An ultrastructural study of the polyp and strobila of Atorella japonica (Cnidaria,Coronatae) with respect to muscles and nerves

Atorella japonica were observed by TEM to examine the nerve plexus in the capitulum of the polyp and the cross-striated muscle cells of the strobila. The nerve plexus included a number of neuromuscular junctions and many interneural synapses. Neuromuscular junctions contained two types of synaptic vesicle: clear and small (ca 75 nm diam.), and dense cored and large (ca 120 nm diam.). The first type of vesicle always appeared near the presynaptic membrane and the second type was distributed behind the former. In interneural synapses, two types of vesicle which were similar to neuromuscular synaptic vesicles were recognized. They were distributed in a pattern similar to that of the neuromuscular synaptic vesicles, but these vesicles were found on both sides of the two synaptic membranes.     

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. III. A morphometric analysis of the number and diameter of intramembrane particles

The intramembrane particles on the presynaptic membrane and on the membrane of synaptic vesicles were studied at freeze-fractured neuromuscular junctions of the frog. The particles on the P face of the presynaptic membrane belong to two major classes: small particles with diameters less than 9 nm and large particles with diameters between 9 and 13 nm. In addition, there were a few extralarge particles with diameters greater than 13 nm. Indirect stimulation of the muscle, or the application of black widow spider venom, decreased the concentration of small particles on the presynaptic membrane but did not change the concentration of large particles. Three similar classes of particles were found on the P face of the membrane of the synaptic vesicles. The concentrations of large and extralarge particles on the vesicle membrane were comparable to the concentrations of these particles on the presynaptic membrane, whereas the concentration of small particles on the vesicle membrane was less than than the concentration of small particles on the presynaptic membrane. These results are compatible with the idea that synaptic vesicles fuse with the presynaptic membrane when quanta of transmitter are released. However, neither the large nor the extralarge particles on the P face of the presynaptic membrane can be used to trace the movement of vesicle membrane that has been incorporated into the axolemma.     

Ultrastructure of calcitonin gene-related peptide-immunoreactive nerve fibres in guinea-pig peribronchial ganglia.

Calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves within guinea-pig peribronchial ganglia were studied at ultrastructural level using pre-embedding immunohistochemistry. Preterminal CGRP-IR axons were unmyelinated and contained singular immunoreactive dense core vesicles. CGRP-IR axon terminals were filled with numerous non-reactive small clear vesicles and few immunoreactive dense core vesicles. Some of these terminals were presynaptic to large neuronal processes emerging from local ganglion cells. Another population of presynaptic varicosities lack CGRP-IR. Within CGRP-IR terminals, non-reactive clear vesicles were clustered at the presynaptic membrane whereas CGRP-IR large vesicles remained in some distance from the synaptic cleft. The present observations indicate that: (1) at least two neurochemically different types of synaptic input exist to guinea-pig peribronchial ganglia. (2) CGRP-IR presynaptic terminals probably utilize a non-peptide transmitter for fast synaptic transmission, whilst the peptides are likely to be released parasynaptically and may act in a modulatory fashion.     

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.     

Quantitative analysis of the structure of the varicose dilatations in the nerve fibers of the rabbit coronary artery (according to the results of three-dimensional reconstruction)]

Axoplasmic vesicles and microtubes in varicosities of axonal plexus in the external sheath of the rabbit coronary artery have been studied. Comparing serial sections and examining three-dimensional reconstruction of small nerve plexus, it was demonstrated that various varicosities differed only in their correlation of the amount of small (30-80 nm) and large (80-180 nm) vesicles. Average diameters in profiles of small and large vesicles are 56.3 nm and 115.6 nm, respectively. There are varicosities containing about 250 or more than 1,000 vesicles. Evenly distributed vesicles throughout the volume of varicosities and lack of specialized structures on the axolemma are supposed to demonstrate the absence of special areas for the mediator removal in the axons studied. The microtubes in the varicosities are peripherally arranged, next to the axolemma and form 1-1.5 wide coils. A suggestion is made that the varicosities in neighbouring axons of the same nerve plexus, with specific structural organization, are special functional units and appear to be peculiar not only for nerve plexus of the coronary artery, but also for other parts of the peripheral vegetative nerve system.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

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.     

The ultrastructure of the innervation of the intestinal wall in the teleosts Myoxocephalus scorpius and Pleuronectes platessa

Summary The innervation of the intestinal wall in the teleosts Myoxocephalus and Pleuronectes was examined electron microscopically. Two classes of axons can be identified. The first, which is in the majority, contains numerous 50–150 nm granular vesicles as well as some 40–50 nm agranular vesicles while the second contains predominantly the 40–50 nm agranular vesicles. Chromate/dichromate staining methods suggest that the first type is aminergic. Both types lie in close association with the perikarya of intrinsic myenteric neurons but only axons containing predominantly agranular vesicles have synaptic membrane specialisations. No axon bundles pass into the longitudinal muscle layer in Myoxocephalus gut and though some do in Pleuronectes, they do not closely approach the smooth muscle cells. Axons containing large granular vesicles lie in intimate contact with the myocytes of the circular muscle layer. Both axon types pass through the submucosa to form a plexus underneath the mucosal epithelium. Varicosities containing agranular or granular vesicles are separated from the epithelial cells by a gap of about 200 nm in which lies a basal lamina.     

