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

Numbers of presynaptic Ca2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such “simple synapses” indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Cav2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Cav2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1–10) and 2.03 at 4 wk (range: 1–4), whereas the mean numbers of Cav2.1 clusters were 2.84 at 2 wk (range: 1–8) and 2.37 at 4 wk (range: 1–5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm2 to 0.0234 μm2), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels

Coupling the Structural and Functional Assembly of Synaptic Release Sites

Information processing in our brains depends on the exact timing of calcium (Ca2+)-activated exocytosis of synaptic vesicles (SVs) from unique release sites embedded within the presynaptic active zones (AZs). While AZ scaffolding proteins obviously provide an efficient environment for release site function, the molecular design creating such release sites had remained unknown for a long time. Recent advances in visualizing the ultrastructure and topology of presynaptic protein architectures have started to elucidate how scaffold proteins establish “nanodomains” that connect voltage-gated Ca2+ channels (VGCCs) physically and functionally with release-ready SVs. Scaffold proteins here seem to operate as “molecular rulers or spacers,” regulating SV-VGCC physical distances within tens of nanometers and, thus, influence the probability and plasticity of SV release. A number of recent studies at Drosophila and mammalian synapses show that the stable positioning of discrete clusters of obligate release factor (M)Unc13 defines the position of SV release sites, and the differential expression of (M)Unc13 isoforms at synapses can regulate SV-VGCC coupling. We here review the organization of matured AZ scaffolds concerning their intrinsic organization and role for release site formation. Moreover, we also discuss insights into the developmental sequence of AZ assembly, which often entails a tightening between VGCCs and SV release sites. The findings discussed here are retrieved from vertebrate and invertebrate preparations and include a spectrum of methods ranging from cell biology, super-resolution light and electron microscopy to biophysical and electrophysiological analysis. Our understanding of how the structural and functional organization of presynaptic AZs are coupled has matured, as these processes are crucial for the understanding of synapse maturation and plasticity, and, thus, accurate information transfer and storage at chemic

Coupling the Structural and Functional Assembly of Synaptic Release Sites

Information processing in our brains depends on the exact timing of calcium (Ca2+)-activated exocytosis of synaptic vesicles (SVs) from unique release sites embedded within the presynaptic active zones (AZs). While AZ scaffolding proteins obviously provide an efficient environment for release site function, the molecular design creating such release sites had remained unknown for a long time. Recent advances in visualizing the ultrastructure and topology of presynaptic protein architectures have started to elucidate how scaffold proteins establish “nanodomains” that connect voltage-gated Ca2+ channels (VGCCs) physically and functionally with release-ready SVs. Scaffold proteins here seem to operate as “molecular rulers or spacers,” regulating SV-VGCC physical distances within tens of nanometers and, thus, influence the probability and plasticity of SV release. A number of recent studies at Drosophila and mammalian synapses show that the stable positioning of discrete clusters of obligate release factor (M)Unc13 defines the position of SV release sites, and the differential expression of (M)Unc13 isoforms at synapses can regulate SV-VGCC coupling. We here review the organization of matured AZ scaffolds concerning their intrinsic organization and role for release site formation. Moreover, we also discuss insights into the developmental sequence of AZ assembly, which often entails a tightening between VGCCs and SV release sites. The findings discussed here are retrieved from vertebrate and invertebrate preparations and include a spectrum of methods ranging from cell biology, super-resolution light and electron microscopy to biophysical and electrophysiological analysis. Our understanding of how the structural and functional organization of presynaptic AZs are coupled has matured, as these processes are crucial for the understanding of synapse maturation and plasticity, and, thus, accurate information transfer and storage at chemical synapses

Modelling active zone calcium dynamics at cerebellar mossy fibre boutons

The rate at which signals can be transmitted between single neurons limits the speed of information processing. Cerebellar mossy fibre boutons are able to maintain synchronous neurotransmitter release at very high action potential frequencies, up to ∼ 1 kHz . The neurotransmitter release occurs at the presynaptic active zone and is controlled by highly localised calcium signals. In order to allow reliable, fast synaptic transmission, calcium ions must be cleared from the active zone. However, the exact mechanisms of calcium clearance remain elusive. Despite the recent advances in imaging technology, it is not yet possible to measure localised calcium signals on the nanometre scale. Nevertheless, it is possible to address the impact of localised calcium signals on neurotransmitter release with use of computational modelling. In this study, I established an experimentally constrained model of an active zone of the cerebellar mossy fibre bouton. My simulations revealed that endogenous fixed buffers that have low calcium binding capacity ( ∼ 15 ) and low affinity for binding calcium in combination with mobile buffers with high affinity for binding calcium enable rapid clearance of calcium from the active zone during high-frequency firing. Moreover, during high-frequency firing, slow endogenous mobile buffers prevent build-up of the intracellular calcium concentration. The results presented in this work suggest that reduced calcium buffering speeds active zone calcium signalling, thus allowing high rates of synaptic transmission

Experimental and Monte Carlo studies of Ca2+ channel function and fast transmitter release at presynaptic active zones of the frog neuromuscular junction

During fast chemical synaptic transmission, neurotransmitter release is triggered by calcium (Ca2+) influx through voltage-gated Ca2+ channels (VGCCs) opened by an action potential (AP) at the nerve terminal. The magnitude and time course of neurotransmitter release is critically determined by the coupling between Ca2+ channels and synaptic vesicles. Studies of the quantitative dependence of transmitter release on the number of VGCCs provide important information for our understanding of the mechanisms that underlie the control and modulation of presynaptic release probability and kinetics. Using high-resolution calcium imaging techniques and variance analysis, I have determined the number of functional VGCCs within individual active zones (AZs) of the adult frog neuromuscular junction (NMJ) and their opening probability in response to single AP stimulation. The results have shown that the average number of VGCCs within individual active zones was relatively small (~28) and the average opening probability of individual Ca2+ channels during a presynaptic AP was very low (~0.24). Therefore, it is predicted that an action potential induced opening of relatively few Ca2+ channels in a single active zone. Furthermore, by combining pharmacological channel block, calcium imaging, postsynaptic recording, and 3D Monte Carlo diffusion-reaction simulations, I have studied the coupling of single Ca2+ channel openings to the triggering of vesicle fusion. I have provided evidence that Ca2+ entry through single open Ca2+ channels at the nerve terminal can be imaged directly and that such Ca2+ flux is sufficient to trigger synaptic vesicle fusion. I have further shown that following a single AP, the Ca2+ influx through a single open channel plays the predominant role in evoking neurotransmitter release, while Ca2+ ions derived from a collection of open Ca2+ channels are rarely required for vesicle exocytosis at this synapse

