Erwin Neher

Single-channel recording

A Practical Guide to Patch Clamping R. Penner. Tightseal Wholecell Recording A. Marty, E. Neher. Guide to Data Acquisition and Analysis S.H. Heinemann. Electronic Design of the Patch Clamp F.J. Sigworth. Low Noise Recording K. Benndorf. Voltage Offsets in Patch Clamp Experiments E. Neher. Techniques for Membrane Capacitance Measurements K.D. Gillis. Patch Pipette Recordings from the Soma, Dendrites and Axon of Neurons in Brain Slices B. Sakmann, G. Stuart. Patchclamp and Calcium Imaging in Brain Slices J. Eilers, et al. Fast Application of Agonists to Isolated Membrane Patches P. Jonas. Electrochemical Detection of Secretion from Single Cells R.H. Chow, L. von Ruden. Technical Approaches to Studying Specific Properties of Ion Channels in Plants R. Hedrich. The Giant Membrane Patch D.W. Hilgemann. A Fast Pressureclamp Technique for Studying Mechanogated Channels D.W. McBride, O.P. Hamill. Electrophysiological Recordings from Xenopus oocytes W. Stuhmer, A.B. Parekh. PCR Analysis of Ion Channel Expression in Single Neurons of Brain Slices H. Monyer, P. Jonas. 3 additional articles. Index.

Calcium gradients and buffers in bovine chromaffin cells.

1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura‐2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage‐gated Ca2+ channels were closed. 3. Dialysis of Fura‐2 into the chromaffin cell caused concentration‐dependent changes in the depolarization‐induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura‐2 competes for Ca2+. 4. The spatially averaged Fura‐2 signal was well described by a model assuming fast competition between Fura‐2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds‐long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98‐99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM‐Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura‐2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura‐2 concentrations (0.4 mM) Fura‐2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.

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 100u2009µM 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 10u2009µM [Ca2+] i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3u2009ms. 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 0.5u2009ms) local elevations of [Ca2+]i to peak values as low as 25u2009µM 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 localxa0[Ca2+]i.

Multiple Roles of Calcium Ions in the Regulation of Neurotransmitter Release

The intracellular calcium concentration ([Ca(2+)]) has important roles in the triggering of neurotransmitter release and the regulation of short-term plasticity (STP). Transmitter release is initiated by quite high concentrations within microdomains, while short-term facilitation is strongly influenced by the global buildup of residual calcium. A global rise in [Ca(2+)] also accelerates the recruitment of release-ready vesicles, thereby controlling the degree of short-term depression (STD) during sustained activity, as well as the recovery of the vesicle pool in periods of rest. We survey data that lead us to propose two distinct roles of [Ca(2+)] in vesicle recruitment: one accelerating molecular priming (vesicle docking and the buildup of a release machinery), the other promoting the tight coupling between releasable vesicles and Ca(2+) channels. Such coupling is essential for rendering vesicles sensitive to short [Ca(2+)] transients, generated during action potentials.

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.

Potassium channels in cultured bovine adrenal chromaffin cells.

K channels of bovine adrenal chromaffin cells were studied using patch‐clamp techniques. Whole‐cell K currents measured near +10 mV were much larger in 1 mM‐external Ca than in Ca‐free saline. Noise analysis suggested that this Ca‐dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside‐out patches (Marty, 1981). The Ca‐dependent K current near +10 mV declined with time due to run‐down of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole‐cell Ca‐dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single‐channel K currents were observed. Under normal ionic conditions (140 mM‐K inside, 140 mM‐Na outside), the unitary conductances measured between ‐20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca‐dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single‐channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.

Fast Vesicle Fusion in Living Cells Requires at Least Three SNARE Complexes

Threes the Charm The molecular machinery mediating membrane fusion during secretion from a cell requires a complex of so-called SNARE protein that forms a coiled bundle of four parallel α-helices. Mohrmann et al. (p. 502, published online 16 September) developed an elegant approach to find out how many SNARE complexes are required to promote secretion of individual secretory vesicles in living chromaffin cells by titrating the ratio of wild-type and mutant SNARE proteins expressed. For fast synchronous release, a minimum of three SNARE complexes per vesicle were required. Fewer SNARE complexes resulted in slower release. Membrane fusion proteins cooperate to promote rapid secretory vesicle exocytosis from neuroendocrine cells. Exocytosis requires formation of SNARE [soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis.

Mechanisms that ensure robust long-term performance of synaptic transmission over a wide range of activity are crucial for the integrity of neuronal networks, for processing sensory information and for the ability to learn and store memories. Recent experiments have revealed that such robust performance requires a tight coupling between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. Distinct presynaptic scaffolding proteins are essential for fulfilling this requirement, providing either ultrastructural coordination or acting as signalling hubs.

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.

Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images

Methods of blind source separation are used in many contexts to separate composite data sets according to their sources. Multiply labeled fluorescence microscopy images represent such sets, in which the sources are the individual labels. Their distributions are the quantities of interest and have to be extracted from the images. This is often challenging, since the recorded emission spectra of fluorescent dyes are environment- and instrument-specific. We have developed a nonnegative matrix factorization (NMF) algorithm to detect and separate spectrally distinct components of multiply labeled fluorescence images. It operates on spectrally resolved images and delivers both the emission spectra of the identified components and images of their abundance. We tested the proposed method using biological samples labeled with up to four spectrally overlapping fluorescent labels. In most cases, NMF accurately decomposed the images into contributions of individual dyes. However, the solutions are not unique when spectra overlap strongly or when images are diffuse in their structure. To arrive at satisfactory results in such cases, we extended NMF to incorporate preexisting qualitative knowledge about spectra and label distributions. We show how data acquired through excitations at two or three different wavelengths can be integrated and that multiple excitations greatly facilitate the decomposition. By allowing reliable decomposition in cases where the spectra of the individual labels are not known or are known only inaccurately, the proposed algorithms greatly extend the range of questions that can be addressed with quantitative microscopy.