Structure of peripheral synapses: autonomic ganglia

Final motor neurons in sympathetic and parasympathetic ganglia receive synaptic inputs from preganglionic neurons. Quantitative ultrastructural analyses have shown that the spatial distribution of these synapses is mostly sparse and random. Typically, only about 1%-2% of the neuronal surface is covered with synapses, with the rest of the neuronal surface being closely enclosed by Schwann cell processes. The number of synaptic inputs is correlated with the dendritic complexity of the target neuron, and the total number of synaptic contacts is related to the surface area of the post-synaptic neuron. Overall, most neurons receive fewer than 150 synaptic contacts, with individual preganglionic inputs providing between 10 and 50 synaptic contacts. This variation is probably one determinant of synaptic strength in autonomic ganglia. Many neurons in prevertebral sympathetic ganglia receive additional convergent synaptic inputs from intestinofugal neurons located in the enteric plexuses. The neurons support these additional inputs via larger dendritic arborisations together with a higher overall synaptic density. There is considerable neurochemical heterogeneity in presynaptic boutons. Some synapses apparently lack most of the proteins normally required for fast transmitter release and probably do not take part in conventional ganglionic transmission. Furthermore, most preganglionic boutons in the ganglionic neuropil do not form direct synaptic contacts with any neurons. Nevertheless, these boutons may well contribute to slow transmission processes that need not require conventional synaptic structures.     

In vivo time-lapse imaging and serial section electron microscopy reveal developmental synaptic rearrangements

Dendrites, axons, and synapses are dynamic during circuit development; however, changes in microcircuit connections as branches stabilize have not been directly demonstrated. By combining in?vivo time-lapse imaging of Xenopus tectal neurons with electron microscope reconstructions of imaged neurons, we report the distribution and ultrastructure of synapses on individual vertebrate neurons and relate these synaptic properties to dynamics in dendritic and axonal arbor structure over hours or?days of imaging. Dynamic dendrites have a high density of immature synapses, whereas stable dendrites have sparser, mature synapses. Axons initiate contacts from multisynapse boutons on stable branches. Connections are refined by decreasing convergence from multiple inputs to postsynaptic dendrites and by decreasing divergence from multisynapse boutons to postsynaptic sites. Visual deprivation or NMDAR antagonists decreased synapse maturation and elimination, suggesting that coactive input activity promotes microcircuit development by concurrently regulating synapse elimination and maturation of remaining contacts.     

The time course of synapse formation of mouse neuroblastoma cells in monolayer cultures

Summary Neuroblastoma cells grown on substrates in culture develop long processes and assume the morphology of normal neurons as judged light microscopically. The development of synapses in the cultured tissue is studied by periodic electron microscopic examination of the areas of contact between cells. The initial expiants are free of any apparent synaptic contacts. After 48 h in culture, simple swellings or boutons are detected at the periphery of the cells or at the end of the fine processes. These initial synaptic profiles contain a few vesicles but lack mitochondria. The synaptic vesicles appear to originate from the smooth endoplasmic reticulum. Further expiants remain primitive, only the number of vesicles in the cytoplasmic swellings or boutons increases. These clusters of vesicles are 40–60 nm in diameter and morphologically distinguishable from the synaptic vesicles of normal neurons. There are no postsynaptic folds or membrane thickenings. Specialized cell contacts between cells are also present.     

Glia and Muscle Sculpt Neuromuscular Arbors by Engulfing Destabilized Synaptic Boutons and Shed Presynaptic Debris

Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived “debris” during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.     

Neuronal and synaptic organization of the lateral geniculate nucleus of the tree shrew,Tupaia glis

Summary The ultrastructural study of the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia glis) revealed two types of neurons: (1) a large thalamocortical relay cell (TCR), which may bear cilia, and (2) a small Golgi type-II interneuron (IN) with an invaginated nucleus. The narrow rim of pale cytoplasm of the IN contains fewer lysosomes and fewer Nissl bodies than the cytoplasm of the TCR. The IN perikarya, which in some cases establish somatosomatic contacts, frequently contain flattened or pleomorphic synaptic vesicles. The ratio of TCR to IN is 31.Three types of axon terminals were observed in the LGN. Two of them contain round synaptic vesicles but differ in size. The large RL boutons undergo dark degeneration after enucleation; they are the terminals of retino-geniculate fibers. The smaller RS boutons show dark degeneration after ablation of the visual cortex; they are the terminals of the cortico-geniculate fibers. The third type of bouton (F₁ does not degenerate after either intervention. The boutons of this type are filled with flattened vesicles and are believed to be intrageniculate terminals. F₂-profiles were interpreted as presynaptic dendrites of the IN. The characteristic synaptic glomeruli found in the LGN contain in their center an optic terminal. These optic terminals establish synaptic contacts with dendrites or spine-like dendritic protrusions of TCRs as well as with presynaptic dendrites. Synaptic triads were also seen. The distribution of the individual types of synaptic contacts in layers 3 and 4 was determined. Layer 4 contains only one third of the retino-geniculate synapses and of the synaptic contacts of F₁-terminals.     

Topological and Statistical Analysis of 3-D Reconstructions of Axon Collaterals

This is the first statistical metrical, topological, and model-oriented study of the spatial properties of collaterals of the primary afferent and reticulo-spinal axons, which monosynaptically innervate motoneurons of the lumbar segments of the frog spinal cord. It has been shown that ramification of both types of the collaterals is significantly asymmetrical. They differ from each other in the degree of asymmetry, which may be used for their topological classification. Spatial distribution of the synaptic boutons of these collaterals corresponds to a compound Poisson scheme, which means that the contacts are aggregated in clusters within 3-D space. We also showed that this distribution is fiducially related to the distribution of the expectation time for contacts measured as the distance along the collateral from a site of the collateral origin to a contact.     

Effect of plasma membrane contacts between cells on uridine incorporation and RNA polymerase activity

The effect of lateral plasma membrane contacts between neighbouring cells on uridine incorporation and endogenous RNA polymerase activity has been studied in kidney epithelial cells growing on a solid substrate in culture. A gradual increase of intercellular membrane contacts was parallelled by a gradual decrease in the rate of uridine incorporation. In contrast, the endogenous RNA polymerase activity remained unaltered when cells established membrane contacts between each other. The results are interpreted as reflecting a decreased uptake of uridine through the plasma membrane rather than a decline in RNA synthesis in cells with intercellular membrane contacts.     

Two different types of dynorphin-A-immunoreactive terminals in rat substantia nigra

Summary The opioid peptide dynorphin A (1–17) is the third transmitter identified in the striatonigral projection, the other two being gamma-aminobutyric acid (GABA) and substance P. The ultrastructural features of the dynorphinergic terminals in substantia nigra/pars reticulata were studied using pre-embedding immunocytochemistry with the classical peroxidase-antiperoxidase-diaminobenzidine-method; these features were compared with GABAergic boutons visualized with an immunogold method. Two distinct types of dynorphin-A-immunoreactive boutons could be identified: (1) type A (81%) possessing characteristics similar to the GA-BAergic nerve endings in this region, i.e., large pleomorphic vesicles and symmetric synaptic contacts, (2) type B (19%) displaying asymmetric synaptic zones and small, mostly round vesicles. These results are in agreement with physiological studies suggesting a dual action of dynorphin A in substantia nigra.     

A peripheral pacemaker drives the circadian rhythm of synaptic boutons in Drosophila independently of synaptic activity

Circadian rhythms in the morphology of neurons have been demonstrated in the fly Drosophila melanogaster. One such rhythm is characterized by changes in the size of synaptic boutons of an identified flight motor neuron, with larger boutons during the day compared with those at night. A more detailed temporal resolution of this rhythm shows here that boutons grow at a time of increased locomotor activity during the morning but become gradually smaller during the day and second period of increased locomotor activity in the evening. We have experimentally manipulated the synaptic activity of the fly during short periods of the day to investigate whether changes in bouton size might be a consequence of the different levels of synaptic activity associated with the locomotion rhythm of the fly. In the late night and early morning, when the flies normally have an intense period of locomotion, the boutons grow independently of whether the flies are active or completely paralyzed. Bouton size is not affected by sleep-deprivation during the early night. The cycle in bouton size persists for 2 days even in decapitated flies, which do not move, reinforcing the notion that it is largely independent of synaptic activity, and showing that a pacemaker other than the main biological clock can drive it.     

