Veratridine-stimulated central synapses in culture: A quantitative ultrastructural analysis

Synapses in explant cultures of fetal rat neocortex at day 18 in vitro were stimulated by veratridine (10^?4M) for 20 min. The cultures were subsequently processed for electron microscopy and the synapses were analyzed by quantitative techniques, incorporating set mathematical treatment. The mean values of area, perimeter, and form factor of the presynaptic elements significantly increased following veratridine stimulation, compared to the values of control synapses. The length of the postsynaptic thickening also increased, while synaptic curvature did not change significantly in the veratridine group. A fivefold reduction was observed in the mean number of synaptic vesicles per presynaptic element and in the vesicle-terminal area ratio, following veratridine stimulation. The cytoplasm-terminal area ratio and the occurrence of vacuoles/cisternae significantly increased after veratridine application. Planar measurement of membranes (boundary length) of different presynaptic organelles revealed that the total membrane did not change significantly in the veratridine group. The data indicated an increase in volume and swelling of the pre- and postsynaptic elements, considerable depletion of synaptic vesicles, and preservation of the total presynaptic membrane following veratridine stimulation in nerve tissue culture.     

Serial synapses in Aplysia

Serial synapses occur between small profiles in the neuropil of Aplysia abdominal ganglion. Material was fixed in phosphate buffered OsO₄, embedded in epon, and sections were stained with uranyl acetate and lead citrate. A class of synapses had the following characteristics: (1) synaptic vesicles clustered against the presynaptic membrane, (2) a widened extracellular space of about 20 nm containing electron-dense material, (3) straightening of the pre- and postsynaptic membranes, and (4) no postsynaptic membrane specialization. Some density between the presynaptic membrane and the adjacent synaptic vesicles was occasionally observed. Synapses occurred between small profiles in the neuropil (typical profile diameters were 1–3 m?m). In this sample of approximately 100 synapses, four serial synapses were identified. The serial synaptic profiles were all small. In addition to the finding of serial synapses, 40% of the postsynaptic profiles contained vesicles similar to the synaptic vesicles seen in presynaptic profile. Serial synapses may be the anatomical substrate of presynaptic inhibition and facilitation and of dishabituation.     

Ultrastructure of nerve plexus in flatworms

Summary Synaptic components from the peripheral nervous system of the polyclad flatworm, Notoplana acticola, are described from electron microscopic observations. Quasineuropile, defined as clusters of neurites containing synaptic vesicles, occurs as scattered islands along the peripheral nerve cords of the plexus. Some neurite clusters only contain one type of synaptic vesicle but others are mixed. The most usual synaptic configuration consists of a single presynaptic element and a pair of postsynaptic neurites sharing a common synaptic cleft. These synapses are polarized and contain clear, 420 Å vesicles. GABA-type synapses are also found. At least two kinds of solid-core vesicles also occur.     

Synaptic relationships in the plexiform layers of carp retina

Summary The synaptic contacts made by carp retinal neurons were studied with electron microscopic techniques. Three kinds of contacts are described: (1) a conventional synapse in which an accumulation of agranular vesicles is found on the presynaptic side along with membrane densification of both pre- and postsynaptic elements; (2) a ribbon synapse in which a presynaptic ribbon surrounded by a halo of agranular vesicles faces two postsynaptic elements; and (3) close apposition of plasma membranes without any vesicle accumulation or membrane densification.In the external plexiform layer, conventional synapses between horizontal cells are described. Horizontal cells possess dense-core vesicles about 1,000 Å in diameter. Membranes of adjacent horizontal cells of the same type (external, intermediate or internal) are found closely apposed over broad regions.In the inner plexiform layer ribbon synapses occur only in bipolar cell terminals. The postsynaptic elements opposite the ribbon may be two amacrine processes or one amacrine process and one ganglion cell dendrite. Amacrine processes make conventional synaptic contacts onto bipolar terminals, other amacrine processes, amacrine cell bodies, ganglion cell dendrites and bodies. Amacrine cells possess dense-core vesicles. Ganglion cells are never presynaptic elements. Serial synapses between amacrine processes and reciprocal synapses between amacrine processes and bipolar terminals are described. The inner plexiform layer contains a large number of myelinated fibers which terminate near the layer of amacrine cells.This work was supported by an N.I.H. grant NB 05404-05 and a Fight for Sight grant G-396 to P.W. and N.I.H. grant NB 05336 to J.E.D. The authors wish to thank Mrs. P. Sheppard and Miss B. Hecker for able technical assistance. P.W. is grateful to Dr. G. K. Smelser, Department of Ophthalmology, Columbia University, for the use of his electron microscope facilities.     

