在被小泡参与突触前后膜特化增厚的形态学研究

Our object was to characterize the morphological changes of coated vesicles and synaptic membranes during synaptogenesis. Neurons from spinal cords of fetal mice were established as isolated cells in primary culture. After a few days in vitro, the neurons extended their neurites and started their interaction. At timed intervals thereafter, cultures were fixed for electron microscopic observation. Coated vesicles were prominent in the neuronal cytoplasm at the time of synaptogenesis (about 7-10 days in vitro). Similar vesicles were seen in continuity with some cisternae in the Golgi regions and there was an increase in number during the synaptogenic period. Indeed it is not established whether the coated vesicles were exocytotic or pinocytotic in nature, but the cisternae which were in continuity with coated vesicles could be labelled by glucose-6-phosphatase (G6Pase) but not by thiamine pyrophosphatase (TPPase). Such vesicles were also seen in continuity with the neuronal plasmalemma near the closest contact site and contributed their undercoating to pre- and postsynaptic densities. The formation of bilateral membrane specialization was described as being structurally similar to synaptic active zones and appeared to be the first definitive sign of synapseformation. It has been suggested that the synaptic dense material may derive wholly or in part from the exocytic coated vesicles which apparently budding off from endoplasmic reticulum cisternae. This incorporation could provide the mechanism for confining specific characteristics of 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.     

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.     

Paralemmin-1, a modulator of filopodia induction is required for spine maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.     

Electron microscope observations on synaptic vesicles in synapses of the retinal rods and cones

The submicroscopic organization of the rod and cone synapses of the albino rabbit has been investigated with the use of the electron microscope. The most common rod synapse consists of an enlarged expansion of the rod fiber (the so called spherule) into which the dendritic postsynaptic fiber of the bipolar cell penetrates and digitates. The membrane surrounding the terminal consists of a double layer, the external of which is interpreted as belonging to the intervening glial cells. The synaptic membrane has a pre- and a postsynaptic layer with a total thickness of 180 to 300 A. The presynaptic layer is frequently denser and is intimately associated with the adjacent synaptic vesicles. The synaptic membrane shows processes constituted by foldings of the presynaptic layer. The entire spherule is filled with synaptic vesicles varying in diameter between 200 and 650 A with a mean of 386 A. In addition, the spherule contains a few large vacuoles near the rod fiber, interpreted as endoplasmic reticulum, and a matrix in which with high resolution a fine filamentous material can be observed. The postsynaptic fiber is homogeneous and usually does not show synaptic vesicles. In animals maintained in complete darkness for 24 hours vesicles appear to accumulate near the synaptic membrane and its processes. After 9 days there is a sharp decrease in size of the synaptic vesicles. A special rod synapse in which the dendritic postsynaptic expansion penetrates directly into the rod cell body has been identified. In line with Cajal's classification this type of synapse could be considered as a somatodendritic one. The cone synapse has a much larger terminal with a more complex relationship with the postsynaptic fiber. However, the same components recognized in the rod synapse can be observed. In animals maintained for 9 days in complete darkness there is also a considerable diminution in size of the synaptic vesicles.     

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.     

Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates

Well-defined ferritin-conjugated lectins were used to map glycoconjugates on the surface of sprouting neurons from rat superior cervical ganglion (SCG) and spinal cord (SC). The cultured neurons were exposed to the markers and processed for electron microscopy, and the number of ferritin particles per unit area of plasmalemma was measured in three different regions: perikaryon, neuritic shaft, and growth cone. Three different binding patterns are observed for different lectin: equal receptor density throughout the plasmalemma of the growing neuron (e.g., Ricinus communis agglutinin I in SCG neurons), gradual decrease (e.g., wheat-germ agglutinin in SCG and SC neurons) and gradual increase (e.g., Ricinus communis agglutinin II in SC neurons) in the density of lectin receptors as one moves from the perikaryon to the growth cone. Furthermore, lectin receptor densities differ in the two types of neurons analyzed. We can conclude that the plasmalemma of the growth cone has biochemical properties different from those of the perikaryon, and that the neuron's structural polarity is expressed in its surface glycoconjugates. This phenomenon may be related to the growth cone's special functional properties and to the process of expansion of the plasma membrane.     

