Physiology of intracellular calcium buffering.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons.

Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

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.

De Novo Missense Variants in SLC32A1 Cause a Developmental and Epileptic Encephalopathy Due to Impaired GABAergic Neurotransmission.

OBJECTIVE: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. METHODS: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model. RESULTS: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity. INTERPRETATION: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism. ANN NEUROL 2022;92:958-973.

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength.

Glutamatergic synapses show large variations in strength and short-term plasticity (STP). We show here that synapses displaying an increased strength either after posttetanic potentiation (PTP) or through activation of the phospholipase-C-diacylglycerol pathway share characteristic properties with intrinsically strong synapses, such as (i) pronounced short-term depression (STD) during high-frequency stimulation; (ii) a conversion of that STD into a sequence of facilitation followed by STD after a few conditioning stimuli at low frequency; (iii) an equalizing effect of such conditioning stimulation, which reduces differences among synapses and abolishes potentiation; and (iv) a requirement of long periods of rest for reconstitution of the original STP pattern. These phenomena are quantitatively described by assuming that a small fraction of "superprimed" synaptic vesicles are in a state of elevated release probability (p ∼ 0.5). This fraction is variable in size among synapses (typically about 30%), but increases after application of phorbol ester or during PTP. The majority of vesicles, released during repetitive stimulation, have low release probability (p ∼ 0.1), are relatively uniform in number across synapses, and are rapidly recruited. In contrast, superprimed vesicles need several seconds to be regenerated. They mediate enhanced synaptic strength at the onset of burst-like activity, the impact of which is subject to modulation by slow modulatory transmitter systems.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

KEY POINTS: The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. ABSTRACT: Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100 µm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500 µm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD  = 543 ± 51 nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38 ± 0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.

Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse.

Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.

When less is more: non-monotonic spike sequence processing in neurons.

Fundamental response properties of neurons centrally underly the computational capabilities of both individual nerve cells and neural networks. Most studies on neuronal input-output relations have focused on continuous-time inputs such as constant or noisy sinusoidal currents. Yet, most neurons communicate via exchanging action potentials (spikes) at discrete times. Here, we systematically analyze the stationary spiking response to regular spiking inputs and reveal that it is generically non-monotonic. Our theoretical analysis shows that the underlying mechanism relies solely on a combination of the discrete nature of the communication by spikes, the capability of locking output to input spikes and limited resources required for spike processing. Numerical simulations of mathematically idealized and biophysically detailed models, as well as neurophysiological experiments confirm and illustrate our theoretical predictions.

Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.

Neuroligins (NL1-NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimulus-coding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.

Similar intracellular Ca2+ requirements for inactivation and facilitation of voltage-gated Ca2+ channels in a glutamatergic mammalian nerve terminal.

Voltage-gated Ca2+ channels (VGCCs) of the P/Q-type, which are expressed at a majority of mammalian nerve terminals, show two types of Ca2+-dependent feedback regulation-inactivation (CDI) and facilitation (CDF). Because of the nonlinear relationship between Ca2+ influx and transmitter release, CDI and CDF are powerful regulators of synaptic strength. To what extent VGCCs inactivate or facilitate during spike trains depends on the dynamics of free Ca2+ ([Ca2+]i) and the Ca2+ sensitivity of CDI and CDF, which has not been determined in nerve terminals. In this report, we took advantage of the large size of a rat auditory glutamatergic synapse--the calyx of Held--and combined voltage-clamp recordings of presynaptic Ca2+ currents (ICa(V)) with UV-light flash-induced Ca2+ uncaging and presynaptic Ca2+ imaging to study the Ca2+ requirements for CDI and CDF. We find that nearly half of the presynaptic VGCCs inactivate during 100 ms voltage steps and require several seconds to recover. This inactivation is caused neither by depletion of Ca2+ ions from the synaptic cleft nor by metabotropic feedback inhibition, because it is resistant to blockade of metabotropic and ionotropic glutamate receptors. Facilitation of ICa(V) induced by repetitive depolarizations or preconditioning voltage steps decays within tens of milliseconds. Since Ca2+ buffers only weakly affect CDI and CDF, we conclude that the Ca2+ sensors are closely associated with the channel. CDI and CDF can be induced by intracellular photo release of Ca2+ resulting in [Ca2+]i elevations in the low micromolar range, implying a surprisingly high affinity of the Ca2+ sensors.

