Microtubules regulate local Ca2+ spiking in secretory epithelial cells.
AuthorsFogarty KE, Kidd JF, Turner A, Skepper JN, Carmichael J, Thorn P
JournalJ Biol Chem
PubMed ID10801885
'The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently ... More
Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution.
AuthorsVyshedskiy A, Lin JW
JournalJ Neurophysiol
PubMed ID10634895
'Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with ... More
Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate.
AuthorsBird GS, Obie JF, Putney JW
JournalJ Biol Chem
PubMed ID1325446
'In saponin-permeabilized mouse lacrimal acinar cells, glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP2) activated the release of sequestered Ca2+ to the same extent as inositol 1,4,5-trisphosphate ((1,4,5)IP3) but with a potency about 1/10 that of (1,4,5)IP3. In lacrimal gland homogenates, [3H]GPIP2 was metabolized to two compounds which upon anion exchange high performance liquid chromatography ... More
Mechanisms of calcium decay kinetics in hippocampal spines: role of spine calcium pumps and calcium diffusion through the spine neck in biochemical compartmentalization.
AuthorsMajewska A, Brown E, Ross J, Yuste R
JournalJ Neurosci
PubMed ID10684874
'Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Although the mechanisms of calcium influx into spines have been explored, it is unknown what determines the calcium decay kinetics in spines. With two-photon microscopy we investigate action potential-induced calcium dynamics in spines from rat CA1 pyramidal neurons ... More
Developmental changes in the distribution of the endoplasmic reticulum and inositol 1,4,5-trisphosphate receptors and the spatial pattern of Ca2+ release during maturation of hamster oocytes.
AuthorsShiraishi K, Okada A, Shirakawa H, Nakanishi S, Mikoshiba K, Miyazaki S
JournalDev Biol
PubMed ID7649386
'During maturation of hamster oocytes, the distribution of the endoplasmic reticulum (ER) and inositol 1,4,5-trisphosphate receptors (InsP3Rs) was found to change dramatically, as observed using confocal microscopy with DiI and electron microscopy for the ER and immunohistochemistry for InsP3Rs. In immature oocytes at the germinal vesicle (GV) stage, ER and ... More
Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.
AuthorsKoester HJ, Baur D, Uhl R, Hell SW
JournalBiophys J
PubMed ID10512842
'The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage ... More
Ca2+ regulation of gelsolin by its C-terminal tail.
AuthorsLin KM, Mejillano M, Yin HL
JournalJ Biol Chem
PubMed ID10862770
'Gelsolin is activated by Ca(2+) to sever actin filaments. Ca(2+) regulation is conferred on the N-terminal half by the C-terminal half. This paper seeks to understand how Ca(2+) regulates gelsolin by testing the "tail helix latch hypothesis," which is based on the structural data showing that gelsolin has a C-terminal ... More
Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals.
AuthorsMartin JL, Stork CJ, Li YV
JournalCell Calcium
PubMed ID16764924
'Investigations into the roles of Ca(2+) and Zn(2+) in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca(2+) or Zn(2+) in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a ... More
Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis.
AuthorsYuste R, Majewska A, Cash SS, Denk W
JournalJ Neurosci
PubMed ID10066251
'Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for ... More
Dopaminergic regulation of dendritic calcium: fast multisite calcium imaging.
AuthorsZhou WL, Oikonomou KD, Short SM, Antic SD,
JournalMethods Mol Biol
PubMed ID23296782
'Optimal dopamine tone is required for the normal cortical function; however it is still unclear how cortical-dopamine-release affects information processing in individual cortical neurons. Thousands of glutamatergic inputs impinge onto elaborate dendritic trees of neocortical pyramidal neurons. In the process of ensuing synaptic integration (information processing), a variety of calcium ... More
NMDA spikes in basal dendrites of cortical pyramidal neurons.