The Ultrastructure of the Nervous System of Gyratrix hermaphroditus (Turbellaria, Rhabdocoela)

The peripheral nervous system and the synapses of G. hermaphroditus are studied with the electron microscope. There is a submuscular as well as a subepithelial plexus. The subepithelial plexus is found among the muscles and between the muscles and the basement membrane. It consists of fibres containing large lucent and lysosome-like vesicles and fibres with only small lucent (synaptic) vesicles. In the deeper lying submuscular plexus also dense and dense-cored vesicles occur in the fibres. Cell bodies are not observed in the plexuses. The separate nerve supplies of the pharynx and the gonads contain nerve cells of the neurosecretory type. Fibres of the same kinds as in the brain are also seen here. The synapses in the neuropile are of two kinds. 1. Symmetrical synapses with an additional presynaptic network are most common. 2. Synapses without thickenings of membranes are observed between lateral membranes of neurites. In the peripheral nervous system are two other kinds of synapses also observed. 1. Asymmertical synapses with a denser and wider postsynaptic thickening and 2. neuromuscular junctions. Neurites containing accumulations of small vesicles against the basement membrane are also described. The organization of the peripheral nervous system is described and discussed in relation to the systematic position of G. hermaphroditus.     

Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles

A polypeptide of Mr 38,000 has been identified as a specific component of the membrane of presynaptic vesicles, using the monoclonal antibody SY38. This protein, which is acidic (isoelectric at approximately pH 4.8) and glycosylated, appears to be an integral membrane protein, as suggested by its solubilization with the nonionic detergent Triton X-100 and the finding that the epitope recognized by antibody SY38 is located on the cytoplasmic surface of those vesicles. It is found in presynaptic vesicles of neurons of the brain, spinal cord, and retina as well as at neuromuscular junctions. It is also found in the adrenal medulla. Its occurrence in diverse vertebrate species indicates its stability during evolution. This protein, for which we propose the name synaptophysin*, provides a molecular marker for the presynaptic vesicle membrane and may be involved in synaptic vesicle formation and exocytosis.     

The relationship between nerves and smooth muscle cells in the rat iris

Summary The dilatator muscle cells form short projections into the stroma of the iris. Close to these projections run several nerve bundles. The unmyelinated axons often show enlargements (varicosities) containing mitochondria and vesicles. Several of the varicosities are partly denuded of the Schwann cell and are covered only by a basement membrane. The varicosities are then separated from the muscle cells only by basement membranes and a 0.1–1 stromal space. The ultrastructure of the iris dilatator muscle thus also fits the view that the autonomic ground plexus with its varicosities forms the real innervation apparatus.The smallest space between axon and muscle has a width of 700–900 Å and is cemented with basement membrane material. It is suggested that the main function of these contact sites is not to transmit a nerve impulse but to anchor the nerves to their effector organ.This study has been supported by grants from the Swedish State Research Council (U 267) and the United States Public Health Service (N B 2854-04).     

The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart

Summary An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed.Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma.Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

The fine structure of a multiterminal innervation of an insect muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

The Fine Structure of a Multiterminal Innervation of an Insect Muscle

The detailed structure of nerve branches, neuromuscular junctions, and muscle fibers of a multiterminal innervation of cockroach abdominal muscle has been studied with the electron microscope. The muscle fiber is of the banded myofibril type; with paired mitochondria and abundant endoplasmic reticulum. The peripheral nerve branches are multiaxonal with large central axon and several small peripheral tunicated axons. Tracheoblasts closely accompany the nerve branches. The multiple neuromuscular junctions show typical axonal vesicles, muscle aposynaptic granules, and close plasma membrane apposition with no interposition of basement membrane material.     

The nervous environment of individual smooth muscle cells of the guinea pig vas deferens

Smooth muscle cells of the external longitudinal coat of the guinea pig vas deferens were followed for 480 mu at 4.5-mu intervals. Muscle bundles and fibers interwove, facilitating intermuscular and neuromuscular contacts. The ribbon- or rodlike muscle cells were about 450 mu long, 3,000 mu3 in volume, and 4,500 mu2 in area. The thickened nuclear zone day anywhere along the middle one-third of the cell. Intercellular distances were 500-800 A. Intrusions were rare, and tight-junctions absent. At any level in a field of 80 muscle fibers there were 10-15 nerve bundles, each containing several varicose axons. Bundles and axons divided. Axons, en passage, were frequently within 500-1,000 A of a muscle fiber. En passage close contacts were rate. Axon terminations were bare, and bare axons invariably terminated. Bare terminations had scattered vesicle-laden varicosities and were from 10-60 mu in length, and all ended within 500 A of muscle fibers. Some made close contact with muscle fibers. Less than half of the muscle cells received this close contact, but some cells were approached by more than one termination. Most terminations involved more than one cell. Some cells had little or no innervation. Some groups of cells had a rich innervation. There was very little evidence of sensory innervation. These conclusions are not valid for other smooth muscles.     

THE LOCALIZATION OF ACETYLCHOLINESTERASE AT THE AUTONOMIC NEUROMUSCULAR JUNCTION

Acetylcholinesterase has been localized at the autonomic neuromuscular junction in the bladder of the toad (Bufo marinus) by the Karnovsky method. High levels of enzyme activity have been demonstrated in association with the membranes of cholinergic axons and the adjacent membranes of the accompanying Schwann cells. The synaptic vesicles stained in occasional cholinergic axons. After longer incubation times, the membrane of smooth muscle cells close to cholinergic axons also stained. Axons with only moderate acetylcholinesterase activity or with no activity at all were seen in the same bundles as cholinergic axons, but identification of the transmitter in these axons was not possible.     

Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction

Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se , adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron‐dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher.     

Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7-1 and presynaptic Munc13-1.

Msec7-1, a mammalian homologue of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family. Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles. However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known. In the present study, we demonstrate that msec7-1 interacts directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones. Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, we suggest that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover.