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Erwin Neher

Publications and source records attributed to Erwin Neher.

At least 19 recordsLinked to original sources
PubMed2006

A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse.

It has been known since the work of Katz and collaborators in the early 1950s that an increase in intracellular Ca(++) concentration ([Ca(++)]) is the immediate trigger for neurotransmitter release. Later work has shown that, next to Ca(++), many other signaling pathways, particularly via cyclic AMP, modulate the release of both neurotransmitters and hormones. However, regulated secretion is a multistep process and the signaling mechanisms involved act at many stages. Biochemical and traditional electrophysiological techniques very often cannot distinguish whether a change in secretion is caused by regulation of ion channels, vesicle trafficking, or the exocytic process itself. My laboratory has made an effort to dissect the stimulus secretion pathway by developing assays in chromaffin cells (for catecholamine release) and at a glutamatergic central nervous synapse (the calyx of Held, a component of the auditory pathway), which permit the study of secretion in single cells under voltage clamp conditions. This enables us to clearly distinguish between consequences of changes in electrical signaling, from those regarding the process of vesicle recruitment or the process of exocytosis.

Adrenal Medulla↗
PubMed2005

Release kinetics, quantal parameters and their modulation during short-term depression at a developing synapse in the rat CNS.

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size (q) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5-7 synapses by 10-100 Hz stimulation, q declined rapidly to 40-12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12-14), but quite severe for frequencies > or = 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of approximately 480 ms in P5-7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8-10 quanta s(-1) per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of approximately 50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.

Action Potentials↗
PubMed2005

Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage.

Neurotransmitter release is triggered by calcium ions and depends critically on the correct function of three types of SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins. With use of the large calyx of Held presynaptic terminal from rats, we found that cleavage of different SNARE proteins by clostridial neurotoxins caused distinct kinetic changes in neurotransmitter release. When elevating calcium ion concentration directly at the presynaptic terminal with the use of caged calcium, cleavage of SNAP-25 by botulinum toxin A (BoNT/A) produced a strong reduction in the calcium sensitivity for release, whereas cleavage of syntaxin using BoNT/C1 and synaptobrevin using tetanus toxin (TeNT) produced an all-or-nothing block without changing the kinetics of remaining vesicles. When stimulating release by calcium influx through channels, a difference between BoNT/C1 and TeNT emerged, which suggests that cleavage of synaptobrevin modifies the coupling between channels and release-competent vesicles.

Action Potentials↗
PubMed2005

Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells.

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase I, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.

Animals↗
PubMed2005

Presynaptic calcium and control of vesicle fusion.

Vesicle fusion and transmitter release at synapses is driven by a highly localized Ca2+ signal that rapidly builds up around open Ca2+-channels at and near presynaptic active zones. It has been difficult to estimate the amplitude and the kinetics of this 'microdomain' signal by direct Ca2+-imaging approaches. Recently, Ca2+ uncaging at large CNS synapses, among them the calyx of Held, has shown that the intrinsic cooperativity of Ca2+ in inducing vesicle fusion is high, with 4-5 Ca2+ ions needed to trigger vesicle fusion. Given the Ca2+-sensitivity of vesicle fusion as determined by Ca2+-uncaging, it was found that a surprisingly small (10-25 microM) and brief (<1 ms) local Ca2+ signal is sufficient to achieve the amount, and the kinetics of the physiological transmitter release. The high cooperativity of Ca2+ in inducing vesicle fusion and the non-saturation of the Ca2+-sensor for vesicle fusion renders small changes of the local Ca2+-signal highly effective in changing the release probability; an insight that is important for our understanding of short-term modulation of synaptic strength.

Calcium↗
PubMed2004

Applying spectral fingerprinting to the analysis of FRET images.

Förster resonance energy transfer (FRET) allows one to study interactions between two fluorescently labeled molecules (donors and acceptors) at distances on the order of 5 nm. Many studies have described methods of how to measure the efficiency of FRET. However, few have addressed the question of how fluorescence from unpaired donors and acceptors can be determined in addition to that from FRET-pairs and how the signal-to-noise ratio (SNR) of such estimates depends on the presence of the partner species. Such knowledge, however, is essential for many biological applications, in which-after initial characterization of the spectral properties of a well-defined donor-acceptor complex-the in vivo affinity and stoichiometry of the complex is of interest. Here, we provide a theoretical analysis on how spectral fingerprinting can be applied to separate fluorescence of FRET pairs from that originating from unpaired donors and acceptors and how to select imaging parameters to optimize the SNR of the estimates. Thereby, we uncover a fundamental problem in this application and discuss ways to evade its adverse consequences. We compare the expected resolution of traditional FRET measures with that of optimized spectral fingerprinting and analyze the resolution of a method for FRET measurements that combines spectral with fluorescence lifetime information.

