Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.     

SOME FEATURES OF THE SUBMICROSCOPIC MORPHOLOGY OF SYNAPSES IN FROG AND EARTHWORM

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

INNERVATION OF THE FIBRILLAR FLIGHT MUSCLE OF AN INSECT: TENEBRIO MOLITOR (COLEOPTERA)

The structure of peripheral nerves, and the organization of the myoneural junctions in flight muscle fibers of a beetle is described. The uniaxonal presynaptic nerve branches display the "tunicated" structure reported in the case of other insect nerves and the relationship between the axon and the lemnoblast folds is discussed. The synapsing nerve terminal shows many similarities with that of central and peripheral junctions of other insects and of vertebrates (e.g., the intra-axonal synaptic vesicles) but certain important differences have been noted between this region in Tenebrio flight muscle and in other insect muscles. Firstly, the axon discards the lemnoblast before the junction is established and the axon effects a circumferential synapse with the plasma membrane of the fiber, which alone shows the increased thickness often observed in both pre- and postsynaptic elements. Secondly, in addition to the synaptic vesicles within the axon are present, in the immediately adjacent sarcoplasm, great numbers of larger postsynaptic vesicles which, it is tentatively suggested, may represent the sites of storage of the enzymatic destroyer of the activating substance similarly quantized within the intra-axonal vesicles. The spatial relationship between the peripherally located junctions and the portion of the fiber plasma membrane internalized as circumtracheolar sheaths is considered, and the possible significance of this with respect to impulse conduction is discussed briefly.     

Ultrastructural elements of CNS development in the Stage 15 Drosophila (Diptera: Drosophilidae) embryo

The ultrastructural neuroanatomy of the wild-type Drosophila melanogaster (Diptera: Drosophilidae) embryo at Stage 15 was examined with the transmission electron microscope. This particular embryonic stage is an approximate midpoint of neurogenesis. No blood-brain barrier has yet formed in the CNS as illustrated by lanthanum tracer infiltration into the neuropil. First structural signs of axo-axonal synapses, in the embryonic neuropil are seen as electron-dense plaques on the cytoplasmic sides of apposing pre- and postsynaptic membranes. Very few clear synaptic vesicles (30–40 nm in diameter) are present, and none of these is clustering, although some are docking on the presynaptic membrane. In the perikaryal rind of the ventral ganglion, several glial somata are shown surrounding a single neuronal soma. Finger-like processes of the glial somata extend into extracellular spaces and contact the surface of the neuron. The functional significance of these findings is discussed.     

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.     

Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes)

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400- A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K- rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine- depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.     

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.     

THE ORGANIZATION OF SYNAPTIC AXOPLASM IN THE LAMPREY (PETROMYZON MARINUS) CENTRAL NERVOUS SYSTEM

The fine structure of synapses in the central nervous system of lamprey (Petromyzon marinus) ammocoetes has been investigated. Both synapses within the neuropil and synaptic links between giant fibers (including Müller cells) and small postsynaptic units are described. The distribution of neurofilaments and microtubules in nerve profiles over a wide diameter range is described, and the possible role of these structures in intracellular transport is discussed. Electron micrographs indicate that small lucent "synaptic vesicles" occur sparsely throughout the axoplasm and in regular arrays in association with microtubules in the vicinity of synapses. Within a synaptic focus, immediately adjoining the presynaptic membrane, vesicles are randomly arranged and are not associated with microtubules. Neurofilaments are present, generally in large numbers, but these are not associated with vesicles or other particulates. The structural findings are considered in terms of current concepts of fast and slow transport in neurons and the mechanochemical control of intracellular movement of materials.     

SUBMICROSCOPIC CHANGES OF THE SYNAPSE AFTER NERVE SECTION IN THE ACOUSTIC GANGLION OF THE GUINEA PIG. AN ELECTRON MICROSCOPE STUDY

