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Sample records for 2-photon calcium imaging

  1. Imaging calcium in neurons.

    PubMed

    Grienberger, Christine; Konnerth, Arthur

    2012-03-08

    Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.

  2. Control of Local Intracellular Calcium Concentration with Dynamic-Clamp Controlled 2-Photon Uncaging

    PubMed Central

    Idoux, Erwin; Mertz, Jerome

    2011-01-01

    The variations of the intracellular concentration of calcium ion ([Ca2+]i) are at the heart of intracellular signaling, and their imaging is therefore of enormous interest. However, passive [Ca2+]i imaging provides no control over these variations, meaning that a full exploration of the functional consequences of [Ca2+]i changes is difficult to attain. The tools designed so far to modify [Ca2+]i, even qualitatively, suffer drawbacks that undermine their widespread use. Here, we describe an electro-optical technique to quantitatively set [Ca2+]i, in real time and with sub-cellular resolution, using two-photon Ca2+ uncaging and dynamic-clamp. We experimentally demonstrate, on neurons from acute olfactory bulb slices of Long Evans rats, various capabilities of this technique previously difficult to achieve, such as the independent control of the membrane potential and [Ca2+]i variations, the functional knocking-in of user-defined virtual voltage-dependent Ca2+ channels, and the standardization of [Ca2+]i patterns across different cells. Our goal is to lay the groundwork for this technique and establish it as a new and versatile tool for the study of cell signaling. PMID:22216105

  3. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  4. In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy

    PubMed Central

    Lee, Martin; Downes, Andy; Chau, You-Ying; Serrels, Bryan; Hastie, Nick; Elfick, Alistair; Brunton, Valerie; Frame, Margaret; Serrels, Alan

    2015-01-01

    The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche. PMID:28243514

  5. Calcium and bones (image)

    MedlinePlus

    ... for the growth, maintenance, and reproduction of the human body. Bones, like other tissues in the body, are continually being re-formed and incorporate calcium into their structure. Calcium is essential for the formation of and maintenance of healthy teeth.

  6. Calcium imaging of infrared-stimulated activity in rodent brain.

    PubMed

    Cayce, Jonathan Matthew; Bouchard, Matthew B; Chernov, Mykyta M; Chen, Brenda R; Grosberg, Lauren E; Jansen, E Duco; Hillman, Elizabeth M C; Mahadevan-Jansen, Anita

    2014-04-01

    Infrared neural stimulation (INS) is a promising neurostimulation technique that can activate neural tissue with high spatial precision and without the need for exogenous agents. However, little is understood about how infrared light interacts with neural tissue on a cellular level, particularly within the living brain. In this study, we use calcium sensitive dye imaging on macroscopic and microscopic scales to explore the spatiotemporal effects of INS on cortical calcium dynamics. The INS-evoked calcium signal that was observed exhibited a fast and slow component suggesting activation of multiple cellular mechanisms. The slow component of the evoked signal exhibited wave-like properties suggesting network activation, and was verified to originate from astrocytes through pharmacology and 2-photon imaging. We also provide evidence that the fast calcium signal may have been evoked through modulation of glutamate transients. This study demonstrates that pulsed infrared light can induce intracellular calcium modulations in both astrocytes and neurons, providing new insights into the mechanisms of action of INS in the brain.

  7. Calcium imaging perspectives in plants.

    PubMed

    Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Occhipinti, Andrea; Maffei, Massimo E

    2014-03-04

    The calcium ion (Ca2+) is a versatile intracellular messenger. It provides dynamic regulation of a vast array of gene transcriptions, protein kinases, transcription factors and other complex downstream signaling cascades. For the past six decades, intracellular Ca2+ concentration has been significantly studied and still many studies are under way. Our understanding of Ca2+ signaling and the corresponding physiological phenomenon is growing exponentially. Here we focus on the improvements made in the development of probes used for Ca2+ imaging and expanding the application of Ca2+ imaging in plant science research.

  8. Calcium Imaging of Sonoporation of Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Sabens, David; Aehle, Matthew; Steyer, Grant; Kourennyi, Dmitri; Deng, Cheri X.

    2006-05-01

    Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real-time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved understanding of sonoporation mechanism. Our experimental results demonstrated the feasibility of calcium imaging of sonoporation in Chinese Hamster Ovary (CHO) cells. We have measured the changes in the intracellular calcium concentration using Fura-2, a fluorescent probe, which indicate influx or flow of Calcium across the cell membrane. Analysis of data identified key aspects in the dynamic sonoporation process including the formation of pores in the cell membrane, and the relative temporal duration of the pores and their resealing. These observations are obtained through the analysis of the rate the calcium concentration changes within the cells, making it possible to visualize membrane opening and repair in real-time through such changes in the intracellular calcium concentration.

  9. Imaging action potentials with calcium indicators.

    PubMed

    MacLean, Jason N; Yuste, Rafael

    2009-11-01

    The understanding of neuronal circuits has been, and will continue to be, greatly advanced by the simultaneous imaging of action potentials in neuronal ensembles. This protocol describes "bulk" loading of brain slices with acetoxymethyl (AM) ester calcium indicators in order to monitor action potential activity in large populations of neurons simultaneously. The imaging of calcium influx into neurons provides an indirect, but accurate, measure of action potential generation in individual neurons. Single-cell resolution, and thus the easy identification of every active cell, is the key advantage of the technique.

  10. Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation

    NASA Astrophysics Data System (ADS)

    Klinger, Antje; Krapf, Lisa; Orzekowsky-Schroeder, Regina; Koop, Norbert; Vogel, Alfred; Hüttmann, Gereon

    2015-11-01

    Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.

  11. Functional calcium imaging in developing cortical networks.

    PubMed

    Dawitz, Julia; Kroon, Tim; Hjorth, J J Johannes; Meredith, Rhiannon M

    2011-10-22

    , synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry. In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

  12. Denoising two-photon calcium imaging data.

    PubMed

    Malik, Wasim Q; Schummers, James; Sur, Mriganka; Brown, Emery N

    2011-01-01

    Two-photon calcium imaging is now an important tool for in vivo imaging of biological systems. By enabling neuronal population imaging with subcellular resolution, this modality offers an approach for gaining a fundamental understanding of brain anatomy and physiology. Proper analysis of calcium imaging data requires denoising, that is separating the signal from complex physiological noise. To analyze two-photon brain imaging data, we present a signal plus colored noise model in which the signal is represented as harmonic regression and the correlated noise is represented as an order autoregressive process. We provide an efficient cyclic descent algorithm to compute approximate maximum likelihood parameter estimates by combing a weighted least-squares procedure with the Burg algorithm. We use Akaike information criterion to guide selection of the harmonic regression and the autoregressive model orders. Our flexible yet parsimonious modeling approach reliably separates stimulus-evoked fluorescence response from background activity and noise, assesses goodness of fit, and estimates confidence intervals and signal-to-noise ratio. This refined separation leads to appreciably enhanced image contrast for individual cells including clear delineation of subcellular details and network activity. The application of our approach to in vivo imaging data recorded in the ferret primary visual cortex demonstrates that our method yields substantially denoised signal estimates. We also provide a general Volterra series framework for deriving this and other signal plus correlated noise models for imaging. This approach to analyzing two-photon calcium imaging data may be readily adapted to other computational biology problems which apply correlated noise models.

  13. Immunohistochemical and calcium imaging methods in wholemount rat retina.

    PubMed

    Sargoy, Allison; Barnes, Steven; Brecha, Nicholas C; Pérez De Sevilla Müller, Luis

    2014-10-13

    In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

  14. Fast online deconvolution of calcium imaging data.

    PubMed

    Friedrich, Johannes; Zhou, Pengcheng; Paninski, Liam

    2017-03-01

    Fluorescent calcium indicators are a popular means for observing the spiking activity of large neuronal populations, but extracting the activity of each neuron from raw fluorescence calcium imaging data is a nontrivial problem. We present a fast online active set method to solve this sparse non-negative deconvolution problem. Importantly, the algorithm 3progresses through each time series sequentially from beginning to end, thus enabling real-time online estimation of neural activity during the imaging session. Our algorithm is a generalization of the pool adjacent violators algorithm (PAVA) for isotonic regression and inherits its linear-time computational complexity. We gain remarkable increases in processing speed: more than one order of magnitude compared to currently employed state of the art convex solvers relying on interior point methods. Unlike these approaches, our method can exploit warm starts; therefore optimizing model hyperparameters only requires a handful of passes through the data. A minor modification can further improve the quality of activity inference by imposing a constraint on the minimum spike size. The algorithm enables real-time simultaneous deconvolution of O(105) traces of whole-brain larval zebrafish imaging data on a laptop.

  15. Fast online deconvolution of calcium imaging data

    PubMed Central

    Zhou, Pengcheng; Paninski, Liam

    2017-01-01

    Fluorescent calcium indicators are a popular means for observing the spiking activity of large neuronal populations, but extracting the activity of each neuron from raw fluorescence calcium imaging data is a nontrivial problem. We present a fast online active set method to solve this sparse non-negative deconvolution problem. Importantly, the algorithm 3progresses through each time series sequentially from beginning to end, thus enabling real-time online estimation of neural activity during the imaging session. Our algorithm is a generalization of the pool adjacent violators algorithm (PAVA) for isotonic regression and inherits its linear-time computational complexity. We gain remarkable increases in processing speed: more than one order of magnitude compared to currently employed state of the art convex solvers relying on interior point methods. Unlike these approaches, our method can exploit warm starts; therefore optimizing model hyperparameters only requires a handful of passes through the data. A minor modification can further improve the quality of activity inference by imposing a constraint on the minimum spike size. The algorithm enables real-time simultaneous deconvolution of O(105) traces of whole-brain larval zebrafish imaging data on a laptop. PMID:28291787

  16. Dopaminergic regulation of dendritic calcium: fast multisite calcium imaging.

    PubMed

    Zhou, Wen-Liang; Oikonomou, Katerina D; Short, Shaina M; Antic, Srdjan D

    2013-01-01

    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 transients are generated in remote dendritic compartments. In order to understand the cellular mechanisms of dopaminergic modulation it is important to know whether and how dopaminergic signals affect dendritic calcium transients. In this chapter, we describe a relatively inexpensive method for monitoring dendritic calcium fluctuations at multiple loci across the pyramidal dendritic tree, at the same moment of time (simultaneously). The experiments have been designed to measure the amplitude, time course and spatial extent of action potential-associated dendritic calcium transients before and after application of dopaminergic drugs. In the examples provided here the dendritic calcium transients were evoked by triggering the somatic action potentials (backpropagation-evoked), and puffs of exogenous dopamine were applied locally onto selected dendritic branches.

