The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions ...underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.
•Number of presynaptic calcium channels (CaV) does not correlate with synaptic strength•Weak synapses are more sensitive to competition with exogenous Ca2+ chelators•EM immunogold labeling of CaV2.1 and Munc13-1 shows synapse-specific nanotopographies•Different nanoscale CaV-synaptic vesicle arrangements explain functional differences
Rebola et al. identified two distinct nanoscale topographies of calcium channels and synaptic vesicles that underlie functional differences in synaptic strength and neuromodulation in cerebellum. These motifs provide new insight into the macromolecular organization mediating synaptic transmission.
Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing ...fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.
Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a ...quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local Ca2+ imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.
Interneurons are critical for neuronal circuit function, but how their dendritic morphologies and membrane properties influence information flow within neuronal circuits is largely unknown. We ...studied the spatiotemporal profile of synaptic integration and short-term plasticity in dendrites of mature cerebellar stellate cells by combining two-photon guided electrical stimulation, glutamate uncaging, electron microscopy, and modeling. Synaptic activation within thin (0.4 μm) dendrites produced somatic responses that became smaller and slower with increasing distance from the soma, sublinear subthreshold input-output relationships, and a somatodendritic gradient of short-term plasticity. Unlike most studies showing that neurons employ active dendritic mechanisms, we found that passive cable properties of thin dendrites determine the sublinear integration and plasticity gradient, which both result from large dendritic depolarizations that reduce synaptic driving force. These integrative properties allow stellate cells to act as spatiotemporal filters of synaptic input patterns, thereby biasing their output in favor of sparse presynaptic activity.
► We found that thin dendrites of small cerebellar SCs filter their excitatory inputs ► Passive cable properties of SCs produce sublinear synaptic integration ► This sublinear dendritic behavior generates a gradient of short-term plasticity ► SC activity is biased towards sparse rather than clustered input patterns
Stellate cells are critical sources of inhibition in the cerebellum, but how their dendrites integrate excitatory synaptic inputs is unknown. Abrahamsson et al. show that thin dendrites and passive membrane properties of SCs promote sublinear synaptic summation and distance-dependent short-term plasticity.
Synaptic transmission between neurons is governed by a cascade of stochastic calcium ion reaction-diffusion events within nerve terminals leading to vesicular release of neurotransmitter. Since ...experimental measurements of such systems are challenging due to their nanometer and sub-millisecond scale, numerical simulations remain the principal tool for studying calcium-dependent neurotransmitter release driven by electrical impulses, despite the limitations of time-consuming calculations. In this paper, we develop an analytical solution to rapidly explore dynamical stochastic reaction-diffusion problems based on first-passage times. This is the first analytical model that accounts simultaneously for relevant statistical features of calcium ion diffusion, buffering, and its binding/unbinding reaction with a calcium sensor for synaptic vesicle fusion. In particular, unbinding kinetics are shown to have a major impact on submillisecond sensor occupancy probability and therefore cannot be neglected. Using Monte Carlo simulations we validated our analytical solution for instantaneous calcium influx and that through voltage-gated calcium channels. We present a fast and rigorous analytical tool that permits a systematic exploration of the influence of various biophysical parameters on molecular interactions within cells, and which can serve as a building block for more general cell signaling simulators.
The starburst amacrine cell in the mouse retina presents an opportunity to examine the precise role of sensory input location on neuronal computations. Using visual receptive field mapping, glutamate ...uncaging, two-photon Ca2+ imaging, and genetic labeling of putative synapses, we identify a unique arrangement of excitatory inputs and neurotransmitter release sites on starburst amacrine cell dendrites: the excitatory input distribution is skewed away from the release sites. By comparing computational simulations with Ca2+ transients recorded near release sites, we show that this anatomical arrangement of inputs and outputs supports a dendritic mechanism for computing motion direction. Direction-selective Ca2+ transients persist in the presence of a GABA-A receptor antagonist, though the directional tuning is reduced. These results indicate a synergistic interaction between dendritic and circuit mechanisms for generating direction selectivity in the starburst amacrine cell.
•In starburst amacrine cells, excitatory inputs are skewed away from release sites•This skewed distribution enhances direction selectivity in a computational model•Direction selectivity of release sites depends on their dendritic locations•The dendritic direction computation is enhanced by GABAergic lateral inhibition
Starburst amacrine cells present an opportunity to examine the precise role of sensory input location on dendritic computations. Vlasits et al. find that the excitatory input distribution is skewed away from outputs, which supports a dendritic computation of motion direction.
Synaptic heterogeneity is widely observed but its underpinnings remain elusive. We addressed this issue using mature calyx of Held synapses whose numbers of bouton-like swellings on stalks of the ...nerve terminals inversely correlate with release probability (Pr). We examined presynaptic Ca
currents and transients, topology of fluorescently tagged knock-in Ca
channels, and Ca
channel-synaptic vesicle (SV) coupling distance using Ca
chelator and inhibitor of septin cytomatrix in morphologically diverse synapses. We found that larger clusters of Ca
channels with tighter coupling distance to SVs elevate Pr in stalks, while smaller clusters with looser coupling distance lower Pr in swellings. Septin is a molecular determinant of the differences in coupling distance. Supported by numerical simulations, we propose that varying the ensemble of two morphological modules containing distinct Ca
channel-SV topographies diversifies Pr in the terminal, thereby establishing a morpho-functional continuum that expands the coding capacity within a single synapse population.
The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca
2+
channels (VGCCs) and Ca
2+
sensors for exocytosis. This ...VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca
2+
chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca
2+
reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca
2+
buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca
2+
influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca
2+
sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca
2+
entry durations are not considered when estimating VGCC–sensor coupling distances.
SIGNIFICANT STATEMENT
The coupling distance between voltage-gated Ca
2+
channels and Ca
2+
sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca
2+
chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca
2+
entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction–diffusion simulations of Ca
2+
-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.
The ability of the brain to rapidly process information from multiple pathways is critical for reliable execution of complex sensory-motor behaviors, yet the cellular mechanisms underlying a neuronal ...representation of multimodal stimuli are poorly understood. Here we explored the possibility that the physiological diversity of mossy fiber (MF) to granule cell (GC) synapses in the mouse vestibulocerebellum may contribute to the processing of coincident multisensory information at the level of individual GCs. We found that the strength and short-term dynamics of individual MF-GC synapses can act as biophysical signatures for primary vestibular, secondary vestibular and visual input pathways. Most GCs receive inputs from different modalities, which, when coactivated, produced enhanced GC firing rates and distinct first spike latencies. Thus, pathway-specific synaptic response properties permit temporal coding of correlated multisensory inputs by single GCs, thereby enriching sensory representation and facilitating pattern separation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca(2+) channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a ...quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local Ca(2+) imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca(2+) buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca(2+) sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.
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