AuthorsSchiller J, Major G, Koester HJ, Schiller Y
JournalNature
PubMed ID10749211
'Basal dendrites are a major target for synaptic inputs innervating cortical pyramidal neurons. At present little is known about signal processing in these fine dendrites. Here we show that coactivation of clustered neighbouring basal inputs initiated local dendritic spikes, which resulted in a 5.9 +/- 1.5 mV (peak) and 64.4 ... More
Robust coding of flow-field parameters by axo-axonal gap junctions between fly visual interneurons.
AuthorsCuntz H, Haag J, Forstner F, Segev I, Borst A
JournalProc Natl Acad Sci U S A
PubMed ID17551009
'Complex flight maneuvers require a sophisticated system to exploit the optic flow resulting from moving images of the environment projected onto the retina. In the fly''s visual course control center, the lobula plate, 10 so-called vertical system (VS) cells are thought to match, with their complex receptive fields, the optic ... More
Electroporation loading of calcium-sensitive dyes into the CNS.
AuthorsBonnot A, Mentis GZ, Skoch J, O'Donovan MJ
JournalJ Neurophysiol
PubMed ID15509647
'Calcium imaging of neural network function has been limited by the extent of tissue labeled or the time taken for labeling. We now describe the use of electroporation-an established technique for transfecting cells with genes-to load neurons with calcium-sensitive dyes in the isolated spinal cord of the neonatal mouse in ... More
In vivo dendritic calcium dynamics in neocortical pyramidal neurons.
AuthorsSvoboda K, Denk W, Kleinfeld D, Tank DW
JournalNature
PubMed ID8990119
'The dendrites of mammalian pyramidal neurons contain a rich collection of active conductances that can support Na+ and Ca2+ action potentials (for a review see ref. 1). The presence, site of initiation, and direction of propagation of Na+ and Ca2+ action potentials are, however, controversial, and seem to be sensitive ... More
Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.
AuthorsO'Donovan MJ, Ho S, Sholomenko G, Yee W
JournalJ Neurosci Methods
PubMed ID8474261
'Membrane-impermeant calcium indicator dyes were used to retrogradely label dorsal root ganglia, spinal motoneurons and interneurons in the spinal cord of the chick embryo. The dyes were also used to label anterogradely primary afferent axons in the spinal cord and synaptic endings in the ciliary ganglion. Labelled neurons were imaged ... More
Axonal calcium entry during fast 'sodium' action potentials in rat cerebellar Purkinje neurones.
AuthorsCallewaert G, Eilers J, Konnerth A
JournalJ Physiol
PubMed ID8887772
'1. Using laser-scanning confocal microscopy, fast Ca2+ transients were recorded in individual not yet myelinated axons of Purkinje neurones in cerebellar slices from young rats. Axonal Ca2+ transients could be detected during a single action potential and had progressively larger amplitudes when the number of action potentials was increased. 2. ... More
Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.
AuthorsClark HA, Kopelman R, Tjalkens R, Philbert MA
JournalAnal Chem
PubMed ID10565275
'Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide ... More
In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons.
AuthorsHelmchen F, Svoboda K, Denk W, Tank DW
JournalNat Neurosci
PubMed ID10526338
'Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, ... More
Elementary events of InsP3-induced Ca2+ liberation in Xenopus oocytes: hot spots, puffs and blips.
AuthorsParker I, Choi J, Yao Y
JournalCell Calcium
PubMed ID8889202
'Liberation of sequestered Ca2+ ions in Xenopus oocytes by the second messenger inositol 1,4,5-trisphosphate (InP3) occurs from functionally discrete sites, which are spaced at intervals of several microns and probably represent clusterings of InsP3 receptor/channels (InsP3R) in the endoplasmic reticulum. As well as requiring InsP3, opening of release channels is ... More
Confocal microscopy of intracellular calcium dynamics during fertilization.
AuthorsStricker SA
JournalBiotechniques
PubMed ID10997262
Fluorescent probes for living cells.
AuthorsJohnson I
JournalHistochem J
PubMed ID10188922
'The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellular ion ... More
Confocal imaging of dendritic Ca2+ transients in hippocampal brain slices during simultaneous current- and voltage-clamp recording.
AuthorsJaffe DB, Brown TH
JournalMicrosc Res Tech
PubMed ID7841500
'Changes in the intracellular Ca2+ concentration ([Ca2+]i) within CA1 hippocampal pyramidal neurons were imaged using confocal laser scanning microscopy in conjunction with Ca(2+)-sensitive fluorescent indicators. The imaging was performed in thick hippocampal brain slices while simultaneously measuring or controlling electrical activity with sharp microelectrodes or whole-cell patch-clamp electrodes. The combination ... More
Acceleration of intracellular calcium waves in Xenopus oocytes by calcium influx.
AuthorsGirard S, Clapham D
JournalScience
PubMed ID8385801
'Many cell membrane receptors stimulate the phosphoinositide (PI) cycle, which produces complex intracellular calcium signals that regulate diverse processes such as secretion and transcription. A major messenger of this cycle, inositol 1,4,5-triphosphate (IP3), stimulates its receptor channel on the endoplasmic reticulum to release calcium into the cytosol. Activation of the ... More
The role of Ca2+ feedback in shaping InsP3-evoked Ca2+ signals in mouse pancreatic acinar cells.
AuthorsKidd JF, Fogarty KE, Tuft RA, Thorn P
JournalJ Physiol
PubMed ID10517811
'1. Cytosolic Ca2+ has been proposed to act as both a positive and a negative feedback signal on the inositol trisphosphate (InsP3) receptor. However, it is unclear how this might affect the Ca2+ response in vivo. 2. Mouse pancreatic acinar cells were whole-cell patch clamped to record the Ca2+-dependent chloride ... More
'Our understanding of the underlying mechanisms of Ca2+ signaling as well as our appreciation for its ubiquitous role in cellular processes has been rapidly advanced, in large part, due to the development of fluorescent Ca2+ indicators. In this chapter, we discuss some of the most common chemical Ca2+ indicators that ... More
Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria.
AuthorsPacher P, Csordás P, Schneider T, Hajnóczky G
JournalJ Physiol
PubMed ID11118489
'Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. To measure the amount ... More
NMDA receptors amplify calcium influx into dendritic spines during associative pre- and postsynaptic activation.
AuthorsSchiller J, Schiller Y, Clapham DE
JournalNat Neurosci
PubMed ID10195125
'Long-term potentiation (LTP) of synaptic strength can be induced by synchronous pre- and postsynaptic activation, and a rise in postsynaptic calcium is essential for induction of LTP. Calcium can enter through both voltage-dependent Ca2+ channels and NMDA-type glutamate receptors, but the relative contributions of these pathways is not known. We ... More
Odor-evoked calcium signals in dendrites of rat mitral cells.
AuthorsCharpak S, Mertz J, Beaurepaire E, Moreaux L, Delaney K
JournalProc Natl Acad Sci U S A
PubMed ID11158622
'Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca(2+)]) and define timing for dendritic release. Here we investigated in vivo, how ... More
A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells.
AuthorsFogarty KE, Kidd JF, Tuft RA, Thorn P
JournalBiophys J
PubMed ID10777728
'InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have ... More
Photon upmanship: why multiphoton imaging is more than a gimmick.
AuthorsDenk W, Svoboda K
JournalNeuron
PubMed ID9115730
Practical aspects of measuring [Ca2+] with fluorescent indicators.
AuthorsKao JP
JournalMethods Cell Biol
PubMed ID8201975
Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis.
One of the hallmarks of apoptosis is the redistribution of phosphatidylserine (PS) from the inner-to-outer plasma membrane (PM) leaflet, where it functions as a ligand for phagocyte recognition and the suppression of inflammatory responses. The mechanism by which apoptotic cells externalize PS has been assumed to involve ... More
Photo-physical properties of Ca2+-indicator dyes suitable for two-photon fluorescence-lifetime recordings.
AuthorsWilms CD, Eilers J,
JournalJ Microsc
PubMed ID17371443
We analyzed the suitability of various Ca2+-indicator dyes for quantitative two-photon fluorescence-lifetime imaging. Although fura-2, fluo-3, BTC and calcein did not show useful Ca2+-dependent lifetime changes, calcium orange, calcium green-1, oregon green-2 and -5N, as well as magnesium green allowed to quantify the Ca2+-free and Ca2+-bound dye fractions by a ... More
Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs.
AuthorsGalione A, McDougall A, Busa WB, Willmott N, Gillot I, Whitaker M
JournalScience
PubMed ID8392748
Propagating Ca2+ waves are a characteristic feature of Ca(2+)-linked signal transduction pathways. Intracellular Ca2+ waves are formed by regenerative stimulation of Ca2+ release from intracellular stores by Ca2+ itself. Mechanisms that rely on either inositol trisphosphate or ryanodine receptor channels have been proposed to account for Ca2+ waves in various ... More
Dendritic integration and its role in computing image velocity.
AuthorsSingle S, Borst A
JournalScience
PubMed ID9743497
The mechanisms underlying visual motion detection can be studied simultaneously in different cell compartments in vivo by using calcium as a reporter of the spatiotemporal activity distribution in single motion-sensitive cells of the fly. As predicted by the Reichardt model, local dendritic calcium signals are found to indicate the direction ... More
An optical submicrometer calcium sensor with conductance sensing capability.
AuthorsShalom S, Strinkovski A, Peleg G, Druckmann S, Krauss A, Lewis A, Linial M, Ottolenghi M
JournalAnal Biochem
PubMed ID9025941
The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous ... More
Optical recording of light-evoked calcium signals in the functionally intact retina.
AuthorsDenk W, Detwiler PB
JournalProc Natl Acad Sci U S A
PubMed ID10359834
Using two-photon excitation of fluorescent indicator dyes, we measured calcium concentration transients in retinal ganglion and amacrine cells without destroying the light sensitivity of the retina by maximally activating or bleaching the photoreceptors. This allowed an immediate assessment of the cellular morphology and study of the calcium signals evoked by ... More
Two-dimensional model of calcium waves reproduces the patterns observed in Xenopus oocytes.
AuthorsGirard S, Lückhoff A, Lechleiter J, Sneyd J, Clapham D
JournalBiophys J
PubMed ID1547335
Biological excitability enables the rapid transmission of physiological signals over distance. Using confocal fluorescence microscopy, we previously reported circular, planar, and spiral waves of Ca2+ in Xenopus laevis oocytes that annihilated one another upon collision. We present experimental evidence that the excitable process underlying wave propagation depends on Ca2+ diffusion ... More
A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.
AuthorsHelmchen F, Fee MS, Tank DW, Denk W
JournalNeuron
PubMed ID11580892
Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned ... More
Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.
AuthorsStricker SA, Centonze VE, Paddock SW, Schatten G
JournalDev Biol
PubMed ID1730391
Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with ... More
Increased frequency of calcium waves in Xenopus laevis oocytes that express a calcium-ATPase.
AuthorsCamacho P, Lechleiter JD
JournalScience
PubMed ID8385800
When inositol 1,4,5-triphosphate (IP3) receptors are activated, calcium is released from intracellular stores in excitatory propagating waves that annihilate each other upon collision. The annihilation phenomenon suggests the presence of an underlying refractory period that controls excitability. Enhanced calcium-adenosine triphosphatase (ATPase) activity might alter the refractory period of calcium release. ... More
An ensemble and single-molecule fluorescence spectroscopy investigation of Calcium Green 1, a calcium-ion sensor.
