Signal regulatory proteins (SIRPS) are secreted presynaptic organizing molecules

Formation of chemical synapses requires exchange of organizing signals between the synaptic partners. Using synaptic vesicle aggregation in cultured neurons as a marker of presynaptic differentiation, we purified candidate presynaptic organizers from mouse brain. A major bioactive species was the extracellular domain of signal regulatory protein alpha (SIRP-alpha), a transmembrane immunoglobulin superfamily member concentrated at synapses. The extracellular domain of SIRP-alpha is cleaved and shed in a developmentally regulated manner. The presynaptic organizing activity of SIRP-alpha is mediated in part by CD47. SIRP-alpha homologues, SIRP-beta and -gamma also have synaptic vesicle clustering activity. The effects of SIRP-alpha are distinct from those of another presynaptic organizer, FGF22: the two proteins induced vesicle clusters of different sizes, differed in their ability to promote neurite branching, and acted through different receptors and signaling pathways. SIRP family proteins may act together with other organizing molecules to pattern synapses.     

The distribution of and interactions between GABA-immunoreactive and non-immunoreactive processes presynaptic to afferents from campaniform sensilla on the trochanter of the locust leg

Summary Campaniform sensilla on the trochanter of the mesothoracic legs of the locust were backfilled with cobalt salts or horseradish peroxidase for light and electron microscopy. The distribution of the terminal branches of afferent neurones in the thoracic ganglia were described from wholemount preparations and from thick slices through the ganglia. Ultrathin sections of identified branches were processed with GABA antibodies using a post-embedding immunogold technique and examined in the electron microscope. Input synapses were observed on fine varicose branches in all regions of the terminal arborisations close to the sites of afferent output. The major branches neither make nor receive synapses. Seventy-two percent of the input synapses are made by processes immunoreactive for GABA. Immunoreactive and non-immunoreactive processes synapse onto afferent terminals in close proximity. In some instances GABA-immunoreactive processes presynaptic to an afferent are also presynaptic to a non-immunoreactive presynaptic processes strongly suggesting that different presynaptic influences can interact directly with each other.     

Single vesicle tracking for studying synaptic vesicle dynamics in small central synapses

Sustained neurotransmission is driven by a continuous supply of synaptic vesicles to the release sites and modulated by synaptic vesicle dynamics. However, synaptic vesicle dynamics in synapses remain elusive because of technical limitations. Recent advances in fluorescence imaging techniques have enabled the tracking of single synaptic vesicles in small central synapses in living neurons. Single vesicle tracking has uncovered a wealth of new information about synaptic vesicle dynamics both within and outside presynaptic terminals, showing that single vesicle tracking is an effective tool for studying synaptic vesicle dynamics. Particularly, single vesicle tracking with high spatiotemporal resolution has revealed the dependence of synaptic vesicle dynamics on the location, stages of recycling, and neuronal activity. This review summarizes the recent findings from single synaptic vesicle tracking in small central synapses and their implications in synaptic transmission and pathogenic mechanisms of neurodegenerative diseases.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.     

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.     

Assembling the presynaptic active zone: a characterization of an active one precursor vesicle

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.     

SYNAPTIC VESICLE DEPLETION AND RECOVERY IN CAT SYMPATHETIC GANGLIA ELECTRICALLY STIMULATED IN VIVO : Evidence for Transmitter Secretion by Exocytosis

This study examined the ultrastructure of presynaptic terminals after short periods of vigorous acetylcholine (ACh) secretion in the cat superior cervical ganglion in vivo. Experimental trunks of cats anesthetized with chloralose-urethane were stimulated supra-maximally for periods of 15–30 min and at several frequencies including the upper physiological range (5–10 Hz). Stimulated and contralateral control ganglia from each animal were fixed by intra-arterial aldehyde perfusion, processed simultaneously, and compared by electron microscopy. Stimulation produced an absolute decrease in the number of synaptic vesicles, an enlargement of axonal surface membrane, and distinct alterations in the shape of presynaptic terminals. Virtually complete recovery occurred within 1 h after stimulation at 10 Hz for 30 min. These results support the hypothesis that ACh release at mammalian axodendritic synapses occurs by exocytosis of synaptic vesicles resulting in the incorporation of vesicle membrane into the presynaptic membrane and that synaptic vesicles subsequently are reformed from plasma membrane.     

