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The basis for silver staining of synapses of the mammalian spinal cord: a light and electron microscope study

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... One is reminded of Ted Jones, friend and support to many, describing the informal meetings in Oxford's Department of Human Anatomy in the 1960s, when recently printed micrographs were passed around and ideas about what on earth they might be looking at were discussed. Incidently, these early ultrastructural studies also demonstrated that one of arguments against chemical transmission -that the synaptic cleft was too wide to account for the brief delays recorded -had been due to the failure of silver impregnation techniques to label the entire presynaptic terminal (Gray and Guillery, 1961). The synaptic cleft was not 1 µm wide -as silver staining of neurofibrils observed with the light microscope suggested -but some 30 to 50 times narroweronly 200-300Å in diameter. ...
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More than a century of dedicated research has resulted in what we now know, and what we think we know, about synapses and neural circuits. This piece asks to what extent some of the major advances – both theoretical and practical – have resulted from carefully considered theory, or experimental design: endeavors that aim to address a question, or to refute an existing hypothesis. It also, however, addresses the important part that serendipity and chance have played. There are cases where hypothesis driven research has resulted in important progress. There are also examples where a hypothesis, a model, or even an experimental approach – particularly one that seems to provide welcome simplification – has become so popular that it becomes dogma and stifles advance in other directions. The nervous system rejoices in complexity, which should neither be ignored, nor run from. The emergence of testable “rules” that can simplify our understanding of neuronal circuits has required the collection of large amounts of data that were difficult to obtain. And although those collecting these data have been criticized for not advancing hypotheses while they were “collecting butterflies,” the beauty of the butterflies always enticed us toward further exploration.
... Since longer formalin fixation times are required for light-microscopical staining, post-osmication is delayed and direct electron-microscopical study of material fixed and stained for light microscopy would be expected to show poor preservation. However, it is not certain to what extent the poor preservation found by Gray & Guillery (1961) with the Bielschowsky method and Guillery & Ralston (1964) with the Nauta method is due to fixation or to damage during staining. Direct correlation after early osmication, as is possible with some Golgi techniques (Stell, 1964;Blackstad, 1965), circumvents these difficulties, but such fixation is unsatisfactory for most other nervous-system stains. ...
Article
The formalin perfusion technique of Pease has been shown to be a satisfactory fixation method for both light and electron microscopy of the nervous system of rat and goldfish, so allowing correlative studies to be made on tissue from the same region of one brain. Immediate post-osmication is not necessary for good ultrastructural preservation and even if itis delayed for 1 week, neuronal structure is adequate for all but detailed cytological work. Unosmicated tissue stained with uranyl acetate and lead citrate showed protein-like structures but not lipids, with membranes appearing as unstained bands. ‘Dark’ glial cells occurred near the surface of the cortex and could not be eliminated by prolonged fixation in situ. ‘Dark’ neurons were found rarely. It is suggested that, with this method of fixation, any poor preservation of tissue in direct light- and electron-microscopical correlative studies is due to the subsequent processing rather than the initial fixation. Preliminary results on material stained by a new Golgi-Kopsch modification and by reduced silver methods and subsequently examined with the electron microscope suggest that such damage is not extensive.
... The word "neurotubules" resurfaced occasionally later on to denote a more defined class of tubules in nerve cells [e.g., Gonatas and Robbins, 1965], but these would eventually be called microtubules in all types of cells (see below). Further study of neuronal fine structure also introduced the term "neurofilaments" [Palay and Palade, 1955], some of which were finally identified as the neurofibrils of old [Gray and Guillery, 1961]. ...
Article
Some unnoticed or seldom remembered precedents of current views on biological motion and its structural bases are briefly outlined, followed by a concise recapitulation of how the present theory has been constructed in the last few decades. It is shown that the evolution of the concept of fibers as main constituents of living matter led to hypothesizing microscopic structures closely resembling microtubules in the 18th century. At the beginning of this period, fibers sliding over each other and driven by interposed moving elements were envisioned as the cause of muscle contraction. In the following century, an account of the mechanism of myofibril contraction visualized longitudinal displacements of myosin-containing submicroscopic rodlets. The existence of fibrils in the protoplasm of non-muscle cells, a subject of long debate in the second half of the 19th century, was virtually discarded as irrelevant or fallacious 100 years ago. The issue resurfaced in the early 1930s as a theoretical notion—the cytosquelette—nearly two decades before intracellular filamentous structures were first observed with electron microscopy. The role originally assumed for such fibrils as signal conductors is nowadays being reappraised, although under new interpretations with a much wider significance including modulation of gene expression, morphogenesis, and even consciousness. Since all of the above ancestral conceptions were eventually abandoned, the corresponding current views are, to a certain extent, recurrent. Cell Motil. Cytoskeleton 46:73–94, 2000 © 2000 Wiley-Liss, Inc.
... The role of these structures awaits a more detailed understanding of the integrated function of the nerve cell than is now available. According to investigations by Gray, Guillery, and Boycott (2,8), the neurofibrillae shown by optical microscopy in nerve cells, processes, and endings correspond to bundles of the neurofilaments shown by electron microscopy. These authors have presented strong evidence for the conclusion that the neurofilaments are responsible for the argyrophilia of neural tissue. ...
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The lateral vestibular nucleus consists of multipolar isodendritic neurons of various sizes The distal segments of some dendrites display broad expansions packed with slender mitochondria and glycogen particles. These distinctive formations are interpreted as being growing tips of dendrites, and the suggestion is advanced that they are manifestations of architectonic plasticity in the mature central nervous system. Unlike large neurons elsewhere, the giant cells (Deiters) contain small Nissl bodies interconnected in a dense mesh-work. The Nissl substance is characterized by randomly arranged cisterns of the endoplasmic reticulum and by a high proportion of free ribosomes. Whether attached or free, ribosomes usually cluster in groups of four to six, and larger polysomal arrays are rare. Free ribosomal clusters also occur in the axon hillock and the initial segment. The neuronal perikarya contain distinctive inclusions consisting of a ball of neurofilaments enveloped by a complex honeycombed membrane. The failure of these fibrillary inclusions to stain with silver suggests that the putative argyrophilia of neurofilaments may reside in an inconstant matrix surrounding them. Giant cells of Deiters are in intimate contact with two kinds of cellular elements—astroglial processes and synaptic terminals. Oligodendroglial cells are only rarely satellites of giant cells; in contrast, they are frequently satellites of small and medium-sized cells.
... Gliofibrils cross over in the cell body and converge to form fascicles within the processes. microscopy with electron microscopy and thin-sectioning technology finally allowed the ultrastructural inspection of silver staining in nervous tissue, which showed that the argyrophilic material constituting the classical neurofibrils corresponds with the neurofilaments of current terminology (Gray and Guillery, 1961; see also Wuerker and Kirkpatrick, 1972). Furthermore, some of the responses of the " neurofibrils " to changes in environmental conditions, as reported by both Cajal and Tello (see above), were also confirmed by early electron microscopy studies (Boycott et al., 1961). ...
Article
One hundred years ago, a novel kind of reticularism threatened to displace the neuron doctrine as the established model of functional organization of the nervous system. The challenging paradigm, championed by Stephan von Apáthy and Albrecht Bethe, held that nerve impulses propagate along neurofibrils connected in a continuous network throughout all nerve cells. Santiago Ramón y Cajal, a leading figure in the conception of the neuron doctrine, headed again the battle against this return of reticularism. Dissatisfied with the available staining techniques, he devised the “reduced silver nitrate method” that even Camillo Golgi recognized as the best at the time for revealing the neurofibrils. In 1904 Cajal already published over a dozen papers in three languages describing neurofibril distributions in the nervous systems of diverse vertebrates and invertebrates, under both normal and experimental conditions. Next he investigated the involvement of neurofibrils in the process of nerve regeneration. This unprecedented survey led him to the conclusion that the neurofibrils are linear “colonies” of particles constituting a semi-solid, dynamic internal skeleton of the nerve cell. Apáthy reacted with a long invective paper that Cajal had no choice but acknowledging. His comprehensive reply, published in 1908, meant the effective end of the renewed reticularist campaign against the neuron doctrine. Along the way, a visionary and today almost forgotten chapter in the history of the cytoskeleton had also been written.
... The morphology of these growth cones was less elaborate than that reported for growth cones in the developing spinal cord of Xenopus tadpoles (Nordlander 1987). We also observed small terminal " rings " (Fig. 3c, d) reported to occur during axonal sprouting in the injured central nervous system (Ramón y Cajal 1913 Cajal –1914) or in some synaptic contacts (Gray and Guillery 1961; Novotny 1979). To confirm the occurrence of growth cones, we performed TEM studies. ...
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In fresh-water turtles, the bridge connecting the proximal and caudal stumps of transected spinal cords consists of regenerating axons running through a glial cellular matrix. To understand the process leading to the generation of the scaffold bridging the lesion, we analyzed the mitotic activity triggered by spinal injury in animals maintained alive for 20-30 days after spinal cord transection. Flow cytometry and bromodeoxyuridine (BrdU)-labeling experiments revealed a significant increment of cycling cells around the lesion epicenter. BrdU-tagged cells maintained a close association with regenerating axons. Most dividing cells expressed the brain lipid-binding protein (BLBP). Cells with BrdU-positive nuclei expressed glial fibrillary acidic protein. As spinal cord regeneration involves dynamic cell rearrangements, we explored the ultra-structure of the bridge and found cells with the aspect of immature oligodendrocytes forming an embryonic-like microenvironment. These cells supported and ensheathed regenerating axons that were recognized by immunocytological and electron-microscopical procedures. Since functional recovery depends on proper impulse transmission, we examined the anatomical axon-glia relationships near the lesion epicenter. Computer-assisted three-dimensional models revealed helical axon-glial junctions in which the intercellular space appeared to be reduced (5-7 nm). Serial-sectioning analysis revealed that fibril-containing processes provided myelinating axon sheaths. Thus, disruption of the ependymal layer elicits mitotic activity predominantly in radial glia expressing BLBP on the lateral aspects of the ependyma. These cycling cells seem to migrate and contribute to the bridge providing the main support and sheaths for regenerating axons.
... and today known as microtubules (De Robertis & Schmitt 1948) and thin intraaxonal filaments (Schmitt & Geren 1950) which were later termed neurofilaments (Gray & Guillery 1961). The cytoskeleton as we know it today is composed of three types of filaments; microtubules, intermediate filaments and microfilaments (Factin). ...
Conference Paper
Schwann cells of peripheral nerves undergo significant changes in morphology and gene expression during development and regeneration of the peripheral nervous system (PNS) after nerve injury. This plasticity is associated with the greater regenerative capability of the PNS compared with the CNS. The transcription factor c-Jun is highly expressed in Schwann cells following nerve injury and is required for the reprogramming of mature Schwann cells to become repair Schwann cells that promote regeneration. Mice with a Schwann cell-specific c-Jun deletion fail to generate these repair cells after nerve injury and consequently have a severe failure of axonal regeneration. One striking phenotype of Schwann cells lacking c- Jun is that in culture they lack normal bi- and tri-polar morphology and in the distal stump of injured nerve they form irregular and flattened profiles. I assessed whether the loss of c-Jun affected their ability to migrate, an important property of repair Schwann cells in vivo after nerve injury. Using two complementary in vitro assays, I revealed a significant reduction in cell migration and found that c-Jun deletion caused changes to the actin cytoskeleton and reduced the formation of focal adhesions. Nevertheless, performing quantitative polymerase chain reaction on a number of genes already known to regulate the cytoskeleton failed to reveal differences between RNA from c-Jun knockout Schwann cells and control RNA. I next investigated whether Schwann cell deletion of c-Jun leads to a reduction of Schwann cell migration and axonal outgrowth across the nerve bridge of the transected nerve. I developed an assay to create a nerve bridge that can be replicated easily in size and proximal-distal location. I achieved this by transecting the peroneal branch of the sciatic nerve while leaving the tibial branch intact to support the two nerve stumps. This method provides a useful model of nerve transection. The results reveal that in the c-Jun cKO mouse at 7 days after injury Schwann cells succeeded in migrating across the nerve bridge, but axons show poor re-entry into the distal nerve stump. This requires further investigation.
Article
It is commonly assumed that most, if not all, neurons contain the intermediate filament protein class known as the neurofilament protein-triplet. The following study investigated the distribution of neurofilament protein-triplet immunoreactivity in selected regions of the guinea-pig central nervous system using monoclonal antibodies directed against phosphorylation-independent epitopes on the three subunits under optimal tissue processing conditions. Neurofilament protein-triplet immunoreactivity was present in distinct subpopulations of neurons in the cerebellar cortex, neocortex, hippocampal formation, retina, striatum and medulla oblongata. In many of these regions, labelled neurons represented only a small proportion of the total. The selective distribution of this intermediate filament protein class was confirmed in double-labelling experiments using antibodies to the neurofilament protein-triplet in combination with antibodies to other neuronal markers. The distribution of neurofilament protein-triplet immunoreactivity also correlated with the distribution of staining observed with a silver impregnation method based on Bielschowsky. The present results in combination with previous observations have demonstrated that the neurofilament protein-triplet is found in specific subclasses of neurons in different regions of the nervous system. Content of this intermediate filament protein class does not appear to be correlated with neuronal size or length of projection. These results also suggest that the selectivity of staining between neuronal classes observed with classical silver impregnation methods may be due to the presence or absence of the neurofilament protein-triplet. The present results may also provide a new perspective on the basis of the selective vulnerability of neurons in degenerative diseases.
Article
Microtubules, neurofilaments, and neurotubules have been studied in neural epithelial cells and differentiating motor neuroblasts in the spinal cord of chick embryos after glutaraldehyde fixation.Microtubules are a prominent feature of neural epithelial cells in the early neural tube (33–40-hour embryos) and in 2- and 3-day embryos. In these cells they are especially conspicuous in the narrow apical ends.Early neuroblasts have both microtubules (neurotubules) and fine filaments. In 2-day embryos, some cells, which may be early neuroblasts forming or about to form axons, have areas of fine filaments and a few microtubules in the basal cytoplasm. As the neuroblasts begin to form processes, microtubules (neurotubules) and fine filaments are seen in the axon and a few microtubules are scattered in the perikaryon. In slightly more advanced neuroblasts (3-day embryos), neurotubules are scattered among other organelles in the cell body and are prominent features of the axon, which sometimes contains neurofilaments also. Motor neuroblasts of 7-day embryos have neurotubules in the perikaryon, dendrites, and axon.The epithelial microtubules and the microtubules of the neuroblasts are similar in appearance to the spindle fibers of the mitotic cells in the neural tube of these embryos.Possible roles of these structures in the differentiating neuroblast are discussed.
Article
The fine structure of the dorsal horn of the cat cord has been compared with the light microscope cytoarchitectural studies. Several distinct laminae may be seen with the electron microscope. Lamina I contains marginal cells of Waldeyer and has its processes oriented in a horizontal direction. Lamina II contains small neurons and processes oriented vertically, and may be distinguished from lamina III by the rich content of myelinated axons in the latter. In laminae I–III there are numerous non-myelinated axons and many axodendritic and axoaxonal synapses but few axosomatic ones. Complex synaptic arrays made up of a central axon synapsing with other axons and dendrites are commonly seen. Laminae IV, V and VI are distinguished by their content of large neurons. They exhibit many axodendritic and axosomatic synapses but few axoaxonal ones and no complex synaptic arrays. Synaptic knobs containing rings of neurofilaments are present only in laminae IV–VI and synapse usually with somata or dendrites of neurons. There are differences in synaptic vesicle configuration and in the density of subsynaptic membranes associated with them. Differentiation of the dorsal horn into horizontal laminae is demonstrated by both light and electron microscope studies. The regional differences in neuronal organization may represent differences in functional organization as well.
Chapter
This brief account is intended merely to serve as an introduction to the phases of neural development that follow primary neural induction. The subject of primary embryonic induction has been reviewed by Saxén and Toivonen (1962) and the classical studies have been dealt with magisterially by Spemann (1938).
Article
Prolonged acrylamide administration produces motor nerve-terminal branch degeneration and impairs axonal outgrowth following nerve crush. It is unclear how early terminal branch degeneration is initiated and whether there is a compensatory regenerative response at the neuromuscular junction (NMJ). A modified Pestronk and Drachman silver-acetylcholinesterase strain was used to carry out a detailed morphometric analysis of the NMJ in soleus and lumbrical muscles. Rats were given 3, 5, or 10 doses of acrylamide, 35 mg/kg/day, by intraperitoneal injection, 5 days/week, and killed 4, 7, or 14 days after the first dose, respectively. Degenerating terminal branches were evident in soleus NMJ after only three doses of acrylamide. Diminished synaptic vesicle content, neurofilament accumulations and tubulo-vesicular profiles were evident after three doses. At later time points, degenerating terminals contained few synaptic vesicles and were engorged with neurofilaments. Endplate lengthening, indicative of denervation supersensitivity, accompanied degeneration. Terminal sprouting proliferated after 3 and 5 doses but was less prominent after 10 doses. Although similar changes occurred in the lumbrical muscle, they were not initiated until after 5 doses. These experiments reveal that pathological changes in terminal branches commence earlier and after a lower cumulative dose of acrylamide than previously reported and suggest that acrylamide exerts a primary effect at motor nerve-terminal branches. Early, vigorous terminal sprouting indicates that acrylamide does not prevent the initiation of regeneration, but with prolonged treatment does cause degeneration of maturing sprouts.
Article
The synaptology of the Cebus lateral geniculate nucleus (LGN) was studied after varying (3–15 days) periods of survival following unilateral and bilateral eye enucleations. Part of the material was processed with the Glees and Nauta techniques for light microscopy while the rest was processed for electron microscope observation. The study revealed a variety of degenerated terminals in the parvocellular portion of the LGN and allowed the differentiation of the retinal from the extraretinal terminals. The most frequent synaptic type of retinal origin is a glomerular large central terminal (up to 20 μ long) which makes axodendritic and axoaxonic synaptic contacts with geniculate dendrites and peripheral small terminals. Simple axodendritic and axosomatic terminals of retinal and extraretinal origin were also found. The early changes affecting the geniculate neurons and astrocytes during the degenerative process are described. These results are discussed in relation to: 1) previous work on the LGN synaptology of cats and macaques; 2) the physiology of the LGN; 3) the phagocytic role of astrocytes; 4) the general problem of degeneration in the central nervous system. In addition, a correlation between the light and electron microscope observations is attempted.
Article
A battery of polyclonal and monoclonal antibodies raised against the triplet of identified neurofilament protein subunits was used to investigate neurofilament protein immunoreactivity in neurons of the guinea-pig coeliac ganglion. Using optimal conditions of fixation and tissue processing for each antibody we found that only 20% of the postganglionic sympathetic neurons in the guinea-pig coeliac ganglion contain neurofilament protein-triplet immunoreactivity. Double labelling with neurofilament protein-triplet antibodies raised in different species demonstrated that all of these antibodies labelled the same population of neurons. Double labelling using mouse monoclonal antibodies against neurofilament proteins in combination with rabbit polyclonals to neuronal markers showed that neurofilament protein-triplet immunoreactivity is restricted to specific chemically coded subpopulations of noradrenergic neurons. Approximately 52% of neurons in the ganglion contain neuropeptide Y and are presumed vasomotor neurons projecting to blood vessels in the submucosa of the small intestine. Virtually none of the neuropeptide Y-containing neurons were labelled with neurofilament protein-triplet antibodies. Neurons that contain somatostatin (21%) project to the submucous ganglia of the small intestine. Approximately two-thirds of neurons containing somatostatin are immunoreactive for the neurofilament protein-triplet. The other postganglionic neurons in the ganglion (27%) project to the myenteric plexus of the small intestine and do not contain either neuropeptide Y or somatostatin. Approximately a quarter of these neurons were labelled with neurofilament protein-triplet antibodies.
Article
Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.
Article
The pattern of normal nerve fibers associated with barrel-shaped structures in the somatosensory cortex of both young and adult mice, has been studied using a reduced silver method. In adult animals, the "barrel" sides and septa can be seen to contain densely packed bundles of nerve fibers running vertically between layers III and V. In parasagittal sections, these fibers appear as very dark bands between adjacent barrels, while in tangenital sections the fibers, cut in cross-section, appear as rings of dark spots concentrated around the barrel edges. In contrast to this, barrels in immature animals have dark, evenly stained centers and pale, cell dense sides. This immature pattern can first be distinguished in 2-day-old animals and persists until 18 to 19 days. Between 18 and 24 days a change from the immature to the adult pattern occurs with the appearance of darkly stained, fine fibers within layer IV, particularly within the barrel sides. It is suggested that the immature pattern is due, primarily, to the staining of thalamic afferents while the adult pattern appears with the development of intracortical and association fibers. Electron microscopy, on tissue previously treated by the silver method, shows that the silver deposits are mainly attached to longitudinal elements of the axoplasm and not associated with myelin. This may explain the success of this method in showing fibers in young, unmyelinated brains.
Article
The filamentous degenerating Purkinje cell axons and boutons have been studied in the light and electron microscope after staining with the Glees, Nauta-Laidlaw, Fink-Heimer (I) and Eager methods.Degenerating filamentous fibers and boutons are stained when the Glees method is used, but local silver deposit is present also in dendrites. Previous observations have shown that dendritic appendages and terminal myelinated axons can be impregnated in Glees sections. No conclusions as regards distribution of boutons or concerning synaptic details can therefore be drawn from light microscopical observations o of Glees sections.Degenerating filamentous fibers and boutons are not stained with the Nauta-Laidlaw and Fink-Heimer techniques. However, the degenerating filamentous fibers start to show argyrophilia when they darken, and are heavily stained with both techniques when they have been transformed to the dark type. The degenerating darkened boutons show affinity to silver in Fink-Heimer sections, and are heavily impregnated when they have reached the dark stage. Degenerating boutons are only occasionally impregnated when the Nauta-Laidlaw method is used.Degenerating filamentous, as well as degenerating dark fibers, are impregnated in Eager sections. Degenerating boutons are, however, only occasionally stained. In the series examined in the present study, this method therefore appears to be similar to the Nauta-Laidlaw technique as regards impregnation of degenerating boutons.However, it is important to note that filamentous as well as dark degenerating fibers are stained in Eager sections.
Article
Cytoskeleton is one of the basic structures of eukaryotic cells. It is a system of fibrillary or tubular proteins of three classes: microtubules, microfilaments and intermediate filaments. Neurofilaments, a member of the last class, occur in neural cells, where they are necessary for the cell to function properly. They are important in supporting and partly controlling the axon diameter and axonal transport. Neurofilaments are probably involved also in regulatory mechanisms, mainly through their extremely rich phosphorylation potential. This article introduces briefly the cytoskeleton in general and focuses on the structure and function of neurofilaments. A review with 189 references.
Article
Previous studies have demonstrated that neurofilament proteins are expressed by type II neurons in the enteric plexuses of a range of species from mouse to human. However, two previous studies have failed to reveal this association in the guinea-pig. Furthermore, immunohistochemistry for neurofilaments has revealed neurons with a single axon and spiny dendrites in human and pig but this morphology has not been described in the guinea-pig or other species. We have used antibodies against high- and medium-weight neurofilament proteins (NF-H and NF-M) to re-examine enteric neurons in the guinea-pig. NF-H immunoreactivity occurred in all type II neurons (identified by their IB4 binding) but these neurons were never NF-M-immunoreactive. On the other hand, 17% of myenteric neurons expressed NF-M. Many of these were uni-axonal neurons with spiny dendrites and nitric oxide synthase (NOS) immunoreactivity. NOS immunoreactivity occurred in surface expansions of the cytoplasm that did not contain neurofilament immunoreactivity. Thus, because of their NOS immunoreactivity, spiny neurons had the appearance of type I neurons. This indicates that the apparent morphologies and the morphological classifications of these neurons are dependent on the methods used to reveal them. We conclude that spiny type I NOS-immunoreactive neurons have similar morphologies in human and guinea-pig and that many of these are inhibitory motor neurons. Both type II and neuropeptide-Y-immunoreactive neurons in the submucosal ganglia exhibit NF-H immunoreactivity. NF-M has been observed in nerve fibres, but not in nerve cell bodies, in the submucosa.