Transients in global Ca2+ concentration induced by electrical activity in a giant nerve terminal.

Giant nerve terminals offer a unique opportunity to learn about dynamic changes in intracellular global Ca(2+) concentration ([Ca(2+)]i) because this quantity can be measured precisely with indicator dyes and the composition of the intra-terminal ionic milieu can be controlled. We review here recent literature on [Ca(2+)]i signalling in the calyx of Held and discuss what these measurements can tell us about endogenous Ca(2+) buffers and Ca(2+) extrusion mechanisms. We conclude that in spite of the favourable experimental conditions, some unresolved questions still remain regarding absolute values for the Ca(2+)-binding ratio, the affinity of the basic fixed buffer and the Ca(2+) affinities of the major endogenous Ca(2+) binding proteins. Uncertainties about some of these presynaptic properties, including the roles of Mg(2+) and ATP (as a Mg(2+) buffer), however, extend to the point that mechanisms controlling the decay of [Ca(2+)]i signals in unperturbed terminals may have to be reconsidered.

Complexins: Ubiquitously Expressed Presynaptic Regulators of SNARE-Mediated Synaptic Vesicle Fusion.

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca2+-triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca2+-triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion.

Erythropoietin re-wires cognition-associated transcriptional networks.

Recombinant human erythropoietin (rhEPO) has potent procognitive effects, likely hematopoiesis-independent, but underlying mechanisms and physiological role of brain-expressed EPO remained obscure. Here, we provide transcriptional hippocampal profiling of male mice treated with rhEPO. Based on ~108,000 single nuclei, we unmask multiple pyramidal lineages with their comprehensive molecular signatures. By temporal profiling and gene regulatory analysis, we build developmental trajectory of CA1 pyramidal neurons derived from multiple predecessor lineages and elucidate gene regulatory networks underlying their fate determination. With EPO as 'tool', we discover populations of newly differentiating pyramidal neurons, overpopulating to ~200% upon rhEPO with upregulation of genes crucial for neurodifferentiation, dendrite growth, synaptogenesis, memory formation, and cognition. Using a Cre-based approach to visually distinguish pre-existing from newly formed pyramidal neurons for patch-clamp recordings, we learn that rhEPO treatment differentially affects excitatory and inhibitory inputs. Our findings provide mechanistic insight into how EPO modulates neuronal functions and networks.

Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1's roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

Presynaptic Ca2+ influx and vesicle exocytosis at the mouse endbulb of Held: a comparison of two auditory nerve terminals.

The functional properties of mammalian presynaptic nerve endings remain elusive since most terminals of the central nervous system are not accessible to direct electrophysiological recordings. In this study, direct recordings were performed for the first time at endbulb of Held terminals to characterize passive membrane properties, voltage-gated Ca(2+) channels (VGCCs) and Ca(2+)-dependent exocytosis. Endbulb of Held terminals arise from endings of auditory nerve fibres contacting spherical bushy cells (SBCs) in the anterior ventral cochlear nucleus (AVCN). These terminals had a high mean input resistance (1.1 G) and a small mean capacitance (4.3 pF). Presynaptic VGCCs were predominantly of the P/Q type (86%) and expressed at a high density with an estimated average number of 6400 channels per terminal. Presynaptic Ca(2+) currents (I(Ca(V))) activated and deactivated rapidly. Simulations of action potential (AP)-driven gating of VGCCs suggests that endbulb APs trigger brief Ca(2+) influx with a mean half-width of 240 µs and a peak amplitude of 0.45 nA which results from the opening of approximately 2600 channels. Unlike Ca(2+) currents at the calyx of Held, I(Ca(V)) of endbulb terminals showed no inactivation during trains of AP-like presynaptic depolarizations. Endbulb terminals are endowed with a large readily releasable vesicle pool (1064 vesicles) of which only a small fraction (<10%) is consumed during a single AP-like stimulus. Fast presynaptic APs together with rapidly gating VGCCs will generate brief intracellular Ca(2+) transients that favour highly synchronous transmitter release. Collectively these characteristics ensure sustained and precise transmission of timing information from auditory stimuli at the endbulbSBC synapse.

Non-negative Matrix Factorization as a Tool to Distinguish Between Synaptic Vesicles in Different Functional States.

