Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat.

Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.     

Electron microscopic cytochemical and morphometric study of the cerebral cortex synapses in postmortem autolysis

Brain tissue staining with phosphotungstic acid was performed to assay neurofilament accumulations in synapses in the molecular layer of the rat cerebral cortex at different intervals after the animals' death. It was found that autolysis began in the dense projections of presynaptic grid. Within 30 min autolysis developed in mature and very young (immature) synapses. By the 90th min autolysis in asymmetric synapses was considerably enhanced. 6 hours later autolysis involved mature and indefinite synapses.     

Para-membrane neurofilament structures of cerebral cortex synapses during ischemia and in the early postischemic period

Using PTA-method, the structure of para-membrane neurofilament density of interneuronal synapses and PTA-positive contacts in neocortical molecular layer were studied in rats during ischemia and postischemic period. Marked reduction of definite contacts (by 25.4%) was recorded by min 90 of ischemia. However, reorganization of asymmetric contacts started during ischemia and continued in postischemic period. Changes in neurofilament density of synapses (primarily, dense projections of presynaptic grid) underlie the early reorganization of synapse architectonics.     

A quantitative study of visual cortex synapses during the postnatal development of dark-reared rats

The method of Aghajanian and Bloom (1967) was applied to the visual cortex of normal and neonatally visually deprived rats. The rats were kept with their mothers in total darkness since birth, in a ventilated and temperature-controlled animal quarter. Controls were rats from the same stock, maintained in a regular 12-h-light/12-h-dark rhythm. The animals were killed at 15, 23, 40, and 65 days of age, and the visual cortices fixed in 4% glutaraldehyde and processed for electron micrography with ethanol-phosphotungstic acid. A total of 6249 synaptic profiles were counted and their numerical density (DS) determined in both conditions. Features of the presynaptic grid were used for classifying the synaptic profiles in: type A [with one presynaptic dense projection (PsDP)]; type B (with two or three PsDPS); and type C (with four or more PsDPS). In the visually deprived rats the DS increases with a rate similar to controls, but the values for each age are slightly lower (P > 0.01). Type B synapses predominate in the visually deprived group while types A and C are scarcer. The differences found between types were maximal at 65 days of age and the results were highly significant (P > 0.001). It is concluded that major effects of dark-rearing are manifested when the individual features of the presynaptic grid are considered. It seems that a selected population of synapses is affected by the alteration of the normal epigenetic influence of early visual experience.     

Diurnal changes in the fine structure of photoreceptors in an abalone,Nordotis discus

Summary The ultrastructure of the synapses in the brain of the monogenean Gastrocotyle trachuri (Platyhelminthes) is described. The synapses consist of one presynaptic terminal separated by a uniformly wide synaptic cleft, from one or more postsynaptic elements. The presynaptic terminals are characterized by the presence of paramembranous dense projections and associated synaptic vesicles. The postsynaptic elements while possessing membrane densities, are usually devoid of vesicles.The structure of the synapses in the brain of Gastrocotyle is compared to synapses from other platyhelminths.     

Novel synaptic structures in the central nervous system of the locust Schlstocerca gregaria

Summary An electron-microscopical study of locust thoracic ganglia reveals that synapses in the neuropily are morphologically heterogeneous. In addition to the conventional dyadic type described frequently in the literature, there is a second type with a complex arrangement of presynaptic dense material and a non-dyadic postsynaptic configuration. Serial-section analysis of these synapses suggests that the presynaptic structures include irregular or curved bars, and small projections.Although the proportion of non-dyadic synapses in the neuropile as a whole is small, a substantial number have been found on the branches of an identified flight motor neurone, labelled intracellularly with metal ions in conjunction with silver intensification. Samples from the arborization of this neurone give some indications of the distribution of non-dyadic synapses on it.The results are discussed in the context of distribution of synapses on other identified locust neurones, and the functional morphology of synapses in other phyla.     

Dendro-dendritic and reciprocal synapses in the primate motor cortex.

Dendro-dendritic synapses have been observed infrequently in the deep layers of the motor cortex. The presynaptic dendrites are of a varicose type and themselves receive a considerable density of synapses both of the asymmetric and symmetrical type. The ultrastructure of the dendro-dendritic synapse itself shows the typical arrangement of presynaptic and postsynaptic membrane densities, often with presynaptic dense projections, and the membrane specialization is of the symmetrical type. There is the usual cleft containing electron-dense material between the presynaptic and postsynaptic profiles. The synaptic vesicles occur in a small cluster confined to a region close to the presynaptic membrane specialization; some of the vesicles are flattened and were shown by tilt analysis to be of the discoid type. Two examples were found of reciprocal dendro-dendritic synapses, both components being of the symmetrical type. A single axon terminal may make a synapse on to both dendrites involved in a dendro-dendritic synapse.     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

Method of ultrastructural analysis with the aid of measuring television complex]

The area of the section of presynaptic dense projections of the active zone of axondendritic synapses of the rat's cortex was measured using a television complex. Tissue specimens were contrasted with phosphotungstic acid. Morphometry was carried out in the regime of error correction in measurement, the errors arising in scanning process of image. The area of the section of mitochondria, lysosome, nucleoli, channels and tanks of the endoplasmic reticulum may be measured by the method suggested. Quantitative data, obtained by the morphometry of the synapse ultrastructure, may serve a basis for understanding the mechanism by which synapses are involved in information processes of the central nervous system.     

