Axonal growth cones and developing axonal collaterals form synaptic junctions in embryonic mouse spinal cord

Summary Axonal growth cones and developing axonal collaterals have been studied in electron microscopic and Golgi preparations of embryonic mouse cervical spinal cord. These structures are first observed on embryonic day 11 (E11), and by E12 both axonal growth cones and developing collaterals are observed to be the presynaptic elements of developing synaptic junctions. These relatively undifferentiated portions of axons comprise nearly one-half of the presynaptic components of the synaptic contacts observed on E12, and they continue to constitute about this same proportion during the rest of the embryonic period examined (E13–16). These early synaptic interactions of developing axonal collaterals may be involved in establishing the basic ramification patterns of mature axons by determining which developing collaterals will continue to grow and differentiate.Coated vesicles are observed fused with axonal collateral plasma membranes at the perimeters of developing synaptic contacts. Occasionally thin extensions of postsynaptic surfaces protrude into these coated vesicles, and similar relationships are also observed between presynaptic surfaces and coated vesicles fused with the membranes of postsynaptic elements. Such coated vesicle/ synaptic surface relationships may represent an early stage in the endocytosis of synaptic membranes by both the pre- and postsynaptic elements of developing synaptic contacts. This might represent a mechanism for the exchange of molecular information between the components of protosynaptic contacts, and the results of such an exchange could be involved in determining whether protosynaptic contacts will differentiate into synaptic junctions.Developing axonal collaterals exhibit a preferential growth toward the sources of potential postsynaptic elements, and this growth implies that the axons present in the embryonic marginal zone are actively searching for appropriate postsynaptic processes rather than passively awaiting their arrival. Many of the collaterals weave around numerous intervening processes in order to reach their postsynaptic destinations, and unusual subaxolemmal lamellae are commonly observed where collaterals make acute curvatures around neurites which block their direct access to postsynaptic processes. The location of these lamellae suggests that they could be involved in producing changes in the direction of growth as developing axonal collaterals search for synaptic partnerships. Main axonal shafts commonly exhibit synaptic contacts adjacent to where they give rise to developing collaterals, and this observation hints that the formation of synapses by axonal shafts might stimulate collateral sprouting. However, other observations (see text) indicate that the formation of synaptic contacts by axonal shafts is not an obligatory prelude to collateral development. Therefore synaptic interactions of main axonal shafts do not appear to be a requirement for the primary induction of collateral development, but they may facilitate collateral growth by providing a confirmation that axons are located in appropriate synaptogenic fields.     

Transitional elements with characteristics of both growth cones and presynaptic terminals observed in cell cultures of cerebellar neurons

Summary As growth cones interact with targets, they become presynaptic terminals by losing growth cone characteristics and acquiring presynaptic characteristics. Results presented here show that transitional elements can be identified in cell cultures of rat cerebellum, which have some characteristics of both growth cones and presynaptic terminals. During the first week in culture, slender growth cones have fine filopodia. Subsequently, many growth cones in contact with the polylysine substrate spontaneously enlarge and become non-motile. In transitional elements, the synaptic vesicle protein p65 extends into the peripheral domain and in some cases, extends into filopodia. Many of these transitional elements have active filopodia but show no movement over the substrate for periods of up to nine days. These transitional elements have lost the actin-rich peripheral domain of the growth cone but retain actin labelling in the filopodia. With electron microscopy, transitional elements were seen to contain accumulations of synaptic vesicles at the site of contact with the substrate. Electron microscopic immunocytochemistry showed these synaptic vesicles labelled for p65 with silver-developed gold particles. Thus, transitional elements have characteristics of both growth cones and presynaptic terminals, suggesting that they may also have functional attributes of both growth cones and presynaptic elements.     