Article
The work was devoted to the ultrastructural analysis of the neurohistological preparations. Sections of the tissue from the precardial parts of the pulmonary and caval dog veins were impregnated with silver salts after Campos and embedded in the araldite by a special method. Electronmicroscopi studies showed reduced silver adsorbed by the tissue of the impregnated preparations to exhibit a granular structure (the granules were 30-400 A in size). The largest of them were revealed in the axoplasm of the myelinated and unmyelinated nerve conductors, whereas the smaller ones found in various cellular and fibrous formations of the tissue substrate; silver granules were as a rule absent within the thickness of the myelin sheath. The noted regularities of adsorption and distribution of the silver granules in the impregnated preparations caused a morphologically expressed phenomenon of argentophilia.
Article
Transverse frozen sections of desheathed rat peripheral nerve were incubated in media of different composition prior to fixation and processing for electron microscopic examination. Neurofilaments remained intact when these tissues were incubated in calcium-free media. A loss of neurofilaments and their replacement by granular debris occurred in myelinated and unmyelinated fibers following incubation in media containing 2 mM calcium. The calcium-mediated disruption of neurofilaments was inhibited by preincubation or incubation with 1 mM p-chloromercuribenzoate (PCMB). The inhibition by preincubation with PCMB could be partially reversed by subsequent preincubation with 10 mM dithioerythritol (DTE). Calcium-mediated breakdown of neurofilaments did not occur after prolonged preincubation in calcium-free media, a finding which suggested that neurofilament disruption was dependent upon a tissue factor which could be lost or inactivated in frozen-sectioned nerve tissues. The findings of the present study provide morphological evidence that neurofilament disruption in mammalian peripheral nerve is mediated by a calcium-activated, PCMB-sensitive enzyme in the axoplasm of myelinated and unmyelinated nerve fibers.
Article
This special issue of the Journal of Eukaryotic Microbiology (JEM) summarizes achievements obtained by generations of researchers with ciliates in widely different disciplines. In fact, ciliates range among the first cells seen under the microscope centuries ago. Their beauty made them an object of scientia amabilis and their manifold reactions made them attractive for college experiments and finally challenged causal analyses at the cellular level. Some of this work was honored by a Nobel Prize. Some observations yielded a baseline for additional novel discoveries, occasionally facilitated by specific properties of some ciliates. This also offers some advantage in the exploration of closely related parasites (malaria). Articles contributed here by colleagues from all over the world encompass a broad spectrum of ciliate life, from genetics to evolution, from molecular cell biology to ecology, from intercellular signaling to epigenetics etc. This introductory chapter, largely based on my personal perception, aims at integrating work presented in this special issue of JEM into a broader historical context up to current research.
Article
Alterations of nerve proteins were studied in desheathed segments of rat sciatic nerves which were freeze-thawed and incubated in solutions containing calcium or EGTA. Calcium caused a selective loss of 69,000, 150,000, and 200,000 MW neurofilament triplet proteins as well as some loss of 55,000–57,000 MW proteins when nerve proteins were examined by SDS gel electrophoresis. The loss of nerve proteins was accompanied by a variable appearance of 25,000, 36,000, and 72,000 MW proteins. The calcium-induced changes did not occur in nerve segments which had been heated to 60°C for 30 min and were prevented by preincubation of tissues with 1 mM p-chloromercuribenzoate (PCMB), N-ethyl-malemide (NEM), or iodoacetamide. The calcium-induced alterations of nerve proteins were attributed to a calcium-activated thiol protease which preferentially degrades neurofilament proteins.Neurofilament protease of rat peripheral nerve was characterized by studying the alterations of nerve proteins under different incubational conditions. Calcium activated proteolysis was demonstrated between pH 6.0 and 8.8 and at 4, 20, and 37°C. Protein breakdown occurred with 50 μM calcium, and could be simulated by strontium and, to a lesser extent, by barium and lanthanum. Other metals had strong (Hg, Zn, Cd, Cu, Co), weak (Pb and Ag), or no (Al, Mg, and Mn) inhibitory effects on enzymatic activity. Proteolysis was also strongly inhibited by TLCK (1 mM) and by TPCK (1 mM) but not by PMSF (1 mM). The properties of neurofilament protease closely resemble those of calcium-activated proteases described in several tissues.
Article
Rainer (Ray) Guillery was a remarkably productive neuroscientist and as such left an indelible mark on the field, both in terms of his direct contributions and also through his success at mentoring and nurturing young scholars who went on to successful careers of their own. Ray's work profoundly advanced our understanding of the related fields of development and thalamocortical functioning; his work was highly imaginative and insightful; and he was a cherished colleague and role model for his many former students and friends in the field. Ray's scholarly efforts were carried out on three continents. He trained initially in London and, after serving on the faculties at the Universities of Wisconsin and Chicago in the United States, he returned to England at the University of Oxford. After retiring from his Oxford post, he went back as a visiting scholar to the University of Wisconsin, and then moved to a post at the University of Marmara in Turkey, which is located in the Asian sector of Istanbul. He finally returned to Oxford in an emeritus capacity and remained there until his death.
Chapter
This material, earlier observed by Arndt (1875), Key and Retzius (1876), and Flemming (1882), was carefully described by Nissl (1889, 1894), who established the importance of alcohol fixation for its visualization. Under the light microscope, this cytoplasmic component occurs in the form of bodies which are stained intensely by means of basic dyes (e.g., methylene blue, toluidine blue, thionine, and cresyl violet). This material, also known as chromophilic substance, chromidial substance, tigroid bodies, etc., exhibits distinctive and consistent characteristics in different types of neuron: for example, it appears as large, rhomboid masses separated by light channels in the motoneurons of the spinal cord (Fig. 5.1) and brainstem and as small granules in some neurons of the sensory ganglia (Fig. 5.1) and in the Mauthner cells of teleosts. In light microscope preparations, it seems to be absent from certain small neurons, such as cerebellar cortex granule cells, retina bipolar cells, and substantia gelatinosa small neurons (see later, this section).