Komposition und Dynamik der Freisetzungsstellen für Neurotransmitter

In den hier vorgestellten Forschungsarbeiten widme ich mich den Freisetzungsstellen für chemische Transmitter in zwei Modellsystemen, Maus chromaffinen Zellen und der Neuromuskulären Endplatte von Drosophila melanogaster Larven. In der ersten Studie (2.1) entwickelte ich ein neues quantitatives mathematisches Modell, das detaillierte Einblicke in die Reaktionsgeschwindigkeiten und molekularen Abhängigkeiten der Transmitterfreisetzung lieferte. In diesem Model wurde die Hypothese getestet, ob sequenzielle, Kalziumkatalysierte Reaktionen der Vesikelreifung ursächlich für die beobachtete biphasische Transmitterfreisetzung sein können, was der Fall ist. Im Vergleich zu klassischen Modellen mit zwei unabhängigen (parallelen) Fusionsreaktionen mit unterschiedlicher Geschwindigkeit waren beide Modelle ähnlich gut geeignet, um die Transmitterfreisetzung aus wildtypischen Maus chromaffinen Zellen zu beschreiben. Allerdings konnten durch das neue, sequenzielle Modell, Effekte der Mutation des vesikulären SNARE Proteins Synaptobrevin-2 etwas leichter (durch die Anpassung weniger Parameter) interpretiert werden und die Kalzium-beschleunigte Wiederauffüllung der freisetzbaren Vesikel nach einem Stimulus genauer vorhergesagt werden. In der zweiten Studie (2.2) identifizierten wir die molekulare Identität von synaptischen Freisetzungsstellen an der glutamatergen neuromuskulären Endplatte von Drosophila melanogaster Larven. Diese Freisetzungsstellen limitieren die Neurotransmitterfreisetzung. Obwohl ihre Existenz über 50 Jahre bekannt war, war ihre molekulare Identität unbekannt geblieben. Mit Hilfe von genetischen Experimenten, elektrophysiologischen Messungen, Lebendmikroskopie und hochauflösender Mikroskopie beschreiben wir eine essentielle Rolle des evolutionär konservierten (M)Unc13 Proteins hierfür. In der dritten Studie (2.3) identifizierten wir eine hierarchische Sequenz molekularer Reaktionen, die eine homöostatische Erhöhung der Neurotransmitterfreisetzung ermöglichen. An der Drosophila melanogaster neuromuskuläern Endplatte zeigen wir, dass diese Potenzierung von (M)Unc13 abhängt. Mit Hilfe hochauflösender Mikroskopie und neuer Algorithmen zur Untersuchung der Ultrastruktur von Synapsen entdeckten wir, dass Synapsen hierfür schnell umgestaltet werden können und zeitlich zwei Potenzierungsphasen unterschieden werden können: Die schnelle Potenzierung beruht zunächst auf dem verfügbaren präsynaptischen Material. Aber dann erfolgt innerhalb von Minuten die zusätzliche Inkorporation mehrerer Proteine ins Zentrum der Synapsen. Dies ist erforderlich, um die Potenzierung über einen Zeitraum von Stunden bis Tagen aufrechtzuerhalten. Wir zeigten, dass sowohl die akute als auch die langanhaltende Potenzierung von (M)Unc13 abhängen und dass die selektive Manipulation (M)Unc13s im Fliegenhirn die Gedächtnisbildung stört, was darauf hindeutet, dass dieses Protein in mehreren Formen der Plastizität fungiert, einschließlich derjenigen, die zum Lernen benötigt werden. In der vierten Studie (2.4) stellten wir das erste schnelle und lichtinduzierte Lipid-Uncaging des Signallipids PI(4,5)P2 vor. Dies wurde mit schnellen elektrophysiologischen Aufzeichnungen der Neurosekretion kombiniert. Wir zeigten eine rasche Regulation (<1 s) der Transmitterfreisetzung durch PI(4,5)P2 und identifizierten in genetischen Experimenten die Proteine Synaptotagmin und (M)Unc13 als essentiell dafür. In der fünften Studie (2.5) untersuchten wir quantitativ die Verteilung der Distanzen zwischen synaptischen Freisetzungsstellen und spannungsgesteuerten Kalziumkanälen. Wir konnten zeigen, dass die Verteilung stark heterogen ist und dass dies direkte Effekte auf die sogenannte Kurzzeitplastizität hat, die schnellen Veränderungen synaptischer Antworten. Mittels stochastischer Simulationen wurden unterschiedliche Modelle der Kurzzeitplastizität untersucht und mit Experimentaldaten verglichen. Unsere Ergebnisse legen nahe, dass die Variabilität synaptischer Antworten und deren Kurzzeitplastizität am ehesten durch eine schnelle Regulation der partizipierenden synaptischen Freisetzungsstellen erklärbar sind. Durch die hier vorgestellten Studien wurden viele Erkenntnisse zum molekularen Aufbau, der Funktion und Regulation synaptischer Freisetzungsstellen gewonnen. Anders als zunächst erwartet, zeichnet sich zum jetzigen Zeitpunkt ab, dass Freisetzungsstellen hochdynamisch durch Kalzium und Signallipide reguliert werden können. Zukünftige Studien können physiologisch wichtige Erkenntnisse zu den molekularen Mechanismen liefern und wie diese synaptische Transmission auf verschiedenen Zeitskalen für Signalverarbeitung, Homöostase und Gedächtnisbildung modulieren