Mobility and cycling of synaptic protein-containing vesicles in axonal growth cone filopodia

The spatial distribution and coordination of vesicular dynamics within growth cones are poorly understood. It has long been thought that membranous organelles are concentrated in the central regions of growth cones and excluded from filopodia; this view has dramatically shaped conceptual models of the cellular mechanisms of axonal growth and presynaptic terminal formation. To begin to test these models, we studied membrane dynamics within axonal growth cones of living rat cortical neurons. We demonstrate that growth cone filopodia contain vesicles that transport synaptic vesicle proteins bidirectionally along filopodia and fuse with the filopodial surface in response to focal stimulation, allowing for both local secretion of vesicular contents and rapid changes in the plasma membrane composition of individual filopodia. Our results suggest a new model in which growth cone filopodia are actively involved in both emitting and responding to local signals related to axon growth and early synapse formation.     

Quantification of synapse formation and maintenance in vivo in the absence of synaptic release

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.     

Cytodifferentiation and synaptogenesis in the neostriatum of fetal and neonatal rhesus monkeys

Summary Cytodifferentiation and synaptogenesis in the neostriatum (caudate nucleus and putamen) were analyzed by the Golgi impregnation method and electron microscopy in 14 fetuses and 8 postnatal rhesus monkeys. During the second fetal month the neostriatum consists primarily of simple, mostly bipolar, immature cells and a small number of undefined profiles ending with growth cones. The first morphologically defined synapses appear in the putamen at embryonic day 60 (E60) and in the head of the caudate nucleus at E65. Synaptic density in both structures is less than one per 1000/m² of neuropil at this stage; synapses are characterized by asymmetric junctions between axonal profiles and immature dendritic shafts, accumulation of an intermembrane web and aggregation of round clear vesicles in presynaptic profiles. During the third fetal month neuronal cell bodies and glial cells enlarge, and axonal and dendritic processes in Golgi preparations become more complex. Although the basic morphology of synapses remains unchanged, their density increases to 9/1000 m² in the putamen and 3.7/1000 m² in the caudate. During the fourth fetal month the four principal cell classes of the neostriatum emerge. Spines on the shafts of dendrites are followed closely by the appearance of axospinous synapses. Synaptic density in the putamen is still significantly higher (10.1/1000 m²) than in the caudate (5.4/1000 m²), but by the end of the fifth fetal month (E150) it is the same (80/1000 m²) in both structures. A dramatic increase in synaptic density to 125/1000 m² occurs before term (E165) with the emergence of the first asymmetric synapses as well is symmetric synapses with flat or pleomorphic vesicles that terminate predominately on dendritic shafts. Synaptic density continues to increase after birth, reaching a plateau of approximately 190/1000 m² at the end of the first postnatal month. Throughout postnatal development the proportions of symmetric and asymmetric synapses on the smooth dendritic shafts undergo systematic fluctuations which may reflect the ingrowth of various afferents as well as local cytological differentiation including the formation of cellular compartments.     

猫三叉神经尾侧脊束核—丘脑—皮质通路在丘脑水平的突触联系

本文采用HRP逆行追踪与顺行溃变结合法对猫三叉神经尾侧脊束核-丘脑-皮质通路在丘脑腹后内侧核内的突触联系型式进行了研究。在电镜下发现,丘脑腹后内侧核內有五种突触联系形式:(1)溃变轴突终末与HRP标记树突形成轴-树突触;(2)溃变轴突终末与HRP标记的胞体形成轴-体突触,上述两类突触型式为该通路在丘脑水平的直接突触联系方式,此外尚有(3)溃变轴突终末与非HRP标记的树突形成的轴-树突触;(4)HRP标记树突与非溃变轴突终末形成轴一树突触;(5)HRP标记树突与非HRP标记的含有突触小泡的突触前树突形成的树-树突触。本文首次报道了三叉丘系纤维与丘脑皮质投射神经元间的直接突触联系方式为轴-树和轴-体突触。同时也发现了以树突为中心的突触复合体,它是该通路在丘脑水平的一个显著特点。     

Synaptogenesis in the dorsal lateral geniculate nucleus of the rat

Summary Synapse formation and maturation were examined in the rat dorsal lateral geniculate nucleus (dLGN) from birth to adulthood. Examination of animals, whose ages were closely spaced in time, showed that the maturation of the synaptic organization of the nucleus takes place chiefly during the first 3 weeks of postnatal life. This period of maturation may be divided into 3 broad stages. During the first stage, which spans the first 4 days of life, there are only a few immature synapses scattered throughout the nucleus; occasionally aggregates of 3 or 4 synapses are encountered. Dendrodendritic synapses first appear at the end of this stage. The second stage, which lasts from the end of the first stage through day 8, is characterized by intensive synaptogenesis as well as extensive growth and degeneration. For the first time, large boutons resembling retinal terminals form multiple synaptic contacts with dendrites and dendritic protrusions; these synaptic arrangements are partially covered by glial processes.A feature characteristic of the developing dLGN during the first 2 postnatal weeks, and particularly during the second stage, is the presence of membrane specializations that resemble vacant postsynaptic densities. These specializations, which may be unapposed or opposite another neuronal process, decrease in frequency as the number of synapses increases. It is not known whether these densities are converted to synapses or whether they result from loss of presynaptic elements.The third stage in the process of synaptogenesis, which spans a period between days 10 and 20, is characterized by myelination and by the diminution of growth cones, degenerating profiles and vacant postsynaptic densities. There is also a very significant increase in the number and maturation of synapses including synaptic glomeruli. However, it is not until the end of this stage that synapses appear qualitatively indistinguishable from synaptic arrangements identified in adult animals.     

Synaptic changes in frog brain after stimulation with potassium chloride

Summary In vitro preparations of frog brains, stimulated by application of KCl were fixed by freeze substitution and examined electron microscopically. Control preparations were bathed in a calcium-free physiological solution with Mg added or in salt solution cooled to 5–10 °C. The isolated brain remains viable in the physiological solution as indicated by the direct cortical responses which can be led off from the forebrain. Control preparations were characterized by a row of vesicles situated close to the presynaptic membrane and by the absence of a well-developed postsynaptic web. In KCl stimulated preparations there were, in addition to synapses resembling those in the controls, synapses exhibiting fusion of synaptic vesicles with the membrane of the axonal ending, synapses in which the vesicles had retreated from the presynaptic membrane but were attached to it by a narrow stalk and synapses exhibiting a pronounced postsynaptic web. The synaptic gap was of a less uniform width than in the control preparations. The KCl stimulated preparations were furthermore characterized by a paucity of extracellular space and often showed invaginations formed by the presynaptic membrane and the plasma membranes of the postsynaptic or adjacent glial structure.     

GABAergic and pallidal terminals in the thalamic reticular nucleus of squirrel monkeys

The ultrastructure of synaptic terminals from the external segment of the globus pallidus and of other synaptic terminals positive for gamma-aminobutyric acid (GABA) was examined in the thalamic reticular nucleus (TRN) of squirrel monkeys. Two GABA-positive terminals types were commonly encountered within the TRN neuropil. The most common type of GABAergic terminals (F terminals) are filled with dispersed pleomorphic synaptic vesicles and clusters of mitochondria. These terminals establish multiple symmetric synapses upon the somata and dendrites of TRN neurons. The external pallidal terminals, labeled with WGA-HRP, arise from thinly myelinated axons and correspond to the medium to large F terminals. A less prevalent population of smaller GABAergic synaptic profiles was also identified. The synaptic profiles in this second group contain considerably fewer pleomorphic synaptic vesicles in small irregular clusters and fewer mitochondria, establish symmetric synapses, are postsynaptic to other axonal terminals, are presynaptic to dendrites and soma, and are unlabeled following pallidal injections of WGA-HRP.     

