A Synaptotagmin Isoform Switch during the Development of an Identified CNS Synapse

Various Synaptotagmin (Syt) isoform genes are found in mammals, but it is unknown whether Syts can function redundantly in a given nerve terminal, or whether isoforms can be switched during the development of a nerve terminal. Here, we investigated the possibility of a developmental Syt isoform switch using the calyx of Held as a model synapse. At mature calyx synapses, fast Ca2+-driven transmitter release depended entirely on Syt2, but the release phenotype of Syt2 knockout (KO) mice was weaker at immature calyces, and absent at pre-calyceal synapses early postnatally. Instead, conditional genetic inactivation shows that Syt1 mediates fast release at pre-calyceal synapses, as well as a fast release component resistant to Syt2 deletion in immature calyces. This demonstrates a developmental Syt1-Syt2 isoform switch at an identified synapse, a mechanism that could fine-tune the speed, reliability, and plasticity of transmitter release at fast releasing CNS synapses

Synaptotagmin2 (Syt2) Drives Fast Release Redundantly with Syt1 at the Output Synapses of Parvalbumin-Expressing Inhibitory Neurons

Parvalbumin-expressing inhibitory neurons in the mammalian CNS are specialized for fast transmitter release at their output synapses. However, the Ca2+ sensor(s) used by identified inhibitory synapses, including the output synapses of parvalbumin-expressing inhibitory neurons, have only recently started to be addressed. Here, we investigated the roles of Syt1 and Syt2 at two types of fast-releasing inhibitory connections in the mammalian CNS: the medial nucleus of the trapezoid body to lateral superior olive glycinergic synapse, and the basket/stellate cell-Purkinje GABAergic synapse in the cerebellum. We used conditional and conventional knock-out (KO) mouse lines, with viral expression of Cre-recombinase and a light-activated ion channel for optical stimulation of the transduced fibers, to produce Syt1-Syt2 double KO synapses in vivo. Surprisingly, we found that KO of Syt2 alone had only minor effects on evoked transmitter release, despite the clear presence of the protein in inhibitory nerve terminals revealed by immunohistochemistry. We show that Syt1 is weakly coexpressed at these inhibitory synapses and must be genetically inactivated together with Syt2 to achieve a significant reduction and desynchronization of fast release. Thus, our work identifies the functionally relevant Ca2+ sensor(s) at fast-releasing inhibitory synapses and shows that two major Syt isoforms can cooperate to mediate release at a given synaptic connection

An Alien Divalent Ion Reveals a Major Role for Ca2+ Buffering in Controlling Slow Transmitter Release

Ca2+-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca2+ buffers and Ca2+ sensors in shaping release kinetics are still controversial. Replacing extracellular Ca2+ by Sr2+ causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr2+ uncaging and made quantitative Sr2+ - and Ca2+ -imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca2+ sensors and Ca2+ buffers in the control of fast and slow release. We show that Sr2+ activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr2+ also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr2+ was less efficient than Ca2+ in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr2+ than for Ca2+ (similar to 5-fold). This, together with an enhanced Sr2+ permeation through presynaptic Ca2+ channels (similar to 2-fold), admits a drastically higher spatially averaged Sr2+ transient compared with Ca2+. Together, despite the lower affinity of Sr2+ at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr2+ transients explain the enhanced late release. This also allows us to conclude that Ca2+ buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca2+

Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP

An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses

Untersuchung zur Vesikel-Andockmodi in neurosecretorischen Zellen mit Totalreflektionsmikroskopie

Nach dem Andocken an die Plasmamembran bereiten sich sekretorische Bläschen (Vesikel) in mehreren Schritten auf ihre Ca2+-abhängige Fusion vor. Die molekularen Vorgänge, die sich zwischen dem ersten morphologischen Kontakt mit der Membran und dem Erreichen des fusionsfähigen ( primed ) Zustands abspielen, sind bisher noch ungeklärt. In dieser Arbeit wurde die Totalreflektionsmikroskopie (TIRFM) verwendet, um in Echtzeit einzelne fluoreszenzmarkierte Large Dense Core -Vesikel (LDCV) an der Plasmamembran intakter Nebennieren-Chromaffinzellen zu beobachten. Die TIRFM-Bildgebung wurde mit dem Verfolgen individueller Partikel und mit einer Korrelations- und Aufenthaltszeitanalyse kombiniert, als auch durch stochastisches Modellieren ergänzt, um die molekularen Vorgänge während des Vesikel-Andockens besser zu charakterisieren. Als Modellsystem wurden Zellen von munc18-1 null-mutanten Mäusen verwendet, da dieses t-SNARE Syntaxin-1a-bindende Protein essentiell für das Andocken und die Fusion von Vesikeln ist.Die durch TIRFM-Messungen bestimmete Dichte NPY-Venus-markierter LDCV in den Zellabdrücken spiegelte Veränderungen beim morphologischen Andocken und bestätigte EM-ultrastrukturelle Unterschiede zwischen munc18-1 Null (M18 KO), Wildtyp (WT) und Null+Munc18-1 (Rescue-) Zellen. Eine Analyse der axialen Zitterbewegung gedockter LDCV durch eine Geschwindigkeits-Autokorrelatonsfunktion zeigte eine eindeutige negative Autokorrelationskomponente für kurze t ~0.5-1 s auf, die in M18 KO-Zellen 4-5 mal kleiner war als in WT-Zellen. Die negative Autokorrelations-Amplitude (NPA) konnte bei Rescue-Zellen vollständig und bei Zellen, die mutiertes Munc18-1D34N;M38V mit einer niedrigen Affinität zu Syntaxin-1a oder Munc18-2 überexprimieren, teilweise wiederhergestellt werden. Bei M18 KO-Zellen konnte der Phorbolester PMA das Andocken und die Sekretion wiederherstellen, die NPA hingegen blieb klein. Computer-Simulationen der LDCV Bewegung ergaben, daß die NPA durch die auf das freie Diffusionsmodell angewendete Einschränkungen bestimmt wird. Eine kleine NPA kann entweder auf eine stark eingeschränkte Bewegung durch Käfigbildung oder Bindungskräfte, oder auf eine nahezu freie Diffusion mit nur schwachen Einschränkungen zurückgeführt werden. Da die Bindungskräfte von der Aktin-Zytomatrix bereitgestellt werden könnten, die in M18 KO-Zellen verdickt ist, wurde die Auswirkung einer pharmakologischen Aktin-Depolymerisation auf die NPA untersucht. Die Entfernung des Aktin-Kortex führte zu einer Wiederherstellung des morphologischen Andockens in M18 KO-Zellen bei unveränderter NPA oder Sekretion und deutet so auf ein schwaches und nicht-funktionelles Binden/Andocken der Vesikel in M18 KO-Zellen hin. Um die Aufgabe von SNAREs aufzuklären, wurden SNAP-25A null-mutante Zellen untersucht und eine Neurotoxin-vermittelte Spaltung von SNAREs durchgeführt. Nur die Überexpression der leichten Kette von BoNT-C1 verringerte die NPA. Durch Entwicklung und Anwendung einer automatisierten Analyse der Vesikel-Aufenthaltszeiten an der Membran konnte gezeigt werden, dass sich annähernde Vesikel nur selten andocken und nur einige dieser Besucher durch mindestens zwei verschiedene Bindungsarten eingefangen werden, die eine niedrige oder eine hohe Affinität aufweisen. Munc18-1 vergrößerte sowohl die Population des letzteren Zustands, als auch die Gesamtrate mit der Vesikel zugeführt werden.Schlußfolgernd konnten drei verschiedene Andockzustände identifiziert werden, in denen andockende Vesikel entweder sofort wieder abkoppeln oder durch minimale Andock/Bindungsprinzipien eingefangen und in einen Munc18-1/Syntaxin-abhängigen, fest gebunden, fusionsbereiten Zustand konvertiert werden

Regulation of transmitter release by Ca2+ and synaptotagmin: insights from a large CNS synapse

