Rate Of Transmitter Release Research Articles

RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

Fusion Pore Selectivity in Chromaffin Cells

Healthy physiological conditions depend on dynamic release of neurotransmitters and hormones by exocytosis, which is initiated by the fusion pore. Previous studies show that reduction of extracellular [NaCl] decreases the amperometric foot current, indicating no co-release of anions with transmitter. It is unknown if this is due to cation selectivity of the fusion pore or due to negatively charged ions, such as ATP, being bound to the granular matrix and unavailable for release. Such selectivity might be expected if the fusion pore is lined by negative charges from lipids and protein. If the initial fusion pore is selective for cations, then its selectivity will affect the rate of transmitter release via the reversal potential across the fusion pore. If the fusion pore is non-selective, then the flux of anions from extracellular space into the vesicle is expected to reduce the rate of release of positively charged catecholamines by making the vesicle lumen more negative. Using amperometry we investigate in chromaffin cells if the transmitter release rate through the fusion pore changes when extracellular chloride is exchanged by the less mobile D-glutamate anion. Electrodiffusion calculations predict that for a non-selective anion permeable fusion pore the average foot current amplitude should increase by ∼30% when chloride is exchanged by D-glutamate with a mobility of 0.25 relative to chloride, whereas no change in foot current is expected for a cation selective fusion pore. Preliminary analysis of all amperometric events in chromaffin cells, results in a mean amperometric foot current of 1.14±0.139 pA in the presence of chloride, and 1.04±0.07 pA for D-glutamate. Further sample collections and analysis will reveal if there is a significant difference in foot current amplitude depending on anion mobility and reveal if the early narrow fusion pore is cation selective.

Use of Push-Pull Superfusion Technique for Identifying Neurotransmitters Involved in Brain Functions: Achievements and Perspectives.

The push-pull superfusion technique (PPST) is a procedure for in vivo examination of transmitter release in distinct brain areas. This technique allows to investigate dynamics of transmitter release both under normal and experimentally evoked conditions. The PPST can be modified so that it is possible to determine release of endogenous transmitters simultaneously with electroencephalogram (EEG) recordings, recordings of evoked potentials or the on-line determination of endogenous nitric oxide (NO) released into the synaptic cleft. Because of the good time resolution, the method provides further the possibility to modify the collection periods of superfusates depending on the neuronal function that is analyzed. For instance, investigation of central cardiovascular control, behavioral tasks or mnemonic processes requires very short collection periods, because changes in transmitter release occur within seconds. Even more important is the time resolution when rates of transmitter release are correlated with evoked extracellular potentials or EEG recordings. This review provides an overview of the different devices which might be combined with the PPST and perspectives for future work.

Ca2+ Uncaging in Nerve Terminals: A Three-Point Calibration Procedure.

Ca(2+) uncaging can be used to create a spatially homogenous elevation of the intracellular free Ca(2+) concentration, [Ca(2+)]i, in cells. When applied to nerve terminals or secretory cells, this technique allows one to elicit transmitter release with a [Ca(2+)]i signal of measurable amplitude, and therefore to directly relate the rate of transmitter release to the measured [Ca(2+)]i. When combined with patch-clamp measurements, Ca(2+) uncaging is done by introducing a Ca(2+)-loaded photolyzable Ca(2+) chelator (like DM-nitrophen) into the cell via the whole-cell patch-pipette. A brief light pulse from a flash lamp or a pulsed laser is used to photolyze the DM-nitrophen. The resulting increase in [Ca(2+)]i is measured with ratiometric fluorescent indicators of suitable Ca(2+) affinity, such as Fura-2, Fura-4F, Fura-2FF, or Fura-6F, depending on the postflash [Ca(2+)]i values. To quantitatively measure [Ca(2+)]i, an accurate calibration of the fluorescent indicator in the presence of the photolyzable Ca(2+) chelator is necessary, which will be described here. Ca(2+) uncaging in nerve terminals has proven useful for investigating Ca(2+)-dependent functions like transmitter release, short-term plasticity, and exocytosis-endocytosis coupling in the presynaptic compartment of neurons.