  17. In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex

    PubMed Central

    Yuryev, Mikhail; Pellegrino, Christophe; Jokinen, Ville; Andriichuk, Liliia; Khirug, Stanislav; Khiroug, Leonard; Rivera, Claudio

    2016-01-01

    The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism. PMID:26778965

  18. Calcium imaging of cortical neurons using Fura-2 AM.

    PubMed

    Barreto-Chang, Odmara L; Dolmetsch, Ricardo E

    2009-01-19

    Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

  19. In vivo neuronal calcium imaging in C. elegans.

    PubMed

    Chung, Samuel H; Sun, Lin; Gabel, Christopher V

    2013-04-10

    The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon and GCaMP allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

  20. Simultaneous patch-clamping and calcium imaging in developing dendrites.

    PubMed

    Kleindienst, Thomas; Lohmann, Christian

    2014-03-01

    Calcium imaging has been used extensively to explore the role of action potential (AP) firing in the development of neuronal structure and synaptic function because increases in intracellular calcium ([Ca(2+)]i) reliably and, within a certain range, linearly reflect neuronal spiking activity. Patterns of APs in individual cells can be deduced from calcium recordings, which have typically been performed at the level of cell bodies. However, neurons are particularly susceptible to phototoxicity when they are illuminated at the soma. Furthermore, for some imaging experiments (e.g., those that address the interactions between dendrites and axons during synapse formation), the cell body of a given neuron may simply not be in the field of view. In these situations, it would be helpful to determine the spiking patterns of a neuron from the calcium activity in its subcellular compartments such as stretches of dendrites or axons. Here, we describe an approach for determining the relationship between AP firing and dendritic calcium transients by simultaneously imaging calcium transients in small dendritic stretches of hippocampal pyramidal neurons in slice cultures from neonatal rats and recording spiking activity with whole-cell patch-clamp recordings in these neurons. These experiments allow us to correlate the electrophysiological spiking pattern with the accompanying changes in the calcium concentration in individual dendritic segments.

  1. Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data

    PubMed Central

    Pnevmatikakis, Eftychios A.; Soudry, Daniel; Gao, Yuanjun; Machado, Timothy A.; Merel, Josh; Pfau, David; Reardon, Thomas; Mu, Yu; Lacefield, Clay; Yang, Weijian; Ahrens, Misha; Bruno, Randy; Jessell, Thomas M.; Peterka, Darcy S.; Yuste, Rafael; Paninski, Liam

    2016-01-01

    SUMMARY We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multineuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data. PMID:26774160

  2. Probing synaptic function in dendrites with calcium imaging.

    PubMed

    Siegel, Friederike; Lohmann, Christian

    2013-04-01

    Calcium imaging has become a widely used technique to probe neuronal activity on the cellular and subcellular levels. In contrast to standard electrophysiological methods, calcium imaging resolves sub- and suprathreshold activation patterns in structures as small as fine dendritic branches and spines. This review highlights recent findings gained on the subcellular level using calcium imaging, with special emphasis on synaptic transmission and plasticity in individual spines. Since imaging allows monitoring activity across populations of synapses, it has recently been adopted to investigate how dendrites integrate information from many synapses. Future experiments, ideally carried out in vivo, will reveal how the dendritic tree integrates and computes afferent signals. For example, it is now possible to directly test the concept that dendritic inputs are clustered and that single dendrites or dendritic stretches act as independent computational units.

  3. Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data.

    PubMed

    Pnevmatikakis, Eftychios A; Soudry, Daniel; Gao, Yuanjun; Machado, Timothy A; Merel, Josh; Pfau, David; Reardon, Thomas; Mu, Yu; Lacefield, Clay; Yang, Weijian; Ahrens, Misha; Bruno, Randy; Jessell, Thomas M; Peterka, Darcy S; Yuste, Rafael; Paninski, Liam

    2016-01-20

    We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multi-neuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data.

  4. Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells.

    PubMed

    Perry, Jacob L; Ramachandran, Nina K; Utama, Budi; Hyser, Joseph M

    2015-11-15

    Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

  5. Simultaneous electrophysiological recording and calcium imaging of suprachiasmatic nucleus neurons.

    PubMed

    Irwin, Robert P; Allen, Charles N

    2013-12-08

    Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca(2+) concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

  6. Simultaneous Sodium and Calcium Imaging from Dendrites and Axons

    PubMed Central

    Miyazaki, Kenichi

    2015-01-01

    Abstract Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca2+]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca2+]i and [Na+]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca2+]i and [Na+]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N). PMID:26730401

  7. Simultaneous Sodium and Calcium Imaging from Dendrites and Axons.

    PubMed

    Miyazaki, Kenichi; Ross, William N

    2015-01-01

    Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca(2+)]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca(2+)]i and [Na(+)]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca(2+)]i and [Na(+)]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).

  8. Combining calcium imaging with other optical techniques.

    PubMed

    Canepari, Marco; Zecevic, Dejan; Vogt, Kaspar E; Ogden, David; De Waard, Michel

    2013-12-01

    Ca(2+) imaging is a commonly used approach for measuring Ca(2+) signals at high spatial resolution. The method is often combined with electrode recordings to correlate electrical and chemical signals or to investigate Ca(2+) signals following an electrical stimulation. To obtain information on electrical activity at the same spatial resolution, Ca(2+) imaging must be combined with membrane potential imaging. Similarly, stimulation of subcellular compartments requires photostimulation. Thus, combining Ca(2+) imaging with an additional optical technique facilitates the study of a number of physiological questions. The aim of this article is to introduce some basic principles regarding the combination of Ca(2+) imaging with other optical techniques. We discuss the design of the optics, the design of experimental protocols, the optical characteristics of Ca(2+) indicators used in combination with an optical probe, and the affinity of the Ca(2+) indicator in relation to the type of measurement. This information will enable the reader to devise an optimal strategy for combined optical experiments.

  9. Imaging of calcium dynamics in pollen tube cytoplasm.

    PubMed

    Barberini, María Laura; Muschietti, Jorge

    2015-01-01

    Cytoplasmic calcium [(Ca(2+))cyt] is a central component of cellular signal transduction pathways. In plants, many external and internal stimuli transiently elevate (Ca(2+))cyt, initiating downstream responses that control different features of plant development. In pollen tubes the establishment of an oscillatory gradient of calcium at the tip is essential for polarized growth. Disruption of the cytosolic Ca(2+) gradient by chelators or channel blockers inhibits pollen tube growth. To quantify the physiological role of (Ca(2+))cyt in cellular systems, genetically encoded Ca(2+) indicators such as Yellow Cameleons (YCs) have been developed. The Cameleons are based on a fluorescence resonance energy transfer (FRET) process. Here, we describe a method for imaging cytoplasmic Ca(2+) dynamics in growing pollen tubes that express the fluorescent calcium indicator Yellow Cameleon 3.6 (YC 3.6), using laser-scanning confocal microscopy.

  10. Calcium imaging in neuron cell death.

    PubMed

    Calvo, María; Villalobos, Carlos; Núñez, Lucía

    2015-01-01

    Intracellular Ca2+ is involved in control of a large variety of cell functions including apoptosis and neuron cell death. For example, intracellular Ca2+ overload is critical in neuron cell death induced by excitotoxicity. Thus, single cell monitoring of intracellular Ca2+ concentration ([Ca2+]cyt ) in neurons concurrently with apoptosis and neuron cell death is widely required. Procedures for culture and preparation of primary cultures of hippocampal rat neurons and fluorescence imaging of cytosolic Ca2+ concentration in Fura2/AM -loaded neurons are described. We also describe a method for apoptosis detection by immunofluorescence imaging. Finally, a simple method for concurrent measurements of [Ca2+]cyt and apoptosis in the same neurons is described.

  11. Calcium

    MedlinePlus

    ... You'll also find calcium in broccoli and dark green, leafy vegetables (especially collard and turnip greens, ... can enjoy good sources of calcium such as dark green, leafy vegetables, broccoli, chickpeas, and calcium-fortified ...

  12. Imaging calcium waves and sparks in central neurons.

    PubMed

    Ross, William N; Manita, Satoshi

    2012-10-01

    Here we describe the use of wide-field charge-coupled device (CCD) camera-based imaging methods to detect the spatial and temporal aspects of calcium release from internal stores in dendrites of neurons in brain slice preparations. This approach is useful for revealing aspects of this signaling system, which is generally invisible to electrical recording. The changes in intracellular calcium ion concentrations, [Ca(2+)](i), sometimes occur as large-amplitude, propagating Ca(2+) waves or as much smaller, localized events (sparks). In this protocol, a cell is loaded with an indicator that responds to Ca(2+), waves or sparks are stimulated in the cell, and the spatial and temporal characteristics of calcium release from internal stores in the cell are detected using wide-field CCD camera-based imaging. Such camera systems have some advantages for detecting and analyzing these [Ca(2+)](i) changes because the waves are spatially extended and the sparks do not always occur at the same locations.

  13. Improved deep two-photon calcium imaging in vivo.

    PubMed

    Birkner, Antje; Tischbirek, Carsten H; Konnerth, Arthur

    2016-12-21

    Two-photon laser scanning calcium imaging has emerged as a useful method for the exploration of neural function and structure at the cellular and subcellular level in vivo. The applications range from imaging of subcellular compartments such as dendrites, spines and axonal boutons up to the functional analysis of large neuronal or glial populations. However, the depth penetration is often limited to a few hundred micrometers, corresponding, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations originating from refractive index inhomogeneties of the tissue are the reasons for these limitations. The depth penetration of two-photon imaging can be enhanced through various approaches, such as the implementation of adaptive optics, the use of three-photon excitation and/or labeling cells with red-shifted genetically encoded fluorescent sensors. However, most of the approaches used so far require the implementation of new instrumentation and/or time consuming staining protocols. Here we present a simple approach that can be readily implemented in combination with standard two-photon microscopes. The method involves an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590 and benefits from the use of ultra-short laser pulses. The approach allows in vivo functional imaging of neuronal populations with single cell resolution in all six layers of the mouse cortex. We demonstrate that stable recordings in deep cortical layers are not restricted to anesthetized animals but are well feasible in awake, behaving mice. We anticipate that the improved depth penetration will be beneficial for two-photon functional imaging in larger species, such as non-human primates.