The ultrastructure of giant fibre and serial synapses in crayfish

Summary The ultrastructure of synapses between the cord giant fibres (lateral and medial) and the motor giant fibres in crayfish, Astacus pallipes, third abdominal ganglia have been examined. These electrotonic synapses are asymmetrical, they have synaptic vesicles only in the presynaptic fibre, and they have synaptic cleft widths normally of about 100 Å but narrowed to about 50 Å in restricted areas. Localized increases in density of the synaptic cleft and adjacent membranes also occur within a synapse, and synaptic vesicles are most tightly grouped at the membrane in such areas. Tight or gap junctions with 30 Å or narrower widths have not been found, but the junctions probably function in a similar way to gap junctions.Three small nerves are closely associated with the synapses between the giant fibres. One of these small nerves has round synaptic vesicles and is thought to be excitatory on morphological grounds; one has flattened vesicles and is thought to be inhibitory; and one is postsynaptic to the lateral giant and the two small presynaptic nerves. It is proposed that these small nerves modulate activity in the much larger giant fibre synapse.     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Synaptogenesis of hippocampal neurons in primary cell culture

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.     

Ganglioside composition of subcellular fractions,including pre- and postsynaptic membranes,fromTorpedo electric organ

Gangliosides were isolated from four subcellular fractions of the electric organ ofTorpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postyynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 g of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.Abbreviations SVs synaptic vesicles - TLC thin-layer chromatography - cholera B-HRP B subunit of cholera toxin linked to horseradish peroxidase     

Fine structure of nerve-melanophore contacts in the angelfish Pterophyllum scalare

Contacts between small unmyelinated nerve fibres and dermal melanophores of the angelfish, Pterophyllum scalare, exhibit several features characteristic of synapses, including small synaptic vesicles and dense core vesicles, a narrow synaptic cleft, electron-dense material at the postsynaptic membrane (cell membrane of the melanophore) and, occasionally, presynaptic densities. An analysis of serial thin sections shows that the synapses described here represent varicosities of an otherwise more or less straight nerve fibre. A single axon thereby may form several en passant synapses with a single melanophore. It is suggested that the synaptic contacts described here not only represent sites of transmitter release but also play a role as sites of firm attachment between nerves and melanophores which guarantee a stable arrangement of nerve fibres and melanophores.Supported by the Deutsche Forschungsgemeinschaft     

Some peculiar synaptic complexes in the first visual ganglion of the fly,Musca domestica

Summary In the lamina ganglionaris, the first optic ganglion of the fly, the inventory of cell types as well as the patterns of their connections are well known from light microscopic investigations. Even the synaptic contacts are known with relative completeness. However, the structural details visible on electron micrographs are very difficult to interpret in functional terms. This paper concentrates on two aspects: 1) the synaptic complex between a retinula cell axon and four postsynaptic elements, arranged in a constant elongated array (it is suggested that all synapses in which the retinula cell is presynaptic are of this kind), and 2) the gnarl complex in which a presynaptic specialization in one neuron is separated from another neuron by a complicated glial invagination. The participation of glia at postsynaptic sites seems to be quite common in this ganglion. Occasionally it seems that a glia cell is the only postsynaptic partner facing a presynaptic specialization within a neuron.     

The fine structure of the central synaptic contacts on an identified crustacean motoneuron

Summary Small nerve terminals in the neuropile of the brain of the crab Scylla serrata make close contact with the secondary, tertiary and higher order central branches of the reflex eye-withdrawal motoneurons. Most contacts have the characteristics of chemically transmitting synapses in that the presynaptic terminals contain agranular vesicles of 25 to 50 nm in diameter and are separated from the motoneuron by a synaptic cleft of about 16 nm. Some terminals contain synaptic ribbons, others contain a mixture of larger (50 to 80 nm) agranular and also dense cored vesicles. In addition large blunt-ended contacts unaccompanied by vesicles, occur between neurons in the neuropile and the motoneuron. It is suggested that the absence of synaptic contacts over the large primary branches of the motoneuron could explain previous physiological findings that little or no resistance changes can be detected in this part of the neuron during excitation or inhibition.We thank Mrs. Joan Goodrum for the preparation of Fig. 1.     

