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E Neher

Publications and source records attributed to E Neher.

At least 37 records · Page 2Linked to original sources
PubMed2000

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells.

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.

Animals↗
PubMed2000

Kinetics of Ca2+ binding to parvalbumin in bovine chromaffin cells: implications for [Ca2+] transients of neuronal dendrites.

kappaS1. The effect of parvalbumin (PV) on [Ca2+] transients was investigated by perfusing adrenal chromaffin cells with fura-2 and fluorescein isothiocyanate (FITC)-labelled PV. As PV diffused into cells, the decay of [Ca2+] transients was transformed from monophasic into biphasic. The proportion of the initial fast decay phase increased in parallel with the fluorescence intensity of FITC, indicating that PV is responsible for the initial fast decay phase. The relationship between the fast decay phase and the [Ca2+] level was investigated using depolarizing trains of stimuli. Within a train the relative amplitude of the fast decay phase was inversely dependent on the [Ca2+] level preceding a given stimulus. Based on these observations, we estimated the Ca2+ binding ratio of PV (kappaP), the apparent dissociation constant of PV for Ca2+ (Kdc, app), and the unbinding rate constant of Ca2+ from PV (kc-) in the cytosol of chromaffin cells. Assuming free [Mg2+] to be 0.14 mM, we obtained values of 51.4 +/- 2.0 nM (n = 3) and 0.95 +/- 0.026 s-1 (n = 3), for Kdc,app and kc-, respectively. With the parameters obtained in the perfusion study, we simulated [Ca2+] transients, using two different Ca2+ extrusion rates (gamma) - 20 and 300 s-1 - which represent typical values for chromaffin cells and neuronal dendrites, respectively. The simulation indicated that Ca2+ is pumped out before it is equilibrated with PV, when gamma is comparable to the equilibration rates between PV and Ca2+, resulting in the fast decay phase of a biexponential [Ca2+] transient. From these results we conclude that Ca2+ buffers with slow kinetics, such as PV, may cause biexponential decays in [Ca2+] transients, thereby complicating the analysis of endogenous Ca2+ binding ratios (kappaS) based on time constants. Nevertheless, estimates of kappaS based on Ca2+ increments provide reasonable estimates for Ca2+ binding ratios before equilibration with PV.

Animals↗
PubMed2000

Differences in Ca2+ buffering properties between excitatory and inhibitory hippocampal neurons from the rat.

Endogenous calcium binding ratios (kappaS) in dendrites of cultured hippocampal neurons were estimated according to the single compartment model for transients in intracellular Ca2+ concentration ([Ca2+]). In addition, the electrophysiological characteristics of neurons were classified by their autaptic currents and intrinsic firing patterns. These data were analysed in order to determine whether a correlation between Ca2+ buffers and electrophysiological type exists. Ca2+ binding ratios of endogenous buffers were estimated by eliciting [Ca2+] transients with short depolarizations, while cells were loaded with fura-2. Two types of estimates could be obtained: one termed kappaS(tau), based on analysing time constants (tau) of [Ca2+] transients, and another termed kappaS(dCa), derived from an analysis of initial amplitudes of [Ca2+] transients. Values for kappaS(tau) and kappaS(dCa) were estimated as 57 +/- 10 (mean +/- s.d., n = 10) and 60 +/- 14 (n = 10), respectively, in excitatory neurons, and 130 +/- 50 (n = 11) and 150 +/- 70 (n = 11), respectively, in inhibitory neurons. The kappaS values of excitatory and inhibitory cells were significantly different from each other, regardless of the measurement method (Student's t test, P < 0.01). However, there was no significant difference in kappaS between the groups classified according to firing patterns. Although kappaS(tau) values were well matched to those of kappaS(dCa) in most excitatory cells, the two values did not agree in three out of the fourteen inhibitory cells investigated. In these cells, the first few [Ca2+] transients after obtaining the whole cell configuration displayed a double exponential decay, suggesting that buffers with slow binding kinetics, such as parvalbumin, are involved. This hypothesis is further explored in an accompanying paper.

Animals↗
PubMed2000

Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25.

The highly conserved SNARE proteins, SNAP-25, syntaxin and synaptobrevin, form a tight ternary complex, which is essential for exocytosis. Crystallization of this complex revealed a four-helix bundle with an unusual hydrophilic layer (zero layer) in its center. In order to evaluate the role of this layer in different kinetic components of secretion, we used the Semliki Forest virus (SFV) system to infect adrenal chromaffin cells with SNAP-25 Q174L, a point mutant in the zero layer. Using combined flash photolysis of caged calcium and membrane capacitance measurements, we investigated its effect on the exocytotic burst and sustained phase of exocytosis with high time resolution. Cells expressing SNAP-25 Q174L displayed a selective reduction in the sustained phase, while the two components of the exocytotic burst remained unaffected. Furthermore, the exocytotic response to the second flash was significantly reduced, indicating a decrease in refilling kinetics. We therefore conclude that the zero layer is critical for the formation of SNARE complexes, but that it plays no role in the dynamic equilibrium between the two exocytosis-competent vesicle pools.

Amino Acid Substitution↗
PubMed1999

Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis.

In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.

Adrenal Glands↗
PubMed1999

Fast scanning and efficient photodetection in a simple two-photon microscope.

Two-photon laser scan microscopy carries many advantages for work on brain slices and bulk tissue. However, it has very low signal levels compared to conventional fluorescence microscopy. This is disadvantageous in fast imaging applications when photon shot noise is limiting. Working on brain slices with excitation powers of 8-10 mW at the specimen plane, the resting signal from cerebellar Purkinje cell somas loaded with 10 microM Oregon Green 488 BAPTA-1 averaged 4 detected photons/micros; axons of interneurons loaded with 200 microM of this indicator yielded about 1 photon/micros. To obtain satisfactory images at high time resolution, long pixel dwell times are required and data collection should be restricted to as few pixels as necessary. Furthermore, a large proportion of total measurement time (duty cycle) should be available for data collection. We therefore developed a method for scanning small regions of interest with line repetition rates two to four times higher than conventional ones and a duty cycle of 70%. We also compared the performance of several photodetectors and found the optimum choice to depend strongly on the photon flux during a given application. For fluxes smaller than 5 photons/micros, the photon counting avalanche photodiode shows the best signal to noise ratio. At larger fluxes, photomultipliers or intensified photodiodes are superior.

Animals↗
PubMed1999

Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells.

NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules.

Adenosine Triphosphate↗
PubMed1999

Properties of a model of Ca++-dependent vesicle pool dynamics and short term synaptic depression.

We explore the properties of models of synaptic vesicle dynamics, in which synaptic depression is attributed to depletion of a pool of release-ready vesicles. Two alternative formulations of the model allow for either recruitment of vesicles from an unlimited reserve pool (vesicle state model) or for recovery of a fixed number of release sites to a release-ready state (release-site model). It is assumed that, following transmitter release, the recovery of the release-ready pool of vesicles is regulated by the intracellular free Ca(++) concentration, [Ca(++)](i). Considering the kinetics of [Ca(++)](i) after single presynaptic action potentials, we show that pool recovery can be described by two distinct kinetic components. With such a model, complex kinetic and steady-state properties of synaptic depression as found in several types of synapses can be accurately described. However, the specific assumption that enhanced recovery is proportional to [Ca(++)](i), as measured with Ca(++) indicator dyes, is not confirmed by experiments at the calyx of Held, in which [Ca(++)](i)-homeostasis was altered by adding low concentrations of the exogenous Ca(++) buffer, fura-2, to the presynaptic terminal. We conclude that synaptic depression at the calyx of Held is governed by localized, near membrane [Ca(++)](i) signals not visible to the indicator dye, or else by an altogether different mechanism. We demonstrate that, in models in which a Ca(++)-dependent process is linearly related to [Ca(++)](i), the addition of buffers has only transient but not steady-state consequences.

Action Potentials↗
PubMed1999

Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse.

The size of a pool of readily releasable vesicles at a giant brainstem synapse, the calyx of Held, was probed with three independent approaches. Using simultaneous pre- and postsynaptic whole-cell recordings, two forms of presynaptic Ca2+ stimuli were applied in rapid succession: uncaging of Ca2+ by flash photolysis and the opening of voltage-gated Ca2+ channels. The ensuing transmitter release showed a nearly complete cross-inhibition between the two stimuli, indicating the depletion of a limited pool of about 700 transmitter quanta. The pool size was confirmed in experiments using enhanced extracellular Ca2+ concentrations, as well as short, high-frequency stimulus trains. The results reveal a surprisingly large pool of functionally available vesicles, of which a fraction of about 0.2 is released by a single presynaptic action potential under physiological conditions.

Action Potentials↗
PubMed1999

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.

Adrenal Glands↗
PubMed1999

Different types of calcium channels and secretion from bovine chromaffin cells.

Bovine chromaffin cells possess several types of Ca2+ channels, and influx of Ca2+ is known to trigger secretion. However, discrepant information about the relative importance of the individual subtypes in secretion has been reported. We used whole-cell patch-clamp measurements in isolated cells in culture combined with fura-2 microfluorimetry and pharmacological manipulation to determine the dependence of secretion on different types of Ca2+ channels. We stimulated cells with relatively long depolarizing voltage-clamp pulses in a medium containing 60 mM CaCl2. We found that, within a certain range of pulse parameters, secretion as measured by membrane capacitance changes was mainly determined by the total cumulative charge of Ca2+ inflow and the basal [Ca2+] level preceding a stimulus. Blocking or reducing the contribution of specific types of Ca2+ channels using either 20 microM nifedipine plus 10 microM nimodipine or 1 microM omegaCTxGVIA (omega-conotoxin GVIA) or 2 microM omegaCTxMVIIC (omega-conotoxin MVIIC) reduced secretion in proportion to Ca2+ charge, irrespective of the toxin used. We conclude that for long-duration stimuli, which release a large fraction of the readily releasable pool of vesicles, it is not so important through which type of channels Ca2+ enters the cell. Release is determined by the total amount of Ca2+ entering and by the filling state of the readily releasable pool, which depends on basal [Ca2+] before the stimulus. This result does not preclude that other stimulation patterns may lead to responses in which subtype specificity of Ca2+ channels matters.

Animals↗
PubMed1998

Two-dimensional determination of the cellular Ca2+ binding in bovine chromaffin cells.

The spatiotemporal profile of intracellular calcium signals is determined by the flux of calcium ions across different biological membranes as well as by the diffusional mobility of calcium and different calcium buffers in the cell. To arrive at a quantitative understanding of the determinants of these signals, one needs to dissociate the flux contribution from the redistribution and buffering of calcium. Since the cytosol can be heterogeneous with respect to its calcium buffering property, it is essential to assess this property in a spatially resolved manner. In this paper we report on two different methods to estimate the cellular calcium binding of bovine adrenal chromaffin cells. In the first method, we use voltage-dependent calcium channels as a source to generate calcium gradients in the cytosol. Using imaging techniques, we monitor the dissipation of these gradients to estimate local apparent calcium diffusion coefficients and, from these, local calcium binding ratios. This approach requires a very high signal-to-noise ratio of the calcium measurement and can be used when well-defined calcium gradients can be generated throughout the cell. In the second method, we overcome these problems by using calcium-loaded DM-nitrophen as a light-dependent calcium source to homogeneously and quantitatively release calcium in the cytosol. By measuring [Ca2+] directly before and after the photorelease process and knowing the total amount of calcium being released photolytically, we get an estimate of the fraction of calcium ions which does not appear as free calcium and hence must be bound to either the indicator dye or the endogenous calcium buffer. This finally results in a two-dimensional map of the distribution of the immobile endogenous calcium buffer. We did not observe significant variations of the cellular calcium binding at a spatial resolution of approximately 2 micron. Furthermore, the calcium binding is not reduced by increasing the resting [Ca2+] to levels as high as 1.1 microM. This is indicative of a low calcium affinity of the corresponding buffers and is in agreement with a recent report on the affinity of these buffers (Xu, T., M. Naraghi, H. Kang, and E. Neher. 1997. Biophys. J. 73:532-545). In contrast to the homogeneous distribution of the calcium buffers, the apparant calcium diffusion coefficient did show inhomogeneities, which can be attributed to restricted diffusion at the nuclear envelope and to rim effects at the cell membrane.

Acetates↗
PubMed1998

Comparison of secretory responses as measured by membrane capacitance and by amperometry.

We have compared capacitance and amperometric measurements in bovine chromaffin cells when secretion was elicited by flash photolysis of caged-calcium or step depolarizations. Total amperometric charge depended linearly on the amount of capacitance increase in both types of experiments. Furthermore, the properties of resolvable amperometric spikes after flashes were comparable to those observed after depolarizations, and their timing was compatible with the rate of capacitance increase. For a more detailed comparison, we used Monte Carlo simulations of multiple amperometric events occurring randomly over the surface of a sphere and summing together, to generate a reference amperometric signal for a given measured capacitance increase. Even after correction for endocytotic processes, the time courses of the integrated experimental records lagged behind the integrated Monte Carlo records by approximately 50 ms in flash and depolarization experiments. This delay was larger by approximately 40 ms than what can be expected from the "pre-foot delay" or the foot duration. Possible sources for the remaining delay could be diffusional barriers like the patch-pipette and the chamber bottom, which are not taken into account in the model. We also applied a novel type of fluctuation analysis to estimate the relative quantum size of an amperometric event. On average the estimates from experimental amperometric traces, in both flash and depolarization experiments, were 3-5 times smaller than estimates from simulated ones. This discrepancy can be due to contributions to the amperometric current from small vesicles, preferred release from cellular regions orientated toward the chamber bottom, or abundance of "foot-only" events. In conclusion, amperometric signals in flash and depolarization experiments displayed similar delayed average time courses and a lower estimate for the relative quantum size compared to the modeled amperometric signals. However, individual amperometric spikes were in agreement with expectations derived from capacitance signals.

Adenosine Triphosphate↗
PubMed1998

Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.

We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) in the whole-cell configuration of the patch clamp technique. We compared the loading time-courses of CG-1 and BS, investigated their intracellular distribution patterns and studied the time course of photobleaching. We determined the apparent dissociation constant of CG-1, both optically and by potentiometric titration. Our findings indicate that: (i) with excitation at 420/488 nm, calibrated fluorescence signals could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellular constituents; and (iii) that calibration requires diffusional equilibration between pipette and the accessible volume of the cell. Spatially resolved recordings of fluorescence excitation spectra revealed elevated fluorescence of CG-1 in the nucleus such that reported [Ca2+]i levels seemed 25% higher compared to cytosolic values. Comparing fluorescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca(2+)-insensitive dye.

Adrenal Glands↗
PubMed1998

Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells.

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.

Animals↗
PubMed1998

Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release.

Munc13-1, a mammalian homolog of C. elegans unc-13p, is thought to be involved in the regulation of synaptic transmission. We now demonstrate that Munc13-1 is a presynaptic high-affinity phorbol ester and diacylglycerol receptor with ligand affinities similar to those of protein kinase C. Munc13-1 associates with the plasma membrane in response to phorbol ester binding and acts as a phorbol ester-dependent enhancer of transmitter release when overexpressed presynaptically in the Xenopus neuromuscular junction. These observations establish Munc13-1 as a novel presynaptic target of the diacylglycerol second messenger pathway that acts in parallel with protein kinase C to regulate neurotransmitter secretion.

Amino Acid Sequence↗