  14. Intravital imaging of podocyte calcium in glomerular injury and disease

    PubMed Central

    Burford, James L.; Villanueva, Karie; Lam, Lisa; Riquier-Brison, Anne; Hackl, Matthias J.; Pippin, Jeffrey; Shankland, Stuart J.; Peti-Peterdi, János

    2014-01-01

    Intracellular calcium ([Ca2+]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca2+]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca2+]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca2+]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca2+]i around the injury site and promoted cell-to-cell propagating podocyte [Ca2+]i waves along capillary loops. [Ca2+]i wave propagation was ameliorated by inhibitors of purinergic [Ca2+]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca2+]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca2+]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca2+]i in glomerular pathology and suggest that purinergic [Ca2+]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney. PMID:24713653

  15. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    PubMed

    Jahn, Karolina; Hille, Carsten

    2014-01-01

    For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-dependent way, unraveling in vitro dissociation constants K(D) of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  16. Calcium imaging of network function in the developing spinal cord.

    PubMed

    O'Donovan, Michael J; Bonnot, Agnès; Wenner, Peter; Mentis, George Z

    2005-05-01

    We have used calcium imaging to visualize the spatiotemporal organization of activity generated by in vitro spinal cord preparations of the developing chick embryo and the neonatal mouse. During each episode of spontaneous activity, we found that chick spinal neurons were activated rhythmically and synchronously throughout the transverse extent of the spinal cord. At the onset of a spontaneous episode, optical activity originated in the ventrolateral part of the cord. Back-labeling of spinal interneurons with calcium dyes suggested that this ventrolateral initiation was mediated by activation of a class of interneurons, located dorsomedial to the motor nucleus, that receive direct monosynaptic input from motoneurons. Studies of locomotor-like activity in the anterior lumbar segments of the neonatal mouse cord revealed the existence of a rostrocaudal wave in the oscillatory component of each cycle of rhythmic motoneuron activity. This finding raises the possibility that the activation of mammalian motoneurons during locomotion may share some of the same rostrocaudally organized mechanisms that evolved to control swimming in fishes.

  17. Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

    PubMed

    Beerman, Rebecca W; Matty, Molly A; Au, Gina G; Looger, Loren L; Choudhury, Kingshuk Roy; Keller, Philipp J; Tobin, David M

    2015-12-15

    Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

  18. Intravital imaging of podocyte calcium in glomerular injury and disease.

    PubMed

    Burford, James L; Villanueva, Karie; Lam, Lisa; Riquier-Brison, Anne; Hackl, Matthias J; Pippin, Jeffrey; Shankland, Stuart J; Peti-Peterdi, János

    2014-05-01

    Intracellular calcium ([Ca²⁺]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²⁺]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²⁺]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²⁺]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²⁺]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²⁺]i waves along capillary loops. [Ca²⁺]i wave propagation was ameliorated by inhibitors of purinergic [Ca²⁺]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²⁺]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²⁺]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²⁺]i in glomerular pathology and suggest that purinergic [Ca²⁺]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

  19. The minimal requirements to use calcium imaging to analyze ICRAC.

    PubMed

    Alansary, Dalia; Kilch, Tatiana; Holzmann, Christian; Peinelt, Christine; Hoth, Markus; Lis, Annette

    2014-06-02

    Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca(2+) imaging technologies to analyze CRAC-dependent Ca(2+) influx. However, Ca(2+) signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of ICRAC is always preferred. Since many laboratories use Ca(2+) imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca(2+) signals not only depend on the net Ca(2+) influx through CRAC channels but also depend on other Ca(2+) influx mechanisms, K(+) channels or Cl(-) channels (which determine the membrane potential), Ca(2+) export mechanisms like plasma membrane Ca(2+) ATPase (PMCA), sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or Na(+)-Ca(2+) exchangers, and (local) Ca(2+) buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca(2+) imaging experiments, including the ability to rule out Ca(2+) signals from other sources.

  20. Direct Imaging of Hippocampal Epileptiform Calcium Motifs Following Kainic Acid Administration in Freely Behaving Mice

    PubMed Central

    Berdyyeva, Tamara K.; Frady, E. Paxon; Nassi, Jonathan J.; Aluisio, Leah; Cherkas, Yauheniya; Otte, Stephani; Wyatt, Ryan M.; Dugovic, Christine; Ghosh, Kunal K.; Schnitzer, Mark J.; Lovenberg, Timothy; Bonaventure, Pascal

    2016-01-01

    Prolonged exposure to abnormally high calcium concentrations is thought to be a core mechanism underlying hippocampal damage in epileptic patients; however, no prior study has characterized calcium activity during seizures in the live, intact hippocampus. We have directly investigated this possibility by combining whole-brain electroencephalographic (EEG) measurements with microendoscopic calcium imaging of pyramidal cells in the CA1 hippocampal region of freely behaving mice treated with the pro-convulsant kainic acid (KA). We observed that KA administration led to systematic patterns of epileptiform calcium activity: a series of large-scale, intensifying flashes of increased calcium fluorescence concurrent with a cluster of low-amplitude EEG waveforms. This was accompanied by a steady increase in cellular calcium levels (>5 fold increase relative to the baseline), followed by an intense spreading calcium wave characterized by a 218% increase in global mean intensity of calcium fluorescence (n = 8, range [114–349%], p < 10−4; t-test). The wave had no consistent EEG phenotype and occurred before the onset of motor convulsions. Similar changes in calcium activity were also observed in animals treated with 2 different proconvulsant agents, N-methyl-D-aspartate (NMDA) and pentylenetetrazol (PTZ), suggesting the measured changes in calcium dynamics are a signature of seizure activity rather than a KA-specific pathology. Additionally, despite reducing the behavioral severity of KA-induced seizures, the anticonvulsant drug valproate (VA, 300 mg/kg) did not modify the observed abnormalities in calcium dynamics. These results confirm the presence of pathological calcium activity preceding convulsive motor seizures and support calcium as a candidate signaling molecule in a pathway connecting seizures to subsequent cellular damage. Integrating in vivo calcium imaging with traditional assessment of seizures could potentially increase translatability of pharmacological

  1. Calcium

    MedlinePlus

    ... milligrams) of calcium each day. Get it from: Dairy products. Low-fat milk, yogurt, cheese, and cottage ... lactase that helps digest the sugar (lactose) in dairy products, and may have gas, bloating, cramps, or ...

  2. Simultaneous measurement of neural spike recordings and multi-photon calcium imaging in neuroblastoma cells.

    PubMed

    Kim, Suhwan; Jung, Unsang; Baek, Juyeong; Kang, Shinwon; Kim, Jeehyun

    2012-11-08

    This paper proposes the design and implementation of a micro-electrode array (MEA) for neuroblastoma cell culturing. It also explains the implementation of a multi-photon microscope (MPM) customized for neuroblastoma cell excitation and imaging under ambient light. Electrical signal and fluorescence images were simultaneously acquired from the neuroblastoma cells on the MEA. MPM calcium images of the cultured neuroblastoma cell on the MEA are presented and also the neural activity was acquired through the MEA recording. A calcium green-1 (CG-1) dextran conjugate of 10,000 D molecular weight was used in this experiment for calcium imaging. This study also evaluated the calcium oscillations and neural spike recording of neuroblastoma cells in an epileptic condition. Based on our observation of neural spikes in neuroblastoma cells with our proposed imaging modality, we report that neuroblastoma cells can be an important model for epileptic activity studies.

  3. Calcium imaging with genetically encoded indicators in behaving primates

    PubMed Central

    Seidemann, Eyal; Chen, Yuzhi; Bai, Yoon; Chen, Spencer C; Mehta, Preeti; Kajs, Bridget L; Geisler, Wilson S; Zemelman, Boris V

    2016-01-01

    Understanding the neural basis of behaviour requires studying brain activity in behaving subjects using complementary techniques that measure neural responses at multiple spatial scales, and developing computational tools for understanding the mapping between these measurements. Here we report the first results of widefield imaging of genetically encoded calcium indicator (GCaMP6f) signals from V1 of behaving macaques. This technique provides a robust readout of visual population responses at the columnar scale over multiple mm2 and over several months. To determine the quantitative relation between the widefield GCaMP signals and the locally pooled spiking activity, we developed a computational model that sums the responses of V1 neurons characterized by prior single unit measurements. The measured tuning properties of the GCaMP signals to stimulus contrast, orientation and spatial position closely match the predictions of the model, suggesting that widefield GCaMP signals are linearly related to the summed local spiking activity. DOI: http://dx.doi.org/10.7554/eLife.16178.001 PMID:27441501

  4. Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue

    PubMed Central

    Morley, John W.; Tapson, Jonathan; Breen, Paul P.; van Schaik, André

    2016-01-01

    Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45–150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6–8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals. PMID:27183102

  5. Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

    PubMed Central

    Kubitscheck, U; Pratsch, L; Passow, H; Peters, R

    1995-01-01

    The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed. Images FIGURE 3 FIGURE 4 FIGURE 5 PMID:7669907

  6. Reconstruction of burst activity from calcium imaging of neuronal population via Lq minimization and interval screening

    PubMed Central

    Quan, Tingwei; Lv, Xiaohua; Liu, Xiuli; Zeng, Shaoqun

    2016-01-01

    Calcium imaging is becoming an increasingly popular technology to indirectly measure activity patterns in local neuronal networks. Based on the dependence of calcium fluorescence on neuronal spiking, two-photon calcium imaging affords single-cell resolution of neuronal population activity. However, it is still difficult to reconstruct neuronal activity from complex calcium fluorescence traces, particularly for traces contaminated by noise. Here, we describe a robust and efficient neuronal-activity reconstruction method that utilizes Lq minimization and interval screening (IS), which we refer to as LqIS. The simulation results show that LqIS performs satisfactorily in terms of both accuracy and speed of reconstruction. Reconstruction of simulation and experimental data also shows that LqIS has advantages in terms of the recall rate, precision rate, and timing error. Finally, LqIS is demonstrated to effectively reconstruct neuronal burst activity from calcium fluorescence traces recorded from large-size neuronal population. PMID:27375930

  7. Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

    PubMed

    Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

    2015-01-01

    Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

  8. Imaging of chondrocalcinosis: calcium pyrophosphate dihydrate (CPPD) crystal deposition disease -- imaging of common sites of involvement.

    PubMed

    Magarelli, N; Amelia, R; Melillo, N; Nasuto, M; Cantatore, F; Guglielmi, G

    2012-01-01

    Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is characterised by the accumulation of pyrophosphate dihydrate crystals in articular and periarticular tissues and it can be classified as sporadic, hereditary or secondary. The diagnosis frequently rests on radiographic findings. Computed tomography scanning can detect well mineralised deposits in joints and also ultrasound may be useful in detecting CPPD crystal deposits. About MRI recent studies have demonstrated the utility of high field in depiction of CPPD crystal deposits. The aim of this review is to focus on the clinical-classificative and radiological aspects of CPPD, particularly the contribution of the different imaging techniques.

  9. Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.

    PubMed

    Yang, Helen H; St-Pierre, François; Sun, Xulu; Ding, Xiaozhe; Lin, Michael Z; Clandinin, Thomas R

    2016-06-30

    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

  10. Zinc modulation of calcium activity at the photoreceptor terminal: a calcium imaging study.

    PubMed

    Anastassov, Ivan; Shen, Wen; Ripps, Harris; Chappell, Richard L

    2013-07-01

    There is abundant experimental evidence that zinc ions (Zn(2+)) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 μM-2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 μM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 μM of membrane-permeable TPEN or 500 μM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca(2+)] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 μM nicardipine or 100 μM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.

  11. Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge.

    PubMed

    Michel, K; Michaelis, M; Mazzuoli, G; Mueller, K; Vanden Berghe, P; Schemann, M

    2011-12-15

    Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

  12. Continuous Fluorescence Imaging of Intracellular Calcium by Use of Ion-Selective Nanospheres with Adjustable Spectra.

    PubMed

    Yang, Chenye; Qin, Yu; Jiang, Dechen; Chen, Hong-Yuan

    2016-08-10

    Continuous fluorescence imaging of intracellular ions in various spectral ranges is important for biological studies. In this paper, fluorescent calcium-selective nanospheres, including calix[4]arene-functionalized bodipy (CBDP) or 9-(diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine (ETH 5350) as the chromoionophore, were prepared to demonstrate intracellular calcium imaging in visible or near-IR regions, respectively. The fluorescence of the nanospheres was controlled by the chromoionophore, and thus the spectral range for detection was adjustable by choosing the proper chromoionophore. The response time of the nanospheres to calcium was typically 1 s, which allowed accurate measurement of intracellular calcium. These nanospheres were loaded into cells through free endocytosis and exhibited fluorescence for 24 h, and their intensity was correlated with the elevation of intracellular calcium upon stimulation. The successful demonstration of calcium imaging by use of ion-selective nanospheres within two spectral ranges in 24 h supported that these nanospheres could be applied for continuous imaging of intracellular ions with adjustable spectra.

  13. High-Speed Fluorescence Imaging and Intensity Profiling of Femtosecond-Induced Calcium Transients

    PubMed Central

    Cranfield, Charles G.; Gu, Min

    2006-01-01

    We have demonstrated a combined imaging system, where the physiology of biological specimens can be imaged and profiled at 10–20 frames per second whilst undergoing femtosecond laser irradiation. Individual GH3 cells labeled with the calcium fluorophore Fluo-3 were stimulated using a counter-propagating focused femtosecond beam with respect to the imaging system. As a result of the stimulation, calcium waves can be generated in COS cells, and laser-induced calcium oscillations are initiated in the GH3 cells. Single-photon fluorescence images and intensity profiles of the targeted specimens are sampled in real-time using a modified PerkinElmer UltraView LCI microscope. PMID:23165061

  14. Calcium imaging of individual erythrocytes: problems and approaches.

    PubMed

    Kaestner, Lars; Tabellion, Wiebke; Weiss, Erwin; Bernhardt, Ingolf; Lipp, Peter

    2006-01-01

    Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.

  15. Microscopic imaging of intracellular calcium in live cells using lifetime-based ratiometric measurements of Oregon Green BAPTA-1.

    PubMed

    Lattarulo, Carli; Thyssen, Diana; Kuchibholta, Kishore V; Hyman, Bradley T; Bacskaiq, Brian J

    2011-01-01

    Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer's disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantages to using a ratiometric approach, which allows quantitative determinations of calcium that are independent of dye concentration. Taking advantage of the distinct lifetime contrast of the calcium-free and calcium-bound forms of OGB, we used time-domain lifetime measurements to generate calibration curves for OGB lifetime ratios as a function of calcium concentration. In summary, we demonstrate approaches using commercially available tools to measure calcium concentrations in live cells at multiple scales using lifetime contrast. These approaches are broadly applicable to other fluorescent readouts that exhibit lifetime contrast and serve as powerful alternatives to spectral or intensity readouts in multiplexing experiments.

  16. In vivo two-photon calcium imaging in the visual system.

    PubMed

    Ohki, Kenichi; Reid, R Clay

    2014-04-01

    Two-photon imaging of calcium-sensitive dyes in vivo has become a common tool used by neuroscientists, largely because of the development of bolus loading techniques, which can label every neuron in a local circuit with calcium-sensitive dye. Like multielectrode recordings, two-photon imaging paired with bolus loading provides a method for monitoring many neurons at once, but, in addition, it provides a means for determining the precise location of every neuron. Thus, it is an ideal method for studying the fine-scale functional architecture of the cortex and guiding the experimenter to individual neurons that can be targeted for further anatomical study. Two-photon calcium imaging enables study of the fine structure of functional maps in the visual cortex in cats and rodents. In mice, it can allow the characterization of specific cell types when paired with transgenic or retrograde labeling.

  17. Calcium imaging in the Drosophila olfactory system with a genetic indicator.

    PubMed

    Root, Cory M; Wong, Allan M; Flores, Jorge; Wang, Jing W

    2013-11-01

    Insects show sophisticated odor-mediated behaviors controlled by an olfactory system that is genetically and anatomically simpler than that of vertebrates, providing an attractive system to investigate the mechanistic link between behavior and odor perception. Advances in neuroscience have been facilitated by modern optical imaging technologies--both in instrumentation and in probe design--that permit the visualization of functional neural circuits. Imaging calcium activity in genetically defined populations of neurons provides an important tool for investigating the function of neural circuits. This article describes a two-photon imaging system for monitoring neural activity in the Drosophila antennal lobe. Odor-evoked calcium activity is followed by measuring the specific expression of the calcium-sensitive green fluorescent protein G-CaMP in Drosophila antennae-brain preparations.

  18. Analysis of potassium and calcium imaging to assay the function of opioid receptors.

    PubMed

    Spahn, Viola; Nockemann, Dinah; Machelska, Halina

    2015-01-01

    As the activation of opioid receptors leads to the modulation of potassium and calcium channels, the ion imaging represents an attractive method to analyze the function of the receptors. Here, we describe the imaging of potassium using the FluxOR™ potassium ion channel assay, and of calcium using Fura-2 acetoxymethyl ester. Specifically, we (1) characterize the activation of the G-protein-coupled inwardly rectifying potassium 2 channel by agonists of μ- and δ-opioid receptors with the aid of the FluxOR™ assay in cultured mouse dorsal root ganglion neurons, and (2) describe calcium imaging protocols to measure capsaicin-induced transient receptor potential vanilloid 1 channel activity during opioid withdrawal in transfected human embryonic kidney 293 cells.

  19. Non-rigid estimation of cell motion in calcium time-lapse images

    NASA Astrophysics Data System (ADS)

    Hachi, Siham; Lucumi Moreno, Edinson; Desmet, An-Sofie; Vanden Berghe, Pieter; Fleming, Ronan M. T.

    2016-03-01

    Calcium imaging is a widely used technique in neuroscience permitting the simultaneous monitoring of electro- physiological activity of hundreds of neurons at single cell resolution. Identification of neuronal activity requires rapid and reliable image analysis techniques, especially when neurons fire and move simultaneously over time. Traditionally, image segmentation is performed to extract individual neurons in the first frame of a calcium sequence. Thereafter, the mean intensity is calculated from the same region of interest in each frame to infer calcium signals. However, when cells move, deform and fire, this segmentation on its own generates artefacts and therefore biased neuronal activity. Therefore, there is a pressing need to develop a more efficient cell tracking technique. We hereby present a novel vision-based cell tracking scheme using a thin-plate spline deformable model. The thin-plate spline warping is based on control points detected using the Fast from Accelerated Segment Test descriptor and tracked using the Lucas-Kanade optical flow. Our method is able to track neurons in calcium time-series, even when there are large changes in intensity, such as during a firing event. The robustness and efficiency of the proposed approach is validated on real calcium time-lapse images of a neuronal population.

  20. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    PubMed

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  1. Investigation of mechanosensation in C. elegans using light field calcium imaging

    PubMed Central

    Shaw, Michael; Elmi, Muna; Pawar, Vijay; Srinivasan, Mandayam A.