Article
Nervous tissue staining is a critical experimental technique which is necessary to investigate neurodegenerative diseases. However, due to the complexity of staining processes and the interference of external factors, it always leads to the instability of staining consequence. This article summarizes the principle and application of nervous tissue staining methods, including Nissl staining, neuron staining, glial staining, myelin stai-ning, synapse staining and nerve fibers staining, which have been successfully established around the world. And their strengths and weaknesses are also described, aiming to guide the researchers to select appropriate staining method.
Chapter
The idea of a neurofilamentous skeleton inside nerve cells is not new. Almost 100 years ago Schultze first expressed the view regarding the presence of neurofibrillar networks within neurons. Many workers doubted the existence of these structures because the methods used to visualize them were capricious, different results being found in different laboratories (see review by Parker, 1929). In the 1890s few were able to emulate the techniques of Apáthy whose skills were widely acclaimed. It was not until the development of the novel and reproducible methods for silver staining by Bielschowsky and Ramón y Cajal that neurofibrils were routinely found. With the advent of high-resolution electron microscopic techniques, we now know that the neurofibrillary structures seen by these early microscopists were composed of the 100-Å-diameter intermediate filaments characteristic of nerve cells—neurofilaments (Boycott et. al., 1961; Gray and Guillery, 1961). In this review, we discuss the more recent history regarding neurofilaments and focus on current unresolved questions regarding their architecture, enzymatic modification, and development. While being a fairly general account of the neurofilament literature, especially over the past decade, we dwell in depth on key issues—like the nature of neurofilament sidearms, and the phosphorylation dependence of many neurofilament antibodies that will be helpful for both specialist and more general readers.
Chapter
There is little, if any, correlation between the histology of cutaneous nerve terminal structures and their physiological specificity. Cutaneous nerve terminals are associated with an organized epidermal or dermal structure. Specificity is conferred on the nerve by the non-nervous structure, which is considered as a specific transducer or at least as responsible for the specific sensitivity of the nerve. In addition to touch, pressure, heat, and cold, the fifth classical modality of cutaneous sensation is pain. Although probably resulting from damage of the skin, pain as such cannot be correlated with forms of external energy, as the other cutaneous sensory modalities can be; the existence of pain is purely subjective. A valid differentiation can be made between pricking pain and diffused pain. In brief, pricking pain is well localized, travels rapidly to consciousness, and does not outlast the provoking stimulus. Diffused pain takes some time to reach consciousness, is poorly localized (particularly in the absence of concomitant tactile stimuli), and usually considerably outlasts the provoking stimulus. It appears that the peripheral fibers that conduct impulses generated by painful stimuli lie not only in the C spectrum, but also in the smaller myelinated group.
Article
Neurofibrillar rings and spheres one-half to 2 μ in diameter occur throughout the dorsal lateral geniculate nucleus of the cat. Counts confirm that they occur predominantly in laminae A and A1, and only rarely in lamina B and the central interlaminar nucleus. The rings lie in zones of lightly argyrophilic tissue between perikarya and rarely contact cell bodies. The argyrophilic zones are 5–20 μ wide, forming a continuous reticulum throughout laminae A and A1. Electron micrographs show rings and nests of neurofilaments that match the neurofibrillar structures in size and distribution. These filaments are not found in axons, but occur in spheroid dendritic appendages, which have previously been described on Golgi preparations. The appendages lie in synaptic zones which are 5–20 μ wide and encapsulated by glial lamellae. Within the zones two major types of specialized contact are recognizable. “Regular” contacts (axo-dendritic and axo-axonal) resemble synaptic contacts in other parts of the CNS. “Filamentous” contacts (axo-dendritic and dendro-dendritic) show neurofilaments in the dendritic cytoplasm and show no concentration of vesicles on the axonal side. Within the appendages filaments from adjacent filamentous contacts join to form a nest around a group of mitochondria. Comparison with previous studies suggests that these dendritic appendages, which receive regular and filamentous contacts, form a major receptive site for the retinal afferents.
Chapter
This chapter summarizes a number of electron microscope studies on the hippocampal region, carried out in the laboratory and discusses their various stages of completion. Various junctions are observed during these studies on the hippocampal region. The cell bodies and initial part of the dendritic shafts of pyramidal cells of the regio superior are discussed. The surface is smooth here, and boutons of normal size are flattened against it; occasionally moderate membrane specialization as in Gray's type 2 synapses is seen. Perhaps these simple synapses correspond to the septohippocampal fibres, which are claimed to end at this level and which are very efficient in activating the pyramidal cells. Axo-somatic contacts in the granular layer of the fascia dentata are without conspicuous membrane specializations but a peculiarity of form is observed. Small spine-like buds with thin necks emerging from the granular somata invaginate many of the boutons ending on these somata. In the stratum radiatum of the regio superior boutons of normal size end with type 1 contact on dendritic branches, as well as on spines.
Article
Neurofibrils, identified after staining with Cajal's reduced silver nitrate, for example, were thought by many senior histologists in the nineteenth and early-twentieth centuries to conduct action potentials. There was no basis for this popular idea, although it was the impetus for intense study of the "neurofibrillar network" within neurons by Golgi, Cajal, Freud, and many others. Here, I trace the way in which this "excitable neurofibrillary" hypothesis led to major problems in the attempt by histologists to identify the central excitatory synapse, postulated by Sherrington on functional grounds and eventually described by Berkley.
