scholarly journals RIM1 and RIM2 redundantly determine Ca2+ channel density and readily releasable pool size at a large hindbrain synapse

2015 ◽  
Vol 113 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Yunyun Han ◽  
Norbert Babai ◽  
Pascal Kaeser ◽  
Thomas C. Südhof ◽  
Ralf Schneggenburger
Keyword(s):  
Pdz Domain ◽  
Decisive Role ◽  
Scaffolding Proteins ◽  
Calyx Of Held ◽  
C2 Domains ◽  
Voltage Gated ◽  
Channel Density ◽  
Readily Releasable Pool ◽  
Vesicle Pool ◽  
Kinetics Of

The localization and density of voltage-gated Ca2+ channels at active zones are essential for the amount and kinetics of transmitter release at synapses. RIM proteins are scaffolding proteins at the active zone that bind to several other presynaptic proteins, including voltage-gated Ca2+ channel α-subunits. The long isoforms of RIM proteins, which contain NH2-terminal Rab3- and Munc13-interacting domains, as well as a central PDZ domain and two COOH-terminal C2 domains, are encoded by two genes, Rim1 and Rim2. Here, we used the ideal accessibility of the large calyx of Held synapse for direct presynaptic electrophysiology to investigate whether the two Rim genes have redundant, or separate, functions in determining the presynaptic Ca2+ channel density, and the size of a readily releasable vesicle pool (RRP). Quantitative PCR showed that cochlear nucleus neurons, which include calyx of Held generating neurons, express both RIM1 and RIM2. Conditional genetic inactivation of RIM2 at the calyx of Held led to a subtle reduction in presynaptic Ca2+ current density, whereas deletion of RIM1 was ineffective. The release efficiency of brief presynaptic Ca2+ “tail” currents and the RRP were unaffected in conditional single RIM1 and RIM2 knockout (KO) mice, whereas both parameters were strongly reduced in RIM1/2 double KO mice. Thus, despite a somewhat more decisive role for RIM2 in determining presynaptic Ca2+ channel density, RIM1 and RIM2 can overall replace each other's presynaptic functions at a large relay synapse in the hindbrain, the calyx of Held.

scholarly journals Quantitative analysis of vesicle recycling at the calyx of Held synapse

2015 ◽  
Vol 112 (15) ◽  
pp. 4779-4784 ◽  
Author(s):  
Xufeng Qiu ◽  
Qianwen Zhu ◽  
Jianyuan Sun
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Kinetic Model ◽  
Temporal Resolution ◽  
Nerve Terminal ◽  
Calyx Of Held ◽  
Vesicle Recycling ◽  
Readily Releasable Pool ◽  
Vesicle Pool ◽  
Kinetics Of

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).

The Pool of Fast Releasing Vesicles Is Augmented by Myosin Light Chain Kinase Inhibition at the Calyx of Held Synapse

2008 ◽  
Vol 99 (4) ◽  
pp. 1810-1824 ◽  
Author(s):  
Geetha Srinivasan ◽  
Jun Hee Kim ◽  
Henrique von Gersdorff
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Regulatory Protein ◽  
Developmental Studies ◽  
Calyx Of Held ◽  
Release Probability ◽  
Readily Releasable Pool ◽  
Action Potential Waveform ◽  
Vesicle Pool

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca2+ currents or action potential waveform. Moreover, Ca2+ imaging experiments showed that [Ca2+]i transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca2+ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.

scholarly journals Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas A. Courtney ◽  
Huan Bao ◽  
Joseph S. Briguglio ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Major Focus ◽  
C2 Domains ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain

1992 ◽  
Vol 262 (4) ◽  
pp. R712-R715 ◽  
Author(s):  
M. A. Perez-Pinzon ◽  
M. Rosenthal ◽  
T. J. Sick ◽  
P. L. Lutz ◽  
J. Pablo ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Tissue Oxygenation ◽  
Mammalian Brain ◽  
Survival Strategy ◽  
Voltage Gated ◽  
Channel Density ◽  
Spike Threshold ◽  
Channel Arrest ◽  
Key Factor

In contrast to mammalian brain, which exhibits rapid degeneration during anoxia, the brains of certain species of turtles show an extraordinary capacity to survive prolonged anoxia. The decrease in energy expenditure shown by the anoxic turtle brain is likely to be a key factor for anoxic survival. The "channel arrest" hypothesis proposes that ion channels, which regulate brain electrical activity in normoxia, may be altered during anoxia in the turtle brain as a mechanism to spare energy. Goals of present research were to test this hypothesis and to determine whether down-regulation of sodium channels is a possible explanation for spike threshold shifts seen during anoxia in isolated turtle cerebellum. We report here that anoxia induced a significant (42%) decline in voltage-gated sodium channel density as determined by studies of the binding of a sodium channel ligand, [3H]brevetoxin. This study demonstrates that sodium channel densities in brain may be regulated by tissue oxygenation or by physiological events associated with anoxia. Moreover, it also suggests that downregulation of sodium channels may be a basis for changes in action potential thresholds, the electrical depression and energy conservation that provide the unique anoxic tolerance of turtle brain.