Synaptic transmission is impaired at neuronal autonomic synapses in agrin-null mice

Neuronal synapse formation is a multistep process regulated by several pre- and postsynaptic adhesion and signaling proteins. Recently, we found that agrin acts as one such synaptogenic factor at neuronal synapses in the PNS by demonstrating that structural synapse formation is impaired in the superior cervical ganglia (SCG) of z+ agrin-deficient mice and in SCG cultures derived from those animals. Here, we tested whether synaptic function is defective in agrin-null (AGD-/-) ganglia and began to define agrin's mechanism of action. Our electrophysiological recordings of compound action potentials showed that presynaptic stimulation evoked action potentials in approximately 40% of AGD-/- ganglionic neurons compared to 90% of wild-type neurons; moreover, transmission could not be potentiated as in wild-type or z+ agrin-deficient ganglia. Intracellular recordings also showed that nerve-evoked excitatory postsynaptic potentials in AGD-/- neurons were only 1/3 the size of those in wild-type neurons and mostly subthreshold. Consistent with these defects in transmission, we found an approximately 40-50% decrease in synapse number in AGD-/- ganglia and cultures, and decreased levels of differentiation at the residual synapses in culture. Furthermore, surface levels of acetylcholine receptors (AChRs) were equivalent in cultured AGD-/- and wild-type neurons, and depolarization reduced the synaptic localization of AChRs in AGD-/- but not wild-type neurons. These findings provide the first direct demonstration that agrin is required for proper structural and functional development of an interneuronal synapse in vivo. Moreover, they suggest a novel role for agrin, in stabilizing the postsynaptic density of nAChR at nascent neuronal synapses.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Phagocytosis and endocytosis in the colorless flagellate Thaumatomonas lauterborni

Phago- and endocytosis have been studied in the colourless flagellate T. lauterborni using electron microscope. The coated pits are formed on the dorsal surface of the cells and in the flagellar pocket; then they are transformed into coated vesicles and transported into the ventral part of the cell loosing their clathrin coat. The storing of small vesicles in the ventral groove region is constant. To begin to feed a flagellate stops and produces within several seconds long ramified filopodia from the ventral groove. These filopodia serve to phagocyte bacteria. Small ventral vesicles represent the membrane pull which is necessary for a quick formation of the vast surface of filopodia. By means of peroxidase reaction in was shown that these vesicles were of endocytotic origin, rather than being the product of the Golgi apparatus functioning.     

Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines

Endocytosis is a fundamental mechanism by which neurons control intercellular signaling, nutrient uptake, and synaptic transmission. This process is carried out by the assembly of clathrin coats and the budding of clathrin-coated vesicles from the neuronal plasma membrane. Here, we demonstrate that in young neurons, clathrin assembly and disassembly occur rapidly, locally, and repeatedly at "hot spots" throughout dendrites and at the tips of dendritic filopodia. In contrast, clathrin coats in mature dendrites reside in stable, long-lasting zones at sites of endocytosis, where clathrin undergoes continuous exchange with local cytosolic pools. In dendritic spines, endocytic zones lie lateral to the postsynaptic density (PSD) where they develop and persist independent of synaptic activity, akin to the PSD itself. These results reveal the presence of a novel specialization dedicated to endocytosis near the postsynaptic membrane.     

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.     

Agrin plays an organizing role in the formation of sympathetic synapses

Agrin is a nerve-derived factor that directs neuromuscular synapse formation, however its role in regulating interneuronal synaptogenesis is less clear. Here, we examine agrin's role in synapse formation between cholinergic preganglionic axons and sympathetic neurons in the superior cervical ganglion (SCG) using agrin-deficient mice. In dissociated cultures of SCG neurons, we found a significant decrease in the number of synapses with aggregates of presynaptic synaptophysin and postsynaptic neuronal acetylcholine receptor among agrin-deficient neurons as compared to wild-type neurons. Moreover, the levels of pre- and postsynaptic markers at the residual synapses in agrin-deficient SCG cultures were also reduced, and these defects were rescued by adding recombinant neural agrin to the cultures. Similarly, we observed a decreased matching of pre- and postsynaptic markers in SCG of agrin-deficient embryos, reflecting a decrease in the number of differentiated synapses in vivo. Finally, in electrophysiological experiments, we found that paired-pulse depression was more pronounced and posttetanic potentiation was significantly greater in agrin-deficient ganglia, indicating that synaptic transmission is also defective. Together, these findings indicate that neural agrin plays an organizing role in the formation and/or differentiation of interneuronal, cholinergic synapses.     