Ultrastructural features of neurons and synaptic contacts in the posterodorsal medial amygdala of adult male rats

The aim of the present study was to describe the ultrastructure of neurons (from eight animals) and to analyse the synaptic terminal distribution (from two animals) in the posterodorsal subnucleus of the medial amygdala (MePD) of adult male rats. Using transmission electron microscopy, it was possible to identify many spiny and aspiny dendrites, unmyelinated axonal bundles, single axonal processes, a few myelinated axons, blood vessels and glial processes in the neuropil. Axodendritic synapses were the most frequently observed (67.5%), appearing to be of either the inhibitory or the excitatory types. The presynaptic region contained round or flattened vesicles that occurred either singly or with dense-cored vesicles (DCVs). The dendrites often received many synapses on a single shaft, and axon terminals displayed synaptic contacts with one or more postsynaptic structures. Dendritic spines showed different morphologies and the synapses on them (23.1%) formed a single and apparently excitatory synaptic contact with round, electron-lucid vesicles alone or, less frequently, with DCVs. Inhibitory and excitatory axosomatic synapses (8.2%) and excitatory axoaxonic synapses (1.2%) were also identified. The present report provides new findings relevant to the study of the MePD cellular organization and could be combined with other morphological data in order to reveal the functional activity of this area in male rats.     

Serial and triadic synapses in the cerebellar nuclei of the cat

Summary A quantitative electron microscopic investigation of the nucleus interpositus in cat cerebellum reveals that about 1.5% of all observed synapses are established between synaptic vesicle-bearing profiles. It is shown by serial sections that 70% of these synaptic complexes are triadic arrangements and 30% are serial synapses. Further analysis discloses that the first presynaptic element in the triadic and serial synapses may be one of four different axonal types: (A) Purkinje-cell axons; (B) and (C) afferent fibers containing large round vesicles and originating from the brain stem (probably mossy and climbing fiber collaterals); and (D) axon terminals containing small round vesicles. Indirect evidence suggests that type D profiles are the recurrent axon collaterals of the projective neurons. The second, postsynaptic and presynaptic, vesicle-bearing process in these complexes is either a class D terminal, or a somewhat more dendrite-like profile (Class E) containing flattened vesicles, and identified as belonging to processes of local Golgi type II interneurons.     

Effects of polyunsaturated fatty acids and hormones on synaptogenesis in serum-free medium cultures of mouse fetal hypothalamic cells

The effects of soluble factors on synaptogenesis by mouse fetal hypothalamic cells cultured in chemically defined conditions have been examined using transmission electron microscopy. Hypothalami taken on the 16th day of gestation were mechanically dissociated and cells were seeded in a minimum serum-free medium supplemented or not with the following components: triiodothyronine, corticosterone and a mixture of polyunsaturated fatty acids (arachidonic acid plus docosahexaenoic acid bound to defatted bovine serum albumin). In the minimum serum free medium synapses were found after 10 days in culture. However, the development of synaptic vesicles was very limited, whereas that of the presynaptic and postsynaptic densities was apparently normal. Supplementation of the minimum serum-free medium with triiodothyronine, corticosterone and polyunsaturated fatty acids added simultaneously, permitted a full development of synapses as attested to by the increase in number and the regular shape and diameter of synaptic vesicles as well as by the complexity and diversity of synapse configurations. Among those three factors, polyunsaturated fatty acids clearly played a key role. The ability of synapses formed in culture to respond to potassium evoked depolarization was examined on cultures grown for 12 days in the simultaneous presence of the three above mentioned supplements. Exposure for 3 min to 60 mM potassium chloride induced in synaptic boutons vesicular depletion, apposition of vesicle clusters onto the presynaptic grid, appearance of a rich filamentous network and of some coated vesicles. Return to 3mM potassium chloride induced in 3 min a massive restoration of the population of vesicles which slightly differed from synaptic vesicles in control cultures. These results show that: (1) the formation of synaptic vesicles in this system is regulated by soluble factors among which polyunsaturated fatty acids play a major role, and (2) synapses formed de novo in chemically defined conditions of culture display the same ability to respond to and to recover from potassium evoked depolarization as adult axon terminals. Thus, they offer a suitable model for analysis of the mechanisms involved in membrane traffic in central neurons.     