Transmitter release at synapses is driven by elevated intracellular Ca2+ concentration ([Ca2+](i)) near the sites of vesicle fusion. [Ca2+](i) signals of profoundly different amplitude and kinetics drive the phasic release component during a presynaptic action potential, and asynchronous release at later times. Studies using direct control of [Ca2+](i) at a large glutamatergic terminal, the calyx of Held, have provided significant insight into how intracellular Ca2+ regulates transmitter release over a wide concentration range. Synaptotagmin-2 (Syt2), the major isoform of the Syt1/2 Ca2+ sensors at these synapses, triggers highly Ca2+-cooperative release above 1 mu M [Ca2+](i) but suppresses release at low [Ca2+](i). Thus, neurons utilize a highly sophisticated release apparatus to maximize the dynamic range of Ca2+-evoked versus spontaneous release

Developmental regulation of the intracellular Ca2+ sensitivity of vesicle fusion and Ca2+–secretion coupling at the rat calyx of Held

Developmental refinement of synaptic transmission can occur via changes in several pre- and postsynaptic factors, but it has been unknown whether the intrinsic Ca2+ sensitivity of vesicle fusion in the nerve terminal can be regulated during development. Using the calyx of Held, a giant synapse in the auditory pathway, we studied the presynaptic mechanisms underlying the developmental regulation of Ca2+–secretion coupling, comparing a time period before, and shortly after the onset of hearing in rats. We found an ∼2-fold leftward shift in the relationship between EPSC amplitude and presynaptic Ca2+ current charge (QCa), indicating that brief presynaptic Ca2+ currents become significantly more efficient in driving release. Using a Ca2+ tail current protocol, we also found that the high cooperativity between EPSC amplitude and QCa was slightly reduced with development. In contrast, in presynaptic Ca2+ uncaging experiments, the intrinsic Ca2+ cooperativity of vesicle fusion was identical, and the intrinsic Ca2+ sensitivity was slightly reduced with development. This indicates that the significantly enhanced release efficiency of brief Ca2+ currents must be caused by a tighter co-localization of Ca2+ channels and readily releasable vesicles, but not by changes in the intrinsic properties of Ca2+-dependent release. Using the parameters of the intrinsic Ca2+ sensitivity measured at each developmental stage, we estimate that during a presynaptic action potential (AP), a given readily releasable vesicle experiences an about 1.3-fold higher ‘local’ intracellular Ca2+ concentration ([Ca2+]i) signal with development. Thus, the data indicate a tightening in the Ca2+ channel–vesicle co-localization during development, without a major change in the intrinsic Ca2+ sensitivity of vesicle fusion

Ca2+ channels and transmitter release at the active zone

Ca2+-dependent transmitter release is the most important signaling mechanism for fast information transfer between neurons. Transmitter release takes places at highly specialized active zones with sub-micrometer dimension, which contain the molecular machinery for vesicle docking and -fusion, as well as a high density of voltage-gated Ca2+ channels. In the absence of direct evidence for the ultrastructural localization of Ca2+ channels at CNS synapses, important insights into Ca2+ channel-vesicle coupling has come from functional experiments relating presynaptic Ca2+ current and transmitter release, at large and accessible synapses like the calyx of Held. First, high slope values in log-log plots of transmitter release versus presynaptic Ca2+ current indicate that multiple Ca2+ channels are involved in release control of a single vesicle. Second, release kinetics in response to step-like depolarizations revealed fast- and slowly releasable sub-pools of vesicles. FRP and SRP, which, according to the "positional" model, are distinguished by a differential proximity to Ca2+ channels. Considering recent evidence for a rapid conversion of SRP- to FRP vesicles, however, we highlight that multivesicular release events and clearance of vesicle membrane from the active zone must be taken into account when interpreting kinetic release data. We conclude that the careful kinetic analysis of transmitter release at presynaptically accessible and molecularly targeted synapses has the potential to yield important insights into the molecular physiology of transmitter release. (C) 2012 Elsevier Ltd. All rights reserved