without neuritic plaques nor tangles in a psychometrically assessed, non-demented person

An 88-year-old mentally normal woman (Blessed test score=27) had very large amounts (397/mm 2) of deposits stained by anti-βA4 serum in the first temporal gyrus. Senile plaques and neurofibrillary tangles were lacking on sections stained with the Bodian's silver method, with anti-tau and anti-paired helical filament (anti-PHF) antibodies. The following βA4 deposits were found in decreasing order of frequency: diffuse (64.8%), stellate (24.4%), primitive (10.2%), classic (0.6%) plaques. Compact plaques were not observed. Diffuse deposits predominated in layers I, III and IV. On the contrary, the rare classic plaques were located in layers II and III. No amyloid angiopathy was seen with Congo red stain although βA4 deposits were seen in vessel walls with immunocytochemistry. These data indicate that severe diffuse βA4 deposits in the neocortex do not induce dementia. They suggest that the development of senile plaques composed of βA4 amyloid and of degenerating neurites is not related solely to the density of the diffuse βA4 deposits. Nor does it depend on the regional susceptibility of the nervous tissue since βA4 deposits were seen in highly vulnerable cortical areas. Some other, as yet unknown, factors seem necessary. In addition, determination of βA4 level in the neocortex is not sufficient for the diagnosis of dementia of Alzheimer type.

Tau-reactive neurofibrillary tangles in cerebellar cortex from patients with Alzheimer's disease

In 4 cases of Alzheimer's disease (AD) a tau antiserum immunostained thin, round or flame-shaped profiles disposed around the nuclei of the cerebellar fusiform-type Golgi cells. In adjacent sections either a Bodian silver method or Congo red failed to reveal any abnormal structures. Since normal tau immunoreactivity is located on axons and is absent in formalin-fixed tissue, the tau-reactive profiles are likely to correspond to small masses of abnormal filaments, antigenically similar to those composing neurofibrillary tangles (NFT). This observation indicates that in AD the NFT formation is more diffuse than that showed with conventional histological methods.

Paired helical filaments from Alzheimer disease patients contain cytoskeletal components

Abstract Neurofibrillary tangles from Alzheimer disease patients share antigenic determinants with neurofilaments and microtubule-associated proteins, as shown by light microscopy immunocytology. The present study addresses the issue of whether these determinants are located on the paired helical filaments or on other components of the neurofibrillary tangle. Sections from postmortem brains from Alzheimer disease patients were stained by using Bodian's silver method or immunostained by using poly- and monoclonal antibodies to neurofilaments and polyclonal antibodies to microtubules. Bodian's silver stain has an intense affinity for neurofibrillary tangles and has been shown to bind to specific domains of neurofilament subunits. The antibodies to neurofilaments used here immunostain most or all of the neurofibrillary tangles present in the sections whereas the antiserum to microtubule protein immunoreacted with about half of the neurofibrillary tangles. All of the antibodies as well as Bodian's silver stain reacted with the paired helical filaments. The epitopes that we have shown to be present in the paired helical filament, in contrast to the corresponding epitopes present in normal neuronal cytoskeleton, are insoluble in ionic detergent. It is concluded that these epitopes are integral components of the paired helical filaments and that, at least in part, paired helical filaments are derived from altered elements of the normal neuronal cytoskeleton.

Paired helical filaments from Alzheimer disease patients contain cytoskeletal components.

Neurofibrillary tangles from Alzheimer disease patients share antigenic determinants with neurofilaments and microtubule-associated proteins, as shown by light microscopy immunocytology. The present study addresses the issue of whether these determinants are located on the paired helical filaments or on other components of the neurofibrillary tangle. Sections from postmortem brains from Alzheimer disease patients were stained by using Bodian's silver method or immunostained by using poly- and monoclonal antibodies to neurofilaments and polyclonal antibodies to microtubules. Bodian's silver stain has an intense affinity for neurofibrillary tangles and has been shown to bind to specific domains of neurofilament subunits. The antibodies to neurofilaments used here immunostain most or all of the neurofibrillary tangles present in the sections whereas the antiserum to microtubule protein immunoreacted with about half of the neurofibrillary tangles. All of the antibodies as well as Bodian's silver stain reacted with the paired helical filaments. The epitopes that we have shown to be present in the paired helical filament, in contrast to the corresponding epitopes present in normal neuronal cytoskeleton, are insoluble in ionic detergent. It is concluded that these epitopes are integral components of the paired helical filaments and that, at least in part, paired helical filaments are derived from altered elements of the normal neuronal cytoskeleton.

Bodian's silver method reveals molecular variation in the evolution of neurofilament proteins

The recent demonstration that Bodian's silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (IFs), provides a specific marker for the identification of neurofilament polypeptides. We have applied the Bodian stain to SDS-PAGE separated polypeptides in nervous tissues from 9 species, representing neuronal evolution in 4 major phyla: chordata, mollusca, arthropoda and annelida. Every species tested except the arthropod showed intense silver staining of a set of polypeptides, each subsequently identified as NFs by immunomethods. These results demonstrate that the affinity of NFs for Bodian's silver stain is conserved during the evolution of nervous systems in a diverse spectrum of animals. Further, considerable variation in the molecular weight of NF subunits was found among the 6 vertebrates studied. This variation suggests that the molecular weight of NFs has not been conserved during evolution, a quality which appears to be unusual for a structural protein.

Bodian's silver method stains neurofilament polypeptides.

Bodian's silver method was used to stain polypeptides of rat spinal cord or peripheral nerve separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands corresponding to the three polypeptide subunits of the neurofilaments were intensely impregnated. Two other polypeptides were stained inconsistently and less intensely. The tubulin band was stained weakly or not at all; other polypeptides, including glial fibrillary acidic protein, actin, and vimentin, remained unstained. This novel application of Bodian's method provides indirect proof that neurofilaments are the neuronal subcellular structure stained by the technique.

Morphological and biochemical development of chick cerebrum cultured in vitro

Explants of 10–12 day-old embryonic chick cerebrum were cultured for up to 21 days by the “flying coverslip roller tube” method. By the middle of the second week and thereafter, closely packed neurons and a rich meshwork of myelin sheaths were observed in the living explants. Neurons in bipolar, tripolar or multipolar form were demonstrated by Bodian's silver method. Morphological development of cultures as shown by the presence of well-differentiated neurons, myelinated axons, and structurally complex synapses, was seen by electron microscopic examination. Seven enzymes of direct importance in neurotransmitter metabolism were studied by quantitative microchemical methods in cultures grown for 7–21 days. Increased activities of five enzymes, choline acetyltransferase, acetylcholinesterase, dihydroxyphenylalanine decarboxylase, 5-hydroxytryptophan decarboxylase, and catechol- O-methyltransferase, were detected over a 21-day period in culture. The activity of two other enzymes, glutamic acid decarboxylase and monoamine oxidase, decreased over the same period. Apart from glutamic acid decarboxylase and monoamine oxidase, enzymes involved in neurotransmitter metabolism showed an increase in activity concomitant with the morphological development of the cerebrum cultures and particularly with maturation of the synapses.

Morphological development of neonatal mouse hippocampus cultured in vitro

Explants of neonatal mouse hippocampus cultured for up to 65 days were studied by Bodian's silver method and electron microscopy. Two principal types of neurons usually found in vivo, the hippocampal pyramidal cells and the dentate granule cells, were identified in the cultured explants by these techniques. Electron microscopic observations demonstrate that the fine structure of neurons and synapses found in the explants faithfully corresponds to that of mature neurons in vivo. Besides axosomatic and axodendritic synapses, the presence of large complex axonal endings, which are probably those of mossy fibers, is also demonstrated and the morphological features of oligodendrocytes and astrocytes are described.

