Impact of spatiotemporal calcium dynamics within presynaptic active zones on synaptic delay at the frog neuromuscular junction.

The spatiotemporal calcium dynamics within presynaptic neurotransmitter release sites (active zones, AZs) at the time of synaptic vesicle fusion is critical for shaping the dynamics of neurotransmitter release. Specifically, the relative arrangement and density of voltage-gated calcium channels (VGCCs) as well as the concentration of calcium buffering proteins can play a large role in the timing, magnitude, and plasticity of release by shaping the AZ calcium profile. However, a high-resolution understanding of the role of AZ structure in spatiotemporal calcium dynamics and how it may contribute to functional heterogeneity at an adult synapse is currently lacking. We demonstrate that synaptic delay varies considerably across, but not within, individual synapses at the frog neuromuscular junction (NMJ). To determine how elements of the AZ could contribute to this variability, we performed a parameter search using a spatially realistic diffusion reaction-based computational model of a frog NMJ AZ (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751-2763, 2013; Ma J, Kelly L, Ingram J, Price TJ, Meriney SD, Dittrich M. J Neurophysiol 113: 71-87, 2015). We demonstrate with our model that synaptic delay is sensitive to significant alterations in the spatiotemporal calcium dynamics within an AZ at the time of release caused by manipulations of the density and organization of VGCCs or by the concentration of calcium buffering proteins. Furthermore, our data provide a framework for understanding how AZ organization and structure are important for understanding presynaptic function and plasticity. NEW & NOTEWORTHY The structure of presynaptic active zones (AZs) can play a large role in determining the dynamics of neurotransmitter release across many model preparations by influencing the spatiotemporal calcium dynamics within the AZ at the time of vesicle fusion. However, less is known about how different AZ structural schemes may influence the timing of neurotransmitter release. We demonstrate that variations in AZ structure create different spatiotemporal calcium profiles that, in turn, lead to differences in synaptic delay.

Transmitter release site organization can predict synaptic function at the neuromuscular junction.

We have investigated the impact of transmitter release site (active zone; AZ) structure on synaptic function by physically rearranging the individual AZ elements in a previously published frog neuromuscular junction (NMJ) AZ model into the organization observed in a mouse NMJ AZ. We have used this strategy, purposefully without changing the properties of AZ elements between frog and mouse models (even though there are undoubtedly differences between frog and mouse AZ elements in vivo), to directly test how structure influences function at the level of an AZ. Despite a similarly ordered ion channel array substructure within both frog and mouse AZs, frog AZs are much longer and position docked vesicles in a different location relative to AZ ion channels. Physiologically, frog AZs have a lower probability of transmitter release compared with mouse AZs, and frog NMJs facilitate strongly during short stimulus trains in contrast with mouse NMJs that depress slightly. Using our computer modeling approach, we found that a simple rearrangement of the AZ building blocks of the frog model into a mouse AZ organization could recapitulate the physiological differences between these two synapses. These results highlight the importance of simple AZ protein organization to synaptic function. NEW & NOTEWORTHY A simple rearrangement of the basic building blocks in the frog neuromuscular junction model into a mouse transmitter release site configuration predicted the major physiological differences between these two synapses, suggesting that transmitter release site structure and organization is a strong predictor of function.

Unbiased Rare Event Sampling in Spatial Stochastic Systems Biology Models Using a Weighted Ensemble of Trajectories.

The long-term goal of connecting scales in biological simulation can be facilitated by scale-agnostic methods. We demonstrate that the weighted ensemble (WE) strategy, initially developed for molecular simulations, applies effectively to spatially resolved cell-scale simulations. The WE approach runs an ensemble of parallel trajectories with assigned weights and uses a statistical resampling strategy of replicating and pruning trajectories to focus computational effort on difficult-to-sample regions. The method can also generate unbiased estimates of non-equilibrium and equilibrium observables, sometimes with significantly less aggregate computing time than would be possible using standard parallelization. Here, we use WE to orchestrate particle-based kinetic Monte Carlo simulations, which include spatial geometry (e.g., of organelles, plasma membrane) and biochemical interactions among mobile molecular species. We study a series of models exhibiting spatial, temporal and biochemical complexity and show that although WE has important limitations, it can achieve performance significantly exceeding standard parallel simulation--by orders of magnitude for some observables.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

New insights into short-term synaptic facilitation at the frog neuromuscular junction.

