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. However, this model has not been quantitatively tested over physiological timescales. Here we describe an experimentally constrained computational modelling framework to quantitatively assess how the molecular architecture of the fusion clamp affects SV exocytosis. Our results argue that the 'release-of-inhibition' model can indeed account for fast calcium-activated SV fusion, and that dual binding of synaptotagmin-1 and synaptotagmin-7 to the same SNARE complex enables synergistic regulation of the kinetics and plasticity of neurotransmitter release. The developed framework provides a powerful and adaptable tool to link the molecular biochemistry of presynaptic proteins to physiological data and efficiently test the plausibility of calcium-activated neurotransmitter release models

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. We found that asynchronous release at MF–CA3 synapses can last on the order of seconds following repetitive MF stimulation. Elevating the stimulation frequency or the external Ca2+ concentration increased the rate of asynchronous release, thus, arguing that presynaptic Ca2+ dynamics is the major determinant of asynchronous release rate. Direct MF bouton Ca2+ imaging revealed slow Ca2+ decay kinetics of action potential (AP) burst‐evoked Ca2+ transients. Finally, we observed that asynchronous release was preferentially mediated by Ca2+ influx through P/Q‐type voltage‐gated Ca2+ channels, while the contribution of N‐type VGCCs was limited. Overall, our results uncover the determinants of long‐lasting asynchronous release from MF terminals and suggest that asynchronous release could influence CA3 pyramidal cell firing up to seconds following termination of granule cell bursting

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. However, this model has not been quantitatively tested over physiological timescales. Here we describe an experimentally constrained computational modelling framework to quantitatively assess how the molecular architecture of the fusion clamp affects SV exocytosis. Our results argue that the ‘release-of-inhibition’ model can indeed account for fast calcium-activated SV fusion, and that dual binding of synaptotagmin-1 and synaptotagmin-7 to the same SNARE complex enables synergistic regulation of the kinetics and plasticity of neurotransmitter release. The developed framework provides a powerful and adaptable tool to link the molecular biochemistry of presynaptic proteins to physiological data and efficiently test the plausibility of calcium-activated neurotransmitter release models

Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release

Release of neurotransmitters relies on submillisecond coupling of synaptic vesicle fusion to the triggering signal: AP-evoked presynaptic Ca2+ influx. The key player that controls exocytosis of the synaptic vesicle is the Ca2+ sensor synaptotagmin 1 (Syt1). While the Ca2+ activation of Syt1 has been extensively characterized, how Syt1 reversibly clamps vesicular fusion remains enigmatic. Here, using a targeted mutation combined with fluorescence imaging and electrophysiology, we show that the structural feature of Syt1 to self-oligomerize provides the molecular basis for clamping of spontaneous and asynchronous release but is not required for triggering of synchronous release. Our findings propose a mechanistic model that explains how Syt1 oligomers regulate different modes of transmitter release in neuronal synapses

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. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that the synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses – the timing and the overall efficacy of neurotransmitter release

Alternative Splicing of P/Q-Type Ca2+ 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 Ca2+ channel (VGCC) a1 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. Here, we investigate how two major splice isoforms of P/Q-type VGCCs (Cav2.1[EFa/b]) regulate presynaptic plasticity in hippocampal neurons. We find that the efficacy of P/Q-type VGCC isoforms in supporting synaptic transmission is markedly different, with Cav2.1[EFa] promoting synaptic depression and Cav2.1[EFb] synaptic facilitation. Following a reduction in network activity, hippocampal neurons upregulate selectively Cav2.1[EFa], the isoform exhibiting the higher synaptic efficacy, thus effectively supporting presynaptic homeostatic plasticity. Therefore, the balance between VGCC splice variants at the synapse is a key factor in controlling neurotransmitter release and presynaptic plasticity

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. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that the synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses – the timing and the overall efficacy of neurotransmitter release

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. We calculate the stress a nanopipet exerts on a cell as the sum of the intrinsic pressure between the tip face and the plasma membrane plus its direct pressure on any glycocalyx, both evaluated from the gap size in terms of the ion current decrease. A survey of cell types confirms that an intracellular pressure of approximately 120 Pa begins to detach the plasma membrane from the cytoskeleton and reveals that the first 0.66 ± 0.09 μm of compression of a neuron cell body is much softer than previous methods have been able to detect.Biotechnology and Biological Sciences Research Council (Grant ID: BB/L006227/1), Engineering and Physical Sciences Research Council (Grant ID: EP/H01098X/1), Medical Research Council (Grant IDs: G0701057, MR/K501372/1), Herchel Smith Postdoctoral Fellowship, Royal Society of Chemistry Analytical Chemistry Trust Fun

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. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures, and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of Episodic Ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1;; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-­associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels

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. Here, we show that glutamate release from mossy fiber terminals triggers action potential firing of the target CA3 pyramidal neurons independently of the average granule cell burst frequency, a phenomenon we term action potential counting. We find that action potential counting in mossy fibers gates glutamate release over a broad physiological range of frequencies and action potential numbers. Using rapid Ca imaging we also show that the magnitude of evoked Ca influx stays constant during action potential trains and that accumulated residual Ca is gradually extruded on a time scale of several hundred milliseconds. Using experimentally constrained 3D model of presynaptic Ca influx, buffering, and diffusion, and a Monte Carlo model of Ca -activated vesicle fusion, we argue that action potential counting at mossy fiber boutons can be explained by a unique interplay between Ca dynamics and buffering at release sites. This is largely determined by the differential contribution of major endogenous Ca buffers calbindin-D and calmodulin and by the loose coupling between presynaptic voltage-gated Ca channels and release sensors and the relatively slow Ca extrusion rate. Taken together, our results identify a previously unexplored information-coding mechanism in the brain