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.     

The postnatal development of the synapse: A morphological approach utilizing synaptosomes

Summary Synaptosomes derived from 2–21 days postnatal rat cerebral cortex have been examined following glutaraldehyde fixation and block PTA staining, with the aim of investigating the maturation of the paramembranous densities at the contact region between the pre- and postsynaptic components. The internal coats of pre- and postsynaptic membranes first appear as undifferentiated plaque-like thickenings, which gradually develop into, or are replaced by, dense projections and postsynaptic focal densities respectively. Both sets of densities pass through an interconnected phase before starting to emerge as discrete entities at 5–7 days. The external coats of the pre- and postsynaptic membranes coalesce to form a plate-like structure which breaks down during development to form the cleft densities or transverse bars of the adult contact region. Although for the first few days of postnatal development only one type of synaptosome can be identified, from 5 days onwards two types corresponding to types A and B of adult life become recognizable.Increase in height of the dense projections has been correlated with increase in the number of synaptic vesicles per synaptosome during postnatal development, indicating that the synaptic vesicles may play a role in the formation and maturation of dense projections. The possible importance of other factors in this process is also discussed.We would like to thank Professors J. Z. Young, F. R. S., and E. G. Gray for their advice, and Mr. S. Waterman for expert photographic assistance.     

Further studies on synaptic junctions

Summary Synaptic junctions in intact rat cerebral cortex have been examined following glutaraldehyde fixation and phosphotungstic acid (PTA) staining. In the presynaptic ending the network has a hexagonal arrangement, while the dense projections are regularly placed along the presynaptic membrane. Cleft densities occupy the intracleft region. The postsynaptic thickening extends uninterrupted along the length of the junction. Qualitatively, the majority of junctions fall into the discontinuous-continuous category, in which the internal coat of the presynaptic membrane together with its associated dense projections is discontinuous along the length of the junction, whereas the postsynaptic thickening is continuous. By contrast, a small number of junctions are continuous-continuous.In an attempt to analyze the junctions quantitatively, nine indices were measured. Histograms of the size distributions of seven of these appear to be bimodal, and from this it is concluded that two junction populations may be distinguishable on quantitative grounds. It is also shown that the distance separating dense projections at the presynaptic membrane is of the order of 10–15 nm. This surprisingly low value has consequences for current ideas on the relationship between synaptic vesicles and dense projections, and these are discussed at length.We would like to acknowledge the technical assistance of Mrs. C. Blackshaw, Mrs. G. Kay and Mr. D. Stuart.     

Axonal protrusions in the small multiple endings in the extraocular muscles of the rat

Summary A special type of myoneural junction has been observed in the extraocular muscles of the rat with electron microscopy. These axon terminals are derived from unmyelinated nerves and contain synaptic vesicles and mitochondria. The terminals are invested by teloglia cells and separated by a synaptic cleft of about 500 Å from a slow-type muscle fibre. From the nerve ending a pseudopod-like evagination projects into the muscle cell. The membranes of this evagination and the muscle cells are only separated by a narrow cleft of about 100 Å, which is devoid of the basement membrane-like material typical of ordinary myoneural junctions. The evagination contains fewer axonal vesicles than other regions of the terminal axoplasm and the postsynaptic part of the muscle plasma membrane in this special region does not exhibit the postsynaptic thickening characteristic of ordinary myoneural junctions.The author thanks ProfessorAntti Telkkä, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.     

An ultrastructural and electrophysiological study of the development of neuro-muscular junctions between previously dissociated foetal rat cells in vitro

Summary The development of neuro-muscular junctions between previously dissociated foetal rat spinal cord and somatic muscle has been investigated. The first indications of junction formation, both ultrastructurally and electrophysiologically, were observed after circa 18 days in vitro. The junctions contained numerous vesicles, but no secondary folds were developed even after 6 weeks in culture, and synaptic densities were not well marked. Functional endplates were found, and action potentials, endplate potentials and miniature endplate potentials recorded.The authors wish to thank Mr. D. Fraser, B. Sc., for valued technical help, and Mr. S. Waterman for photographic printing.     

