Studying synaptic efficiency by post-hoc immunolabelling (original) (raw)
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Quantification of synapse formation and maintenance in vivo in the absence of synaptic release
Neuroscience, 2004
Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.
Journal of Cell Science, 2010
Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca v 2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca 2+ oscillations sensitive to the inhibitor -conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.
2021
The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs (presynaptic boutons) from single pyramidal cells in culture with 4 millisecond temporal resolution, and localise vesicular release sites with ~ 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among presynaptic boutons supplied by the same axon, and that asynchronous release fraction is elevated in parallel with short-term facilitation at synapses with low release probability. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the presynaptic release area than synchronous sites. These findings are consistent with a model in which functional presynaptic properties are regulated via a synapsespecific adjustment of the coupling distance between presynaptic Ca 2+ channels and releaseready synaptic vesicles. Together our results reveal a universal relationship between the two major functional properties of synapses-the timing and the overall probability of neurotransmitter release. Main text Synaptic transmission provides the basis for neuronal communication. When an actionpotential propagates through the axonal arbour, it activates voltage-gated Ca 2+ channels (VGCCs) located in the vicinity of release-ready synaptic vesicles docked at the presynaptic active zone 1. Ca 2+ ions enter the presynaptic terminal and activate the vesicular Ca 2+ sensor Synaptotagmin 1 (Syt1, or its isoforms Syt2 and Syt9), thus triggering exocytosis of synaptic vesicles filled with neurotransmitter molecules. Neurotransmitter diffuses across the synaptic cleft, binds postsynaptic receptors and evokes further electrical or chemical signalling in the postsynaptic target cell. This whole process occurs on a time scale of a few milliseconds. Recent data demonstrate that such speed and precision are in large part achieved via the formation of nanocomplexes that include presynaptic VGCCs, vesicles belonging to a readily releasable pool (RRP) and postsynaptic neurotransmitter receptors 2-4. In addition to fast, synchronous release, which keeps pace with action potentials, many synapses also exhibit delayed asynchronous release that persists for tens to hundreds of milliseconds 1, 5. Asynchronous release is potentiated during repetitive presynaptic firing and is triggered via activation of multiple sensors with both low (e.g. Syt1) and high (e.g. Syt7) Ca 2+ affinity 6. Accumulating evidence demonstrates that the balance between synchronous and asynchronous release plays an important role in coordinating activity within neuronal networks, for example, by increasing the probability of postsynaptic cell firing and/or modulating action potential precision 7-10. It is well established that asynchronous release levels vary among different types of neurons 1, 10, 11. Interestingly, recent data show that the ratio between asynchronous and synchronous release can also be differentially regulated among presynaptic boutons supplied by the same axon and depends on the identity of the postsynaptic cell, which contributes to target cell-specific communication in the brain 7, 8. The mechanisms that control the relative contributions of synchronous and asynchronous release at the level of single synapses are however poorly understood. Variability in asynchronous release among different neuronal types has been attributed to differences in
Potentiation of Evoked Vesicle Turnover at Individually Resolved Synaptic Boutons
Neuron, 1996
summed behavior of synaptic populations, which are usually large and of uncertain homogeneity. Many fun-and Stephen J Smith Department of Molecular and Cellular Physiology damental questions thus remain unanswered. Is a given form of plasticity expressed in a graded or an all-or-none Beckman Center Stanford Medical School fashion at the individual synapse? How does plasticity vary from synapse to synapse within a given population? Stanford, California 94305 What factors might govern or predict the probability or extent of plasticity at a given synapse? A single-bouton analysis of plasticity, using immunocytochemical Summary means, was reported recently by Malgaroli et al. (1995), but this study addressed only spontaneous release We have studied synaptic plasticity in hippocampal mechanisms. The applicability of this approach and its cell cultures using a new imaging approach that allows results to the plasticity of evoked release was not estabunambiguous discrimination of presynaptic function lished. at the level of single synaptic boutons. Employing a To study the plasticity of evoked vesicle turnover at protocol designed to test for use-dependent plasticsingle synapses, with no possibility of mistaking