Short-term synaptic facilitation occurs during high-frequency stimulation, is known to be dependent on presynaptic calcium ions, and persists for tens of milliseconds after a presynaptic action potential. We have used the frog neuromuscular junction as a model synapse for both experimental and computer simulation studies aimed at testing various mechanistic hypotheses proposed to underlie short-term synaptic facilitation. Building off our recently reported excess-calcium-binding-site model of synaptic vesicle release at the frog neuromuscular junction (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751-2763, 2013), we have investigated several mechanisms of short-term facilitation at the frog neuromuscular junction. Our studies place constraints on previously proposed facilitation mechanisms and conclude that the presence of a second class of calcium sensor proteins distinct from synaptotagmin can explain known properties of facilitation observed at the frog neuromuscular junction. We were further able to identify a novel facilitation mechanism, which relied on the persistent binding of calcium-bound synaptotagmin molecules to lipids of the presynaptic membrane. In a real physiological context, both mechanisms identified in our study (and perhaps others) may act simultaneously to cause the experimentally observed facilitation. In summary, using a combination of computer simulations and physiological recordings, we have developed a stochastic computer model of synaptic transmission at the frog neuromuscular junction, which sheds light on the facilitation mechanisms in this model synapse.

An excess-calcium-binding-site model predicts neurotransmitter release at the neuromuscular junction.

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.

Organization and function of transmitter release sites at the neuromuscular junction.

The neuromuscular junction is known as a strong and reliable synapse. It is strong because it releases an excess of chemical transmitter, beyond what is required to bring the postsynaptic muscle cell to threshold. Because the synapse can sustain suprathreshold muscle activation during short trains of action potentials, it is also reliable. The presynaptic mechanisms that lead to reliability during short trains of activity have only recently been elucidated. It appears that there are relatively few calcium channels in individual active zones, that channels open with a low probability during action potential stimulation and that even if channels open the resulting calcium flux only rarely triggers vesicle fusion. Thus, each synaptic vesicle may only associate with a small number of calcium channels, forming an unreliable single vesicle release site. Strength and reliability of the neuromuscular junction emerge as a result of its assembly from thousands of these unreliable single vesicle release sites. Hence, these synapses are strong while at the same time only releasing a small subset of available docked vesicles during each action potential, thus conserving transmitter release resources. This prevents significant depression during short trains of action potential activity and confers reliability.

Single-pixel optical fluctuation analysis of calcium channel function in active zones of motor nerve terminals.

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca(2+)) channels during an action potential and the number of such Ca(2+) channels within active zones of frog neuromuscular junctions. Analysis revealed ∼36 Ca(2+) channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (∼200-250). The probability that each channel opened during an action potential was only ∼0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca(2+) channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca(2+) influx through individual Ca(2+) channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones.

Use of the Airtraq laryngoscope for emergency intubation in the prehospital setting: a randomized control trial.

OBJECTIVES: The optical Airtraq laryngoscope (Prodol Meditec, Vizcaya, Spain) has been shown to have advantages when compared with direct laryngoscopy in difficult airway patients. Furthermore, it has been suggested that it is easy to use and handle even for inexperienced advanced life support providers. As such, we sought to assess whether the Airtraq may be a reliable alternative to conventional intubation when used in the prehospital setting. DESIGN, SETTING, AND PATIENTS: Prospective, randomized control trial in emergency patients requiring endotracheal intubation provided by anesthesiologists or emergency physicians responding with an emergency medical service helicopter or ground unit associated with the Department of Anesthesiology, General Hospital, Wiener Neustadt, Austria. MEASUREMENTS AND MAIN RESULTS: During the 18-month study period, 212 patients were enrolled. When the Airtraq was used as first-line airway device (n=106) vs. direct laryngoscopy (n=106), success rate was 47% vs. 99%, respectively (p<.001). Reasons for failed Airtraq intubation were related to the fiber-optic characteristic of this device (i.e., impaired sight due to blood and vomitus, n=11) or to assumed handling problems (i.e., cuff damage, tube misplacement, or inappropriate visualization of the glottis, n=24). In 54 of 56 patients where Airtraq intubation failed, direct laryngoscopy was successful on the first attempt; in the remaining two and in one additional case of failed direct laryngoscopy, the airway was finally secured employing the Fastrach laryngeal mask. There was no correlation between success rates and body mass index, age, indication for airway management, emergency medical service unit, or experience of the physicians. CONCLUSIONS: Based on these results, the use of the Airtraq laryngoscope as a primary airway device cannot be recommended in the prehospital setting without significant clinical experience obtained in the operation room. We conclude that the clinical learning process of the Airtraq laryngoscope is much longer than reported in the anesthesia literature.