The degenerative changes of the synaptic regions after nerve section have been studied with the electron microscope in the interneuronal synapse of the ventral ganglion of the acoustic nerve of the guinea pig. Fixation with buffered osmic tetroxide was carried out 22, 44, and 48 hours after destruction of the cochlea on one side; the contralateral ganglion being used as control. The submicroscopic organization of normal axosomatic and axodendritic synapses is described. In the synaptic ending four morphological components are recognized: the membrane, the mitochondria, the synaptic vesicles (19, 20), and the cytoplasmic matrix. The intimate contact of glial processes with the endings and with the surface of the nerve cell is described. At the level of the synaptic junction there is a direct contact of the limiting membranes of the ending and of the cell body or dendrite. Both contacting membranes constitute the synaptic one with a total thickness of about 250 A. This membrane has regions of higher electron density where the synaptic vesicles come into intimate contact and fuse with it. Definite degenerative submicroscopic changes in the nerve endings were observed after 22 hours of destruction of the cochlea and were much more conspicuous after 44 and 48 hours. After 22 hours there is swelling of the ending and decreased electron density of the matrix. Most synaptic vesicles have disappeared or seem to undergo a process of clumping and dissolution. Some mitochondria also show signs of degeneration. After 44 hours the synaptic vesicles have practically disappeared; mitochondria are in different stages of lysis; the membrane of the ending becomes irregular in shape, and there is shrinkage and in some cases detachment of the ending. No changes in the postsynaptic cytoplasm were observed. These observations and particularly the rapid lysis of the synaptic vesicles are discussed in correlation with data from the literature indicating the early alteration of synaptic function and the biochemical changes occurring after section of the afferent nerve. The hypothesis that the synaptic vesicles may be carriers of acetylcholine or other active substances (19, 20) and that they may act as biochemical units in synaptic transmission is also discussed.²     

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.     

Ultrastructural observations on rapid formation of neuro-muscular junctions in vitro

Summary The development of neuro-muscular junctions (mouse, rat) from the time of first contact between neurons and myotubes in culture and the changes which lead to the formation of functional synaptic contacts have been investigated using light microscopy and ultrastructural techniques.An extensive basal lamina was present when the neuronal cell population was added to the developing myotubes in culture. The nerve cells were initially strongly attracted to each other and nerve cell aggregates formed rapidly. It was only when nerve fibres began to grow out of these aggregates to contact developing myotubes that changes within the cytoplasm of the two adjacent cells were observed. These developments included accumulations of filaments, membrane densities, mitochondria and large clear vesicles within both cells in the region of contact. In addition, collections of glycogen granules and an extensive membrane reticular complex were found within myotubes, and an extensive granular material filled many of the nerve processes. The basal lamina within the intercellular space appeared more electron-dense than elsewhere and was traversed by strands linking the two cell membranes. These features all appeared to be stages in the initial formation of neuro-muscular junctions. It was only after these events had occurred that presynaptic vesicles gradually appeared within the future nerve terminal. The results of this paper therefore support the view that synaptic transmission at developing mammalian neuromuscular junctions is not necessarily dependent on the presence of presynaptic vesicles.     

Electron microscopy of stomatogastric ganglion in the lobster Homarus americanus

The stomatogastric ganglion and two of the associated afferent and efferent nerve trunks (stomatogastric and dorsal ventricular nerves) from Homarus americanus have been examined with light and electron microscopy after glutaraldehyde-osmium tetroxide fixation. The dorsally located neuron somata, rich in ribosomes and glycogen, are encased in multi-layered glial and fibrous sheaths. The synaptic neuropil regions occur scattered throughout the central and ventral part of the ganglion, interspersed amonglarger nerve fibres of extrinsic and intrinsic origin from which the neuropil is derived. Neural processes containing masses of small clear vesicles plus larger dense-core vesicles make apparent synaptic contacts at points of increased membrane density with smaller, non-vesicle-containing or sometimes other vesicle-containing nerve fibres.     

Some features of the submicroscopic morphology of synapses in frog and earthworm

Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.     

A freeze-fracture study of afferent and efferent synapses of hair cells in the sensory epithelium of the organ of Corti in the guinea pig

Summary Afferent and efferent synapses of hair cells in the organ of Corti of the guinea pig have been examined in freeze-fracture replicas.Afferent synapse In the inner hair cells, intramembranous particles 10 nm in diameter are aggregated on the ridge on the P-face of the presynaptic membrane directly beneath the synaptic rod. In the outer hair cells, in which the synaptic rod is located in the presynaptic cytoplasm underneath the presynaptic membrane, small aggregations of intramembranous particles 10 nm in diameter can be found on the P-face of the presynaptic membrane corresponding to the site of the presynaptic dense projection. Intramembranous particles 10 nm in diameter are also densely aggregated on the P-face of the postsynaptic membrane of the outer hair cells.Efferent synapse of the outer hair cells Large intramembranous particles 13 nm in diameter are distributed in clusters composed of four to ten particles on the P-face of the presynaptic membrane. In the P-face of the postsynaptic membrane, disc-like aggregations of intramembranous particles 9 nm in diameter are found. The subsynaptic cistern covers the cytoplasmic surface of the postsynaptic membrane of the efferent synapse; it may cover more than one postsynaptic membrane when several efferent synapses are in close proximity to one another.     