Activity dependent modulation of synaptic transmission by presynaptic calcium stores: A dichotomy of short-term depression and facilitation

Ongoing electrical activity can initiate a positive feedback loop via Inositol Triphosphate Receptors (IP3Rs) and Ryanodine Receptors (RyRs) on the Endoplasmic Receptors (ER) that can lead to release of calcium from the ER. We investigated how the presence of this additional source of calcium in the presynaptic terminals in addition to the Voltage Dependent Calcium Channels (VDCCs) can regulate synaptic transmission. We carried out 3D Monte Carlo simulations of all the molecular interactions that govern transmitter release in a 1) Canonical CA3-CA1 synapse 2) A synapse reconstructed from serial section Transmission Electron Microscope images. The relatively simple geometry of CA3-CA1 synapses allows activity-dependent local calcium at the active zone and the related transmitter release profiles to be quantitatively analyzed. In paired-pulse stimulation, the presence of the molecular pathways that regulate the calcium stores increased the calcium buffering capacity of the synapse, which decreased release probability and enhanced paired-pulse facilitation. In contrast, a high-frequency stimulus could trigger the activation of presynaptic RyRs and Metabotropic Glutamate Receptors (mGluRs) leading to IP3 production and ultimately to release of calcium from the SER. IP3Rs and RyRs operated at a much slower time scale, on the order of seconds compared to the millisecond timescale of the VDCCs. This led to an increase in the basal level of intracellular calcium and enhanced transmitter release rates. In reconstructions of hippocampal neuropil, the ER appears in a majority of the presynaptic terminals in varying degrees of abundance. We further explored the functional implications of the range of ER geometries and therefore the calcium carrying capacity, observed in the synaptic ultrastructure and the effect of different arrangements between IP3Rs and VDCCs on synaptic plasticity. The synaptic ultrastructure precisely orchestrated the degree of facilitation and depression at the synapse and the existence of presynaptic calcium stores provided the synapse with an additional intrinsic time scale that could be regulated by activity.

Rapid Synaptic Vesicle Endocytosis in Cone Photoreceptors of Salamander Retina

Following synaptic vesicle exocytosis, neurons retrieve the fused membrane by a process of endocytosis to provide a supply of vesicles for subsequent release and maintain the presynaptic active zone. Rod and cone photoreceptors use a specialized structure called the synaptic ribbon that enables them to sustain high rates of neurotransmitter release. They must also employ mechanisms of synaptic vesicle endocytosis capable of keeping up with release. While much is known about endocytosis at another retinal ribbon synapse, that of the goldfish Mb1 bipolar cell, less is known about endocytosis in photoreceptors. We used capacitance recording techniques to measure vesicle membrane fusion and retrieval in photoreceptors from salamander retinal slices. We found that application of brief depolarizing steps (<100 ms) to cones evoked exocytosis followed by rapid endocytosis with a time constant ∼250 ms. In some cases, the capacitance trace overshot the baseline, indicating excess endocytosis. Calcium had no effect on the time constant, but enhanced excess endocytosis resulting in a faster rate of membrane retrieval. Surprisingly, endocytosis was unaffected by blockers of dynamin, suggesting that cone endocytosis is dynamin independent. This contrasts with synaptic vesicle endocytosis in rods, which was inhibited by the dynamin inhibitor dynasore and GTPγS introduced through the patch pipette, suggesting that the two photoreceptor types employ distinct pathways for vesicle retrieval. The fast kinetics of synaptic vesicle endocytosis in photoreceptors likely enables these cells to maintain a high rate of transmitter release, allowing them to faithfully signal changes in illumination to second-order neurons.

Effects of Presynaptic Calcium Stores on Short-Term Synaptic Plasticity

In reconstructions of hippocampal neuropil, Smooth Endoplasmic Reticulum (SER) appears in a majority of the presynaptic terminals. In the presence of ongoing electrical activity, Inositol Triphosphate Receptors (IP3Rs) on the SER initiate a positive feedback loop that can lead to release of calcium from the SER via an IP3-mediated pathway. We investigated how the presence of this additional source of calcium in addition to the Voltage Dependent Calcium Channels (VDCCs) can regulate synaptic transmission. We carried out 3D Monte Carlo simulations of the molecular interactions that govern transmitter release in a 1) Canonical CA3-CA1 synapse 2) Synapse reconstructed from serial section Transmission Electron Microscope images. The relatively simple geometry of CA3-CA1 synapses allows activity-dependent local calcium at the active zone and the related transmitter release profiles to be quantitatively analyzed. In paired-pulse stimulation, the presence of molecular pathways that regulate the calcium stores increased the calcium buffering capacity of the synapse, which decreased the initial release probability and enhanced paired-pulse facilitation. In contrast, a high-frequency stimulus could trigger the activation of presynaptic Metabotropic Glutamate Receptors (mGluRs) leading to IP3 production and ultimately to release of calcium from the SER. IP3Rs operated at a much slower time scale, on the order of seconds compared to the millisecond timescale of the VDCCs. This led to an increase in the basal level of intracellular calcium and enhanced transmitter release rates. We further explored the functional implications of the range of SER geometries observed in the synaptic ultrastructure and the effect of different arrangements between IP3Rs and VDCCs on synaptic plasticity. The synaptic ultrastructure precisely orchestrated the degree of facilitation and depression and the existence of presynaptic calcium stores provided the synapse with an additional intrinsic time scale that could be regulated by activity.

Altered neurotransmitter release machinery in mice deficient for the deubiquitinating enzyme Usp14

Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles.

SNARE Force Synchronizes Synaptic Vesicle Fusion and Controls the Kinetics of Quantal Synaptic Transmission

Neuronal communication relies on rapid and discrete intercellular signaling but neither the molecular mechanisms of the exocytotic machinery that define the timing of the action potential-evoked response nor those controlling the kinetics of transmitter release from single synaptic vesicles are known. Here, we investigate how interference with the putative force transduction between the complex-forming SNARE (soluble N-ethylamide-sensitive factor attachment protein receptor) domain and the transmembrane anchor of synaptobrevin II (SybII) affects action potential-evoked currents and spontaneous, quantal transmitter release at mouse hippocampal synapses. The results indicate that SybII-generated membrane stress effectively determines the kinetics of the action potential-evoked response and show that SNARE force modulates the concentration profile of cleft glutamate by controlling the rate of transmitter release from the single synaptic vesicle. Thus, multiple SybII actions determine the exquisite temporal regulation of neuronal signaling.

Synaptotagmin Has an Essential Function in Synaptic Vesicle Positioning for Synchronous Release in Addition to Its Role as a Calcium Sensor

A multitude of synaptic proteins interact at the active zones of nerve terminals to achieve the high temporal precision of neurotransmitter release in synchrony with action potentials. Though synaptotagmin has been recognized as the Ca2+ sensor for synchronous release, it may have additional roles of action. We address this question at the calyx of Held, a giant presynaptic terminal, that allows biophysical dissection of multiple roles of molecules in synaptic transmission. Using high-level expression recombinant adenoviruses, in conjunction with a stereotactic surgery in postnatal day 1 rats, we overcame the previous inability to molecular perturb the calyx by overexpression of a mutated synaptotagmin. We report that this mutation leaves intrinsic Ca2+ sensitivity of vesicles intact while it destabilizes the readily releasable pool of vesicles and loosens the tight coupling between Ca2+ influx and release, most likely by interfering with the correct positioning of vesicles with respect to Ca2+ channels.

Mechanisms contributing to synaptic Ca 2+ signals and their heterogeneity in hair cells

Sound coding at hair cell ribbon synapses is tightly regulated by Ca(2+). Here, we used patch-clamp, fast confocal Ca(2+) imaging and modeling to characterize synaptic Ca(2+) signaling in cochlear inner hair cells (IHCs) of hearing mice. Submicrometer fluorescence hotspots built up and collapsed at the base of IHCs within a few milliseconds of stimulus onset and cessation. They most likely represented Ca(2+) microdomains arising from synaptic Ca(2+) influx through Ca(V)1.3 channels. Synaptic Ca(2+) microdomains varied substantially in amplitude and voltage dependence even within single IHCs. Testing putative mechanisms for the heterogeneity of Ca(2+) signaling, we found the amplitude variability unchanged when blocking mitochondrial Ca(2+) uptake or Ca(2+)-induced Ca(2+) release, buffering cytosolic Ca(2+) by millimolar concentrations of EGTA, or elevating the Ca(2+) channel open probability by the dihydropyridine agonist BayK8644. However, we observed substantial variability also for the fluorescence of immunolabeled Ca(V)1.3 Ca(2+) channel clusters. Moreover, the Ca(2+) microdomain amplitude correlated positively with the size of the corresponding synaptic ribbon. Ribbon size, previously suggested to scale with the number of synaptic Ca(2+) channels, was approximated by using fluorescent peptide labeling. We propose that IHCs adjust the number and the gating of Ca(V)1.3 channels at their active zones to diversify their transmitter release rates.