Article
Available records indicate that the human body has always been conceived, in different periods and cultures, as spanned by multiple channels for internal communication and coherent functioning as a unit-"meridians" in treatises of Chinese medicine, metu in Egyptian papyri, srotas in Ayurvedic Indian texts, and neura in the Western scientific heritage from ancient Greece. Unfortunately, the earliest extant figurative depictions of such pathways of general control, complementary to the blood vessels, are late medieval copies of old crude sketches that attempted to show the main anatomico-physiological systems. The scarcity of adequate illustrations was more than compensated in the Renaissance, when the efforts of both artists and anatomists for the first time produced basically correct renditions of the human nervous system and many other bodily structures. As attention was next focused on microscopic structure as a requisite to understand physiological mechanisms, during the Enlightenment the nerves were revealed to consist of numerous thin tubes or fibers aligned in parallel. Improved microscopy techniques in the nineteenth century led to discovering and delineating still finer fibrils coursing along the cores of the nerve fibers themselves. Electron microscopy, developed throughout the twentieth century, recognized some of these fibrils within nerve fibers as being also tubular. All the progressive stages in understanding nerve construction, at increasingly more detailed scales, have been accompanied by technological advances and by debate about the structure and function relationship. And every step has been a source of amazing imagery.
Article
Bouton degeneration studies in the cat spinal cord have suggested a possible explanation for the phenomena of spinal shock. Degeneration of terminal boutons on a cell is not limited to those which have been isolated from their parent cell bodies, but spreads from these to neighboring boutons. These, however, are only temporarily affected. It is thus suggested that if the synaptic zone of a nerve cell is profoundly altered by degeneration of a critical number of boutons termineaux, the whole of the zone is disorganized and activity in the cells is lost. With time, the boutons with severed fibers will degenerate, and those with intact fibers will reorganize after a variable period of time depending on the state of the synaptic zone. Reflexes return in an altered form because the organization of the synaptic zone is altered.
Article
After lesions in the anterior olfactory areas, sections of the olfactory bulb stained by the method of Nauta and Gygax showed a massive centrifugal projection to the granule cells, and a direct projection to the perimeter of the glomeruli. Fine and coarse centrifugal fibers degenerated, some of the latter running in the lateral olfactory tract, whereas the commissural projection from the contralateral olfactory bulb consists of fine fibers to the granule cells only. Numerous terminal boutons could be stained by the method of Glees in the granule cell layers on the side of the lesion only. Small numbers of terminal boutons were seen in the external plexiform layer of the retina in Glees preparations after lesions of the optic nerve, but centrifugal fibers in the latter were demonstrated only with difficulty by the Nauta method. Lesions were made in the brain to identify the sources of the centrifugal fibers, and it was found that three separate components of the supraoptic commissures could be distinguished by position in the optic chiasma. These components arose, respectively, in the superior colliculus, dorsolateral thalamus, and mesencephalic reticular formation. Some of these fibers may send collaterals to the retina, but only suggestive evidence of this has been obtained by the methods of Nauta and Glees.
Article
Otto Deiters, for whom the lateral vestibular nucleus and the supporting cells of the outer auditory hair cells were named, died in 1863 aged 29. He taught in the Bonn Anatomy Department, had an appointment in the University Clinic and ran a small private practice. He published articles on the cell theory, the structure and development of muscle fibers, the inner ear, leukemia and scarlet fever. He was the second of five surviving children in an academic family whose private correspondence revealed him as a young man with limited social skills and high ambitions to complete a deeply original study of the brainstem and spinal cord. However, first his father then his younger brother died, leaving him and his older brother responsible for a suddenly impecunious family as he failed to gain academic promotion. Otto died of typhus two years after his younger brother's death, leaving his greatest scientific achievement to be published posthumously. This showed that most nerve cells have a single axon and several dendrites; he recognized the possibility that nerve cells might be functionally polarized and produced the first illustrations of synaptic inputs to dendrites from what he termed a second system of nerve fibers. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.
Article
The degeneration of the boutons, following rhizotomy of the C2 roots, has been studied in cats' spinal cords fixed in Susa and stained by albumin-silver impregnation on slides (Mihálik, '50). It was found that, three days after the lesion, large rosette shaped argyrophilic structures appeared around the nerve cells and their dendrites. These structures disappeared the fourth day. It was concluded that the presynaptic end-branches of the severed postior root fibers do not undergo a granular degeneration in situ but first retract and metamorphose into “rosettes” and then disappear by further degeneration and absorbtion. This rosette forming is considered to be an agony reaction at the terminals of the severed posterior root fibers, and it is considered to be homologous to the metamorphic agony reaction (at the stump of the amputated part of the peripheral nerves) described by Cajal.
Article
A reduced silver stain was used to examine the development of complexity of motor nerve terminals in the postnatal period. Terminals in three histochemically different muscles were examined in mice aged 12 days to 3 years. The total number and total length of intraterminal axon branches increases with age, but only until animals are 3 months old. Terminals become longest and most branched in the histochemically glycolytic tensor fascia latae (TFL) muscle, shortest and least branched in the oxidative diaphragm, and intermediate in the histochemically mixed gluteus muscle. In addition, myelinated terminal branches develop in TFL and to a lesser extent in the gluteus between 1 and 3 months of age. These myelinated branches appear to be produced by nodal sprouting from the penultimate node of Ranvier of the terminal axon, and also by myelination of pre-existing terminal branches. The diameter of muscle fibres also increases until animals are 3 months old, and there is a good correlation between mean fibre diameter and either mean terminal length or mean number of terminal branches when all muscles at all ages are compared. This suggests that terminal growth could be determined by muscle fibre growth; however, within any given muscle there is little or no correlation between the diameter of a muscle fibre and either the length or number of branches of its nerve terminal, suggesting that terminal morphology is not controlled solely by muscle fibre growth. The presence of a myelinated branch in a nerve terminal is also unrelated to fibre diameter within a given muscle, but again when means are compared there are good, but significantly different correlations for the three different muscles. Thus some kind of muscle or nerve type-specific property additional to a general effect of muscle fibre size influences the development of myelinated terminal branches. Between 3 and 12 months of age terminal complexity remains constant or may decrease slightly. At 19 months or older, when mice are becoming senile, a large proportion of synapses have terminal sprouts and muscle fibres become innervated by two or more distinct axons. These changes can be attributed to the death of some motor neurons and sprouting of the remaining axons.