Microtubule-synaptic vesicle associations in cultured rat spinal cord neurons

Summary This paper describes new ultrastructural features of neural processes and of synapses in cultured CNS tissue treated with albumin before fixation using a modification of the technique recently introduced by Gray (1975). Nerve fibre bundles in explants of foetal spinal cord grown in vitro for 15–18 days were transected microsurgically. After transection the cultures were exposed to 20% albumin in distilled water and then fixed in unbuffered osmium tetroxide followed by unbuffered glutaraldehyde.In this material, but not in controls (injured but not exposed to albumin; exposed to albumin without injury) microtubules were found within many axonal varicosities, often situated close to presynaptic membrane specializations. These microtubules were closely associated with vesicles resembling synaptic vesicles, which were occasionally aligned in rows along the microtubules. Similar vesicle-microtubule associations were also found in non-terminal axons. Microtubules were also observed very close to some postsynaptic densities.The possibility that the microtubule-vesicle associations are involved in vesicle movements (along axons and/or within axon terminals) is discussed. A more direct involvement of microtubules in terminals in the mechanism of transmitter release is also considered.The author wishes to thank Dr. A.R. Lieberman for his help and advice, Mr. Derek Fraser and Mr. Peter Felton for their technical assistance, Mr. Stuart Waterman for the photographic prints, and Professor D.W. James for laboratory facilities     

Electrotonic synapses in the mammalian spinal cord

By means of light and electron microscopy methods structural peculiarities of motor nuclei have been studied in the rat spinal cord (17 animals) on the 1st-3d and on the 10th-18th days of postnatal ontogenesis. Synaptic junctions of the gap type are revealed; they are considered as electrotonic synapses. Dendro-somatic and dendrodendritic synaptic junctions of the gap type are found. Together with the electrotonic synapses, morphologically mixed synapses of axo-somatic and axo-axonal types are disclosed; they contain, besides organells, specific for chemical synapses, close opposition areas of pre- and postsynaptic membranes of the gap junction type. Morphologically mixed synapses occur in neuropil of the motor nuclei of the spinal cord in young rats of all age groups studied. Homologous synapses are detected in the motor nuclei of the white mouse spinal cord. Synaptic junctions of the gap type in the mammalian spinal cord could be a substrate of electrical interaction between its motor neurons.     

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.     

An analysis of some aspects of synaptosomal ultrastructure by the use of serial sections

Summary Synaptosomes and synaptic junctions have been examined employing serial sections, with emphasis placed on four areas of investigation. 1. Starting from unequivocal synaptosomal profiles and tracing them through consecutive sections to the periphery of the synaptosomes, it is clear that vesicles are the one constant feature of the presynaptic terminal. In no instance was it possible to identify an empty membranous profile as synaptosomal. 2. Following a similar procedure it was found that the criteria required to predict the existence of a junctional region within a synaptosomal profile are: the accumulation of synaptic vesicles at one locus within its presynaptic component, and the presence of a postsynaptic profile characterized by two or more junctional features. 3. Serial sections of non-osmicated, PTA stained synaptic junctions confirm the regularity and orderliness of dense projection distribution along the length of the junction. 4. Complex vesicles can usually be followed in two and sometimes three adjacent sections, appearing either as intact vesicles or empty shells. Further observations confirmed that the latter profiles may be sections through the periphery of intact vesicles or through isolated shell fragments. They are more common in the latter form in unbuffered material.This work was supported in part by the Australian Research Grants Committee. We would like to thank Mr. David Stuart and Mrs. Zel Gobby for assistance with the photography.     

小鼠脊髓内存在抑制性含锌神经元

目的探讨小鼠脊髓中是否含有抑制性的含锌神经元。方法应用锌金属自显影技术、免疫电镜技术和共聚焦激光扫描显微术,研究游离锌离子、锌转运蛋白(zinc transporter 3,ZnT3)与(glutamic acid decarboxylate,GAD)在小鼠脊髓内的共存情况。结果小鼠脊髓内至少有三种含锌神经元轴突终末,其中大多数为GAD阳性即γ-氨基丁酸能含锌神经元轴突终末,另外两种分别为GAD阴性含扁圆形小泡的甘氨酸能含锌神经元轴突终末和含圆形清亮小泡的兴奋性谷氨酸能含锌神经元轴突终末。结论在哺乳动物脊髓内存在大量的抑制性含锌神经元。锌离子从抑制性含锌神经元轴突终末释放到突触间隙内,作为神经调质作用于突触后的GABA受体或甘氨酸受体,参与脊髓运动和感觉功能的调控。     

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.     

Spillover of glutamate onto synaptic AMPA receptors enhances fast transmission at a cerebellar synapse

Diffusion of glutamate from the synaptic cleft can activate high-affinity receptors, but is not thought to contribute to fast AMPA receptor-mediated transmission. Here, we show that single AMPA receptor EPSCs at the cerebellar mossy fiber-granule cell connection are mediated by both direct release of glutamate and rapid diffusion of glutamate from neighboring synapses. Immunogold localization revealed that AMPA receptors are located exclusively in postsynaptic densities, indicating that spillover of glutamate occurs between synaptic contacts. Spillover currents contributed half the synaptic charge and exhibited little trial-to-trial variability. We propose that spillover of glutamate improves transmission efficacy by both increasing the amplitude and duration of the EPSP and reducing fluctuations arising from the probabilistic nature of transmitter release.     

Fast removal of synaptic glutamate by postsynaptic transporters

Glutamate transporters are believed to remove glutamate from the synaptic cleft only slowly because they cycle slowly. However, we show that when glutamate binds to postsynaptic transporters at the cerebellar climbing fiber synapse, it evokes a conformation change and inward current that reflect glutamate removal from the synaptic cleft within a few milliseconds, a time scale much faster than the overall cycle time. Contrary to present models, glutamate removal does not require binding of an extracellular proton, and the time course of transporter anion conductance activation differs from that of glutamate removal. The charge movement associated with glutamate removal is consistent with the majority of synaptically released glutamate being removed from the synaptic cleft by postsynaptic transporters.     

Characterization of the cyclic 3',5'-nucleotide phosphodiesterase actitivty associated with synaptosomal plasma membranes and synaptic junctions.

Some characteristics of the cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a Km of 2.10 19(-4) M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca2+ while no Ca2+ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.     

Molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with rat brain synaptic functions.

The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.     

GABAA receptor kinetics in the cerebellar nuclei: evidence for detection of transmitter from distant release sites

Neurons of the cerebellar nuclei receive GABAergic input from Purkinje cells. Purkinje boutons have several closely spaced presynaptic densities without GABA transporters, raising the possibility that neurotransmitter released by one presynaptic site diffuses to multiple postsynaptic sites. To test whether such local spillover may contribute to transmission, we studied gating of GABA(A) receptors at 31-33 degrees C in cerebellar nuclear neurons acutely dissociated from mice. Currents were evoked by rapid application of long steps, brief pulses, and high-frequency trains of GABA to outside-out patches. Receptors desensitized and deactivated rapidly, and dose-response measurements estimated an EC(50) of approximately 30 microM. From these data, a kinetic scheme was developed that replicated the recorded currents. Next, we simulated diffusion of GABA in the synaptic cleft, constrained by previous electron microscopic data, and drove the kinetic GABA(A) receptor model with modeled concentration transients. Simulations predicted receptor occupancies of approximately 100% directly opposite the release site and approximately 50% at distant postsynaptic densities, such that receptors up to 700 nm from a release site opened on the timescale of the inhibitory postsynaptic currents before desensitizing. Further simulations of probabilistic release from multiple-site boutons suggested that local spillover-mediated transmission slows the onset and limits the extent of depression during high-frequency signaling.     

Characterization of the cyclic 3′,5′-nucleotide phosphodiesterase activity associated with synaptosomal plasma membranes and synaptic junctions

Some characteristics of the cyclic 3′,5′-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a K_m of 2 · 10^?4 M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca²⁺ while no Ca²⁺ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.     

Synaptic morphology and cytoplasmic densities: Rapid post-mortem effects

In the study of nervous tissue, the shift from the living state to the post-mortem condition affects the ultrastructure of the neuron in ways incompletely understood. We have observed rapid post-mortem changes associated with the synaptic region in the molecular layer of the rat dentate gyrus. These changes are evident in tissue taken from anesthetized animals in which perfusion was begun while the heart was beating but breathing had stopped. Such alterations were rarely in evidence if perfusion was started while the animal was breathing, either with or without artificial means. The following morphological alterations were observed: (a) an increase in spherical electron densities seen near the synapse as well as in the perinuclear cytoplasm; (b) a variation in the amount of density attached to the thickening of post-synaptic membrane; (c) a change in the curvature of the synaptic cleft. Because of these rapid alterations, caution is recommended in the interpretation of the in vivo morphology of the synapse and associated densities.     

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.     

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.