Synaptic vesicles (SVs) undergo multiple steps of functional maturation (priming) before being fusion competent. We present an analysis technique, which decomposes the time course of quantal release during repetitive stimulation as a sum of contributions of SVs, which existed in distinct functional states prior to stimulation. Such states may represent different degrees of maturation in priming or relate to different molecular composition of the release apparatus. We apply the method to rat calyx of Held synapses. These synapses display a high degree of variability, both with respect to synaptic strength and short-term plasticity during high-frequency stimulus trains. The method successfully describes time courses of quantal release at individual synapses as linear combinations of three components, representing contributions from functionally distinct SV subpools, with variability among synapses largely covered by differences in subpool sizes. Assuming that SVs transit in sequence through at least two priming steps before being released by an action potential (AP) we interpret the components as representing SVs which had been 'fully primed', 'incompletely primed' or undocked prior to stimulation. Given these assumptions, the analysis reports an initial release probability of 0.43 for SVs that were fully primed prior to stimulation. Release probability of that component was found to increase during high-frequency stimulation, leading to rapid depletion of that subpool. SVs that were incompletely primed at rest rapidly obtain fusion-competence during repetitive stimulation and contribute the majority of release after 3-5 stimuli.

Munc13-1 is a Ca2+-phospholipid-dependent vesicle priming hub that shapes synaptic short-term plasticity and enables sustained neurotransmission.

During ongoing presynaptic action potential (AP) firing, transmitter release is limited by the availability of release-ready synaptic vesicles (SVs). The rate of SV recruitment (SVR) to release sites is strongly upregulated at high AP frequencies to balance SV consumption. We show that Munc13-1-an essential SV priming protein-regulates SVR via a Ca2+-phospholipid-dependent mechanism. Using knockin mouse lines with point mutations in the Ca2+-phospholipid-binding C2B domain of Munc13-1, we demonstrate that abolishing Ca2+-phospholipid binding increases synaptic depression, slows recovery of synaptic strength after SV pool depletion, and reduces temporal fidelity of synaptic transmission, while increased Ca2+-phospholipid binding has the opposite effects. Thus, Ca2+-phospholipid binding to the Munc13-1-C2B domain accelerates SVR, reduces short-term synaptic depression, and increases the endurance and temporal fidelity of neurotransmission, demonstrating that Munc13-1 is a core vesicle priming hub that adjusts SV re-supply to demand.

Ultrastructural Imaging of Activity-Dependent Synaptic Membrane-Trafficking Events in Cultured Brain Slices.

Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation ("flash-and-freeze") and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and "flash-and-freeze" electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation.

Synaptic vesicles in mature calyx of Held synapses sense higher nanodomain calcium concentrations during action potential-evoked glutamate release.

During development of the calyx of Held synapse, presynaptic action potentials (APs) become substantially faster and briefer. Nevertheless, this synapse is able to upregulate quantal output triggered by arriving APs. Briefer APs lead to less effective gating of voltage-gated Ca(2+) channels (VGCCs). Therefore, mechanisms downstream of Ca(2+) entry must effectively compensate for the attenuated Ca(2+) influx associated with shorter APs in more mature calyces. This compensation could be achieved by tighter spatial coupling between VGCCs and synaptic vesicles, so that the latter are exposed to higher intracellular Ca(2+) concentration ([Ca(2+)](i)). Alternatively or additionally, the Ca(2+) sensitivity of the release apparatus may increase during synapse development. To differentiate between these possibilities, we combined paired patch-clamp recordings with Ca(2+) imaging and flash photolysis of caged Ca(2+) and estimated the [Ca(2+)](i) requirements for vesicle release in the developing mouse calyx of Held synapse. Surprisingly, the dose-response relationship between [Ca(2+)](i) and release rate was shifted slightly to the right in more mature calyces, rendering their vesicles slightly less sensitive to incoming Ca(2+). Taking into account the time course and peak rates of AP-evoked release transients for the corresponding developmental stages, we estimate the local [Ca(2+)](i)"seen" by the Ca(2+) sensors on synaptic vesicles to increase from 35 to 56 mum [from postnatal day 9 (P9)-P11 to P16-P19]. Our results reinforce the idea that developmental tightening of the spatial coupling between VGCCs and synaptic vesicles plays a predominant role in enhancing quantal output at this synapse and possibly other central synapses.