Morphological evidence for neuronal regulation of luteinizing hormone-releasing hormone-containing neurons by neuropeptide Y in the rat septo-preoptic area

Using a preembedding double immunolabeling technique, synaptic contacts were found between luteinizing hormone-releasing hormone (LHRH)-containing neurons and neuropeptide Y-containing axonal fibers in the rat septo-preoptic area. In demonstrating LHRH neurons, we used mainly an antiserum generated against rat gonadotrophic hormone-releasing hormone-associated peptide. Although many diaminobenzidine-labeled neuropeptide Y-containing fibers were seen around silver-gold-labeled LHRH cell bodies, synapses with synaptic membrane specialization were scarce. The fiber terminals usually contained many small clear vesicles and some large cored vesicles. The synapses were characterized with the presynaptic accumulation of the small clear vesicles and symmetric thickenings of the synaptic membranes.     

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

Summary Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.     

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: dendritic differentiation, synaptic maturation, and formation of cell-class specific features

 The morphological differentiation of E16 murine Purkinje cells (PCs) in dissociated cerebellar cultures was analyzed by light and electron microscopic immunocytochemistry after 2–5 weeks in vitro (wiv), with particular emphasis on dendritic differentiation, synaptic maturation, and formation of stereotypical fine structural features. This study complements a companion paper on the features of PCs after 1 wiv. After 2 wiv, the PCs have an eccentric nucleus and the cytoplasmic organelles appear immature; the axon has a distinct initial segment and beaded axon collaterals but its boutons still contain sparse synaptic vesicles; dendrites show few bifurcations and tufts of spiny branchlets. After 3 wiv, the PCs display a centered nucleus, an extensive hypolemmal cisternal system, and stacks of up to four cisterns of granular endoplasmic reticulum; there is an increased number of dendritic bifurcations, spiny branchlets, mature spines, and axonal branches; dendritic tips still contain vesicle clusters, suggesting growth, and many synapses and afferent boutons continue to display immature features. After 4 wiv, elaborate perinucleolar coiled body rosettes, subsurface cistern-mitochondrion complexes and large stacks of granular endoplasmic reticulum finally appear within the soma; dendrites show a further increase in the numbers of bifurcations, segments and spines; most spines are synaptic and show mature features; afferent synapses are differentially distributed; PC boutons consistently display mature features and show a considerable degree of target specificity, although naked spines and reduced glial sheaths persist. After 5 wiv, PCs do not show further maturation and some dystrophic features appear. We conclude that under standard conditions and despite the disruption of normal tissue organization, PCs in dissociated cultures differentiate maximally after 4 wiv, at which stage they display many of the light and electron microscopic features that characterize mature PCs in situ. This prolonged developmental time-frame resembles that in the normal cerebellum. In view of the increasing usage of dissociated cerebellar cultures to study aspects of neuronal differentiation, synaptic activation and neuronal-glial interactions, an elucidation of the neurocytology of dissociated cerebellar cultures as presented in this study provides important clues for the interpretation of experimental data. Accepted: 30 June 1997     

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.     

