Minimal presynaptic protein machinery governing diverse kinetics of calcium-evoked neurotransmitter release.

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic calcium influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically relevant conditions to delineate the minimal protein machinery sufficient to account for various modes of calcium-triggered vesicle fusion dynamics.

A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity.

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca-triggered vesicle fusion and short-term facilitation.

The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses.

Calcium-evoked release of neurotransmitters from synaptic vesicles (SVs) is catalysed by SNARE proteins. The predominant view is that, at rest, complete assembly of SNARE complexes is inhibited ('clamped') by synaptotagmin and complexin molecules. Calcium binding by synaptotagmins releases this fusion clamp and triggers fast SV exocytosis.

The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses.

Calcium-evoked release of neurotransmitters from synaptic vesicles (SVs) is catalysed by SNARE proteins. The predominant view is that, at rest, complete assembly of SNARE complexes is inhibited ('clamped') by synaptotagmin and complexin molecules. Calcium binding by synaptotagmins releases this fusion clamp and triggers fast SV exocytosis.

Asynchronous glutamate release is enhanced in low release efficacy synapses and dispersed across the active zone.

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 from single pyramidal cells with 4 millisecond temporal and 75 nm spatial resolution.

Forward genetics identifies a novel sleep mutant with sleep state inertia and REM sleep deficits.

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants.

Slow-decaying presynaptic calcium dynamics gate long-lasting asynchronous release at the hippocampal mossy fiber to CA3 pyramidal cell synapse.

Action potentials trigger two modes of neurotransmitter release, with a fast synchronous component and a temporally delayed asynchronous release. Asynchronous release contributes to information transfer at synapses, including at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse where it controls the timing of postsynaptic CA3 pyramidal neuron firing. Here, we identified and characterized the main determinants of asynchronous release at the MF-CA3 synapse.

Fluorescence lifetime imaging reveals regulation of presynaptic Ca by glutamate uptake and mGluRs, but not somatic voltage in cortical neurons.

Brain function relies on vesicular release of neurotransmitters at chemical synapses. The release probability depends on action potential-evoked presynaptic Ca entry, but also on the resting Ca level. Whether these basic aspects of presynaptic calcium homeostasis show any consistent trend along the axonal path, and how they are controlled by local network activity, remains poorly understood.

Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release.

Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca activation mechanism has been well-characterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function.

Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis.

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca-dependent release has been unclear. Here, we report that the Ca-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca-coupled vesicular release.

Action potential counting at giant mossy fiber terminals gates information transfer in the hippocampus.

Neuronal communication relies on action potential discharge, with the frequency and the temporal precision of action potentials encoding information. Hippocampal mossy fibers have long been recognized as conditional detonators owing to prominent short-term facilitation of glutamate release displayed during granule cell burst firing. However, the spiking patterns required to trigger action potential firing in CA3 pyramidal neurons remain poorly understood.

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

Background: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.

Results: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations.

Synergistic control of neurotransmitter release by different members of the synaptotagmin family.

Quantal neurotransmitter release at nerve terminals is tightly regulated by the presynaptic Ca concentration. Here, we summarise current advances in understanding how the interplay between presynaptic Ca dynamics and different Ca release sensors shapes action potential-evoked release on a timescale from hundreds of microseconds to hundreds of milliseconds. In particular, we review recent studies that reveal the synergistic roles of the low Ca affinity/fast release sensors synaptotagmins 1, 2 and 9 and the high affinity/slow release sensor synaptotagmin 7 in the regulation of synchronous and asynchronous release and of short-term synaptic plasticity.

Alternative Splicing of P/Q-Type Ca Channels Shapes Presynaptic Plasticity.

Alternative splicing of pre-mRNAs is prominent in the mammalian brain, where it is thought to expand proteome diversity. For example, alternative splicing of voltage-gated Ca channel (VGCC) α subunits can generate thousands of isoforms with differential properties and expression patterns. However, the impact of this molecular diversity on brain function, particularly on synaptic transmission, which crucially depends on VGCCs, is unclear.

Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization.

Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog-digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization.

Low Stress Ion Conductance Microscopy of Sub-Cellular Stiffness.

Directly examining subcellular mechanics whilst avoiding excessive strain of a live cell requires the precise control of light stress on very small areas, which is fundamentally difficult. Here we use a glass nanopipet out of contact with the plasma membrane to both exert the stress on the cell and also accurately monitor cellular compression. This allows the mapping of cell stiffness at a lateral resolution finer than 100 nm.

Action potential broadening in a presynaptic channelopathy.

Brain development and interictal function are unaffected in many paroxysmal neurological channelopathies, possibly explained by homoeostatic plasticity of synaptic transmission. Episodic ataxia type 1 is caused by missense mutations of the potassium channel Kv1.1, which is abundantly expressed in the terminals of cerebellar basket cells.

Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation.

Action potential-dependent release of synaptic vesicles and short-term synaptic plasticity are dynamically regulated by the endogenous Ca(2+) buffers that shape [Ca(2+)] profiles within a presynaptic bouton. Calmodulin is one of the most abundant presynaptic proteins and it binds Ca(2+) faster than any other characterized endogenous neuronal Ca(2+) buffer. Direct effects of calmodulin on fast presynaptic Ca(2+) dynamics and vesicular release however have not been studied in detail.

Lambert-Eaton syndrome IgG inhibits transmitter release via P/Q Ca2+ channels.

Objective: To determine whether immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome (LEMS) decreases action potential–evoked synaptic vesicle exocytosis,and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs).

Methods: IgG was obtained from 4 patients with LEMS (3 males, 1 female), including 2 patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all 4 patients, and against N-type VGCCs in 2.

Differential triggering of spontaneous glutamate release by P/Q-, N- and R-type Ca2+ channels.

The role of voltage-gated Ca2+ channels (VGCCs) in spontaneous miniature neurotransmitter release is incompletely understood. We found that stochastic opening of P/Q-, N- and R-type VGCCs accounts for ∼50% of all spontaneous glutamate release at rat cultured hippocampal synapses, and that R-type channels have a far greater role in spontaneous than in action potential-evoked exocytosis. VGCC-dependent miniature neurotransmitter release (minis) showed similar sensitivity to presynaptic Ca2+ chelation as evoked release, arguing for direct triggering of spontaneous release by transient spatially localized Ca(2+) domains.

Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels.

Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤ 1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100-150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell).

Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources.

Tonic GABA type A (GABAA) conductance is a key factor regulating neuronal excitability and computation in neuronal networks. The magnitude of the tonic GABAA conductance depends on the concentration of ambient GABA originating from vesicular and non-vesicular sources and is tightly regulated by GABA uptake. Here we show that the transport system regulating ambient GABA responsible for tonic GABAA conductances in hippocampal CA1 interneurons depends on its source.

Independent regulation of basal neurotransmitter release efficacy by variable Ca²+ influx and bouton size at small central synapses.

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient.

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an "adhesion G-protein-coupled receptor," LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1's extracellular domain to purify its endogenous ligand.

Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors.

The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins.