Development of electrophysiological properties of fusiform neurons from the dorsal cochlear nucleus of mice before and after hearing onset.

The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature DCN fusiform neurons fall into two qualitatively distinct types: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the prehearing phase (P4-P13), we found that most fusiform neurons are quiet, with active neurons emerging after hearing onset at P14. Subthreshold properties underwent significant changes before hearing onset, whereas changes to the action potential waveform occurred mainly after P14, with the depolarization and repolarization phases becoming markedly faster and half-width significantly decreased. The activity threshold in posthearing neurons was more negative than in prehearing cells. Persistent sodium current (INaP) was increased after P14, coinciding with the emergence of spontaneous firing. Thus, we suggest that posthearing expression of INaP leads to hyperpolarization of the activity threshold and the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.NEW & NOTEWORTHY Auditory brainstem neurons express unique electrophysiological properties adapted for their complex physiological functions that develop before hearing onset. Fusiform neurons of the DCN present two firing states, quiet and active, but the origin of these states is not known. Here, we showed that the quiet and active states develop after hearing onset at P14, along with changes in action potentials, suggesting an influence of auditory input on the refining of fusiform neuron's excitability.

Thiamine Deficiency Increases Intrinsic Excitability of Mouse Cerebellar Purkinje Cells.

Thiamine deficiency is associated with cerebellar dysfunction; however, the consequences of thiamine deficiency on the electrophysiological properties of cerebellar Purkinje cells are poorly understood. Here, we evaluated these parameters in brain slices containing cerebellar vermis. Adult mice were maintained for 12-13 days on a thiamine-free diet coupled with daily injections of pyrithiamine, an inhibitor of thiamine phosphorylation. Morphological analysis revealed a 20% reduction in Purkinje cell and nuclear volume in thiamine-deficient animals compared to feeding-matched controls, with no reduction in cell count. Under whole-cell current clamp, thiamine-deficient Purkinje cells required significantly less current injection to fire an action potential. This reduction in rheobase was not due to a change in voltage threshold. Rather, thiamine-deficient neurons presented significantly higher input resistance specifically in the voltage range just below threshold, which increases their sensitivity to current at these critical membrane potentials. In addition, thiamine deficiency caused a significant decrease in the amplitude of the action potential afterhyperpolarization, broadened the action potential, and decreased the current threshold for depolarization block. When thiamine-deficient animals were allowed to recover for 1 week on a normal diet, rheobase, threshold, action potential half-width, and depolarization block threshold were no longer different from controls. We conclude that thiamine deficiency causes significant but reversible changes to the electrophysiology properties of Purkinje cells prior to pathological morphological alterations or cell loss. Thus, the data obtained in the present study indicate that increased excitability of Purkinje cells may represent a leading indicator of cerebellar dysfunction caused by lack of thiamine.

Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells.

The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by ∼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with ß-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, ß-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, ß-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Thiamine Deficiency Increases Ca2+ Current and CaV1.2 L-type Ca2+ Channel Levels in Cerebellum Granular Neurons.

Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer's disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca2+ current density and CaV1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca2+ dynamics, two factors that have been implicated in neurodegeneration.

Functional and structural properties of ion channels at the nerve terminal depends on compact myelin.

KEY POINTS: In the present study, we document the role of compact myelin in regulating the structural and functional properties of ion channels at the nerve terminals, using electrophysiology, dynamic Na(+) imaging and immunohistochemistry. The subcellular segregation of Na(+) channel expression and intracellular Na(+) dynamics at the heminode and terminal was lost in the dysmyelinated axon from Long-Evans shaker rats, which lack compact myelin. In Long-Evans shaker rats, loss of the Nav ß4 subunit specifically at the heminode reduced resurgent and persistent Na(+) currents, whereas K(+) channel expression and currents were increased. The results of the present study suggest that there is a specific role for compact myelin in dictating protein expression and function at the axon heminode and in regulating excitability of the nerve terminal. ABSTRACT: Axon myelination increases the conduction velocity and precision of action potential propagation. Although the negative effects of demyelination are generally attributed to conduction failure, accumulating evidence suggests that myelination also regulates the structural properties and molecular composition of the axonal membrane. In the present study, we investigated how myelination affects ion channel expression and function, particularly at the last axon heminode before the nerve terminal, which regulates the presynaptic excitability of the nerve terminal. We compared the structure and physiology of normal axons and those of the Long-Evans shaker (LES) rat, which lacks compact myelin. The normal segregation of Na(+) channel expression and dynamics at the heminode and terminal was lost in the LES rat. Specifically, NaV -α subunits were dispersed and NaV ß4 subunit was absent, whereas the density of K(+) channels was increased at the heminode. Correspondingly, resurgent and persistent Na(+) currents were reduced and K(+) current was increased. Taken together, these data suggest a specific role for compact myelin in the orchestration of ion channel expression and function at the axon heminode and in regulating excitability of the nerve terminal.

Glycolysis selectively shapes the presynaptic action potential waveform.

Mitochondria are major suppliers of cellular energy in neurons; however, utilization of energy from glycolysis vs. mitochondrial oxidative phosphorylation (OxPhos) in the presynaptic compartment during neurotransmission is largely unknown. Using presynaptic and postsynaptic recordings from the mouse calyx of Held, we examined the effect of acute selective pharmacological inhibition of glycolysis or mitochondrial OxPhos on multiple mechanisms regulating presynaptic function. Inhibition of glycolysis via glucose depletion and iodoacetic acid (1 mM) treatment, but not mitochondrial OxPhos, rapidly altered transmission, resulting in highly variable, oscillating responses. At reduced temperature, this same treatment attenuated synaptic transmission because of a smaller and broader presynaptic action potential (AP) waveform. We show via experimental manipulation and ion channel modeling that the altered AP waveform results in smaller Ca2+ influx, resulting in attenuated excitatory postsynaptic currents (EPSCs). In contrast, inhibition of mitochondria-derived ATP production via extracellular pyruvate depletion and bath-applied oligomycin (1 µM) had no significant effect on Ca2+ influx and did not alter the AP waveform within the same time frame (up to 30 min), and the resultant EPSC remained unaffected. Glycolysis, but not mitochondrial OxPhos, is thus required to maintain basal synaptic transmission at the presynaptic terminal. We propose that glycolytic enzymes are closely apposed to ATP-dependent ion pumps on the presynaptic membrane. Our results indicate a novel mechanism for the effect of hypoglycemia on neurotransmission. Attenuated transmission likely results from a single presynaptic mechanism at reduced temperature: a slower, smaller AP, before and independent of any effect on synaptic vesicle release or receptor activity.

Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.

INTRODUCTION: Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol triphosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. METHODS: We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine, 2-aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U- 73122). RESULTS: Ryanodine decreases paired-pulse facilitation for intervals <20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals of <20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired-pulse depression for intervals <20 ms. CONCLUSIONS: Ryanodine, IP3 receptors, and phospholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets for improving the safety factor of neuromuscular transmission during repetitive activity of the neuromuscular junction.

Characteristics of dorsal root ganglia neurons sensitive to Substance P.

BACKGROUND: Substance P modulates ion channels and the excitability of sensory neurons in pain pathways. Within the heterogeneous population of Dorsal Root Ganglia (DRG) primary sensory neurons, the properties of cells that are sensitive to Substance P are poorly characterized. To define this population better, dissociated rat DRG neurons were tested for their responsiveness to capsaicin, ATP and acid. Responses to ATP were classified according to the kinetics of current activation and desensitization. The same cells were then tested for modulation of action potential firing by Substance P. RESULTS: Acid and capsaicin currents were more frequently encountered in the largest diameter neurons. P2X3-like ATP currents were concentrated in small diameter neurons. Substance P modulated the excitability in 20 of 72 cells tested (28%). Of the Substance P sensitive cells, 10 exhibited an increase in excitability and 10 exhibited a decrease in excitability. There was no significant correlation between sensitivity to capsaicin and to Substance P. Excitatory effects of Substance P were strongly associated with cells that had large diameters, fired APs with large overshoots and slowly decaying after hyperpolarizations, and expressed acid currents at pH 7. No neurons that were excited by Substance P presented P2X3-like currents. In contrast, neurons that exhibited inhibitory effects of Substance P fired action potentials with rapidly decaying after hyperpolarizations. CONCLUSION: We conclude that excitatory effects of Substance P are restricted to a specific neuronal subpopulation with limited expression of putative nociceptive markers.

Long-term application of adrenergic agonists modulates nociceptive ion channels.