Light and electron microscope study of neurons and synapses in neonatal mouse olfactory bulb cultured in vitro

Explants of neonatal mouse olfactory bulb were maintained for 7–60 days in tissue culture, and the morphology of neurons and synapses were studied by Bodian's silver method and electron microscopy. In Bodian silver preparations two principal types of neurons, mitral cells and granule cells, were identified. Electron microscopic observations revealed that the neurons contained the orgelles usually found in adult neurons. Besides conventional axosomatic and axodendritic synapses, four types of atypical synaptic junctions were demonstrated: (a) Somatoaxonic or somatodendritic synapses; (b) reciprocal synapses which were identified as the dendrodendritic synapses; (c) serial synapses; (d) synapses in which synaptic vesicles were localized on both sides of synaptic junctions. These findings, particularly the occurrence of the atypical synaptic junctions, indicate that isolated fragments of mouse olfactory bulb can be maintained for extended periods of time and achieve a remarkable degree of morphological development under tissue culture conditions.

The intermediate root of the trigeminal nerve in the dog (Canis familaris).

AbstractThe intermediate root of the trigeminal nerve in the dog has been investigated both macroscopically and microscopically. Sixty‐two trigeminal complexes (trigeminal ganglion, trigeminal roots and the portion of the pons to which the roots were attached) in the dog were dissected out and removed. Each of the complexes was fixed in 10% formalin, dehydrated and embedded in paraffin. The paraffin blocks were cut serially at 10 μ. Every other slide was either stained with Luxol Fast Blue or impregnated with Bodian's silver method. In all cases, between the motor and sensory roots an intermediate root composed of one distinct rootlet was identified. Most frequently the intermediate root was attached to the pons from 0.5 to 3.0 mm lateral to the motor root and rostral to the sensory root from 0.5 to 2.0 mm. From its pontine attachment the intermediate root extended anteromedially for a distance of from 2.0 to 5.0 mm before it became incorporated in the lateral aspect of the free motor root. Closer to the trigeminal ganglion the motor root and the intermediate root fused with the expanding sensory root. The fibers in the intermediate root ranged from 1.5 to 7.5 μ in diameter with the majority of fibers (60 to 70%) having a diameter of from 4.0 to 6.0 μ. Approximately 10% of the fibers were unmyelinated. The total number of fibers in the intermediate root varied from 170 to 416 with an average of 266 fibers. The morphological data obtained in an experimental animal such as presented in this paper may provide a basis for future experimental work on the clarification of the functional role of the trigeminal intermediate root.

Intramural neural elements in components of the carotid bifurcation. A histological basis for differential function.

The histology of the specialized region of the carotid bifurcation in man was studied with Orcein stain for elastic tissue, Masson's trichrome for muscle and connective tissue, and Bodian's silver method for neural elements. Four distinct regions exist: the common carotid, which appears to be solely a conduit; the carotid sinus, which is thin and very elastic with its baroreceptors in the medial wall; the external carotid, which is highly muscular and presumably active; and the internal carotid, also highly muscular and presumably active. The transition between zones is abrupt. Prominent clusters of up to 30 or 40 multipolar nerve cells, 15 to 25 microns in diameter, were found in the subintimal region of the internal carotid artery, some in the common, and a few in the external carotid. Occasional rounder cells of similar size with only one process were seen, possibly of sensory type. No cell bodies were seen in the adventitia of any vessel. No relation to the perivascular plexus was established. It is speculated that the neurons may be related to the reported local differential response to environment by the internal and external carotid systems.

STUDIES ON THE INNERVATION OF THE STAPEDIUS MUSCLE OF THE CAT.

AbstractA study of nerve‐muscle relations and fine innervation patterns is reported for the stapedius muscle of the cat. Gross relations of the muscle to its associated nerves and other middle ear structures are shown by illustrations. Histologic features of innervation are studied with the Bodian silver method. the thiocholine technique, and iron hematoxylin and osmic acid stains.The muscle is richly supplied by 3 to 4 facial nerve branches in which no axons are found above 8 μ and the majority are unimodally grouped at 2–4 μ. The auricular nerve of the vagus does not penetrate the muscle. Three to four distinct zone is of fasciculi are found whose muscle fibers are 14–20 μ in diameter. Each zone is supplied by primary nerves whose branches do not exhibit terminal overlap or multiple innervation of muscle fibers. Short terminal axons lead to motor and plates from all intramuscular nerve branches. Nerve branches and motor end plates are confined to the basal third of the muscle. Sensory endings are not detected within the muscle or its tendon. It is estimated that each motor neuron supplies no more than five muscle fibers but more probably three or less.