AuthorsLu Y, Paige MF,
JournalJ Fluoresc
PubMed ID17638063
The calcium-ion indicator dye, Calcium Green 1 (CG-1), has been characterized using a combination of ensemble and single-molecule optical spectroscopy measurements. In terms of ensemble measurements, CG-1 demonstrated a strong increase in fluorescence emission as a function of increasing [Ca(2+)]. This was accompanied by a change in the relative proportions ... More
Differential modulation of SERCA2 isoforms by calreticulin.
AuthorsJohn LM, Lechleiter JD, Camacho P
JournalJ Cell Biol
PubMed ID9722609
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by ... More
Dye loading with patch pipettes.
AuthorsEilers J, Konnerth A,
JournalCSH Protoc
PubMed ID20147143
This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction ... More
Dynamic fluorescence measurements of two-state systems: applications to calcium-chelating probes.
AuthorsHirshfield KM, Toptygin D, Packard BS, Brand L
JournalAnal Biochem
PubMed ID8470792
A fluorescence technique for characterizing ligand binding is evaluated. This technique uses the combination of steady-state and time-resolved methods to recover molar concentrations and overcomes errors inherent in the use of either method alone. The technique is applicable to time-resolved measurements made either with time-domain or frequency-domain instrumentation. A straightforward ... More
Patterns of elevated free calcium and calmodulin activation in living cells.
AuthorsHahn K, DeBiasio R, Taylor DL
JournalNature
PubMed ID1436037
The temporal and spatial dynamics of intracellular signals and protein effectors are being defined as a result of imaging using fluorescent reagents within living cells. We have described a new class of fluorescent analogues termed optical biosensors, which sense chemical or molecular events through their effects on protein transducers. One ... More
Imaging neuronal calcium fluorescence at high spatio-temporal resolution.
AuthorsCanepari M, Mammano F
JournalJ Neurosci Methods
PubMed ID10065988
A rapid fluorescence imaging system was developed and utilised to investigate the time-course of intracellular calcium concentration ([Ca2+]i) gradients generated by action potentials in CA1-CA3 pyramidal cells within brain slices of the rat hippocampus. The system, which is based on a fast commercial CCD camera, can acquire hundreds of 128 ... More
Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo.
AuthorsSvoboda K, Helmchen F, Denk W, Tank DW
JournalNat Neurosci
PubMed ID10195182
In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics ... More
Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function.
AuthorsWang SS, Augustine GJ
JournalNeuron
PubMed ID7576625
Chemical signals generated at synapses are highly limited in both spatial range and time course, so that experiments studying such signals must measure and manipulate them in both these dimensions. We describe an optical system that combines confocal laser scanning microscopy, to measure such signals, with focal photolysis of caged ... More
Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.
AuthorsDenk W, Delaney KR, Gelperin A, Kleinfeld D, Strowbridge BW, Tank DW, Yuste R
JournalJ Neurosci Methods
PubMed ID7869748
Light scattering by brain tissue and phototoxicity are major obstacles to the use of high-resolution optical imaging and photo-activation ('uncaging') of bioactive compounds from inactive ('caged') precursors in intact and semi-intact nervous systems. Optical methods based on 2-photon excitation promise to reduce these obstacles (Denk, 1994; Denk et al., 1990, ... More
Networks of coactive neurons in developing layer 1.
AuthorsSchwartz TH, Rabinowitz D, Unni V, Kumar VS, Smetters DK, Tsiola A, Yuste R
JournalNeuron
PubMed ID9539127
Spontaneous neuronal activity plays an important role in the development of cortical circuitry, yet its spatio-temporal dynamics are poorly understood. Cajal-Retzius (CR) neurons in developing layer 1 are necessary for correct cortical lamination and are strategically located to coordinate early circuit activity. To characterize the spontaneous activity of CR and ... More
Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.