An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling

To identify structures involved in the translocation of the synaptic vesicles towards the presynaptic membrane, an ultrastructural study has been undertaken by means of (1) the E-PTA stain and (2) the HMM-labeling procedure. Using serial sections of E-PTA stained nervous tissue, especially those made in transversal and tangential planes, the geometric order of the presynaptic grid and of its constituents has been described in detail. It consisted of dense projections having the shape of small truncated pyramids cut parallel to their hexagonal bases which rested on the electron-lucent presynaptic membrane. The dense projections were arranged at the points of equilateral triangles. Around each dense projection, six asymmetric hexagonal holes were seen to be arrayed in an hexagonal pattern, forming thus the presynaptic sieve. From the spiny tops of the dense projections, which appeared as specialized structures of the dense material coating the inner surface of the plasma membrane at the level of the synaptic cleft, fine filaments, 40--60 A in diameter, radiated and formed a three-dimensional meshwork pervading the presynaptic bag. The dense cytoplasmic coating delineating the plasma membrane served as anchor points for these microfilaments. Upon incubation with rabbit skeletal muscle HMM the microfilaments underwent specific structural changes, consisting of: (1) a striking increase in diameter; (2) the association of periodic and polarized substructures with their surfaces. The synaptic vesicles and mitochondria were seen to be attached to the numerous HMM-decorated filaments or enmeshed in the network formed by these filaments. The actin-like filaments were anchored to the plasma membrane at many points and to the presynaptic dense projections. Following incubation in the buffer alone or in buffer HMM solutions containing Na+ pyrophosphate or ATP, no arrowheaded structures were observed. Thus, a network consisting of actin-like filaments was demonstrated in the presynaptic bag. Of particular interest was the structural relation of the actin-like filaments with the occasional, tapered myosin-like filaments. The role of the presynaptic actin-like network in the transport of synaptic vesicles towards the presynaptic membrane by a mechanism of chemomechanical transduction is discussed. In the postsynaptic dendrite or dendritic spine, a filamentous network was observed to be attached to the subsynaptic web by means of the E-PTA stain and of the HMM-labeling procedure. The occurrence of an actin-like meshwork in the postsynaptic region is suggested to produce changes in the macromolecular configuration of the postsynaptic membrane by a "mechanoenzyme" system similar to that described in the mitochondrial membrane.     

A note on the structure of synapses in the ventral nerve cord of the onychophoran peripatoides leuckarti

Summary The fine structure of synapses, their distribution and arrangement in the ventral nerve cord of Peripatoides leuckarti (phylum Onychophora) is described. The asymmetric synaptic junctions show a well developed synaptic cleft (300Å) and pre- and subsynaptic electron dense apposition. They frequently show an array of presynaptic projections and a subsynaptic cisterna of endoplasmic reticulum. The onychophoran synapses differ from those of annelids and insects.Supported by the Deutsche Forschungsgemeinschaft, grant Schu 374/1     

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.     

Further studies on synaptic junctions

Summary Synaptic junctions in intact rat cerebral cortex have been examined following glutaraldehyde fixation and phosphotungstic acid (PTA) staining. In the presynaptic ending the network has a hexagonal arrangement, while the dense projections are regularly placed along the presynaptic membrane. Cleft densities occupy the intracleft region. The postsynaptic thickening extends uninterrupted along the length of the junction. Qualitatively, the majority of junctions fall into the discontinuous-continuous category, in which the internal coat of the presynaptic membrane together with its associated dense projections is discontinuous along the length of the junction, whereas the postsynaptic thickening is continuous. By contrast, a small number of junctions are continuous-continuous.In an attempt to analyze the junctions quantitatively, nine indices were measured. Histograms of the size distributions of seven of these appear to be bimodal, and from this it is concluded that two junction populations may be distinguishable on quantitative grounds. It is also shown that the distance separating dense projections at the presynaptic membrane is of the order of 10–15 nm. This surprisingly low value has consequences for current ideas on the relationship between synaptic vesicles and dense projections, and these are discussed at length.We would like to acknowledge the technical assistance of Mrs. C. Blackshaw, Mrs. G. Kay and Mr. D. Stuart.     

Innervation des cellules du pancréas endocrine du Poisson Téléostéen Xiphophorus helleri H.

Summary The pancreatic islet tissue of the bony fish Xiphophorus helleri H. is innervated by amyelinic fibers. Neuroglandular junctions on the islet cells show a structure similar to that of the synapses of the central nervous system. Presynaptic projections, intrasynaptic lines, and postsynaptic bands are to be observed. In the nerve endings two kinds of vesicles occur: large granular vesicles (diameter:850–1150 Å) and small clear vesicles (diameter: 500–650 Å). Synaptic vesicles are gathered around the presynaptic dense projections. This precise organization suggests the existence of a direct nervous control involved in the metabolism of the pancreatic hormones.