Relais cells and afferent axons in the dorsal part of the lateral geniculate body in the albino rat under geometrical aspects]

1. The average volumes of dendritic domaines of relay neurons (P-neurons) were calculated and the quantitative relations to the neuronal elements situated in this area were investigated. Likewise we carried out measurements and calculations at the terminal parts of afferent axons, to find a conception concerning possible contacts between axons and P-neurons considering quantitative aspects. 2. The dendrites of one P-neuron are distributed in an area of about 0,008 mum3. In this area there are located somata of at least 120 other P-neurons and dendrites of altogether about 900 P-neurons. 3. The type-1-axons (cortical afferents) run almost linearly in the longitudinal system of the CGLd. Traversing a distance adequate to the diameter of a P-neuron (250 mum) the dendrites of 150 to 170 P-neurons may cross the course of one axon. At this distance the axon, however, has just set up about 50 boutons, thus synaptic contacts may be established with one third at most of the existing cells. A type-1-axon that is bifurcating in the entrance area into the CGLd is altogether of about 2000 mum in length and is able to develop about 420 presynaptic profiles. 4. The type-2-axons (retinal afferents) show a distinct terminal branching zone. The Golgi-Kopsch impregnated terminals of type-2a-axons are distributed in a space of 147000 mum3 capacity, the corresponding terminals of type-2b-axons in a space of 443000 mum3. The type-2a-axons having an average number of 23 boutons, may contact the dendritic branching zones of 25 P-neurons. There is a good reason to assume that type-2b-axons are in contact also with terminal dendritic parts of P-neurons. Thus the number of P-cells, which spread their dendrites into the terminal branching zone of one type-2b-axon may amount to 540. The average number of boutons of one type-2b-terminal, however, is only about 160. This means that synaptic contacts may be developed to the P-neurons-dendrites not exceeding 30% of them. 5. Various aspects of divergence of axon terminals in the albino rat's CGLd are discussed.     

Synchronization of electrical field potentials in neocortex of rat after isolation of a cortex island in the contralateral brain hemisphere

A correlation between the number of boutons and synchronization of electrical activity in two sites of the intact right somatosensory cortex of rats was anakyzed at different stages of axonal sprouting elicited by isolation of a cortex slab in the left cortex. Time delay between the development of epileptiform field potentials in two sites of intact cortex located at a distance of 4 mm from each other was determined as a parameter of synchronization. The analysis was carried out in 30 and 90 days after the complete isolation of the neural island in a symmetrical site of the contralateral cortex. Epileptiform activity was induced by penicillin. A significant increase in the number of boutons in the II and V layers of the intact cortex observed 90 days after the isolation of neural island in a symmetrical site of the cortex corresponded to a significant decrease in the delay of electrical activity development. Similar effects were observed in the V layer of the island 30 days after the isolation. The results suggest that the cortex lesion activates formation of new synaptic boutons in a contralateral site and increases a degree of synchronization of electrical activity, which may affect the epileptogenesis. The data suggest that pyramids of the III and, most probably, V layers form a neuronal network in the rat neocortex thus providing synchronization of epileptiform field potentials.     

Species differences in the coexistence of 5-hydroxytryptamine and substance P in presynaptic boutons in the cervical ventral horn

Summary Substance P (SP)-and 5-hydroxytryptamine (5-HT)-containing presynaptic boutons in the cervical ventral horn were studied in chicken, hamster, rat and monkey spinal cords, using PAP and protein A-gold double-labeling techniques in conjunction with monoclonal antibodies. In the chicken, the PAP method demonstrated that SP-immunoreactive boutons contained large spherical dense-cored vesicles (DCVs) whereas 5-HT-immunoreactive boutons displayed both elongated and spherical DCVs. Using the protein A-gold double-labeling technique, 10-nm gold particles for SP were localized over the spherical DCV-containing boutons whereas 15-nm gold particles for 5-HT were localized on elongated DCV-containing boutons. On the other hand, in the other species investigated, both SP-and 5-HT-immunoreactive boutons had similar morphological features as shown by the PAP method; both contained elongated and spherical DCVs. The two differentsized gold particles, each of which labeled either 5-HT or SP, were found together over DCVs in a single bouton. These results indicate that 5-HT and SP are contained in different presynaptic boutons in the chicken, although in the hamster, rat and Japanese macaque, the two neurotransmitters/modulators coexist in the same DCVs in a single bouton. Species differences have thus been demonstrated for the coexistence of 5-HT and SP in the spinal ventral horn.     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

Intercellular adhesion between juvenile liver cells : A method to measure the formation of stable lateral contacts between cells attached to a collagen gel

Liver cells, isolated from young rats by a collagenase perfusion technique, have been seeded on gels consisting of reconstituted collagen type I fibres. The cells attached rapidly to the collagen and formed contacts with each other over large regions of their lateral margins. Digestion of the collagen gels with bacterial collagenase released the cells, which had now formed stable cell-cell bonds, into suspension. The collagenase did not destroy the intercellular contacts. By measuring the remaining single cells with an electronic particle counter and the total number of cells from the assay of lactate dehydrogenase activity, the degree of aggregation (formation of intercellular contacts) could be determined quantitatively. It was demonstrated that formation of stable contacts do not require serum, but that calcium ions play a significant role. This method advantageous over other methods for the determination of cell adhesion, in that it measures the formation of bonds between cells attached to a solid surface and thus closely mimics the in vivo situation.     

