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E Neher

Publications and source records attributed to E Neher.

At least 19 recordsLinked to original sources
PubMed2004

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image.

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal-to-noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal-to-noise ratio or optimize a newly defined 'figure of merit', which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.

Bacterial Proteins↗
PubMed2001

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.

Action Potentials↗
PubMed2001

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.

Animals↗
PubMed2001

Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis.

At the large excitatory calyx of Held synapse, the quantal size during an evoked EPSC and the number of active zones contributing to transmission are not known. We developed a nonstationary variant of EPSC fluctuation analysis to determine these quantal parameters. AMPA receptor-mediated EPSCs were recorded in slices of young (postnatal 8-10 d) rats after afferent fiber stimulation, delivered in trains to induce synaptic depression. The means and the variances of EPSC amplitudes were calculated across trains for each stimulus number. During 10 Hz trains at 2 mm Ca(2+) concentration ([Ca(2+)]), we found linear EPSC variance-mean relationships, with a slope that was in good agreement with the quantal size obtained from amplitude distributions of spontaneous miniature EPSCs. At high release probability with 10 or 15 mm [Ca(2+)], competitive antagonists were used to partially block EPSCs. Under these conditions, the EPSC variance-mean plots could be fitted with parabolas, giving estimates of quantal size and of the binomial parameter N. With the rapidly dissociating antagonist kynurenic acid, quantal sizes were larger than with a slowly dissociating antagonist, suggesting that the effective glutamate concentration was increased at high release probability. Considering the possibility of multivesicular release and moderate saturation of postsynaptic AMPA receptors, we conclude that the binomial parameter N (637 +/- 117; mean +/- SEM) represents an upper limit estimate of the number of functional active zones. We estimate that during normal synaptic transmission, the probability of vesicle fusion at single active zones is in the range of 0.25-0.4.

Animals↗
PubMed2001

Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I.

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca(2+)-dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca(2+)-uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca(2+)-dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca(2+)-sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.

Adrenal Glands↗
PubMed2001

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.

Animals↗
PubMed2001

Transmitter release modulation by intracellular Ca2+ buffers in facilitating and depressing nerve terminals of pyramidal cells in layer 2/3 of the rat neocortex indicates a target cell-specific difference in presynaptic calcium dynamics.

1. In connections formed by nerve terminals of layer 2/3 pyramidal cells onto bitufted interneurones in young (postnatal day (P)14-15) rat somatosensory cortex, the efficacy and reliability of synaptic transmission were low. At these connections release was facilitated by paired-pulse stimulation (at 10 Hz). In connections formed by terminals of layer 2/3 pyramids with multipolar interneurones efficacy and reliability were high and release was depressed by paired-pulse stimulation. In both types of terminal, however, the voltage-dependent Ca2+ channels that controlled transmitter release were predominantly of the P/Q- and N-subtypes. 2. The relationship between unitary EPSP amplitude and extracellular calcium concentration ([Ca2+]o) was steeper for facilitating than for depressing terminals. Fits to a Hill equation with nH = 4 indicated that the apparent KD of the Ca2+ sensor for vesicle release was two- to threefold lower in depressing terminals than in facilitating ones. 3. Intracellular loading of pyramidal neurones with the fast and slowly acting Ca2+ buffers BAPTA and EGTA differentially reduced transmitter release in these two types of terminal. Unitary EPSPs evoked by pyramidal cell stimulation in bitufted cells were reduced by presynaptic BAPTA and EGTA with half-effective concentrations of approximately 0.1 and approximately 1 mM, respectively. Unitary EPSPs evoked in multipolar cells were reduced to one-half of control at higher concentrations of presynaptic BAPTA and EGTA (approximately 0.5 and approximately 7 mM, respectively). 4. Frequency-dependent facilitation of EPSPs in bitufted cells was abolished by EGTA at concentrations of > or = 0.2 mM, suggesting that accumulation of free Ca2+ is essential for facilitation in the terminals contacting bitufted cells. In contrast, facilitation was unaffected or even slightly increased in the terminals loaded with BAPTA in the concentration range 0.02-0.5 mM. This is attributed to partial saturation of exogenously added BAPTA. However, BAPTA at concentrations > or = 1 mM also abolished facilitation. 5. Frequency-dependent depression of EPSPs in multipolar cells was not significantly reduced by EGTA. With BAPTA, the depression decreased at concentrations > 0.5 mM, concomitant with a reduction in amplitude of the first EPSP in a train. 6. An analysis is presented that interprets the effects of EGTA and BAPTA on synaptic efficacy and its short-term modification during paired-pulse stimulation in terms of changes in [Ca2+] at the release site ([Ca2+]RS) and that infers the affinity of the Ca2+ sensor from the dependence of unitary EPSPs on [Ca2+]o. 7. The results suggest that the target cell-specific difference in release from the terminals on bitufted or multipolar cells can be explained by a longer diffusional distance between Ca2+ channels and release sites and/or lower Ca2+ channels density in the terminals that contact bitufted cells. This would lead to a lower [Ca2+] at release sites and would also explain the higher apparent K(D) of the Ca2+ sensor in facilitating terminals.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

Quantitative relationship between transmitter release and calcium current at the calyx of held synapse.

A newly developed deconvolution method (Neher and Sakaba, 2001) allowed us to resolve the time course of neurotransmitter release at the calyx of Held synapse and to quantify some basic aspects of transmitter release. First, we identified a readily releasable pool (RRP) of synaptic vesicles. We found that the size of the RRP, when tested with trains of strong stimuli, was constant regardless of the exact stimulus patterns, if stimuli were confined to a time interval of approximately 60 msec. For longer-lasting stimulus patterns, recruitment of new vesicles to the RRP made a substantial contribution to the total release. Second, the cooperativity of transmitter release as a function of Ca(2+) current was estimated to be 3-4, which confirmed previous results (Borst and Sakmann, 1999; Wu et al., 1999). Third, an initial small Ca(2+) influx increased the efficiency of Ca(2+) currents in subsequent transmitter release. This type of facilitation was blocked by a high concentration of EGTA (0.5 mm). Fourth, the release rates of synaptic vesicles at this synapse turned out to be heterogeneous: once a highly Ca(2+)-sensitive population of vesicles was consumed, the remaining vesicles released at lower rates. These components of release were more clearly separated in the presence of 0.5 mm EGTA, which prevented the buildup of residual Ca(2+). Conversely, raising the extracellular Ca(2+) concentration facilitated the slower population such that its release characteristics became more similar to those of the faster population under standard conditions. Heterogeneous release probabilities are expected to support the maintenance of synaptic transmission during high-frequency stimulation.

Animals↗
PubMed2001

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held.

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Estimating synaptic parameters from mean, variance, and covariance in trains of synaptic responses.

Fluctuation analysis of synaptic transmission using the variance-mean approach has been restricted in the past to steady-state responses. Here we extend this method to short repetitive trains of synaptic responses, during which the response amplitudes are not stationary. We consider intervals between trains, long enough so that the system is in the same average state at the beginning of each train. This allows analysis of ensemble means and variances for each response in a train separately. Thus, modifications in synaptic efficacy during short-term plasticity can be attributed to changes in synaptic parameters. In addition, we provide practical guidelines for the analysis of the covariance between successive responses in trains. Explicit algorithms to estimate synaptic parameters are derived and tested by Monte Carlo simulations on the basis of a binomial model of synaptic transmission, allowing for quantal variability, heterogeneity in the release probability, and postsynaptic receptor saturation and desensitization. We find that the combined analysis of variance and covariance is advantageous in yielding an estimate for the number of release sites, which is independent of heterogeneity in the release probability under certain conditions. Furthermore, it allows one to calculate the apparent quantal size for each response in a sequence of stimuli.

Analysis of Variance↗
PubMed2001

Highly nonlinear photodamage in two-photon fluorescence microscopy.

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).

Adrenal Glands↗
PubMed2001

Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse.

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.

Animals↗
PubMed2001

Calcium signals and synaptic short-term plasticity in the central nervous system.

Plastic changes in the connectivity between neurons underly the adaptive information processing of the central nervous system. The shortest forms of such plasticity are synaptic depression and facilitation, which happen on the subsecond time scale. New techniques allow to study synaptic transmission at unprecedented resolution and to dissect its various components, such as presynaptic Ca++ currents, Ca++ signals, and transmitter stores. Precise knowledge on the components, which are altered during plastic changes, is essential for understanding these important mechanisms.

Action Potentials↗
PubMed2000

R-Type Ca2+ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells.

Patch-clamp measurements of Ca(2+) currents and membrane capacitance were performed on slices of mouse adrenal glands, using the perforated-patch configuration of the patch-clamp technique. These recording conditions are much closer to the in vivo situation than those used so far in most electrophysiological studies in adrenal chromaffin cells (isolated cells maintained in culture and whole-cell configuration). We observed profound discrepancies in the quantities of Ca(2+) channel subtypes (P-, Q-, N-, and L-type Ca(2+) channels) described for isolated mouse chromaffin cells maintained in culture. Differences with respect to previous studies may be attributable not only to culture conditions, but also to the patch-clamp configuration used. Our experiments revealed the presence of a Ca(2+) channel subtype never before described in chromaffin cells, a toxin and dihydropyridine-resistant Ca(2+) channel with fast inactivation kinetics, similar to the R-type Ca(2+) channel described in neurons. This channel contributes 22% to the total Ca(2+) current and controls 55% of the rapid secretory response evoked by short depolarizing pulses. Our results indicate that R-type Ca(2+) channels are in close proximity with the exocytotic machinery to rapidly regulate the secretory process.

Adrenal Glands↗
PubMed2000

The readily releasable pool of vesicles in chromaffin cells is replenished in a temperature-dependent manner and transiently overfills at 37 degrees C.

Maturation of exocytic vesicles to the release-ready state is regulated by several factors, including intracellular calcium concentration ([Ca(2+)](int)) and the state of protein phosphorylation. Here we investigated the effects of temperature on the recovery from depletion of the readily releasable pool (RRP) of vesicles in adrenal chromaffin cells. Exocytosis and [Ca(2+)](int) were monitored by combined membrane capacitance and fura-2 measurements. At higher temperatures, a faster pool refilling and a larger RRP size were observed. The time constants of the recovery from depletion ranged from 3.6 to 1.1 sec (22 and 37 degrees C, respectively) yielding a Q(10) of 2.3. The changes of the Ca(2+) signal between the different temperatures could not account for the differences in recovery kinetics. At 32 and 37 degrees C, we observed a transient overfilling of the RRP after pool depletion, which stands in clear contrast to the sustained secretory depression seen at lower temperatures. The overshoot in RRP size was very prominent in cells with lower basal [Ca(2+)](int), hence with a large difference between prestimulus and poststimulus [Ca(2+)](int). In cells with higher basal [Ca(2+)](int), the pool was larger under steady-state conditions but showed less overfilling on stimulation. We conclude that vesicle maturation is markedly accelerated at physiological temperature, thus allowing for a rapid adaptation of the pool size to the relatively short-lived Ca(2+) transient.

Animals↗
PubMed2000

Intracellular calcium dependence of transmitter release rates at a fast central synapse.

Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central nervous system, however, there are no experimental estimates of this local [Ca2+]i signal. Here we show, by using calcium ion uncaging in the large synaptic terminals of the calyx of Held, that step-like elevations to only 10 microM [Ca2+]i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3 ms. Kinetic analysis of transmitter release rates after [Ca2+]i steps revealed the rate constants for calcium binding and vesicle fusion. These show that transient (around 0.5 ms) local elevations of [Ca2+]i to peak values as low as 25 microM can account for transmitter release during single presynaptic action potentials. The calcium sensors for vesicle fusion are far from saturation at normal release probability. This non-saturation, and the high intracellular calcium cooperativity in triggering vesicle fusion, make fast synaptic transmission very sensitive to modulation by changes in local [Ca2+]i.

Action Potentials↗