Synaptic plasticity in the oedematous human cerebral cortex

Synaptic plastic changes are fundamental events which occur spontaneously during development, maturity and aging processes or can be induced by injury or trauma. To examine lesion-induced synaptic plasticity, cortical biopsies were taken from the frontal, parietal, temporal and occipital cortex of living patients during neurosurgical treatment of brain trauma, brain tumours and vascular malformations, and processed for transmission electron microscopy. Enlargement of both pre- and postsynaptic endings, irregularly shaped, lobulated, stellate and bifurcated presynaptic endings and conformational changes of dendritic spines were observed. Numerous flat, curved and invaginated axodendritic and axospinous asymmetric synapses were distinguished and a smaller proportion of axodendritic and axosomatic symmetric synapses. Activated or sensitized synapses showed numerous frontline spheroid synaptic vesicles, prominent dense presynaptic dense projections and increased length of synaptic membrane complex. Perforated synapses, multiple synapses and serial synapses were also found evincing synaptic splitting and formation of new synaptic connections. The overall images suggest increased number of excitatory circuits, which were correlated with the tonico-clonic convulsion or post-traumatic seizures observed in some patients. Numerous coated vesicles were observed in pre- and postsynaptic structures. Increased number of polyribosomes were found in the dendritic shafts. The dilated spine apparatus, the coated vesicles and the increased number of polyribosomes seem to represent a system for synthesis, transport and storage of synaptic proteins for the formation of new synapses. Coexisting synaptic plasticity and synaptic degeneration were observed in the patients under study. Dendritic and astrocyte synapse-like junctions were also characterized.     

Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.     

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: neuronal polarity

 Cerebellar Purkinje cells (PC) display a highly distinctive form of polarity. We have cultured murine PCs from dissociated E16 cerebellar anlagen for 1 week to investigate the early stages of neuronal compartmentalization and synaptic interactions, features which are important for the establishment of neuronal polarity. To unequivocally identify the PCs we utilized light and electron microscopic immunocytochemistry with an antiserum to the cell class-specific marker L7/pcp2 gene product. The PCs typically show a single, long axon, numerous short appendages classified as filopodia and protospines, and a small number of protodendrites. The nucleus is positioned asymmetrically in both the horizontal and vertical axes of the soma. The Golgi apparatus, coated and uncoated vesicles, and mitochondria are prominent ultrastructural features, while the endoplasmic reticulum is highly fragmented. The cell body receives rudimentary synapses on its smooth surfaces and appendages and no consistent morphological differences were detected between these elementary contacts. The axon is clearly identifiable; it emanates from either the cell body or a protodendrite, bifurcates at predominantly right angles, forms beaded collaterals, and terminates with relatively large growth cones. The varicosities of the PC axon contain pleomorphic synaptic vesicles and form rudimentary synapses primarily with the dendritic shafts of immunonegative neurons. The protodendrites are short, quickly tapering and sparsely branched; they emit numerous filopodia and immature spines and terminate with small growth cones. Rudimentary synapses are received on the proximal dendritic shafts and filopodia, and more mature synapses occur frequently on protospines. With few exceptions, PCs lie atop an astrocytic bed layer and glial processes are apposed to the various aspects of the PC body left free by the afferent axons. By contrast, PC processes are largely free of glial sheaths. We conclude that the ”stellate stage” of PC development in situ is replicated rather faithfully in culture and that PCs have established polarity and have begun to form intercellular contacts by 1 week in vitro. Moreover, the PCs are already morphologically distinct from other cell types in the 1-week cultures, although they have yet to develop the differentiated features that distinguish mature PCs. Accepted: 30 June 1997     

Thirty years of synaptosome research

Summary Detached synapses (synaptosomes), first isolated by the author in 1958 and identified as such in 1960, are sealed presynaptic nerve terminals often with a portion of the target cell — sometimes amounting to a complete dendritic spine — adhering to their external surface. They can be prepared in high yield from brain tissue and also in decreasing yield from spinal cord, retina, sympathetic ganglia, myenteric plexus and electric organs. They are sealed structures which, under metabolizing conditions, respire, take up oxygen and glucose, extrude Na⁺, accumulate K⁺, maintain a normal membrane potential and, on depolarization, release transmitter in a Ca²⁺-dependent manner. They thus provide an excellent preparation with which to investigate synaptic function without the complications encountered with synapsesin situ. They also serve as the parent fraction for preparations of synaptic vesicles and other synaptic components.