Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.

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 ( p fusion ) 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 p fusion of SVs with a mature release machinery but reflect both p fusion 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.

Recent progress in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) research led to high-purity preparations of human cardiomyocytes (CMs) differentiated from these two sources-suitable for tissue regeneration, in vitro models of disease, and cardiac safety pharmacology screening. We performed a detailed characterization of the effects of nifedipine, cisapride, and tetrodotoxin (TTX) on Cor.4UÒ human iPSC-CM, using automated whole-cell patch-clamp recordings with the CytoPatch Ô 2 equipment, within a complex assay combining multiple voltage-clamp and current-clamp protocols in a well-defined sequence, and quantitative analysis of several action potential (AP) parameters. We retrieved three electrical phenotypes based on AP shape: ventricular, atrial/nodal, and Stype (with ventricular-like depolarization and lack of plateau). To suppress spontaneous firing, present in many cells, we injected continuously faint hyperpolarizing currents of -10 or -20 pA. We defined quality criteria (both seal and membrane resistance over 1 GO), and focused our study on cells with ventricular-like AP. Nifedipine induced marked decreases in AP duration (APD): APD90 (49.8% and 40.8% of control values at 1 and 10 lM, respectively), APD50 (16.1% and 12%); cisapride 0.1 lM increased APD90 to 176.2%; and tetrodotoxin 10 lM decreased maximum slope of phase to 33.3% of control, peak depolarization potential to 76.3% of control, and shortened APD90 on average to 80.4%. These results prove feasibility of automated voltage-and current-clamp recordings on human iPSC-CM and their potential use for in-depth drug evaluation and proarrhythmic liability assessment, as well as for diagnosis and pharmacology tests for cardiac channelopathy patients.

Non-technical summary The release of neurotransmitter from presynaptic nerve endings is triggered by Ca 2+ influx through voltage-gated Ca 2+ channels (VGCCs) that open when an action potential (AP) invades the presynaptic terminal. The functional properties of VGCCs expressed in presynaptic terminals remain elusive because most terminals are too small to be accessible to electrophysiological recordings. We performed direct presynaptic recordings to characterize Ca 2+ channels and transmitter release in a large mammalian presynaptic terminal, the endbulb of Held. Endbulb terminals are formed by the endings of auditory nerve fibres that contact bushy cells located in the anterior ventral cochlear nucleus. We find that endbulb terminals are endowed with >1000 readily releasable vesicles and express an average number of >6000 VGCCs. About half of the VGCCs open during a single AP. Thus, multiple Ca 2+ channels control the release of a single transmitter vesicle at the endbulb of Held.Abstract 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 endbulb→SBC synapse.