Domain motion of individual F1-ATPase ß-subunits during unbiased molecular dynamics simulations.

F(1)-ATPase is the catalytic domain of F(1)F(o)-ATP synthase and consists of a hexameric arrangement of three noncatalytic α and three catalytic ß subunits. We have used unbiased molecular dynamics simulations with a total simulation time of 900 ns to investigate the dynamic relaxation properties of isolated ß-subunits as a step toward explaining the function of the integral F(1) unit. To this end, we simulated the open (ß(E)) and the closed (ß(TP)) conformations under unbiased conditions for up to 120 ns each using several samples. The simulations confirm that nucleotide-free ß(E) retains its open configuration over the course of the simulations. The same is true when the neighboring α subunits are included. The nucleotide-depleted as well as the nucleotide-bound isolated ß(TP) subunits show a significant trend toward the open conformation during our simulations, with one trajectory per case opening completely. Hence, our simulations suggest that the equilibrium conformation of a nucleotide-free ß-subunit is the open conformation and that the transition from the closed to the open conformation can occur on a time scale of a few tens of nanoseconds.

One nerve suffices: A clinically guided nerve ultrasound protocol for the differentiation of multifocal motor neuropathy (MMN) and amyotrophic lateral sclerosis (ALS).

OBJECTIVE: To investigate diagnostic accuracy of a nerve ultrasound (US) protocol that is individualized to a patient's clinical deficits for the differentiation of amyotrophic lateral sclerosis with predominant lower motoneuron disease (ALS/LMND) and multifocal motor neuropathy (MMN). METHODS: Single-center, prospective, examiner-blinded, diagnostic study in two cohorts. Cohort I (model development): Convenience sample of subjects with ALS/LMND or MMN according to revised El-Escorial or EFNS guidelines. Cohort II (model validation): Consecutively recruited treatment-naïve subjects with suspected diagnosis of ALS/LMND or MMN. Cutoffs for 28 different US values were determined by Receiver Operating Curve (ROC) in cohort I. Area Under The Curve (AUC) of US was compared to nerve conduction studies (NCS). Diagnostic accuracy of US protocols, individualized according to clinical deficits, was compared to former rigid non-individualized protocols and to random examination site selection in cohort II. RESULTS: 48 patients were recruited. In cohort I (28 patients), US had higher ROC AUCs than NCS, US 0.82 (0.12) (mean (standard deviation)), NCS (compound muscle action potential (CMAP) 0.60 (0.09), p < .001; two-sided t-test). US models based on the nerve innervating the clinically most affected muscles had higher correct classification rates (CCRs, 93%) in cohort II than former rigid protocols (85% and 80%), or models with random measurement site selection (66% and 80%). CONCLUSIONS: Clinically guided US protocols for differentiation of ALS/LMND from MMN increase diagnostic accuracy when compared to clinically unguided protocols. They also require less measurements sites to achieve this accuracy.

Rapid creation, Monte Carlo simulation, and visualization of realistic 3D cell models.

Spatially realistic diffusion-reaction simulations supplement traditional experiments and provide testable hypotheses for complex physiological systems. To date, however, the creation of realistic 3D cell models has been difficult and time-consuming, typically involving hand reconstruction from electron microscopic images. Here, we present a complementary approach that is much simpler and faster, because the cell architecture (geometry) is created directly in silico using 3D modeling software like that used for commercial film animations. We show how a freely available open source program (Blender) can be used to create the model geometry, which then can be read by our Monte Carlo simulation and visualization softwares (MCell and DReAMM, respectively). This new workflow allows rapid prototyping and development of realistic computational models, and thus should dramatically accelerate their use by a wide variety of computational and experimental investigators. Using two self-contained examples based on synaptic transmission, we illustrate the creation of 3D cellular geometry with Blender, addition of molecules, reactions, and other run-time conditions using MCell's Model Description Language (MDL), and subsequent MCell simulations and DReAMM visualizations. In the first example, we simulate calcium influx through voltage-gated channels localized on a presynaptic bouton, with subsequent intracellular calcium diffusion and binding to sites on synaptic vesicles. In the second example, we simulate neurotransmitter release from synaptic vesicles as they fuse with the presynaptic membrane, subsequent transmitter diffusion into the synaptic cleft, and binding to postsynaptic receptors on a dendritic spine.

PcrA helicase, a prototype ATP-driven molecular motor.