    2016-01-01

    We describe a new experimental approach to investigate touch sensation in the model organism C. elegans using light field deconvolution microscopy. By combining fast volumetric image acquisition with controlled indentation of the organism using a high sensitivity force transducer, we are able to simultaneously measure activity in multiple touch receptor neurons expressing the calcium ion indicator GCaMP6s. By varying the applied mechanical stimulus we show how this method can be used to quantify touch sensitivity in C. elegans. We describe some of the challenges of performing light field calcium imaging in moving samples and demonstrate that they can be overcome by simple data processing. PMID:27446713

  2. An image-based model of calcium waves in differentiated neuroblastoma cells.

    PubMed Central

    Fink, C C; Slepchenko, B; Moraru, I I; Watras, J; Schaff, J C; Loew, L M

    2000-01-01

    Calcium waves produced by bradykinin-induced inositol-1,4, 5-trisphosphate (InsP(3))-mediated release from endoplasmic reticulum (ER) have been imaged in N1E-115 neuroblastoma cells. A model of this process was built using the "virtual cell," a general computational system for integrating experimental image, biochemical, and electrophysiological data. The model geometry was based on a cell for which the calcium wave had been experimentally recorded. The distributions of the relevant cellular components [InsP(3) receptor (InsP(3)R)], sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pumps, bradykinin receptors, and ER] were based on 3D confocal immunofluorescence images. Wherever possible, known biochemical and electrophysiological data were used to constrain the model. The simulation closely matched the spatial and temporal characteristics of the experimental calcium wave. Predictions on different patterns of calcium signals after InsP(3) uncaging or for different cell geometries were confirmed experimentally, thus helping to validate the model. Models in which the spatial distributions of key components are altered suggest that initiation of the wave in the center of the neurite derives from an interplay of soma-biased ER distribution and InsP(3) generation biased toward the neurite. Simulations demonstrate that mobile buffers (like the indicator fura-2) significantly delay initiation and lower the amplitude of the wave. Analysis of the role played by calcium diffusion indicated that the speed of the wave is only slightly dependent on the ability of calcium to diffuse to and activate neighboring InsP(3) receptor sites. PMID:10866945

  3. Regional calcium distribution and ultrasound images of the vessel wall in human carotid arteries

    NASA Astrophysics Data System (ADS)

    Szikszai, Z.; Kertész, Zs.; Uzonyi, I.; Szíki, G. Á.; Magyar, M. T.; Molnár, S.; Ida, Y.; Csiba, L.

    2005-04-01

    Arterial calcification can take place at two sites in the vessel wall: the intima and the media. Intimal calcification occurs exclusively within atherosclerotic plaques, while medial calcification may develop independently. Extensive calcified plaques in the carotid arteries can be easily detected by B-mode ultrasonic imaging. The calcium content might correlate with the ultrasound reflectance of the vessel wall, and could be a surrogate marker for arteriosclerosis. In this study, segments of human carotid arteries collected at autopsy were examined by ultrasonography in vitro and calcium distributional maps of sections from the same segments were determined by particle induced X-ray emission. Our aim was to make a first step towards investigating the relationship between the calcium distributional maps and the respective ultrasound images.

  4. Direct injection of indicators for calcium imaging at the Drosophila larval neuromuscular junction.

    PubMed

    Macleod, Gregory T

    2012-07-01

    Calcium imaging is a technique in which Ca(2+)-binding molecules are loaded into live cells and as they bind Ca(2+) they "indicate" the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. This article describes the direct injection of Ca(2+) indicators at the Drosophila larval neuromuscular junction (NMJ). This technique allows rapid loading of most Ca(2+) indicators, but there are drawbacks in that it is a difficult technique to master and requires additional electrophysiological equipment. Also, Ca(2+) indicators that are easily injected are usually susceptible to compartmentalization.

  5. Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.

    PubMed

    Renault, Renaud; Sukenik, Nirit; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Peyrin, Jean-Michel; Bottani, Samuel; Monceau, Pascal; Moses, Elisha; Vignes, Maéva

    2015-01-01

    In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

  6. Imaging of calcium transients in skeletal muscle fibers.

    PubMed Central

    Vergara, J; DiFranco, M; Compagnon, D; Suarez-Isla, B A

    1991-01-01

    Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures. Images FIGURE 4 FIGURE 5 FIGURE 6 PMID:2015378

  7. Twenty Years of Calcium Imaging: Cell Physiology to Dye For

    PubMed Central

    Knot, Harm J.; Laher, Ismail; Sobie, Eric A.; Guatimosim, Silvia; Gomez-Viquez, Leticia; Hartmann, Hali; Song, Long-Sheng; Lederer, W.J.; Graier, Wolfgang F.; Malli, Roland; Frieden, Maud; Petersen, Ole H.

    2016-01-01

    The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca2+ in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca2+ signaling is accomplished. In excitable cells, the coordination of changing Ca2+ concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca2+ signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes. PMID:15821159

  8. Presynaptic imaging of projection fibers by in vivo injection of dextran-conjugated calcium indicators.

    PubMed

    Brenowitz, Stephan D; Regehr, Wade G

    2012-04-01

    Dextran-conjugated calcium indicators are stably retained within neurons. As a result, they are well suited to measuring presynaptic calcium at physiological temperatures. In addition, dextran indicators can be used to label neurons and their presynaptic boutons in vivo. This has allowed measurements of calcium in the presynaptic boutons of projection fibers that cannot be stably loaded with other types of indicators. This protocol describes a technique for in vivo loading of the climbing fiber projection to the cerebellum with dextran-conjugated indicators for subsequent presynaptic calcium imaging in brain slices. This technique is applicable to studies of projection fibers in many species from which brain slices can be prepared. The dextran indicator is injected into the inferior olive using a stereotaxic device. After a period of 1-3 d, cerebellar slices are prepared and presynaptic calcium transients are measured at physiological temperature in labeled climbing fibers. The protocol also discusses important considerations for using dextran-conjugated indicators to measure presynaptic calcium.

  9. Classification of calcium in intravascular OCT images for the purpose of intervention planning

    NASA Astrophysics Data System (ADS)

    Shalev, Ronny; Bezerra, Hiram G.; Ray, Soumya; Prabhu, David; Wilson, David L.

    2016-03-01

    The presence of extensive calcification is a primary concern when planning and implementing a vascular percutaneous intervention such as stenting. If the balloon does not expand, the interventionalist must blindly apply high balloon pressure, use an atherectomy device, or abort the procedure. As part of a project to determine the ability of Intravascular Optical Coherence Tomography (IVOCT) to aid intervention planning, we developed a method for automatic classification of calcium in coronary IVOCT images. We developed an approach where plaque texture is modeled by the joint probability distribution of a bank of filter responses where the filter bank was chosen to reflect the qualitative characteristics of the calcium. This distribution is represented by the frequency histogram of filter response cluster centers. The trained algorithm was evaluated on independent ex-vivo image data accurately labeled using registered 3D microscopic cryo-image data which was used as ground truth. In this study, regions for extraction of sub-images (SI's) were selected by experts to include calcium, fibrous, or lipid tissues. We manually optimized algorithm parameters such as choice of filter bank, size of the dictionary, etc. Splitting samples into training and testing data, we achieved 5-fold cross validation calcium classification with F1 score of 93.7+/-2.7% with recall of >=89% and a precision of >=97% in this scenario with admittedly selective data. The automated algorithm performed in close-to-real-time (2.6 seconds per frame) suggesting possible on-line use. This promising preliminary study indicates that computational IVOCT might automatically identify calcium in IVOCT coronary artery images.

  10. Determination of calcium concentrations in cells and tissue with fluorescence lifetime imaging (FILM)

    NASA Astrophysics Data System (ADS)

    Gensch, T.; Wirth, M.

    2011-03-01

    The determination of ion concentrations in cells - in particular in neurons - is very important for understanding cell function and life. Calcium is an ubiquitous messenger in almost all cell types. Fluorescence lifetime imaging (FLIM) can be of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. Several organic chromophores have been tested in cuvette experiments as well as in living cells and cell tissue with respect to their applicability in FLIM studies. The calcium concentration changes in several cell types were investigated by FLIM with two-photon excitation.

  11. Inference of neuronal network spike dynamics and topology from calcium imaging data.

    PubMed

    Lütcke, Henry; Gerhard, Felipe; Zenke, Friedemann; Gerstner, Wulfram; Helmchen, Fritjof

    2013-01-01

    Two-photon calcium imaging enables functional analysis of neuronal circuits by inferring action potential (AP) occurrence ("spike trains") from cellular fluorescence signals. It remains unclear how experimental parameters such as signal-to-noise ratio (SNR) and acquisition rate affect spike inference and whether additional information about network structure can be extracted. Here we present a simulation framework for quantitatively assessing how well spike dynamics and network topology can be inferred from noisy calcium imaging data. For simulated AP-evoked calcium transients in neocortical pyramidal cells, we analyzed the quality of spike inference as a function of SNR and data acquisition rate using a recently introduced peeling algorithm. Given experimentally attainable values of SNR and acquisition rate, neural spike trains could be reconstructed accurately and with up to millisecond precision. We then applied statistical neuronal network models to explore how remaining uncertainties in spike inference affect estimates of network connectivity and topological features of network organization. We define the experimental conditions suitable for inferring whether the network has a scale-free structure and determine how well hub neurons can be identified. Our findings provide a benchmark for future calcium imaging studies that aim to reliably infer neuronal network properties.

  12. Simultaneous imaging of structural plasticity and calcium dynamics in developing dendrites and axons.

    PubMed

    Siegel, Friederike; Lohmann, Christian

    2013-11-01

    During nervous system development, the formation of synapses between pre- and postsynaptic neurons is a remarkably specific process. Both structural and functional plasticity are critical for the selection of synaptic partners and for the establishment and maturation of synapses. To unravel the respective contributions of structural and functional mechanisms as well as their interactions during synaptogenesis, it is important to directly observe structural changes and functional signaling simultaneously. Here, we present an imaging approach to simultaneously follow changes in structure and function. Differential labeling of individual cells and the neuronal network with distinct dyes allows the study of structural plasticity and changes in calcium signaling associated with neural activity at the same time and with high resolution. This is achieved by bulk loading of neuronal populations with a calcium-sensitive indicator in combination with electroporation of individual cells with a calcium indicator and an additional noncalcium-sensitive dye with a different excitation spectrum. Recordings of the two differently labeled structures can be acquired simultaneously using confocal microscopy. Thus, structural plasticity and calcium dynamics of the individually labeled neuron and the surrounding network can be related to each other. This combined imaging approach can be applied to virtually all systems of neuronal networks to study structure and function. We provide a comprehensive description of the labeling procedure, the imaging parameters, and the important aspects of analysis for simultaneous recordings of structure and function in individual neurons.