A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation

Formation of glutamatergic synapses entails development of "silent" immature contacts into mature functional synapses. To determine how this transformation occurs, we investigated the development of neurotransmission at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, followed accumulation of synapsin I. During this period, synaptic transmission was primarily mediated by activation of NMDA receptors, suggesting that most synapses were functionally silent. However, local glutamate application to silent synapses indicated that these synapses contained functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverted functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release.     

Synaptic terminal parameters in unanaesthetized rat cerebral cortex

The ultrastructure of synapses from the molecular layer of parietal cortex was examined in two groups of unanesthetized rats. Rats of the first group were killed by stunning across the back of the neck, and those of the second group by the introduction of fixative through a preimplanted carotid artery cannula. Comparison of synapses from the two groups revealed that the distribution of synaptic types was the same. A larger percentage of synapses of the cannulated group has vesicle attachment sites than did those of the stunned group. The area and perimeter of the presynaptic terminals were significantly larger in synapses from the cannulated group, although the equivalent length of the postsynaptic thickening was less. The mean value for synaptic curvature was greater in the cannulated group, although over 80% of synapses in both groups had positive curvatures. No significant differences were found between the groups for the relationships between presynaptic terminal area and synaptic vesicle number, and between postsynaptic thickening length and synaptic curvature. Membrane recycling is suggested as a mechanism of accounting for the differences. The preponderance of postively-curved synapses in unanesthetized material may indicate a preponderance of functioning synapses.     

Short term synaptic depression imposes a frequency dependent filter on synaptic information transfer

Depletion of synaptic neurotransmitter vesicles induces a form of short term depression in synapses throughout the nervous system. This plasticity affects how synapses filter presynaptic spike trains. The filtering properties of short term depression are often studied using a deterministic synapse model that predicts the mean synaptic response to a presynaptic spike train, but ignores variability introduced by the probabilistic nature of vesicle release and stochasticity in synaptic recovery time. We show that this additional variability has important consequences for the synaptic filtering of presynaptic information. In particular, a synapse model with stochastic vesicle dynamics suppresses information encoded at lower frequencies more than information encoded at higher frequencies, while a model that ignores this stochasticity transfers information encoded at any frequency equally well. This distinction between the two models persists even when large numbers of synaptic contacts are considered. Our study provides strong evidence that the stochastic nature neurotransmitter vesicle dynamics must be considered when analyzing the information flow across a synapse.     

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.     

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

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

Scribble Interacts with β-Catenin to Localize Synaptic Vesicles to Synapses

An understanding of how synaptic vesicles are recruited to and maintained at presynaptic compartments is required to discern the molecular mechanisms underlying presynaptic assembly and plasticity. We have previously demonstrated that cadherin–β-catenin complexes cluster synaptic vesicles at presynaptic sites. Here we show that scribble interacts with the cadherin–β-catenin complex to coordinate vesicle localization. Scribble and β-catenin are colocalized at synapses and can be coimmunoprecipitated from neuronal lysates, indicating an interaction between scribble and β-catenin at the synapse. Using an RNA interference approach, we demonstrate that scribble is important for the clustering of synaptic vesicles at synapses. Indeed, in scribble knockdown cells, there is a diffuse distribution of synaptic vesicles along the axon, and a deficit in vesicle recycling. Despite this, synapse number and the distribution of the presynaptic active zone protein, bassoon, remain unchanged. These effects largely phenocopy those observed after ablation of β-catenin. In addition, we show that loss of β-catenin disrupts scribble localization in primary neurons but that the localization of β-catenin is not dependent on scribble. Our data supports a model by which scribble functions downstream of β-catenin to cluster synaptic vesicles at developing synapses.     