presynity resembling N-methyl-D-aspartate receptor-depenaptic and postsynaptic components, we have used a dent long-term potentiation (NMDA-type LTP), we find new functional imaging approach. An optical technique, that brief tetanic stimuli induce a potentiation of originally developed by Betz and colleagues (Betz and evoked synaptic vesicle turnover that lasts for at least Bewick, 1992, 1993; Betz et al., 1992) assays presynap-1 hr. Induction of this clearly presynaptic potentiation tic function by quantitative fluorescence imaging of a is blocked by putative postsynaptic glutamate recepdye (e.g., FM 1Ϫ43) that can be trapped in recycling tor antagonists, suggesting that a retrograde inducsynaptic vesicles (Henkel et al., 1996). The elegant studtion signal might be involved. Potentiation appears to ies of the Betz group demonstrated that this optical occur approximately equally at boutons of low and technique allows accurate measurement of activityhigh initial release probabilities, and evidently does dependent vesicle release at motor nerve terminals. not involve an increase in the size of the total recycling Subsequent work established that FM 1Ϫ43 can be used synaptic vesicle pool. in a similar fashion at synapses in hippocampal cell cultures (Ryan et al., 1993; Reuter, 1995; Ryan and Smith, 1995). The fact that FM 1Ϫ43 measurements can
2010
Release probability (p r ) is a fundamental presynaptic parameter which is critical in defining synaptic strength. Knowledge of how synapses set and regulate their p r is a fundamental step in understanding synaptic transmission and communication between neurons. Despite its importance, p r is difficult to measure directly at single synapses. One important strategy to achieve this has relied on the application of fluorescence-based imaging methods, but this is always limited by the lack of detailed information on the morphological and structural properties of the individual synapses under study, and thus precludes an investigation of the relationship between p r and synaptic anatomy. Here we outline a powerful methodology based on using FM-styryl dyes, photoconversion and correlative ultrastructural analysis in dissociated hippocampal cultured neurons, which provides both a direct readout of p r as well as nanoscale detail on synaptic organization and structure. We illustrate the value of this approach by investigating, at the level of individual reconstructed terminals, the relationship between release probability and defined vesicle pools. We show that in our population of synapses, p r is highly variable, and while it is positively correlated with the number of vesicles docked at the active zone it shows no relationship with the total number of synaptic vesicles. The lack of a direct correlation between total synaptic size and performance in these terminals suggests that factors other than the absolute magnitude of the synapse are the most important determinants of synaptic efficacy.
The kinetics of synaptic vesicle recycling measured at single presynaptic boutons
Neuron, 1993
We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a tt/z = 60 s (-24OC); once internalized, vesicles become reavailable for exocytosis in-30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Caz+.
eLife, 2013
The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca 2+ entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C 2 A domain that mediates protein homo-or hetero-meric interactions. Here, we show that the C 2 A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca 2+ entry sites, mediated by the C 2 A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C 2 A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C 2 A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone.
Vesicular release probability sets the strength of individual Schaffer collateral synapses
2020
Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. Analyzing the variability of quantal parameters, we conclude that the vesicular release probability rather t...
Differentially poised vesicles underlie fast and slow components of release at single synapses
Journal of General Physiology, 2020
In several types of central mammalian synapses, sustained presynaptic stimulation leads to a sequence of two components of synaptic vesicle release, reflecting the consecutive contributions of a fast-releasing pool (FRP) and of a slow-releasing pool (SRP). Previous work has shown that following common depletion by a strong stimulation, FRP and SRP recover with different kinetics. However, it has remained unclear whether any manipulation could lead to a selective enhancement of either FRP or SRP. To address this question, we have performed local presynaptic calcium uncaging in single presynaptic varicosities of cerebellar interneurons. These varicosities typically form “simple synapses” onto postsynaptic interneurons, involving several (one to six) docking/release sites within a single active zone. We find that strong uncaging laser pulses elicit two phases of release with time constants of ∼1 ms (FRP release) and ∼20 ms (SRP release). When uncaging was preceded by action potential–e...