  13. [Olfactory perception and learning in the honey bee (Apis mellifera): calcium imaging in the antenna lobe].

    PubMed

    Sandoz, Jean-Christophe

    2003-01-01

    Honey bees are a key-model in the study of learning and memory, because they show considerable learning abilities, their brain is well described and is accessible to a wide range of physiological recordings and treatments. We use in vivo calcium imaging to study olfactory perception in the bee brain, and combine this method to appetitive olfactory conditioning to unravel the neural substrates of olfactory learning. Odours are detected by receptor neurons on the antennae. Each receptor neuron projects to the first-order neuropile of the olfactory pathway, the antennal lobe, connecting to projection neurons in one of its 160 functional units, the glomeruli. In calcium imaging experiments, each odour elicits a particular activity pattern of antennal lobe glomeruli, according to a code conserved between individuals. The antennal lobe is also a site where the olfactory memory is formed. Using optical imaging, two studies have shown modulations of odour representation in the antennal lobe after learning, with different effects depending on the type of conditioning used. While simple differential conditioning (A + B- training) showed an increased calcium response to the reinforced odour, side-specific conditioning (A + B-/B + A- training) decorrelated the calcium responses of odours between brain sides. This difference may owe to the formation of different memories, which will be addressed in future work. By specifically staining antennal lobe neuronal subpopulations, we hope to be able in the future to study synaptic plasticity in the honey bee.

  14. Inference of neuronal network spike dynamics and topology from calcium imaging data

    PubMed Central

    Lütcke, Henry; Gerhard, Felipe; Zenke, Friedemann; Gerstner, Wulfram; Helmchen, Fritjof

    2013-01-01

    Two-photon calcium imaging enables functional analysis of neuronal circuits by inferring action potential (AP) occurrence (“spike trains”) from cellular fluorescence signals. It remains unclear how experimental parameters such as signal-to-noise ratio (SNR) and acquisition rate affect spike inference and whether additional information about network structure can be extracted. Here we present a simulation framework for quantitatively assessing how well spike dynamics and network topology can be inferred from noisy calcium imaging data. For simulated AP-evoked calcium transients in neocortical pyramidal cells, we analyzed the quality of spike inference as a function of SNR and data acquisition rate using a recently introduced peeling algorithm. Given experimentally attainable values of SNR and acquisition rate, neural spike trains could be reconstructed accurately and with up to millisecond precision. We then applied statistical neuronal network models to explore how remaining uncertainties in spike inference affect estimates of network connectivity and topological features of network organization. We define the experimental conditions suitable for inferring whether the network has a scale-free structure and determine how well hub neurons can be identified. Our findings provide a benchmark for future calcium imaging studies that aim to reliably infer neuronal network properties. PMID:24399936

  15. Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator.

    PubMed

    Minderer, Matthias; Liu, Wenrui; Sumanovski, Lazar T; Kügler, Sebastian; Helmchen, Fritjof; Margolis, David J

    2012-01-01

    In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal precision, and furthermore, can be measured repeatedly in separate imaging sessions over multiple weeks. This method opens new possibilities for the longitudinal study of stability and plasticity of cortical sensory representations.

  16. Combined analysis of intracellular calcium with dual excitation fluorescence photometry and imaging

    NASA Astrophysics Data System (ADS)

    Uttenweiler, Dietmar; Wojciechowski, Reinhold; Makabe, Makoto; Veigel, Claudia; Fink, Rainer H.

    1995-10-01

    We have developed an integrated microscopy system combining fast dual-excitation fluorescence photometry and digital image analysis with high spatial resolution, based mainly on standard components. With the combination of these well-established techniques in one setup it is possible to monitor intracellular calcium with both sufficiently high temporal and high spatial resolution on the same preparation for many biological applications. Our system consists of a commercially available dual-excitation photometric system, an attached ICCD camera, and a frame grabber board. With this integrated setup one can easily switch between the fast photometric mode and the imaging mode. We used the system to record Fura-2 calcium images (340/380 nm ratios), which were correlated with the faster spot measurements and were analyzed by means of image processing. As an example for its application we reconstructed caffeine-induced calcium transient released from the sarcoplasmic reticulum of isolated and permeabilized skeletal muscle fiber preparations. Such a combined technique will also be important for cellular studies using other fluorescence indicators. Additionally, the described system has an external trigger facility that enables combination with other cell physiological methods, e.g., electrophysiological techniques.

  17. Pixel timing correction in time-lapsed calcium imaging using point scanning microscopy.

    PubMed

    Boiroux, Dimitri; Oke, Yoshihiko; Miwakeichi, Fumikazu; Oku, Yoshitaka

    2014-11-30

    In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.

  18. Association of Coronary Aortic Calcium with Abdominal Aortic Calcium Detected on Lateral Dual Energy X-Ray Absorptiometry Spine Images

    PubMed Central

    Schousboe, John T.; Claflin, Diane; Barrett-Connor, Elizabeth

    2009-01-01

    The association of abdominal aortic calcium (AAC) on lateral spine bone densitometry with coronary artery calcium (CAC) has not been reported. We studied 33 men and 73 women who had CAC scored with electron beam computed tomography at the 8th visit of the Rancho Bernardo study and lateral spine dual energy x-ray absorptiometry (DXA) images fully evaluable for AAC done at the 9th study visit. The association between CAC level and AAC tertile was assessed by ordinal logistic regression. The odds ratio of having a higher level of CAC score for those in the top tertile of AAC score (24-point scale score ≥ 5) was 6.42 (95% C.I. 2.28 – 18.1) and on an 8-point scale (score ≥ 3) was 3.38 (95% C.I. 1.26 – 9.07), compared to those with AAC scores in the bottom tertiles, adjusted for age, sex, systolic blood pressure, total and high density lipoprotein (HDL) cholesterol, smoking status, and diabetes. A 24-point AAC score of ≥ 5 had a sensitivity of 65% and a specificity of 70% to detect a high CAC score (≥ 400 units). An 8-point AAC score ≥ 3 had a sensitivity of 45% and a specificity of 78%. In conclusion, a high level of AAC on lateral spine DXA is strongly associated with coronary artery disease and may be commonly encountered since bone densitometry is indicated in all women age ≥ 65 and all men age ≥ 70. Its presence should be reported to the patient's physician to identify and manage modifiable risk factors. PMID:19616658

  19. Live imaging of calcium spikes during double fertilization in Arabidopsis

    PubMed Central

    Hamamura, Yuki; Nishimaki, Moe; Takeuchi, Hidenori; Geitmann, Anja; Kurihara, Daisuke; Higashiyama, Tetsuya

    2014-01-01

    Ca2+ waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca2+ in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca2+ spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca2+ oscillations on pollen tube arrival. The two synergid cells showed distinct Ca2+ dynamics depending on their respective roles in tube reception. These Ca2+ dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants. PMID:25146889

  20. Imaging calcium carbonate distribution in human sweat pore in vivo using nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Xueqin; Gasecka, Alicja; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-03-01

    Nonlinear microscopies, including two-photon excited autofluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), were used to study individual human sweat pore morphology and topically applied antiperspirant salt penetration inside sweat pore, in vivo on human palms. Sweat pore inner morphology in vivo was imaged up to the depth of 100 μm by TPEF microscopy. The 3D penetration and distribution of "in situ calcium carbonate" (isCC), an antiperspirant salt model, was investigated using CARS microscopy.

  1. Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans.

    PubMed

    Turek, Michal; Besseling, Judith; Bringmann, Henrik

    2015-06-24

    Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

  2. Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Nguyen, Jeffrey; Shipley, Frederick; Linder, Ashley; Plummer, George; Liu, Mochi; Setru, Sagar; Shaevitz, Joshua; Leifer, Andrew

    The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. Acquiring this data, however, is challenging because it is difficult to track and image individual neurons as an animal deforms its posture and moves many body lengths. Here, we present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s are recorded at 6 head-volumes/s using spinning disk confocal microscopy. At the same time, we record low magnification images of the animal to measure the animals behavior and track its head as it moves. We develop a time independent neuronal matching algorithm that uses non-rigid point set registration and machine learning to correctly match neurons across time. Using this method, we are able to observe calcium transients from up to 90 neurons for over 4 min and correlate the neural activity with the animal's behavior.

  3. Estimating background-subtracted fluorescence transients in calcium imaging experiments: a quantitative approach.

    PubMed

    Joucla, Sébastien; Franconville, Romain; Pippow, Andreas; Kloppenburg, Peter; Pouzat, Christophe

    2013-08-01

    Calcium imaging has become a routine technique in neuroscience for subcellular to network level investigations. The fast progresses in the development of new indicators and imaging techniques call for dedicated reliable analysis methods. In particular, efficient and quantitative background fluorescence subtraction routines would be beneficial to most of the calcium imaging research field. A background-subtracted fluorescence transients estimation method that does not require any independent background measurement is therefore developed. This method is based on a fluorescence model fitted to single-trial data using a classical nonlinear regression approach. The model includes an appropriate probabilistic description of the acquisition system's noise leading to accurate confidence intervals on all quantities of interest (background fluorescence, normalized background-subtracted fluorescence time course) when background fluorescence is homogeneous. An automatic procedure detecting background inhomogeneities inside the region of interest is also developed and is shown to be efficient on simulated data. The implementation and performances of the proposed method on experimental recordings from the mouse hypothalamus are presented in details. This method, which applies to both single-cell and bulk-stained tissues recordings, should help improving the statistical comparison of fluorescence calcium signals between experiments and studies.

  4. Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators.

    PubMed

    Weitz, Andrew C; Behrend, Matthew R; Lee, Nan Sook; Klein, Ronald L; Chiodo, Vince A; Hauswirth, William W; Humayun, Mark S; Weiland, James D; Chow, Robert H

    2013-04-01

    Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.

  5. Imaging atrial arrhythmic intracellular calcium in intact heart

    PubMed Central

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R.