Histochemical and cytochemical studies of alkaline phosphatase activity in the synapses of rat brain

Summary Although a number of studies have been carried out on alkaline phosphatase (Al-P), this enzyme has not definitely been detected in synapses at the electron-microscopic level. Recently, we have successfully demonstrated, by perfusing specimens with 1% glutaraldehyde for fixation for as short a time as 8–10 min, that Al-P activity is localized on the presynaptic and postsynaptic membranes of the rat central nervous system (CNS). There were four types of presynaptic membrane: (1) those with the activity only on the membrane, (2) those with the activity only on the synaptic vesicle membrane, (3) those with the activity on both the presynaptic membrane and the synaptic vesicle membrane, and (4) those entirely free of the activity. The postsynaptic membranes were classified into two varieties: (1) those with the activity in the postsynaptic membrane and the postsynaptic thickening, and (2) those entirely without the activity. Thus, the occurrence of the enzyme activity assumed various combinations of presynaptic and postsynaptic involvement. The incidence of synapses either with presynaptic or postsynaptic activity varied distinctly from site to site.     

Bassoon and the synaptic ribbon organize Ca²+ channels and vesicles to add release sites and promote refilling

At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+ channel clustering and synaptic vesicle docking are organized. Here, we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses--mostly lacking the ribbon--showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+ channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+ channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis was reduced, notwithstanding normal coupling of the remaining Ca2+ channels to exocytosis. In vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in vivo data independently supported morphological and functional in vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+ channels and vesicles, and (2) promote vesicle replenishment.     

Histochemical and cytochemical studies of alkaline phosphatase activity in the synapses of rat brain.

Although a number of studies have been carried out on alkaline phosphatase (A1-P), this enzyme has not definitely been detected in synapses at the electron-microscopic level. Recently, we have successfully demonstrated, by perfusing specimens with 1% glutaraldehyde for fixation for as short a time as 8-10 min, that A1-P activity is localized on the presynaptic and postsynaptic membranes of the rat central nervous system (CNS). There were four types of presynaptic membrane: (1) those with the activity only on the membrane, (2) those with the activity only on the synaptic vesicle membrane, (3) those with the activity on both the presynaptic membrane and the synaptic vesicle membrane, and (4) those entirely free of the activity. The postsynaptic membranes were classified into two varieties: (1) those with the activity in the postsynaptic membrane and the postsynaptic thickening, and (2) those entirely without the activity. Thus, the occurrence of the enzyme activity assumed various combinations of presynaptic and postsynaptic involvement. The incidence of synapses either with presynaptic or postsynaptic activity varied distinctly from site to site.     

Changes in the distribution of calcium calmodulin-dependent protein kinase II at the presynaptic bouton after depolarization

Phosphorylation of synapsin I by CaMKII has been reported to mobilize synaptic vesicles from the reserve pool. In the present study, the distributions of α-CaMKII and of synapsin I were compared in synaptic boutons of unstimulated and stimulated hippocampal neurons in culture by immunogold electron microscopy. CaMKII and synapsin I are located in separate domains in presynaptic terminals of unstimulated neurons. Label for α -CaMKII typically surrounds synaptic vesicle clusters and is absent from the inside of the cluster in control synapses. In contrast, intense labeling for synapsin I is found within the vesicle clusters. Following 2 minutes of depolarization in high K⁺, synaptic vesicles decluster and CaMKII label disperses and mingles with vesicles and synapsin I. These results indicate that, under resting conditions, CaMKII has limited access to the synapsin I in synaptic vesicle clusters. The peripheral distribution of CaMKII around vesicle clusters suggests that CaMKII-mediated declustering progresses from the periphery towards the center, with the depth of penetration into the synaptic vesicle cluster depending on the duration of CaMKII activation. Depolarization also promotes a significant increase in CaMKII immunolabel near the presynaptic active zone. Activity-induced redistribution of CaMKII leaves it in a position to facilitate phosphorylation of additional presynaptic proteins regulating neurotransmitter release.     

N-cofilin can compensate for the loss of ADF in excitatory synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.