Synaptology of the rat suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the rat the fine structure of the synapses and some features of their topological arrangement were studied. Five types of synapses could be distinguished with certainty: A. Two types of Gray-type-I (GTI) or asymmetrical synapses (approximately 33%). The presynaptic elements contain strikingly different types of mitochondria. Size of clear vesicles: approximately 450 A. Synapses with subjunctional bodies often occur, among these also "crest synapses". Localization: dendritic shafts and spines, rarely somata. B. Three types of Gray-type-2 (GTII) or symmetrical synapses (approximately 66%):1) Axo-dendritic and -somatic (=AD) synapses. Size of clear vesicles: approximately 500 A. 2) Invaginated axo-dendritic and -somatic (=IAD) synapses with club-like postsynaptic protrusions within the presynaptic elements (PreE1). Size of clear vesicles is very variable: approximately 400-1,000 A. 3) Dendro-dendritic, -somatic and somato-dendritic (=DD) synapses occurring at least partly in reciprocal arrangements. They represent an intrinsic system. Shape of clear vesicles: often oval; sucrose treatment partly produces flattening. Dense core-vesicles (dcv) are found in all GTII- and most of the GTI-synapses after three-dimensional reconstruction. All types of synapses (mostly GTII-synapses) can be enclosed by multilamellar astroglial formations. The synapses often occur in complex synaptic arrangements. Dendrites and somata of females show significantly more multivesiculated bodies than those of males. Further pecularities of presynaptic (PreELs) and postsynaptic elements (PostELs) within the SCN are described and discussed.     

Pattern of synaptic connections in the pineal organ of the ayu,Plecoglossus altivelis (Teleostei)

Summary Synaptic connections were studied by means of electron microscopy in the sensory pineal organ of the ayu, Plecoglossus altivelis, a highly photosensitive teleost species. Three types of specific contacts were observed in the pineal end-vesicle: 1) symmetrically organized gap junctions between the basal processes of adjacent photoreceptor cells; 2) sensory synapses endowed with synaptic ribbons, formed by basal processes of photoreceptor cells and dendrites of pineal neurons; 3) conventional synapses between pineal neurons, containing both clear and dense-core vesicles at the presynaptic site. Based on these findings, the following interpretations are given: (i) The gap junctions may be involved in an enhancement of electric communication and signal encoding between pineal photoreceptor cells. (ii) The sensory synapses transmit photic signals from the photoreceptor cells to pineal nerve cells. (iii) The conventional synapses are assumed to be involved in a lateral interaction and/or summation of information in the sensory pineal organ. A concept of synaptic relationships among the sensory and neuronal elements in the pineal organ of the ayu is presented.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany     

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.     

Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.     

ELECTRON MICROSCOPY OF SYNAPTIC STRUCTURE OF OCTOPUS BRAIN

The well known type of synapse between a presynaptic process containing vesicles and a "clear" postsynaptic process can be commonly observed in the various lobes of the brain of Octopus. The presynaptic vesicles are aggregated near regions of the synaptic membranes which show specialisation and asymmetric "thickening" indicating functional polarisation, and here chemical transmission is presumed to take place. In addition, in the vertical lobe a very interesting serial arrangement of synaptic contacts occurs. Presynaptic bags, formed from varicosities of fibres from the superior frontal lobe, contact the trunks of amacrine cells in the manner just described. The trunks, however, although apparently postsynaptic are themselves packed with synaptic vesicles. The trunks, in turn, make "presynaptic" contacts with clear spinous processes of other neurons of yet undetermined origin. Typical polarised membrane specialisations occur at the contact regions. The trunk vesicles aggregated closest to the contact regions have a shell of particles round their walls. At present, there is no way of telling whether the membrane conductance to the various ions is differently affected at either of the transmission sites, and, if an inhibitory mechanism is involved, whether it is of the presynaptic or postsynaptic variety.     

A further contribution to the study of the contact region of Octopus synaptosomes

Summary Octopus synaptosomes have been examined after glutaraldehyde fixation and phosphotungstic acid (PTA) staining of non-osmicated tissue. The results concentrate on the appearance of the contact region between the presynaptic component of synaptosomes and their postsynaptic processes. Membranes have a triple-layered appearance, consisting of an electronopaque internal coat, an electrontranslucent band and an electronopaque external coat. Good examples of this are found in synaptosomal, dendritic and axonal membranes. At specialized synaptic contact regions the external coats of the pre- and postsynaptic membranes coalesce to form a prominent synaptic plate, which has a width of 18 nm and is subdivided into zones of varying electronopacity. It is suggested that this plate is formed from the specialized external coat of the postsynaptic membrane and the unspecialized external coat of the presynaptic membrane. Presynaptic spicules extend from the internal coat of the presynaptic membrane. They are closely associated with elements of the presynaptic network.It is suggested that the synaptic plate is probably composed of mucopolysaccharides, while the relation of the plate to acetylcholinesterase is discussed. It is proposed that functional localization at the synapse is less precise in octopus than vertebrates.I would like to express my thanks to Professors J. Z. Young, F. R. S. and E. G. Gray for their helpful advice, and also Mr. S. Waterman for photographic assistance.     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.