Dorsal root ganglia (DRG) neurons transduce and convey somatosensory information from the periphery to the central nervous system. Adrenergic mediators are known to modulate nociceptive inputs in DRG neurons, acting as up- or down-regulators of neuronal excitability. They are also important in the development of sympathetic neuropathy. ATP-activated P2X channels and capsaicin-activated TRPV1 channels are directly involved in the transduction of nociceptive stimuli. In this work, we show that long-term (up to 3 days) in vitro stimulation of DRG neurons with selective α1-adrenergic agonist increased slow but not fast ATP-activated currents, with no effect on capsaicin currents. Selective agonists for α2, ß1 and ß3-adrenergic receptors decreased capsaicin activated currents and had no effect on ATP currents. Capsaicin currents were associated with increased neuronal excitability, while none of the adrenergic modulators produced change in rheobase. These results demonstrate that chronic adrenergic activation modulates two nociceptive transducer molecules, increasing or decreasing channel current depending on the adrenergic receptor subtype. These observations aid our understanding of nociceptive or antinociceptive effects of adrenergic agonists.

Central dysmyelination reduces the temporal fidelity of synaptic transmission and the reliability of postsynaptic firing during high-frequency stimulation.

Auditory brain stem circuits rely on fast, precise, and reliable neurotransmission to process auditory information. To determine the fundamental role of myelination in auditory brain stem function, we examined the evoked auditory brain stem response (ABR) from the Long Evans shaker (LES) rat, which lacks myelin due to a genetic deletion of myelin basic protein. In control rats, the ABR evoked by a click consisted of five well-defined waves (denoted waves I-V). In LES rats, waves I, IV, and V were present, but waves II and III were undetectable, indicating disrupted function in the earliest stages of central nervous system auditory processing. In addition, the developmental shortening of the interval between waves I and IV that normally occurs in control rats was arrested and resulted in a significant increase in the central conduction time in LES rats. In brain stem slices, action potential transmission between the calyx of Held terminals and the medial nucleus of the trapezoid body (MNTB) neurons was delayed and less reliable in LES rats, although the resting potential, threshold, input resistance, and length of the axon initial segment of the postsynaptic MNTB neurons were normal. The amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) and the degree of synaptic depression during high-frequency stimulation were not different between LES rats and controls, but LES rats exhibited a marked slow component to the EPSC decay and a much higher rate of presynaptic failures. Together, these results indicate that loss of myelin disrupts brain stem auditory processing, increasing central conduction time and reducing the reliability of neurotransmission.

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction.

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-ß-cyclodextrin (MßCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MßCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MßCD were larger than those from control. In addition, treatment with MßCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MßCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

Microscopy-based cellular contractility assay for adult, neonatal, and hiPSC cardiomyocytes.

This protocol provides instructions to acquire high-quality cellular contractility data from adult, neonatal, and human induced pluripotent stem cell-derived cardiomyocytes. Contractility parameters are key to unravel mechanisms underlying cardiac pathologies, yet difficulties in acquiring data can compromise measurement accuracy and reproducibility. We provide optimized steps for microscope and camera setup, as well as cellular selection criteria for different cardiomyocyte cell types, aiming to obtain robust and reliable data. Moreover, we use CONTRACTIONWAVE software to analyze and show the optimized results. For complete details on the use and execution of this profile, please refer to Scalzo et al. (2021).

Hippocampal neurons maintain a large PtdIns(4)P pool that results in faster PtdIns(4,5)P2 synthesis.