scholarly journals Determinants of synaptic strength vary across an axon arbor

2012 ◽  
Vol 107 (9) ◽  
pp. 2430-2441 ◽  
Author(s):  
Xiaoyu Peng ◽  
Thomas D. Parsons ◽  
Rita J. Balice-Gordon
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Synaptic Strength ◽  
Pool Size ◽  
Spatial Modulation ◽  
Readily Releasable Pool ◽  
Single Axon ◽  
Individual Vesicle ◽  
Highly Correlated ◽  
Vesicle Pool

We used synaptophysin-pHluorin expressed in hippocampal neurons to address how functional properties of terminals, namely, evoked release, total vesicle pool size, and release fraction, vary spatially across individual axon arbors. Consistent with previous reports, over short arbor distances (∼100 μm), evoked release was spatially heterogeneous when terminals contacted different postsynaptic dendrites or neurons. Regardless of the postsynaptic configuration, the evoked release and total vesicle pool size spatially covaried, suggesting that the fraction of synaptic vesicles available for release (release fraction) was similar over short distances. Evoked release and total vesicle pool size were highly correlated with the amount of NMDA receptors and PSD-95 in postsynaptic specialization. However, when individual axons were followed over longer distances (several hundred micrometers), a significant increase in evoked release was observed distally that was associated with an increased release fraction in distal terminals. The increase in distal release fraction can be accounted for by changes in individual vesicle release probability as well as readily releasable pool size. Our results suggest that for a single axon arbor, presynaptic strength indicated by evoked release over short distances is correlated with heterogeneity in total vesicle pool size, whereas over longer distances presynaptic strength is correlated with the spatial modulation of release fraction. Thus the mechanisms that determine synaptic strength differ depending on spatial scale.

scholarly journals Erratum: A Three-Pool Model Dissecting Readily Releasable Pool Replenishment at the Calyx of Held

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jun Guo ◽  
Jian-long Ge ◽  
Mei Hao ◽  
Zhi-cheng Sun ◽  
Xin-sheng Wu ◽  
...  
Keyword(s):  
Calyx Of Held ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Pool Model

scholarly journals Ca2+-Dependent, Stimulus-Specific Modulation of the Plasma Membrane Ca2+ Pump in Hippocampal Neurons

2009 ◽  
Vol 101 (5) ◽  
pp. 2563-2571 ◽  
Author(s):  
Michael J. Ferragamo ◽  
Jessica L. Reinardy ◽  
Stanley A. Thayer
Keyword(s):  
Plasma Membrane ◽  
Neuronal Activity ◽  
Action Potentials ◽  
Hippocampal Neurons ◽  
Cyclopiazonic Acid ◽  
Synaptic Activity ◽  
Voltage Gated ◽  
Clearance Kinetics ◽  
Transient Elevation ◽  
Kinetics Of

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in restoring Ca2+ to basal levels following transient elevation by neuronal activity. Here we examined the effects of various stimuli that increase [Ca2+]i on PMCA-mediated Ca2+ clearance from hippocampal neurons. We used indo-1-based microfluorimetry in the presence of cyclopiazonic acid to study the rate of PMCA-mediated recovery of Ca2+ elevated by a brief train of action potentials. [Ca2+]i recovery was described by an exponential decay and the time constant provided an index of PMCA-mediated Ca2+ clearance. PMCA function was assessed before and for ≥60 min following a 10-min priming stimulus of either 100 μM N-methyl-d-aspartate (NMDA), 0.1 mM Mg2+ (reduced extracellular Mg2+ induces intense excitatory synaptic activity), 30 mM K+, or control buffer. Recovery kinetics slowed progressively following priming with NMDA or 0.1 mM Mg2+; in contrast, Ca2+ clearance initially accelerated and then slowly returned to initial rates following priming with 30 mM K+-induced depolarization. Treatment with 10 μM calpeptin, an inhibitor of the Ca2+ activated protease calpain, prevented the slowing of kinetics observed following treatment with NMDA but had no affect on the recovery kinetics of control cells. Calpeptin also blocked the rapid acceleration of Ca2+ clearance following depolarization. In calpeptin-treated cells, 0.1 mM Mg2+ induced a graded acceleration of Ca2+ clearance. Thus in spite of producing comparable increases in [Ca2+]i, activation of NMDA receptors, depolarization-induced activation of voltage-gated Ca2+ channels and excitatory synaptic activity each uniquely affected Ca2+ clearance kinetics mediated by the PMCA.

scholarly journals Aczonin, a 550-Kd Putative Scaffolding Protein of Presynaptic Active Zones, Shares Homology Regions with Rim and Bassoon and Binds Profilin

1999 ◽  
Vol 147 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Xiaolu Wang ◽  
Mark Kibschull ◽  
Michael M. Laue ◽  
Beate Lichte ◽  
Elisabeth Petrasch-Parwez ◽  
...  
Keyword(s):  
Active Zone ◽  
Pdz Domain ◽  
Amino Acid Sequences ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Molecular Architecture ◽  
C2 Domains ◽  
Cytoskeletal Dynamics ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys4 zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.

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