Mapping of experimental axon degeneration by electron microscopy of golgi preparations

Summary For the mapping of the terminal area of transected axons within the central nervous system, electron microscopy has recently been adopted. A greater accuracy is thereby obtained than with silver impregnation and light microscopy, since it becomes possible to determine the kinds of structure (soma, dendrites, spines) with which the degenerating boutons establish synaptic contact. In the present study this technique was extended by Golgi impregnation of such material with the aim of making possible classification of the postsynaptic neuron. A few days after transection of a pathway (commissural fibres to the hippocampus being used as a model in this study) the brain was fixed by perfusion with phosphate buffered formalin with sucrose. This was followed by immersion in an osmium tetroxide-potassium dichromate mixture (Dalton's fixative without sodium chloride) later replaced by a solution of silver nitrate. Satisfactory Golgi impregnation of nerve cells and processes was obtained. By careful trimming, and reembedding of selected areas, blocks for ultramicrotomy could be obtained which contained only one type of impregnated cell, e.g., hippocampal pyramidal cells.The relation of basal dendrites of such cells to degenerating boutons of commissural fibres was studied. Numerous examples of contact between degenerating boutons and spines belonging to the basal dendrites were seen. Although the Golgi precipitate obscured the postsynaptic substance in the spines, a number of features at the sites of contact were considered strong indication that many of the contacts were synapses and not merely the result of random juxtaposition. This combined procedure is supposed to be applicable to other problems and to other parts of the nervous system as well.This study was supported in part by Grant NB 02215 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. This ais is gratefully acknowledged. The author is indebted to Mrs. J. L. Vaaland and Mr. B. V. Johansen for valuable technical assistance.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry

Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only a number of expected adrenergic characteristics but, paradoxically, also certain cholinergic functions such as the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). The purpose of this study was to determine whether the entire population of cultured neurons was aquiring cholinergic capabilities, or whether this phenomenon was restricted to a subpopulation. After 1--6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for electron microscopy analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE), and DOPA-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1 to 8 wk in culture, the average ChAc activity per neuron increased 1,100-fold, and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was that characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. Considering these data along with available physiological studies, we conclude that these cultures contain one population of neurons that is initially adrenergic. Over time, under conditions of this culture system, this population develops cholinergic mechanisms. That a neuron may, at a given time, express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.     

A Neurosecretory System in the Brain of the Lancelet, Branchiostoma lanceolatum

Ultrastructural and histochemical studies indicate a neurosecretory system exists in the lancelet brain with basal properties resembling a primitive hypothalamic system. A nucleus of secreting neurons, containing peptide granules (115 nm), is prominent in the dorsal walls of the brain. The axons establish contacts with the ventral brain surface, probably releasing their secretory product out of the brain. The neurons are innervated by dopaminergic "boutons en passant" often very active with a high number of electron translucent vesicles as well as dense-core vesicles (90 nm). Ventrally located cellbodies containing what are probably secretory peptide granules (110 nm) establish contacts with their basal processes on the ventral brain surface.     

Should I Stay or Should I Go? Presynaptic Boutons in the Adult Cortex Still Haven't Made Up Their Minds

Previous studies demonstrating turnover of the dendritic spines of cortical neurons have suggested a modest rate of turnover of synaptic connections. Now, two papers in this issue of Neuron address this question from the other side of the synapse, the presynaptic boutons. Both studies use in vivo multiphoton imaging of cortical axons to show that synaptic boutons come and go, just like spines. One of the studies shows remarkable diversity in the lability of boutons depending on the cell type from which they originate, with some boutons displaying nearly complete turnover in just a few months. The other study shows that bouton turnover occurs in primates as well as rodents.     

Subsurface cisterns and lamellar bodies in the granule cells of the guinea-pig fascia dentata

Summary Subsurface cisterns (SSC's) and less frequent lamellar bodies (LB's) were identified in the granule cells of the guinea-pig fascia dentata. Both structures, composed of flattened or collapsed agranular cisterns, are continuous with the regular rough endoplasmic reticulum and occasionally connected with each other forming LB-SSC complexes. The SSC's are apposed to glia, synaptic boutons, and nerve cell processes as well as to neighboring granule cells appearing here singly and in confronting pairs. The quantitative analysis of the various cisternal appositions compared to the distribution of the tissue components on the granule cell soma shows that the overwhelming majority of SSC's are related to glial cells.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.