    2014-01-01

    Abnormalities in intracellular Ca2+ signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca2+ in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca2+ imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca2+ activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca2+ waves and Ca2+ alternans. Moreover, we applied this strategy to analyze Ca2+ signals in the hearts of WT and knock-in mice harboring a ‘leaky’ type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca2+ leak increases the susceptibility to Ca2+ alternans and Ca2+ waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca2+ leak via RyR2 by acute treatment with S107 reduced both Ca2+ alternans and Ca2+ waves, and prevented atrial arrhythmias. PMID:24041536

  6. Imaging atrial arrhythmic intracellular calcium in intact heart.

    PubMed

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R

    2013-11-01

    Abnormalities in intracellular Ca(2+) signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca(2+) in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca(2+) imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca(2+) activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca(2+) waves and Ca(2+) alternans. Moreover, we applied this strategy to analyze Ca(2+) signals in the hearts of WT and knock-in mice harboring a 'leaky' type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca(2+) leak increases the susceptibility to Ca(2+) alternans and Ca(2+) waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca(2+) leak via RyR2 by acute treatment with S107 reduced both Ca(2+) alternans and Ca(2+) waves, and prevented atrial arrhythmias.

  7. Imaging fast calcium currents beyond the limitations of electrode techniques.

    PubMed

    Jaafari, Nadia; De Waard, Michel; Canepari, Marco

    2014-09-16

    The current understanding of Ca(2+) channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca(2+) currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca(2+) channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca(2+) currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca(2+) imaging with low-affinity indicators. The optical measurement of the Ca(2+) current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca(2+) channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca(2+) channels. In particular, we measured the Ca(2+) current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca(2+) channels in their native physiological environment.

  8. Optical Imaging of Voltage and Calcium in Cardiac Cells & Tissues

    PubMed Central

    Herron, Todd J.; Lee, Peter; Jalife, José

    2012-01-01

    Cardiac optical mapping has proven to be a powerful technology for studying cardiovascular function and disease. The development and scientific impact of this methodology are well documented. Because of its relevance in cardiac research, this imaging technology advances at a rapid pace. Here we review technological and scientific developments during the past several years and look also towards the future. First we explore key components of a modern optical mapping setup, focusing on 1) new camera technologies, 2) powerful light-emitting-diodes (from ultraviolet to red) for illumination, 3) improved optical filter technology, 4) new synthetic and optogenetic fluorescent probes, 5) optical mapping with motion and contraction, 6) new multi-parametric optical mapping techniques and 7) photon scattering effects in thick tissue preparations. We then look at recent optical mapping studies in single cells, cardiomyocyte monolayers, atria and whole hearts. Finally, we briefly look into the possible future roles of optical mapping in the development of regenerative cardiac research, cardiac cell therapies, and molecular genetic advances. PMID:22343556

  9. Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes.

    PubMed

    O'Donovan, M J; Ho, S; Sholomenko, G; Yee, W

    1993-02-01

    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 using digital videomicroscopy. Motoneurons and dorsal root ganglion cells exhibited a frequency-dependent change in fluorescence during antidromic stimulation. Single antidromic stimuli resulted in fluorescence transients that could be resolved in individual cells in real time. In addition, fluorescence changes could be recorded in motoneurons during episodes of bursting generated by rhythmic synaptic inputs from premotor networks. Stimulus-induced fluorescence signals were also detected in axons and synaptic endings labelled anterogradely. Optical signals were largely abolished in the absence of extracellular calcium. The results show that calcium changes can now be measured in identified populations of neurons and presynaptic terminals. The strong dependence of these signals on impulse activity suggests that the technique will be useful for monitoring the activity of identified neuronal populations. The calcium-dependent fluorescence signal probably results from cytosolic dye derived from diffusion which may limit the technique to situations in which the dye can be applied close (< 1 cm) to cell bodies.

  10. Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology, calcium imaging and confocal microscopy.

    PubMed

    Galizia, C G; Kimmerle, B

    2004-01-01

    The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.

  11. Chromaffin cell calcium signal and morphology study based on multispectral images

    NASA Astrophysics Data System (ADS)

    Wu, Hongxiu; Wei, Shunhui; Qu, Anlian; Zhou, Zhuan

    1998-09-01

    Increasing or decreasing the internal calcium concentration can promote or prevent programmed cell death (PCD). We therefore performed a Ca2+ imaging study using Ca2+ indicator dye fura-2 and a sensitive cooled-CCD camera with a 12 bit resolution. Monochromatic beams of light with a wavelength of 345,380 nm were isolated from light emitted by a xenon lamp using a monochromator. The concentration of free calcium can be directly calculated from the ratio of two fluorescence values taken at two appropriately selected wavelength. Fluorescent light emitted from the cells was capture using a camera system. The cell morphology study is based on multispectral scanning, with smear images provided as three monochromatic images by illumination with light of 610,535 and 470 nm wavelengths. The nuclear characteristic parameters extracted from individual nuclei by system are nuclear area, nuclear diameter, nuclear density vector. The results of the restoration of images and the performance of a primitive logic for the detection of nuclei with PCD proved the usefulness of the system and the advantages of using multispectral images in the restoration and detection procedures.

  12. Calcium imaging of odor-evoked responses in the Drosophila antennal lobe.

    PubMed

    Silbering, Ana F; Bell, Rati; Galizia, C Giovanni; Benton, Richard

    2012-03-14

    The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide

  13. Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy.

    PubMed

    Quirin, Sean; Vladimirov, Nikita; Yang, Chao-Tsung; Peterka, Darcy S; Yuste, Rafael; Ahrens, Misha B

    2016-03-01

    Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning-removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416×832×160  μm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain.

  14. Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

    PubMed

    Takaoka, Yousuke; Shigenaga, Miyuki; Imai, Masaki; Nukadzuka, Yuuki; Ishimaru, Yasuhiro; Saito, Kei; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ueda, Minoru

    2016-01-01

    In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

  15. Topical application of indicators for calcium imaging at the Drosophila larval neuromuscular junction.

    PubMed

    Macleod, Gregory T

    2012-07-01

    Calcium imaging is a technique in which Ca(2+)-binding molecules are loaded into live cells and as they bind Ca(2+) they "indicate" the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. This article describes the topical application of Ca(2+) indicators at the Drosophila larval neuromuscular junction (NMJ). This loading technique is simple to execute and yields data quickly. The drawback is that the data can be difficult to interpret, primarily because it is difficult to ascertain which cellular and subcellular compartment(s) are loaded (e.g., muscle, nerve, or glia; cytosol, mitochondrion, or endoplasmic reticulum).

  16. Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans.

    PubMed

    Nguyen, Jeffrey P; Shipley, Frederick B; Linder, Ashley N; Plummer, George S; Liu, Mochi; Setru, Sagar U; Shaevitz, Joshua W; Leifer, Andrew M

    2016-02-23

    The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal's position and orientation. Custom software tracks the 3D position of the animal's head in real time and two feedback loops adjust a motorized stage and objective to keep the animal's head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal's behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.

  17. Comparing Analysis Methods in Functional Calcium Imaging of the Insect Brain.

    PubMed

    Balkenius, Anna; Johansson, Anders J; Balkenius, Christian

    2015-01-01

    We investigate four different methods for background estimation in calcium imaging of the insect brain and evaluate their performance on six data sets consisting of data recorded from two sites in two species of moths. The calcium fluorescence decay curve outside the potential response is estimated using either a low-pass filter or constant, linear or polynomial regression, and is subsequently used to calculate the magnitude, latency and duration of the response. The magnitude and variance of the responses that are obtained by the different methods are compared, and, by computing the receiver operating characteristics of a classifier based on response magnitude, we evaluate the ability of each method to detect the stimulus type and conclude that a polynomial approximation of the background gives the overall best result.

  18. Optical imaging of neuronal activity in tissue labeled by retrograde transport of Calcium Green Dextran.

    PubMed

    McPherson, D R; McClellan, A D; O'Donovan, M J

    1997-05-01

    In many neurophysiological studies it is desirable to simultaneously record the activity of a large number of neurons. This is particularly true in the study of vertebrate motor systems that generate rhythmic behaviors, such as the pattern generator for locomotion in vertebrate spinal cord. Optical imaging of neurons labeled with appropriate fluorescent dyes, in which fluorescence is activity-dependent, provides a means to record the activity of many neurons at the same time, while also providing fine spatial resolution of the position and morphology of active neurons. Voltage-sensitive dyes have been explored for this purpose and have the advantage of rapid response to transmembrane voltage changes. However, voltage-sensitive dyes bleach readily, which results in phototoxic damage and limits the time that labeled neurons can be imaged. In addition, the signal-to-noise ratio is typically small, so that averaging of responses is usually required. As an alternative to voltage-sensitive dyes, calcium-sensitive dyes can exhibit large changes in fluorescence. Most neurons contain voltage-sensitive Ca2+ channels, and numerous reports indicate that neuronal activity is accompanied by increased intracellular Ca2+ concentration. In this protocol we describe a method to use retrograde transport of the dextran conjugate of a calcium-sensitive dye (Calcium Green Dextran) to label selectively populations of brain and spinal interneurons in a primitive vertebrate (lamprey), for subsequent video-rate imaging of changes in intracellular fluorescence during neuronal activity. Although described with specific reference to lampreys, the technique has also been applied to embryonic chick spinal cord and larval zebrafish preparations and should be easily adaptable to other systems. The most significant novel feature of the protocol is the use of retrograde axonal transport to selectively fill neurons that have known axonal trajectories. Using lampreys, we have obtained activity

  19. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

    PubMed Central

    Akerboom, Jasper; Carreras Calderón, Nicole; Tian, Lin; Wabnig, Sebastian; Prigge, Matthias; Tolö, Johan; Gordus, Andrew; Orger, Michael B.; Severi, Kristen E.; Macklin, John J.; Patel, Ronak; Pulver, Stefan R.; Wardill, Trevor J.; Fischer, Elisabeth; Schüler, Christina; Chen, Tsai-Wen; Sarkisyan, Karen S.; Marvin, Jonathan S.; Bargmann, Cornelia I.; Kim, Douglas S.; Kügler, Sebastian; Lagnado, Leon; Hegemann, Peter; Gottschalk, Alexander; Schreiter, Eric R.; Looger, Loren L.