PtdIns(4,5)P2 is a signaling lipid central to the regulation of multiple cellular functions. It remains unknown how PtdIns(4,5)P2 fulfills various functions in different cell types, such as regulating neuronal excitability, synaptic release, and astrocytic function. Here, we compared the dynamics of PtdIns(4,5)P2 synthesis in hippocampal neurons and astrocytes with the kidney-derived tsA201 cell line. The experimental approach was to (1) measure the abundance and rate of PtdIns(4,5)P2 synthesis and precursors using specific biosensors, (2) measure the levels of PtdIns(4,5)P2 and its precursors using mass spectrometry, and (3) use a mathematical model to compare the metabolism of PtdIns(4,5)P2 in cell types with different proportions of phosphoinositides. The rate of PtdIns(4,5)P2 resynthesis in hippocampal neurons after depletion by cholinergic or glutamatergic stimulation was three times faster than for tsA201 cells. In tsA201 cells, resynthesis of PtdIns(4,5)P2 was dependent on the enzyme PI4K. In contrast, in hippocampal neurons, the resynthesis rate of PtdIns(4,5)P2 was insensitive to the inhibition of PI4K, indicating that it does not require de novo synthesis of the precursor PtdIns(4)P. Measurement of phosphoinositide abundance indicated a larger pool of PtdIns(4)P, suggesting that hippocampal neurons maintain sufficient precursor to restore PtdIns(4,5)P2 levels. Quantitative modeling indicates that the measured differences in PtdIns(4)P pool size and higher activity of PI4K can account for the experimental findings and indicates that high PI4K activity prevents depletion of PtdIns(4)P. We further show that the resynthesis of PtdIns(4,5)P2 is faster in neurons than astrocytes, providing context to the relevance of cell type-specific mechanisms to sustain PtdIns(4,5)P2 levels.

Presynaptic resurgent Na+ currents sculpt the action potential waveform and increase firing reliability at a CNS nerve terminal.

Axonal and nerve terminal action potentials often display a depolarizing after potential (DAP). However, the underlying mechanism that generates the DAP, and its impact on firing patterns, are poorly understood at axon terminals. Here, we find that at calyx of Held nerve terminals in the rat auditory brainstem the DAP is blocked by low doses of externally applied TTX or by the internal dialysis of low doses of lidocaine analog QX-314. The DAP is thus generated by a voltage-dependent Na(+) conductance present after the action potential spike. Voltage-clamp recordings from the calyx terminal revealed the expression of a resurgent Na(+) current (I(NaR)), the amplitude of which increased during early postnatal development. The calyx of Held also expresses a persistent Na(+) current (I(NaP)), but measurements of calyx I(NaP) together with computer modeling indicate that the fast deactivation time constant of I(NaP) minimizes its contribution to the DAP. I(NaP) is thus neither sufficient nor necessary to generate the calyx DAP, whereas I(NaR) by itself can generate a prominent DAP. Dialysis of a small peptide fragment of the auxiliary ß4 Na(+) channel subunit into immature calyces (postnatal day 5-6) induced an increase in I(NaR) and a larger DAP amplitude, and enhanced the spike-firing precision and reliability of the calyx terminal. Our results thus suggest that an increase of I(NaR) during postnatal synaptic maturation is a critical feature that promotes precise and resilient high-frequency firing.

Dense optical flow software to quantify cellular contractility.

Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics. For this reason, we develop CONTRACTIONWAVE, high-performance software written in Python programming language that allows the user to process large data image files and obtain contractility parameters by analyzing optical flow from images obtained with videomicroscopy. The software was validated by using neonatal, adult-, and human-induced pluripotent stem-cell-derived cardiomyocytes, treated or not with drugs known to affect contractility. Results presented indicate that CONTRACTIONWAVE is an excellent tool for examining changes to cardiac cellular contractility in animal models of disease and for pharmacological and toxicology screening during drug discovery.

Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition.

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

Reliability and precision of the mouse calyx of Held synapse.

Traditionally, the calyx of Held synapse is viewed as a highly reliable relay in the sound localization circuit of the auditory brainstem, with every presynaptic action potential triggering a postsynaptic action potential in vivo. However, this view is at odds with slice recordings that report large short-term depression (STD). To investigate the reliability and precision of this synapse, we compared slice and in vivo recordings from medial nucleus of the trapezoid body neurons of young adult mice. We show that the extracellularly recorded complex waveform can be used to estimate both presynaptic release and postsynaptic excitability. Whereas under standard slice conditions the synapse underwent large STD, both extracellular and whole-cell recordings indicated that in vivo the size of the EPSPs was independent of recent history. The estimated quantal content was typically <20 in vivo, much lower than in the resting synapse under standard slice conditions. However, due to the large quantal size and summation of EPSPs, the safety factor of this synapse was generally still sufficiently large and postsynaptic failures were observed only infrequently in vivo. When present, failures were typically due to stochastic fluctuations in EPSP size or postsynaptic spike depression. In vivo, the calyx of Held synapse thus functions as a tonic synapse. The price it pays for its low release probability is an increase in jitter and synaptic latency and occasional postsynaptic failures.