Synaptic junction development in the spinal cord of an amphibian embryo: An electron microscope study

Summary An electron microscopical study has been made of the cervical spinal cord of Xenopus laevis embryos, from the time that the neural tube closes until the larvae were hatched and could swim. Sections of the whole cord were searched for intercellular junctions during this period. Two nonsynaptic types were found, the first were widely distributed puncta adherentia, the second were rare and similar to gap junctions. Membrane specializations with synaptic vesicles were first found when the neural folds had fused; membrane-vesicle clusters which looked like the presynaptic half of a synaptic junction were present, together with synaptic junctions lacking any postsynaptic membrane thickening or cytoplasm density. About four hours later, mature synaptic junctions with full thickening of the postsynaptic membrane, dense cytoplasm and striated or dense material in the synaptic cleft were present. Presynaptic mitochondria, dense-cored and flattened vesicles, fibre to fibre and fibre to cell body synapses were present from the first, as were synapses onto very fine dendrites which might be filopodia from dendritic growth cones. Synaptogenesis may start with the accumulation of vesicles in dense cytoplasm near a thickened cell membrane; the postsynaptic element becomes associated with this membrane-vesicle cluster and matures by increasing cleft and cytoplasmic density, and by membrane thickening.     

Localization of calmodulin in rat cerebellum by immunoelectron microscopy

Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.     

Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization.

We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity.     

Lesion-Induced synaptogenesis in brain: A study of dynamic changes in neuronal membrane specializations

When incoming fibers to a given brain region are damaged and degenerate, the remaining undamaged fibers can, in some cases, form new synapses, and restore physiologically functional circuitry. Synaptic membrane events underlie this reconstruction: the connection between membranes is broken and reformed. In order to understand these membrane events, it is necessary to know the molecular composition of the synapse and the nature of the interaction between pre- and postsynaptic membranes. The synaptic membranes are probably joined by proteins extending from their surfaces. The postsynaptic membrane has on its outer surface an array of lectin receptors, probably glycoproteins. On its inner surface, juxtaposed to the bilayer, the membrane has an electron-dense structure called the postsynaptic density which, from studies on the isolated structure, is composed of a few polypeptides. On the basis of the molecular composition and structure of CNS synapses and ultrastructural studies of the lesion-induced synaptogenesis, some of the underlying dynamic events at synaptic membranes are inferred. New synapses are formed either by reutilization of the old contact sites or by generation of new ones. The protein and carbohydrates in the cleft are enzymatically degraded and a new synapse is generated in response to ingrowing fibers by the addition or reutilization of the specialized proteins of postsynaptic membrane, which differentiate a small segment of the postsynaptic membrane.     

Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits

Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.     

Morphological changes induced by sodium bromide in murine neuroblastoma cells in vitro

Summary Sodium bromide was applied in vitro to mouse neuroblastoma cells of different ages for short and long periods (2h to 10 days). The changes observed light-and-electron microscopically were similar to those described earlier after GABA treatment. Coated vesicles proliferated and originated by pinching off from the Golgi complex and from the rough endoplasmic reticulum. Numerous coated vesicles were continuous with the plasma membrane, especially near zones in which electron-dense material aggregated at the inner aspect of the plasmalemma. Small invaginations, similar in ultrastructure to coated vesicles, were also formed. It is unclear whether the coated vesicles or the dense plasmalemma invaginations contribute to the undercoating by fusing with the adjacent electron-dense plasma membrane. There was a distinct increase in the number and area of specialized contacts (intermediate junctions and zonulae adhaerentes) between cells and their processes. A floccular or filamentous electron-dense substance varying in amount and appearance was occasionally seen between the contacting membranes. Varicosities of terminal swellings of cell processes contained vesicles of variable size, shape and density, and also profiles of the smooth endoplasmic reticulum. Under the influence of sodium bromide, similar to the effect of GABA, mitochondria appeared within the varicosities, and primitive contacts (intermediate junctions) were formed between the terminal swellings and potential postsynaptic elements, which were absent in controls.Additionally, dense-core vesicles proliferated and aggregated at the cell periphery. They were often arranged linearly below the plasma membranes of perikarya and processes, and surrounded by a highly electron-dense substance. The similarity of the present findings to those obtained after GABA treatment and their relation to synaptogenesis are discussed.