AuthorsTschödrich-Rotter M, Kubitscheck U, Ugochukwu G, Buckley JT, Peters R
JournalBiophys J
PubMed ID8789089
Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single ... More
Effect on sperm-induced activation current and increase of cytosolic Ca2+ by agents that modify the mobilization of [Ca2+]i. I. Heparin and pentosan polysulfate.
AuthorsMohri T, Ivonnet PI, Chambers EL
JournalDev Biol
PubMed ID7589794
The mechanism of the elevation of intracellular free Ca2+ ([Ca2+]i) induced by a single sperm in eggs of the sea urchin Lytechinus variegatus was investigated. Simultaneous measurements of [Ca2+]i, and of the activation current, were carried out on eggs microinjected with Ca Green-1 or Ca Green dextran, and voltage clamped ... More
Calmodulin directly gates gap junction channels.
AuthorsPeracchia C, Sotkis A, Wang XG, Peracchia LL, Persechini A
JournalJ Biol Chem
PubMed ID10852921
Cytosolic changes control gap junction channel gating via poorly understood mechanisms. In the past two decades calmodulin participation in gating has been suggested, but compelling evidence for it has been lacking. Here we show that calmodulin indeed is associated with gap junctions and plays a direct role in chemical gating. ... More
The spine neck filters membrane potentials.
AuthorsAraya R, Jiang J, Eisenthal KB, Yuste R
JournalProc Natl Acad Sci U S A
PubMed ID17093040
Dendritic spines receive most synaptic inputs in the forebrain. Their morphology, with a spine head isolated from the dendrite by a slender neck, indicates a potential role in isolating inputs. Indeed, biochemical compartmentalization occurs at spine heads because of the diffusional bottleneck created by the spine neck. Here we investigate ... More
AuthorsZhou YD, Acker CD, Netoff TI, Sen K, White JA
JournalProc Natl Acad Sci U S A
PubMed ID16365307
Repeated induction of pre- and postsynaptic action potentials (APs) at a fixed time difference leads to long-term potentiation (LTP) or long-term depression (LTD) of the synapse, depending on the temporal order of pre- and postsynaptic activity. This phenomenon of spike-timing-dependent plasticity (STDP) is believed to arise by nonlinear processes that ... More
T-jump study of calcium binding kinetics of calcium chelators.
AuthorsNaraghi M
JournalCell Calcium
PubMed ID9481476
Recent experimental studies have investigated the kinetic competition between calcium chelators and the secretion apparatus at a fast central synapse. Simultaneously, mathematical modelling studies indicate the importance of a quantitative knowledge of the binding kinetics of the chelators in studying fast physiological processes operating on a millisecond time scale. Using ... More
Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
AuthorsXu C, Zipfel W, Shear JB, Williams RM, Webb WW
JournalProc Natl Acad Sci U S A
PubMed ID8855254
Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's ... More
Calcium waves along the cleavage furrows in cleavage-stage Xenopus embryos and its inhibition by heparin.
AuthorsMuto A, Kume S, Inoue T, Okano H, Mikoshiba K
JournalJ Cell Biol
PubMed ID8858172
Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium transients that localized to ... More
Induction of filopodia by direct local elevation of intracellular calcium ion concentration.
AuthorsLau PM, Zucker RS, Bentley D
JournalJ Cell Biol
PubMed ID10366598
In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which ... More
Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.
AuthorsOheim M, Naraghi M, Müller TH, Neher E
JournalCell Calcium
PubMed ID9793690
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 ... More
Morphological control of inositol-1,4,5-trisphosphate-dependent signals.
Inositol-1,4,5-trisphosphate (InsP(3))-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP(3) itself is not generally available. In particular, it has not been determined how the interplay of InsP(3) generation, diffusion, and degradation within complex cellular geometries can control ... More
Calcium binding to fluorescent calcium indicators: calcium green, calcium orange and calcium crimson.
AuthorsEberhard M, Erne P
JournalBiochem Biophys Res Commun
PubMed ID1930217
The recently introduced fluorescent calcium sensitive indicators calcium green, calcium orange and calcium crimson suggest important improvements and advantages to detect small calcium transients at low indicator concentrations. Thermodynamic dissociation constants and dissociation rate constants of calcium green, calcium orange and calcium crimson were measured by use of fluorescence titration ... More
Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.
AuthorsWilding M, Wright EM, Patel R, Ellis-Davies G, Whitaker M
JournalJ Cell Biol
PubMed ID8858173
Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from ... More
Calcium dependence of aequorin bioluminescence dissected by random mutagenesis.
AuthorsTricoire L, Tsuzuki K, Courjean O, Gibelin N, Bourout G, Rossier J, Lambolez B
JournalProc Natl Acad Sci U S A
PubMed ID16769886
Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by ... More
Validity of the rapid buffering approximation near a point source of calcium ions.
AuthorsSmith GD, Wagner J, Keizer J
JournalBiophys J
PubMed ID8744292
In the presence of rapid buffers the full reaction-diffusion equations describing Ca2+ transport can be reduced using the rapid buffering approximation to a single transport equation for [Ca2+]. Here we simulate the full and reduced equations, exploring the conditions necessary for the validity of the rapid buffering approximation for an ... More
Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers.
AuthorsPetit L, Maier E, Gibert M, Popoff MR, Benz R
JournalJ Biol Chem
PubMed ID11278669
Epsilon toxin is a potent toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in animals. One of the main properties of epsilon toxin is the production of edema. We have previously found that epsilon toxin causes a rapid swelling of Madin-Darby ... More
Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.
AuthorsEilers J, Callewaert G, Armstrong C, Konnerth A
JournalProc Natl Acad Sci U S A
PubMed ID7479766
Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a ... More
Highly localized Ca(2+) accumulation revealed by multiphoton microscopy in an identified motoneuron and its modulation by dopamine.
Calcium is essential for synaptic transmission and the control of the intrinsic firing properties of neurons; this makes Ca(2+) channels a prime target for neuromodulators. A combination of multiphoton microscopy and voltage-clamp recording was used to determine the localization of voltage-dependent Ca(2+) accumulation in the two pyloric dilator (PD) neurons ... More
Adenophostin A induces spatially restricted calcium signaling in Xenopus laevis oocytes.
AuthorsBird GS, Takahashi M, Tanzawa K, Putney JW
JournalJ Biol Chem
PubMed ID10400696
The activation of intracellular calcium release and calcium entry across the plasmalemma in response to intracellular application of inositol 2,4,5-trisphosphate and adenophostin A, two metabolically stable agonists for inositol 1,4,5-trisphosphate receptors, was investigated using Xenopus laevis oocytes and confocal imaging. Intracellular injection of inositol 2,4,5-trisphosphate induced a rapidly spreading calcium ... More
Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.
AuthorsZhao M, Hollingworth S, Baylor SM
JournalBiophys J
PubMed ID8789107
Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators ... More
Relation between intracellular Ca2+ signals and Ca(2+)-activated Cl- current in Xenopus oocytes.
AuthorsParker I, Yao Y
JournalCell Calcium
PubMed ID8055544
Activation of inositol 1,4,5-trisphosphate (InsP3) signalling in Xenopus oocytes causes intracellular Ca2+ mobilization and thereby activates a Ca(2+)-dependent Cl- membrane conductance. Measurements of cytosolic Ca2+ levels using fluorescent indicators, however, revealed little correspondence with Cl- currents. Intracellular photorelease of InsP3 from a caged precursor evoked transient currents that peaked while ... More
High Throughput Screening with Multiphoton Excitation.
AuthorsLakowicz JR, Gryczynski I, Gryczynski Z
JournalJ Biomol Screen
PubMed ID10838432
Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons ... More
Hydroxychloroquine inhibits calcium signals in T cells: a new mechanism to explain its immunomodulatory properties.