Ultrastructural organization of the anterior sensory neuropile in the metathoracic ganglion of the locust Locusta migratoria]

The study of serial sections of the metathoracic ganglion of Locusta migratoria showed that the fibres of the tympanal nerve terminate in the dorsal half of the anterior sensory neuropile (ASN). Three types of synaptic endings were found in the ASN. Endings type I contain dense-core vesicles 600-850 A in diameter, more or less uniformly distributed in the axoplasm. They do not form specialized contacts with postsynaptic fibres and are localized only in the most ventral part of the ASN. Endings type II contain clear round vesicles 400-450 A in diameter (rare 250-300 A) and form typical synapses with dense pre-and postsynaptic membranes and synaptic cleft 150-200 A. Four types of contacts formed by these endings with postsynaptic fibres were found: 1 : 1 synapses; convergent, divergent and serial. All of them are well presented in the auditory neuropile. Endings type III contain both dense-core and clear vesicles in different relation. Only clear vesicles of these endings are connected with the active sites of the membrane.     

Morphological evidence for a direct innervation of the mouse vomeronasal glands

Summary The morphological evidence for a direct autonomic innervation of the mouse vomeronasal glands is presented. Axonal varicosities containing a few densecore vesicles and numerous clear vesicles (36–60 nm in diameter) make synaptic contacts with the secretory cells at the base of the glandular acini. The axonal presynaptic membrane is associated with a distinct dense material and it is separated from the secretory cell by a synaptic cleft of about 12–14 nm. At the postsynaptical level, coated vesicles can be found. Additional postsynaptical specializations have not been observed.     

Dynamics of dendritic spines and their afferent terminals: spines are more motile than presynaptic boutons

Previous work has established that dendritic spines, sites of excitatory input in CNS neurons, can be highly dynamic, in later development as well as in mature brain. Although spine motility has been proposed to facilitate the formation of new synaptic contacts, we have reported that spines continue to be dynamic even if they bear synaptic contacts. An outstanding question related to this finding is whether the presynaptic terminals that contact dendritic spines are as dynamic as their postsynaptic targets. Using multiphoton time-lapse microscopy of GFP-labeled Purkinje cells and DiI-labeled granule cell parallel fiber afferents in cerebellar slices, we monitored the dynamic behavior of both presynaptic terminals and postsynaptic dendritic spines in the same preparation. We report that while spines are dynamic, the presynaptic terminals they contact are quite stable. We confirmed the relatively low levels of presynaptic terminal motility by imaging parallel fibers in vivo. Finally, spine motility can occur when a functional presynaptic terminal is apposed to it. These analyses further call into question the function of spine motility, and to what extent the synapse breaks or maintains its contact during the movement of the spine.     

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     

CHANGES IN THE FINE STRUCTURE OF THE NEUROMUSCULAR JUNCTION OF THE FROG CAUSED BY BLACK WIDOW SPIDER VENOM

Application of black widow spider venom to the neuromuscular junction of the frog causes an increase in the frequency of miniature end-plate potentials (min.e.p.p.) and a reduction in the number of synaptic vesicles in the nerve terminal. Shortly after the increase in min.e.p.p. frequency, the presynaptic membrane of the nerve terminal has either infolded or "lifted." Examination of these infoldings or lifts reveals synaptic vesicles in various stages of fusion with the presynaptic membrane. After the supply of synaptic vesicles has been exhausted, the presynaptic membrane returns to its original position directly opposite the end-plate membrane. The terminal contains all of its usual components with the exception of the synaptic vesicles. The only other alteration of the structures making up the neuromuscular junction occurs in the axon leading to the terminal. Instead of completely filling out its Schwann sheath, the axon has pulled away and its axoplasm appears to be denser than the control. The relation of these events to the vesicle hypothesis is discussed.     

Coupling exo- and endocytosis: An essential role for PIP2 at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~ 200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.