Article
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SUMMARY nl ). The concentrations of reducible silver, developed silver, and silver nuclei in the sections were determined, but it is doubtful if the values obtained for silver nuclei are significant. All three forms of silver increased with pH, time, and the concentration of silver in the impregnating solution. Temperature of impregnation had little effect on the uptake of reducible silver, but increased the developed silver, presumably by increasing the silver nuclei. An increase in the temperature of a hydroquinone-sulphite developer increased the amount of reducible silver reduced by the developer. The deposition of silver by a glycine physical developer was shown to follow a curve which was reasonably consistent with the assumption of a typical autocatalytic reaction. The uptake of silver by non-nervous tissues provided evidence that the process is not specific for nerves; the final specificity of staining is determined during develop- ment. The quantitative results are consistent with the hypothesis that the histidine in the sections is responsible for the combination of reducible silver.
Article
Three types of structural change can be produced in the nervous system of lizards by altering the environmental temperature from 19 to 32°C. These changes are reversible and take from 1 to 4 weeks to occur. (1) In the nucleus sphericus the fibre plexus within the zone of cells becomes filled with argyrophilic spheres, 2 to 3 μ in diameter, when the animals are kept at the higher temperature. (2) In the cochlear grey, the superior olivary nucleus and the hippocampus there is a loss of neurofibrillar rings when the animals are kept at the higher temperature. This is accompanied by: (3) a loss of mitochondrial material. Electron microscopical studies show that both these last changes occur in synaptic terminals. Within the terminal bags neurofilaments can be seen orientated in the form of a ring, encircling a dense group of mitochondria. Around the neurofilaments is a zone of pale-staining cytoplasm, which is free from synaptic vesicles. The classical neurofibrillar boutons are seen in the regions that show neurofilaments in electron micrographs. Regions that show no neurofibrillar boutons appear to be free of terminal neurofilaments.
It is possible to reveal all the terminal boutons on the ventral horn cells of the spinal cord after fixation with formalin, mordanting (Weigert-Pal), embedding in carbowax and staining with haematoxylin or by a silver method. The boutons are more numerous than has been supposed and cover the greater part of the surface of the nerve cell body and dendrites. Electron micrographs after osmium fixation show a thin membrane at the surface of the nerve cell body and dendrites. The boutons are closely apposed to this surface and are themselves covered by thin membranes. At the region of contact there is usually no separation visible, with the relatively low magnification used, between pre- and post-synaptic membranes. The boutons contain many bodies that absorb electrons strongly and are presumably mitochondria. The pre-synaptic nerve fibres are provided with relatively thick sheaths, except where they swell out to form boutons. The protoplasm of glia cells fills up all the space between the neuronal elements. No large tissue spaces or intercellular matrix appear. Exchanges between the neurons and capillaries presumably take place through the glial protoplasm.
Article
Spinal cord of cat and rabbit was fixed by perfusion with 10% formalin in physiological salt solution followed by a 2-day immersion in 10% aqueous formalin. Further treatment (postchroming) consisted of a 5-day immersion in: K2Cr2O7, 5 gm; CrFl3-4H2O, 2 gm; distilled water, 100 ml; followed by 5% aqueous K2Cr2O7 at 38–40°C for 2–4 wks. After thorough washing, blocks were embedded by infiltration first with polyethylene glycol 1000 M. E. and then with Nonex 63B (Gemec Chemicals Co., London, E. C. 2), and casting in the Nonex. Sections were stained, either mounted or unmounted, by modifications of the Bielschowsky-Gros method, and mounted sections by Weigert-Pal's hematoxylin or by Silver's Protargol method. All 3 methods gave apparently complete staining of pericellular end feet and showed also an abundance of mitochondria. Cytologic preservation was much better than that seen after the usual procedures for this type of staining. Retention of lipoid material in the sections is considered to be the cause of efficient staining of end feet and mitochondria.
Article
IT has been found that the terminal plexus of the reptilian brain undergoes marked changes with the environmental temperature.
1. Thin sections of representative neurons from intramural, sympathetic and dorsal root ganglia, medulla oblongata, and cerebellar cortex were studied with the aid of the electron microscope. 2. The Nissl substance of these neurons consists of masses of endoplasmic reticulum showing various degrees of orientation; upon and between the cisternae, tubules, and vesicles of the reticulum lie clusters of punctate granules, 10 to 30 mµ in diameter. 3. A second system of membranes can be distinguished from the endoplasmic reticulum of the Nissl bodies by shallower and more tightly packed cisternae and by absence of granules. Intermediate forms between the two membranous systems have been found. 4. The cytoplasm between Nissl bodies contains numerous mitochondria, rounded lipid inclusions, and fine filaments.
Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.
Article
In squid, frog, rat, and human nerves examined in thin sections with the electron microscope the axon contains, in addition to certain other particulates, characteristic filaments. These filaments have diameters ranging from about 100 to 200 A and have indefinite length. They frequently have a nodose appearance due to the presence of discontinuities sometimes fairly regularly spaced along the filaments. This structure differs unmistakably from that of the dense-edged fibrils called "neurotubules" and it is clear that the latter are not axonic constituents. Though dense-edged fibrils can readily be demonstrated in fragmented formalin-fixed nerve preparations, they are seldom observed in thin sections. When such structures were seen in these experiments they were located in the connective tissue sheath. The present evidence offers no support for the view that "neurotubules" are structural entities of normal intact nerves.
Submikroskopische morphologie von Gastropodennerven
MEYER, G. F. (1957). Submikroskopische morphologie von Gastropodennerven. Z. Zellforsch. 45, 343-68.
The vestibular club endings in Ameiurus
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BARTELMEZ, G. W. & HOERR, N. R. (1933). The vestibular club endings in Ameiurus. J. comp. Neurol. 57, 401-28.
The submicroscopic structure of nerve fibres
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Experiments on the mechanism of silver staining IV. Electron microscope studies
PETERS, A. (1955). Experiments on the mechanism of silver staining IV. Electron microscope studies. Quart. J. micr. Sci. 96, 317-22.
1. Electron micrograph. Cat cord. A presynaptic bag containing a ring of neurofilaments surrounding a group of mitochondria; synaptic vesicles are also present
  • Fig
Fig. 1. Electron micrograph. Cat cord. A presynaptic bag containing a ring of neurofilaments surrounding a group of mitochondria; synaptic vesicles are also present. (Below) myelinated axon.