Hydroxychloroquine (HCQ), a lysosomotropic amine, is an immunosuppressive agent presently being evaluated in bone marrow transplant patients to treat graft-versus-host disease. While its immunosuppressive properties have been attributed primarily to its ability to interfere with antigen processing, recent reports demonstrate HCQ also blocks T-cell activation in vitro. To more precisely ... More
A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.
AuthorsThomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P
JournalCell Calcium
PubMed ID11032777
Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. ... More
The induction of long-term potentiation (LTP) is generally assumed to be triggered by Ca2+ entry into dendritic spines via NMDA receptor-gated channels. A previous computational model proposed that spines serve several functions in this process. First, they compartmentalize and amplify increase in [Ca2+]i. Second, they augment the nonlinear relationship between ... More
Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones.
AuthorsLancaster B, Batchelor AM
JournalJ Physiol
PubMed ID10639100
1. Whole-cell recordings were made from rat CA1 neurones in brain slices. When electrodes contained diazo-2 (2 mM) or dibromo BAPTA (1 mM) a large steady-state outward current (hundreds of picoamps) developed within 5 min of breakthrough at a VH of -60 mV. BAPTA itself (1 mM) caused qualitatively similar ... More
alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction.
AuthorsElrick DB, Charlton MP
JournalJ Neurophysiol
PubMed ID10601481
alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which ... More
Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane.
AuthorsGraier WF, Paltauf-Doburzynska J, Hill BJ, Fleischhacker E, Hoebel BG, Kostner GM, Sturek M
JournalJ Physiol
PubMed ID9481676
1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ dyes, such as fura-2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca2+ elevations occur which are controlled by cell membrane Na(+)-Ca2+ exchange ... More
Non-capacitative calcium entry in Chinese hamster ovary cells expressing the platelet-derived growth factor receptor.
AuthorsMathias RS, Zhang SJ, Wilson E, Gardner P, Ives HE
JournalJ Biol Chem
PubMed ID9360982
Platelet-derived growth factor (PDGF) is believed to produce intracellular calcium (Ca2+i) transients by inositol trisphosphate (InsP3)-mediated release of intracellular Ca2+ stores followed by "capacitative" Ca2+ entry due to emptying of these stores. We examined the roles for the phospholipase Cgamma-InsP3 pathway and the emptying of InsP3-dependent intracellular Ca2+ stores in ... More
Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.
AuthorsSuzuki K, Tanaka Y, Nakajima Y, Hirano K, Itoh H, Miyata H, Hayakawa T, Kinosita K
JournalBiophys J
PubMed ID7756541
Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially ... More
Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy.
AuthorsBullen A, Saggau P
JournalPflugers Arch
PubMed ID9716714
The instrumental design and experimental conditions for high-speed, simultaneous optical recording of membrane potential and intracellular Ca2+ with subcellular resolution are presented. This method employs an extended version of a high-speed, random-access, laser-scanning fluorescence microscope designed to record fast physiological signals from small neuronal structures with high spatiotemporal resolution (Bullen, ... More
Kinetics of calcium release from manganese peroxidase during thermal inactivation.
AuthorsTimofeevski SL, Aust SD
JournalArch Biochem Biophys
PubMed ID9185626
It was previously reported that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was susceptible to thermal inactivation because it contains relatively labile Ca2+ ions required for stability and activity [Sutherland and Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. In this work we determined that four Ca2+ ions are present ... More
On the roles of Ca2+ diffusion, Ca2+ buffers, and the endoplasmic reticulum in IP3-induced Ca2+ waves.
AuthorsJafri MS, Keizer J
JournalBiophys J
PubMed ID8580358
We have investigated the effects of Ca2+ diffusion, mobile and stationary Ca2+ buffers in the cytosol, and Ca2+ handling by the endoplasmic reticulum on inositol 1,4,5-trisphosphate-induced Ca2+ wave propagation. Rapid equilibration of free and bound Ca2+ is used to describe Ca2+ sequestration by buffers in both the cytosol and endoplasmic ... More