Despite extensive studies, the mechanisms underlying molecular motor function are still poorly understood. Key to the mechanisms is the coupling of ATP hydrolysis to conformational changes of the motor protein. To investigate this coupling, we have conducted combined quantum mechanical/molecular mechanical simulations of PcrA helicase, a strikingly simple motor that translocates unidirectionally along single-stranded DNA (ssDNA). Our results reveal a close similarity in catalytic site structure and reaction pathway to those of F1-ATPase, and these similarities include a proton relay mechanism important for efficient ATP hydrolysis and an "arginine finger" residue that is key to the coupling of the chemical reaction to protein conformational changes. By means of in silico mutation studies, we identified the residue Q254 as being crucial for the coupling of ssDNA translocation to the actual catalytic event. Based on the present result for PcrA helicase and previous findings for F1-ATPase, we propose a general mechanism of ATP-driven molecular motor function.

Presynaptic mechanisms controlling calcium-triggered transmitter release at the neuromuscular junction.

Calcium-triggered neurotransmission underlies most communication in the nervous system. Yet, despite the conserved and essential nature of this process, the molecular underpinnings of calcium-triggered neurotransmission have been difficult to study directly and our understanding to this date remains incomplete. Here we frame more recent efforts to understand this process with a historical perspective of the study of neurotransmitter release at the neuromuscular junction. We focus on the role of calcium channel distribution and organization relative to synaptic vesicles, as well as the nature of the calcium sensors that trigger release. Importantly, we provide a framework for understanding how the function of neurotransmitter release sites, or active zones, contributes to the function of the synapse as a whole.

Practically applicable nerve ultrasound models for the diagnosis of axonal and demyelinating hereditary motor and sensory neuropathies (HMSN).

PURPOSE: To develop specific diagnostic ultrasound (US) models for hereditary motor and sensory neuropathies (HMSN) in patients with primarily demyelinating or axonal polyneuropathies (PNP) according to standard nerve conduction studies (NCS) criteria. METHODS: Single-centre, examiner-blinded cross-sectional study in acquired PNP (consecutive recruitment strategy) and HMSN patients (convenience sample). Allocation into demyelinating or axonal phenotype via easily applicable NCS criteria. Assessment of single measurements by receiver-operating curve (ROC) analysis, development of diagnostic models based on the best measurement values in ROC. RESULTS: Of 85 enrolled subjects, 53 (62%) had HMSN and 32 (38%) acquired PNPs, and 60 subjects (71%) had demyelinating and 25 (29%) axonal PNP. ROC area under the curve of means of the z-transformed 5 best measurement values was 0.87 for demyelinating and 0.99 for axonal HMSN. Diagnostic models showed high accuracy for both demyelinating (84% sensitivity, 86% specificity) and axonal HMSN (100% sensitivity and specificity). As a measure of variability of morphologic changes, standard deviations of z-transformed measurements were compared for acquired PNP and HMSN. In contrast to previous reports of more homogenous nerve enlargements in HMSN, standard deviations were higher in HMSN than in acquired PNP. Additionally, the performance of previously published models for the diagnosis of HMSN in demyelinating PNP was compared. Previously published models showed lower sensitivities (50-58%), but comparable specificities (91-100%) when applied to NCS-criteria defined demyelinating PNP group. CONCLUSION: Diagnostic ultrasound models for HMSN in patients with demyelinating or axonal neuropathies show high accuracy and can contribute to differential diagnosis in clinical routine.

Diagnostic accuracy of nerve ultrasound in hereditary and sporadic non-entrapment neuropathies.

The objective of this study is to compare the diagnostic accuracy of nerve ultrasound (US) and nerve conduction studies (NCS) for acquired non-entrapment peripheral neuropathies (PNP) and hereditary motor and sensory neuropathies (HMSN) in a routine clinical setting. The methods are based on a single-center, prospective, examiner-blinded cross-sectional study on three subject groups of healthy controls, PNP (both enrolled by a consecutive recruitment strategy), and HMSN patients (convenience sample). A clinical reference standard based on the neuropathy impairment (NIS) and neuropathy symptoms scores (NSS) was used for PNP as the external validation criterion. Diagnostic accuracy was assessed by receiver-operating curve (ROC) analyses of single-nerve measurements and logit models. Of a total of 676 consecutively screened subjects, 107 (15.8 %) were recruited, of which 36 (33.6 %) had a PNP. HMSN group consisted of 53 subjects (30 subjects (56.6 %) with genetic confirmation). AUCs of best diagnostic logit models to distinguish between controls and PNP patients were 0.86 for US and 0.97 for NCS corresponding to an equivalent specificity [US 93 % (95 % CI: 83-98 %), NCS 89 % (95 % CI: 78-95 %)], but inferior sensitivity of US [US 56 % (95 % CI: 35-74 %), NCS 97 % (95 % CI: 84-100 %)]. For differentiation between PNP and HMSN, both methods had equivalent AUCs of 0.95 corresponding to similar sensitivities/specificities. Simpler diagnostic models based on measurement protocols feasible for clinical routine revealed similar diagnostic accuracies. US has an inferior sensitivity than NCS for acquired PNP, but comparable specificity. For identification of HMSN in a PNP population, US and NCS show comparable performance.

Nerve ultrasound in the differentiation of multifocal motor neuropathy (MMN) and amyotrophic lateral sclerosis with predominant lower motor neuron disease (ALS/LMND).

The objective of the study was to investigate nerve ultrasound (US) in comparison to nerve conduction studies (NCS) for differential diagnosis of amyotrophic lateral sclerosis with predominant lower motoneuron disease(ALS/LMND) and multifocal motor neuropathy(MMN). A single-center, prospective, examiner-blinded cross-sectional diagnostic study in two cohorts was carried out. Cohort I: convenience sample of subjects diagnosed with ALS/LMND or MMN (minimal diagnostic criteria:possible ALS (revised EL-Escorial criteria), possible MMN (European Federation of Neurosciences guidelines).Cohort II: consecutive subjects with suspected diagnosis of either ALS/LMND or MMN. Diagnostic US and NCS models were developed based on ROC analysis of 28 different US and 32 different NCS values measured in cohort I. Main outcome criterion was sensitivity/specificity of these models between ALS/LMND and MMN in cohort II.Cohort I consisted of 16 patients with ALS/LMND and 8 patients with MMN. For cohort II, 30 patients were recruited, 8 with ALS/LMND, 5 with MMN, and 17 with other diseases. In cohort I, the three best US measures showed higher mean ± SD areas under the curve than the respective NCS measures (0.99 ± 0.01 vs. 0.79 ± 0.03, p<0.001; two-sided t test). The US model with highest measurement efficacy (8 values) and diagnostic quality reached 100 % sensitivity and 92 % specificity for MMN in cohort II, while the respective NCS model (6 values, including presence of conduction blocks) reached 100 and 52 %. Nerve US is of high diagnostic accuracy for differential diagnosis of ALS/LMND and MMN. It might be superior to NCS in the diagnosis of MMN in hospital-admitted patients with this differential diagnosis.

When light falls in LOV: a quantum mechanical/molecular mechanical study of photoexcitation in Phot-LOV1 of Chlamydomonas reinhardtii.

Plants use sophisticated photosensing mechanisms to maximize their utilization of the available sunlight and to control developmental processes. The plant blue-light receptors of the Phot family mediate plant phototropism and contain two light, oxygen, and voltage (LOV)-sensitive domains as photoactive elements. Here, we report combined quantum mechanical/molecular mechanical simulations of the photocycle of a complete Phot-LOV1 domain from Chlamydomonas reinhardtii. We have investigated the electronic properties and structural changes that follow blue-light absorption. This permitted us to characterize the pathway for flavin-cysteinyl adduct formation, which was found to proceed via a neutral radical state generated by hydrogen atom transfer from the reactive cysteine residue, Cys57, to the chromophore flavin mononucleotide. Interestingly, we find that adduct formation does not cause any larger scale conformational changes in Phot-LOV1, which suggests that dynamic effects mediate signal transmission following the initial photoexcitation event.

CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays.

The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.

Are unreliable release mechanisms conserved from NMJ to CNS?

The frog neuromuscular junction (NMJ) is a strong and reliable synapse because, during activation, sufficient neurotransmitter is released to trigger a postsynaptic action potential (AP). Recent evidence supports the hypothesis that this reliability emerges from the assembly of thousands of unreliable single vesicle release sites. The mechanisms that govern this unreliability include a paucity of voltage-gated calcium channels, a low probability of calcium channel opening during an AP, and the rare triggering of synaptic vesicle fusion even when a calcium channel does open and allows calcium flux. Here, we discuss the evidence that these unreliable single vesicle release sites may be the fundamental building blocks of many types of synapses in both the peripheral and central nervous system (PNS and CNS, respectively).