Cells↗
PubMed2004

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25.

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.

Alanine↗
PubMed2003

Combining deconvolution and fluctuation analysis to determine quantal parameters and release rates.

Analysis methods are described which integrate information from fluctuation analysis with that from deconvolution. Together the two approaches allow to derive a consistent quantitative description of quantal release (both evoked, spontaneous and asynchronous) under conditions in which quantal parameters may change during a repetitively applied stimulation protocol. Specifically, our methods take into account the effects of accumulating transmitter in the synaptic cleft and postsynaptic receptor desensitization, which may develop during strong stimulation. Several ways to handle non-stationarities are described. Examples are provided for the Calyx of Held, a glutamatergic synapse, in which both the pre- and the postsynaptic compartments can be voltage-clamped.

Algorithms↗
PubMed2003

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.

Action Potentials↗
PubMed2003

Direct modulation of synaptic vesicle priming by GABA(B) receptor activation at a glutamatergic synapse.

Second messenger cascades involving G proteins and calcium are known to modulate neurotransmitter release. A prominent effect of such a cascade is the downmodulation of presynaptic calcium influx, which markedly reduces evoked neurotransmitter release. Here we show that G-protein-mediated signalling, such as through GABA (gamma-amino butyric acid) subtype B (GABA(B)) receptors, retards the recruitment of synaptic vesicles during sustained activity and after short-term depression. This retardation occurs through a lowering of cyclic AMP, which blocks the stimulatory effect of increased calcium concentration on vesicle recruitment. In this signalling pathway, cAMP (functioning through the cAMP-dependent guanine nucleotide exchange factor) and calcium/calmodulin cooperate to enhance vesicle priming. The differential modulation of the two forms of synaptic plasticity, presynaptic inhibition and calcium-dependent recovery from synaptic depression, is expected to have interesting consequences for the dynamic behaviour of neural networks.

Animals↗
PubMed2003

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23.

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.

Alternative Splicing↗
PubMed2003

Probing the intracellular calcium sensitivity of transmitter release during synaptic facilitation.

In nerve terminals, residual Ca(2+) remaining from previous activity can cause facilitation of transmitter release by a mechanism that is still under debate. Here we show that the intracellular Ca(2+) sensitivity of transmitter release at the calyx of Held is largely unchanged during facilitation, which leaves an increased microdomain Ca(2+) signal as a possible mechanism for facilitation. We measured the Ca(2+) dependencies of facilitation, as well as of transmitter release, to estimate the required increment in microdomain Ca(2+). These measurements show that linear summation of residual and microdomain Ca(2+) accounts for only 30% of the observed facilitation. However, a small degree of supralinearity in the summation of intracellular Ca(2+) signals, which might be caused by saturation of cytosolic Ca(2+) buffer(s), is sufficient to explain facilitation at this CNS synapse.

Animals↗
PubMed2003

Involvement of actin polymerization in vesicle recruitment at the calyx of Held synapse.

Depletion and replenishment of pools of synaptic vesicles are important determinants of short-term synaptic plasticity, but the underlying molecular mechanisms are not yet clear. As a first step toward understanding the process of vesicle recruitment, we have applied various specific agents directly to the presynaptic terminal of the calyx of Held synapse. Here we show that the nonhydrolyzable ATP analog ATP-gammaS retards the recovery from vesicle pool depletion, as does latrunculin A. Phalloidin has no effects on recovery, suggesting that dynamic actin reorganization is not necessary. Unexpectedly, neither N-ethylmaleimide nor staurosporine affected the recovery, calling into question the role of N-ethylmaleimide-sensitive factor and protein kinases. The results suggest that intact actin polymerization is involved in vesicle recruitment.

Actins↗
PubMed2003

Heterogeneous presynaptic release probabilities: functional relevance for short-term plasticity.

We discuss a model of presynaptic vesicle dynamics, which allows for heterogeneity in release probability among vesicles. Specifically, we explore the possibility that synaptic activity is carried by two types of vesicles; first, a readily releasable pool and, second, a reluctantly releasable pool. The pools differ regarding their probability of release and time scales on which released vesicles are replaced by new ones. Vesicles of both pools increase their release probability during repetitive stimulation according to the buildup of Ca(2+) concentration in the terminal. These properties are modeled to fit data from the calyx of Held, a giant synapse in the auditory pathway. We demonstrate that this arrangement of two pools of releasable vesicles can account for a variety of experimentally observed patterns of synaptic depression and facilitation at this synapse. We conclude that synaptic transmission cannot be accurately described unless heterogeneity of synaptic release probability is taken into account.

Action Potentials↗
PubMed2003

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+.

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.

Animals↗
PubMed2002

Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

Animals↗