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead, we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation, and suggest a prefusion rather than postfusion role for vesicular cation channels.

Optical postsynaptic measurement of vesicle release rates for hippocampal synapses undergoing asynchronous release during train stimulation.

Developing hippocampal neurons in microisland culture were found to undergo rapid depression of excitatory synaptic activity caused by consumption of their readily releasable pool (RRP) of vesicles in response to 20 Hz trains of stimulation. Associated with depression was a switch to an asynchronous release mode that maintained transmission at a high steady-state rate equivalent to approximately 2.1 RRPs per second. We have applied postsynaptic Ca2+ imaging to directly monitor these asynchronous release events to estimate both the steady rate of transmitter release and the number of quanta within the RRP at individual hippocampal synapses. Based on the frequency of asynchronous release measured at individual synapses postsynaptically using Ca2+ imaging (5-17 sec after train stimulation) and with knowledge of the time course by which asynchronous release rates decay, we estimate that individual hippocampal synapses exhibit (in response to train stimulation) peak release rates of up to 21 quanta per second from an RRP that contains, on average, 10 quanta. Use-dependent block of evoked synaptic activity by MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate] confirmed that synapses undergoing asynchronous release are not significantly different from the general population with regard to their composition of NMDA receptor and/or release probability. Given that high-frequency trains deplete the synapse of readily releasable quanta (and that these release rates can only be maintained for a few seconds), these high rates of asynchronous release likely reflect refilling of vesicles from a reserve pool and not necessarily the continuous action of a relatively slow clathrin- and endosome-dependent process.

Increase in the releasable pool of synaptic vesicles underlies facilitation

Facilitation is the ability of presynaptic terminals to release neurotransmitter more efficiently following repetitive stimulation. We demonstrated that facilitation can be explained by Ca 2+-dependent vesicles priming and the increase in the number of synaptic vesicles activated for release. Employing the model with two Ca 2+ sensors, we computed Ca 2+ concentration at the sites of priming and release, the size of the releasable pool of vesicles, and the rate of transmitter release during repetitive nerve stimulation. The calculated rates of vesicle release and the increase in the releasable pool during facilitation were in agreement with the results of electrophysiology experiments.

The timing of phasic transmitter release is Ca2+-dependent and lacks a direct influence of presynaptic membrane potential.

Ca2+ influx through voltage-gated Ca2+ channels and the resulting elevation of intracellular Ca2+ concentration, [Ca2+]i, triggers transmitter release in nerve terminals. However, it is controversial whether in addition to the opening of Ca2+ channels, membrane potential directly affects transmitter release. Here, we assayed the influence of membrane potential on transmitter release at the calyx of Held nerve terminals. Transmitter release was evoked by presynaptic Ca2+ uncaging, or by presynaptic Ca2+ uncaging paired with presynaptic voltage-clamp depolarizations to +80 mV, under pharmacological block of voltage-gated Ca2+ channels. Such a change in membrane potential did not alter the Ca2+ dependence of transmitter release rates or synaptic delays. We also found, by varying the amount of Ca2+ influx during Ca2+ tail-currents, that the time course of phasic transmitter release is not invariant to changes in release probability. Rather, the time difference between peak Ca2+ current and peak transmitter release became progressively shorter with increasing Ca2+ current amplitude. When this time difference was plotted as a function of the estimated local [Ca2+]i at the sites of vesicle fusion, a slope of approximately 100 micros per 10 microM [Ca2+]i was found, in reasonable agreement with a model of cooperative Ca2+ binding and vesicle fusion. Thus, the amplitude and time course of the [Ca2+]i signal at the sites of vesicle fusion controls the timing and the amount of transmitter release, both under conditions of brief periods of Ca2+ influx, as well as during step-like elevations of [Ca2+]i produced by Ca2+ uncaging.

Physiological regulation of Munc18/nSec1 phosphorylation on serine-313.

Increased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18-1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser-313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser-313 phosphorylation. Using phospho-Ser-313-specific antisera, we demonstrate here that Ser-313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser-313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4-aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser-313 phosphorylation. As phosphorylation of nSec1 on Ser-313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long-term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.

Artificial cells: unique insights into exocytosis using liposomes and lipid nanotubes.

Exocytosis is the fundamental process underlying neuronal communication. This process involves fusion of a small neurotransmitter-containing vesicle with the plasma membrane of a cell to release minute amounts of transmitter molecules. Exocytosis is thought to go through an intermediate step involving formation of a small lipid nanotube or fusion pore, followed by expansion of the pore to the final stage of exocytosis. The process of exocytosis has been studied by various methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins relative to the mechanics of lipid-membrane-driven processes and to manipulate system parameters (e.g., membrane composition, pH, ion concentration, temperature, etc.). We describe the use of liposome-lipid nanotube networks to create an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, without protein intervention, can drive expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore.

In VivoProperties of theDrosophila inebriated-Encoded Neurotransmitter Transporter

Altering neurotransmitter levels within the nervous system can cause profound changes in behavior and neuronal function. Neurotransmitter transporters play important roles in regulating neurotransmitter levels by performing neurotransmitter reuptake. It was previously shown that mutations in the Drosophila inebriated (ine)-encoded neurotransmitter transporter cause increased neuronal excitability. Here we report a further functional characterization of Ine. First we show that Ine functions in the short-term (time scale of minutes to a few hours) to regulate neuronal excitability. Second, we show that Ine is able to control excitability from either neurons or glia cells. Third, we show that overexpression of Ine reduces neuronal excitability. Overexpression phenotypes of ine include: delayed onset of long-term facilitation and increased failure rate of transmitter release at the larval neuromuscular junction, reduced amplitude of larval nerve compound action potentials, suppression of the leg-shaking behavior of mutants defective in the Shaker-encoded potassium channel, and temperature-sensitive paralysis. Each of these overexpression phenotypes closely resembles those of loss of function mutants in the para-encoded sodium channel. These data raise the possibility that Ine negatively regulates neuronal sodium channels, and thus that the substrate neurotransmitter of Ine positively regulates sodium channels.

Estimating Transmitter Release Rates from Postsynaptic Current Fluctuations

A method is presented that allows one to estimate transmitter release rates from fluctuations of postsynaptic current records under conditions of stationary or slowly varying release. For experimental applications, we used the calyx of Held, a glutamatergic synapse, in which "residual current," i.e., current attributable to residual glutamate in the synaptic cleft, is present. For a characterization of synaptic transmission, several postsynaptic parameters, such as the mean amplitude of the miniature postsynaptic current and an apparent single channel conductance, have to be known. These were obtained by evaluating variance and two more higher moments of the current fluctuations. In agreement with Fesce et al. (1986), we found both by simulations and by analyzing experimental records that high-pass filtering of postsynaptic currents renders the estimates remarkably tolerant against nonstationarities. We also found that release rates and postsynaptic parameters can be reliably obtained when release rates are low ( approximately 10 events/msec). Furthermore, during a long-lasting stimulus, the transmitter release at the calyx of Held was found to decay to a low, stationary rate of 10 events/msec after depletion of the "releasable pool" of synaptic vesicles. This stationary release rate is compatible with the expected rate of recruitment of new vesicles to the release-ready pool of vesicles. MiniatureEPSC (mEPSC) size is estimated to be similar to the value of spontaneously occurring mEPSC under this condition.

Combining Deconvolution and Noise Analysis for the Estimation of Transmitter Release Rates at the Calyx of Held

The deconvolution method has been used in the past to estimate release rates of synaptic vesicles, but it cannot be applied to synapses where nonlinear interactions of quanta occur. We have extended this method to take into account a nonlinear current component resulting from the delayed clearance of glutamate from the synaptic cleft. We applied it to the calyx of Held and verified the important assumption of constant miniature EPSC (mEPSC) size by combining deconvolution with a variant of nonstationary fluctuation analysis. We found that amplitudes of mEPSCs decreased strongly after extended synaptic activity. Cyclothiazide (CTZ), an inhibitor of glutamate receptor desensitization, eliminated this reduction, suggesting that postsynaptic receptor desensitization occurs during strong synaptic activity at the calyx of Held. Constant mEPSC sizes could be obtained in the presence of CTZ and kynurenic acid (Kyn), a low-affinity blocker of AMPA-receptor channels. CTZ and Kyn prevented postsynaptic receptor desensitization and saturation and also minimized voltage-clamp errors. Therefore, we conclude that in the presence of these drugs, release rates at the calyx of Held can be reliably estimated over a wide range of conditions. Moreover, the method presented should provide a convenient way to study the kinetics of transmitter release at other synapses.