Nanodomain coupling explains Ca^2+ independence of transmitter release time course at a fast central synapse

A puzzling property of synaptic transmission, originally established at the neuromuscular junction, is that the time course of transmitter release is independent of the extracellular Ca2+ concentration ([Ca2+]o), whereas the rate of release is highly [Ca2+]o-dependent. Here, we examine the time course of release at inhibitory basket cell-Purkinje cell synapses and show that it is independent of [Ca2+]o. Modeling of Ca2+-dependent transmitter release suggests that the invariant time course of release critically depends on tight coupling between Ca2+ channels and release sensors. Experiments with exogenous Ca2+ chelators reveal that channel-sensor coupling at basket cell-Purkinje cell synapses is very tight, with a mean distance of 10–20 nm. Thus, tight channel-sensor coupling provides a mechanistic explanation for the apparent [Ca2+]o independence of the time course of release

Experimental and computational studies of calcium-triggered transmitter release

Calcium influx through presynaptic calcium channels triggers transmitter release, but many of the details that underlie calcium-triggered secretion are not well understood. In an attempt to increase our understanding of this process, synaptic transmission at the frog neuromuscular junction has been investigated using physiological experiments and computational modeling. Pharmacological manipulations ((R)-roscovitine and DAP) were used as tools to modulate presynaptic calcium influx and study effects on transmitter release. I showed that (R)-roscovitine predominately slowed deactivation kinetics of calcium current (by 427%), and as a result, increased the integral of calcium channel current evoked by a physiological action potential waveform (by 44%). (R)-roscovitine also increased the quantal content of acetylcholine released from the motor nerve terminals (by 149%) without changing paired-pulse facilitation under low calcium conditions. In contrast, exposure to 3,4-diaminopyridine (which affects transmitter release evoked by partially blocking potassium channels, altering the amplitude of the presynaptic action potential, and indirectly increasing calcium entry) increased paired-pulse facilitation (by 23%). In normal calcium conditions, both pharmacological treatments showed relatively similar effects on paired-pulse facilitation. I used a computational model, constrained by previous reports in the literature and my physiological measurements, to simulate my experimental data. This model faithfully reproduced calcium current with a single action potential, the average number of released synaptic vesicles, and the effects of (R)-roscovitine and DAP on calcium influx and vesicle release. Using this model, I made several predictions about the mechanisms underlying transmitter release. First, calcium ions originating from one or two voltage-gated calcium channels most often contributed to cause the fusion of each vesicle. Second, the calcium channel closest to a vesicle that fuses, provides 77% of calcium ions. My simulation of paired-pulse facilitation using the present model needed more adjustments, and in the process of adjusting the model parameters, various hypotheses that might explain observed short-term synaptic plasticity, including the effects of changes in buffer conditions, the effects of uneven calcium channel distribution, reducing terminal volume by adding vesicles to a storage pool, changes in the second action potential waveform, and possible persistent changes in vesicle release machinery were explored

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

Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton

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

Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.

Optical single-channel recording is a novel tool for the study of individual Ca2+-permeable channels within intact cells under minimally perturbed physiological conditions. As applied to the functioning and spatial organization of IP3Rs, this approach complements our existing knowledge, which derives largely from reduced systems - such as reconstitution into lipid bilayers and patch clamping of IP3Rs on the membrane of excised nuclei - where the spatial arrangement and interactions among IP3Rs via CICR are disrupted. The ability to image the activity of single IP3R channels with millisecond resolution together with localization of their positions with a precision of a few tens of nanometers both raises several intriguing questions and holds promise of answers. In particular, what mechanism underlies the anchoring of puffs and blips to static locations; why do these Ca2+ release events appear to involve only a very small fraction of the IP3Rs within a cell; and how can we reconcile the relative immotility of functional IP3Rs with numerous studies reporting free diffusion of IP3R protein in the ER membrane

Mechanismen der synaptischen Übertragung an der zerebellären Moosfaser-Körnerzell-Synapse

Die Funktion unseres Zentralnervensystems beruht auf der zeitlich präzisen Übertragung elektrischer Signale zwischen Neuronen. Diese synaptische Übertragung findet in weniger als einer tausendstel Sekunde statt. Eine schnelle und hochfrequente Signalübertragung erweitert die Kodierungskapazität und beschleunigt die Verarbeitung von Informationen. Obwohl viele der an synaptischer Übertragung beteiligten Prozesse und Proteine bekannt sind, ist das Verständnis der Mechanismen, die für eine schnelle und hochfrequente Signalübertragung verantwortlich sind, bisher unvollständig. Um die Mechanismen hochfrequenter synaptischer Übertragung zu untersuchen, wurden in dieser Arbeit prä- und postsynaptische Patch-Clamp Ableitungen an der zerebellären Moosfaser-Körnerzell-Synapse in akuten Hirnschnitten der Maus eingesetzt. Es zeigte sich, dass diese Synapse präsynaptische Aktionspotenziale mit einer Frequenz über einem Kilohertz feuern kann und dass Informationen in diesem Frequenzbereich an die postsynaptische Zelle übertragen werden können. Hierbei vermitteln besonders schnelle Natrium- und Kalium-Kanäle eine extrem kurze Dauer der Aktionspotenziale, die dennoch metabolisch relativ effizient sind. Schnelle Kalzium-Kanäle und eine schwache präsynaptische Kalzium-Pufferung ermöglichen eine synchrone Vesikelfreisetzung mit hohen Frequenzen. Zusätzlich greift die Präsynapse auf einen großen Vorrat an freisetzbaren Vesikeln zurück, dessen Auffüllung besonders schnell stattfindet. Aufgrund der hochfrequenten synaptischen Übertragung ist die Moosfaser- Körnerzell-Synapse ideal, um zu untersuchen, wie schnell die auf eine Vesikelfreisetzung folgende Endozytose vonstatten geht. Mit optimierten, hochauflösenden Kapazitätsmessungen konnte an der Moosfaser-Körnerzell- Synapse eine sehr schnelle Endozytose nach einzelnen Aktionspotenzialen gezeigt werden. Die hohe Geschwindigkeit der Endozytose unterstützt somit eine hochfrequente synaptische Übertragung. Diese schnelle Endozytose wird durch die Moleküle Dynamin und Actin vermittelt und ist unabhängig von einer Wirkung von Clathrin. Stärkere Stimuli wie längere Depolarisationen evozieren eine langsamere Form der Endozytose, die zusätzlich Clathrin-abhängig ist. Durch die mechanistische Beschreibung hochfrequenter Signalübertragung an einer zentralen Synapse erweitern die Ergebnisse der vorliegenden Arbeit unser Verständnis von synaptischer Übertragung und Informationsverarbeitung im Zentralnervensystem

Multiquantal Glutamate Release from Rod Photoreceptors

Neurons communicate via Ca2+-dependent release of neurotransmitters packaged into vesicles (quanta). Some CNS neurons, especially sensory synapses, can release multiple vesicles at a time, increasing information transmission and overcoming the unreliability of a stochastic process. Ribbon-bearing neurons, including retinal photoreceptors, face the challenge of encoding sensory receptor potentials into an ever-changing train of vesicle release events. We studied release of glutamate using voltage clamp to measure anion currents activated during glutamate reuptake into presynaptic terminals (IA(glu)) of salamander and mouse rods, finding that each employ distinct mechanisms for multiquantal release. In amphibian rods, we found that 1/3 of the spontaneous IA(glu) fusion events involve synchronous fusion of multiple vesicles. By varying intracellular buffering to localize Ca2+-dependent events, we found that multiquantal release occurs near Ca2+ sources. In photoreceptors, Ca2+ influx occurs just below synaptic ribbons. Vesicles house SNARE machinery so we hypothesized that vesicles on the ribbon undergo homotypic fusion prior to exocytosis. Destruction of ribbons and disruption of the SNARE-protein syntaxin3B prevented spontaneous multiquantal release, suggesting that salamander rods are capable of multivesicular release due to homotypic fusion of vesicles along ribbons. In mouse rods, spontaneous release at −70 mV involved the stochastic fusion of single vesicles. With depolarization, glutamate release increased linearly with voltage-gated Ca2+ currents. As the membrane approached the resting potential in darkness of −40 mV, rods began to release glutamate in multivesicular bursts of 17±7 vesicles every 2801±598 ms. Release evoked by brief depolarizations and bursts both involved the same pool of ribbon-associated vesicles with fusion regulated by the vesicular Ca2+ sensor synaptotagmin-1. A second, slower component of release controlled by synaptotagmin-7 is also present in rods but not cones. We hypothesized a v role for coordinated bursts of release in transmitting single photon signals. The rate of bursting was responsive to small voltage changes of 1.0-3.5 mV and the voltage waveform that triggered bursts most effectively was similar to single photon responses. We propose that multiquantal bursts contribute to mechanisms that filter out small noisy events to improve reliable detection of single photons by the retina

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity

Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using nonnegative tensor factorization and conventional state modeling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release–machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at the calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but are determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming, and fusion. It suggests a mechanism for activity-induced redistribution of synaptic efficacy