Avec la collaboration technique de M. D. Streicher.     

The relationship between complex vesicles, dense-cored vesicles and dense projections in cortical synaptosomes

Cortical synaptosomes fixed in unbuffered OsO4 and glutaraldehyde have been block-stained with phosphotungstic acid (PTA) in order to investigate the relationship between complex vesicles and dense projections. It is concluded that the shell of the complex vesicles contributes to the formation of dense projections and that, in addition, there is a correspondence between this shell and the previously described presynaptic network. The process by which complex vesicles become associated with dense projections appears to be accentuated by electrical stimulation of the synaptosomes.     

New observations on the substructure of the active zone of brain synapses and motor endplates

This study offers a new concept on the origin and function of the hitherto enigmatic presynaptic dense projections (dps) of neurons and motor endplates. After a deuterium oxide-albumin pretreatment (da), brain tissue and motor endplate of rat and frog reveal an intricate association of smooth endoplasmic reticulum (ser), microtubules (mts) and synaptic vesicles (sv) at the presynaptic grid-active zone of synapses. The ser entwines the mts, which are clothed in svs, and impinges directly onto the presynaptic membrane as sacs or 'tubular-fibrillar' extensions. Since no dps are seen in these sections, whereas they do occur in conventionally processed material (i.e. without da pretreatment), it is suggested that the dps of conventional material may, in part, originate from improperly fixed ser at these points. Thus for the first time we demonstrate an in vivo system of ser which, because its 'finger' processes come into intimate contact with the presynaptic membrane, may be implicated in Ca2+ ion translocation, presumably out of the presynaptic bulb. Since no such tubular ser has been demonstrated in what are claimed to be sophisticated techniques (i.e. high-speed slam-freezing-freeze substitution) the actual sophistication of such methods is questioned.     

A critical evaluation of the relationship between the presynaptic network,synaptic vesicles and dense projections in central synapses

Summary Synapses of the oculomotor nucleus of Echidna have been examined ultrastructurally with the aim of integrating data obtained from osmicated and nonosmicated PTA stained material. Particular emphasis has been laid on the relationship between the synaptic vesicles of the osmicated material and the presynaptic network and vesicular grid of the PTA material. This relationship has been explored qualitatively by examining osmicated material of varying qualities of fixation. Such material contains dense projections in addition to synaptic vesicles, and various vesicular network appearances. A variety of measurement techniques have shown that the PTA network is characterised by reticular strands, spaces, and regular hexagonal units smaller than vesicles, these observations prompting the formulation of a vesicle-network coincidence model of the presynaptic terminal. This model has been tested by tracing the profiles of vesicles within the PTA network and comparing their size and shape frequency distributions with those of osmicated synaptic vesicles. The distributions have been found to be essentially similar, suggesting that vesicles can be located within the network, and that the hexagonal network units are formed only in the presence of an underlying vesicular matrix.Additionally, the following points have emerged: 1) the dense projections in the two types of material appear to be equivalent; 2) a loose correlation exists between dense projections and vesicles in osmicated terminals, increase in the area of the dense projections being associated with a decrease in the area of the vesicles; 3) network and dense projection units are similar. In view of the similarity between network and dense projection units, the demonstrated vesicular basis of the network raises the question of whether dense projections are entirely independent structures, or whether they depend in part for their existence on the nearby presence of synaptic vesicles.This work was supported by the Arnold Yeldham and Mary Raine Research Foundation of the University of Western Australia and by the Australian Research Grants Committee and the Nuffield Foundation     

Ultrastructural Study of the Brain of a Typhloplanid Flatworm

The brain of Strongylostoma simplex shows the organization typical of the most evolutionary groups of free-living plathelminths. Three types of neurons, two types of neurosecretory cells, and a certain number of glia-like cells are present. Somato-axonic synapses and axo-dendritic contacts with a “spine apparatus”, like those described in vertebrates, have been identified. These two types of synapses have never been reported in other plathelminth brains. Local thickenings and dense bars attached to the presynaptic membrane are also observed, together with the paramembranous density common in many flatworm synapses.     

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.This work was supported by funds from the University of Virginia Research and Development Committee.     

Plastic restructuring of cerebral cortex synapses in the postischemic period

Using the technique for the cerebral tissue contrasting with phosphoric-tungsten acid, synaptic architectonics of the molecular layer of the rat sensomotor cortex has been studied for 30 days of recirculation after 5 minutes' stop of the systemic circulation resulted from an acute hemorrhage. Gradation of the synapses per groups is performed according to the height of compact projections of the presynaptic network. Dynamics of the synaptic compactness changes with different height is stated to differ essentially during the postischemic period. A conclusion is made that in reconstruction of the synaptic architectonics of the neocortex during the postischemic period a leading role belongs to the change of state of specialized paramembranous microfilamentous compact projections of the presynaptic network. They make a part of the neuronal cytoskeleton and are included into a single integral complex--the system of subcytoskeleton and are included into a single integral complex--the system of subsynaptic units.