    2013-01-01

    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics. PMID:23459413

  20. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

    PubMed

    Collot, Mayeul; Wilms, Christian D; Bentkhayet, Asma; Marcaggi, Païkan; Couchman, Kiri; Charpak, Serge; Dieudonné, Stéphane; Häusser, Michael; Feltz, Anne; Mallet, Jean-Maurice

    2015-03-31

    The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

  1. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging

    PubMed Central

    Collot, Mayeul; Wilms, Christian D; Bentkhayet, Asma; Marcaggi, Païkan; Couchman, Kiri; Charpak, Serge; Dieudonné, Stéphane; Häusser, Michael; Feltz, Anne; Mallet, Jean-Maurice

    2015-01-01

    The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments. DOI: http://dx.doi.org/10.7554/eLife.05808.001 PMID:25824291

  2. Measuring and Modeling Sonoporation Dynamics in Mammalian Cells via Calcium Imaging

    NASA Astrophysics Data System (ADS)

    Kumon, R. E.; Parikh, P.; Sabens, D.; Aehle, M.; Kourennyi, D.; Deng, C. X.

    2007-05-01

    In this study, calcium imaging via the fluorescent indicator Fura-2 is used to characterize the sonoporation of Chinese Hamster Ovarian (CHO) cells in the presence of Optison™ microbubbles. Evolution of the calcium concentration within cells is determined from real-time fluorescence intensity measurements before, during, and after exposure to a 1 MHz ultrasound tone burst (0.2 s, 0.45 MPa). To relate microscopic sonoporation parameters to the measurements, an analytical model that includes sonoporation and plasma membrane transport is developed, assuming rapid mixing (uniform spatial distribution) in the cell. Fitting the measured data to the model provides estimated values for the poration area as a function of poration relaxation rate as well as plasma membrane pump and leakage rates. A modified compartment model that includes the effects of sonoporation, buffering proteins, and transport across the plasma membrane, endoplasmic reticulum, and mitochondria is also investigated. Numerical 3solutions of this model show a variety of behaviors for the calcium dynamics of the cell.

  3. Ultrasmall Nanoplatforms as Calcium-Responsive Contrast Agents for Magnetic Resonance Imaging.

    PubMed

    Moussaron, Albert; Vibhute, Sandip; Bianchi, Andrea; Gündüz, Serhat; Kotb, Shady; Sancey, Lucie; Motto-Ros, Vincent; Rizzitelli, Silvia; Crémillieux, Yannick; Lux, Francois; Logothetis, Nikos K; Tillement, Olivier; Angelovski, Goran

    2015-10-07

    The preparation of ultrasmall and rigid platforms (USRPs) that are covalently coupled to macrocycle-based, calcium-responsive/smart contrast agents (SCAs), and the initial in vitro and in vivo validation of the resulting nanosized probes (SCA-USRPs) by means of magnetic resonance imaging (MRI) is reported. The synthetic procedure is robust, allowing preparation of the SCA-USRPs on a multigram scale. The resulting platforms display the desired MRI activity—i.e., longitudinal relaxivity increases almost twice at 7 T magnetic field strength upon saturation with Ca(2+). Cell viability is probed with the MTT assay using HEK-293 cells, which show good tolerance for lower contrast agent concentrations over longer periods of time. On intravenous administration of SCA-USRPs in living mice, MRI studies indicate their rapid accumulation in the renal pelvis and parenchyma. Importantly, the MRI signal increases in both kidney compartments when CaCl2 is also administrated. Laser-induced breakdown spectroscopy experiments confirm accumulation of SCA-USRPs in the renal cortex. To the best of our knowledge, these are the first studies which demonstrate calcium-sensitive MRI signal changes in vivo. Continuing contrast agent and MRI protocol optimizations should lead to wider application of these responsive probes and development of superior functional methods for monitoring calcium-dependent physiological and pathological processes in a dynamic manner.

  4. Simultaneous optogenetic manipulation and calcium imaging in freely moving C. elegans.

    PubMed

    Shipley, Frederick B; Clark, Christopher M; Alkema, Mark J; Leifer, Andrew M

    2014-01-01

    Understanding how an organism's nervous system transforms sensory input into behavioral outputs requires recording and manipulating its neural activity during unrestrained behavior. Here we present an instrument to simultaneously monitor and manipulate neural activity while observing behavior in a freely moving animal, the nematode Caenorhabditis elegans. Neural activity is recorded optically from cells expressing a calcium indicator, GCaMP3. Neural activity is manipulated optically by illuminating targeted neurons expressing the optogenetic protein Channelrhodopsin. Real-time computer vision software tracks the animal's behavior and identifies the location of targeted neurons in the nematode as it crawls. Patterned illumination from a DMD is used to selectively illuminate subsets of neurons for either calcium imaging or optogenetic stimulation. Real-time computer vision software constantly updates the illumination pattern in response to the worm's movement and thereby allows for independent optical recording or activation of different neurons in the worm as it moves freely. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the worm's mechanosensory circuit. We record and compare calcium transients in the backward locomotion command interneurons AVA, in response to optical activation of the anterior mechanosensory neurons ALM, AVM or both.

  5. Design and synthesis of calcium responsive magnetic resonance imaging agent: Its relaxation and luminescence studies.

    PubMed

    Tanwar, Jyoti; Datta, Anupama; Chauhan, Kanchan; Kumaran, S Senthil; Tiwari, Anjani K; Kadiyala, K Ganesh; Pal, Sunil; Thirumal, M; Mishra, Anil K

    2014-07-23

    Calcium concentration modulation both inside and outside cell is of considerable interest for nervous system function in normal and pathological conditions. MRI has potential for very high spatial resolution at molecular/cellular level. Design, synthesis and evaluation of Gd-DO3A-AME-NPHE, a calcium responsive MRI contrast agent is presented. The probe is comprised of a Gd(3+)-DO3A core coupled to iminoacetate coordinating groups for calcium induced relaxivity switching. In the absence of Ca(2+) ions, inner sphere water binding to the Gd-DO3A-AME-NPHE is restricted with longitudinal relaxivity, r1 = 4.37 mM(-1) s(-1) at 4.7 T. However, addition of Ca(2+) triggers a marked enhancement in r1 = 6.99 mM(-1) s(-1) at 4.7 T (60% increase). The construct is highly selective for Ca(2+) over competitive metal ions at extracellular concentration. The r1 is modulated by changes in the hydration number (0.2 to 1.05), which was confirmed by luminescence emission lifetimes of the analogous Eu(3+) complex. T1 phantom images establish the capability of complex of visualizing changes in [Ca(2+)] by MRI.

  6. Spatiotemporal effects of sonoporation measured by real-time calcium imaging.

    PubMed

    Kumon, R E; Aehle, M; Sabens, D; Parikh, P; Han, Y W; Kourennyi, D; Deng, C X

    2009-03-01

    To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca(2+)](i)) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison microbubbles. At extracellular [Ca(2+)](o) of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of [Ca(2+)](i) in time) with durations of 100-180 s. Immediate [Ca(2+)](i) transients after ultrasound application were induced by ultrasound-mediated microbubble-cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium [Ca(2+)](i) levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca(2+)](i) in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7-20 microm/s over a distance >200 microm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca(2+) for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison microbubbles can generate transient [Ca(2+)](i) changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane.

  7. Non-degenerate 2-photon excitation for fluorescence microscopy in scattering medium (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Fainman, Yeshaiahu; Yang, Mu-Han; Abashin, Maxim; Saisan, Payam; Tian, Peifang; Ferri, Christopher; Devor, Anna

    2016-10-01

    Non-degenerate 2-photon excitation of a fluorophore with two laser beams of different photon energies may offer independent degree of freedom in tuning of the photon flux (i.e., the power) for each beam. Wereport a practical demonstration that the emission intensity of a fluorophore excited in the non-degenerate regime in scattering medium is more efficient than the commonly used degenerate 2-photon excitation. In our experiments we use spatially and temporally aligned Ti:Sapphiremode-locked laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, respectively. The non-degenerate 2-photon excitation mechanism takes advantage of the infrared wavelengths used in 3-photon microscopy to achieve increased penetration depth, while preserving relatively high 2-photon excitation cross section, exceeding that achievable with the 3-photon excitation. Importantly, independent control of power for each beam implies that the flux requirement for the higher photon energy NIR beam, which experiences higher scattering in biological tissue, can be relaxed at the expense of increasing the flux of the lower photon energy SWIR beam which experiences lower scattering, thus promising deeper penetration with higher efficiency of excitation.Applications for in vivo brain imaging will be also discussed.

  8. Imaging and analysis of evoked excitatory-postsynaptic-calcium-transients by individual presynaptic-boutons of cultured Aplysia sensorimotor synapse.

    PubMed

    Malkinson, Guy; Spira, Micha E

    2010-04-01

    The use of the sensory-motor (SN-MN) synapse of the Aplysia gill withdrawal reflex has contributed immensely to the understanding of synaptic transmission, learning and memory acquisition processes. Whereas the majority of the studies focused on analysis of the presynaptic mechanisms, recent studies indicated that as in mammalian synapses, long term potentiation (LTP) formed by Aplysia SN-MN synapse depends on elevation of the postsynaptic free intracellular calcium concentration ([Ca2+](i)). Consistently, injection of the fast calcium chelator BAPTA to the MN prevents the formation of serotonin-induced LTP. Nevertheless, currently there are no published reports that directly examine and document whether evoked synaptic transmission is associated with transient increase in the postsynaptic [Ca2+](i). In the present study we imaged, for the first time, alterations in the postsynaptic [Ca2+](i) in response to presynaptic stimulation and analyzed the underlying mechanisms. Using live imaging of the postsynaptic [Ca2+](i) while monitoring the EPSP, we found that evoked transmitter release generates excitatory postsynaptic calcium concentration transients (EPSCaTs) by two mechanisms: (a) activation of DNQX-sensitive postsynaptic receptors-gated calcium influx and (b) calcium influx through nitrendipine-sensitive voltage-gated calcium channels (VGCCs). Concomitant confocal imaging of presynaptic boutons and EPSCaTs revealed that approximately 86% of the presynaptic boutons are associated with functional synapses.

  9. In vivo photoacoustic neuronal imaging of odor-evoked calcium signals in the drosophila brain (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhang, Ruiying; Rao, Bin; Rong, Haoyang; Raman, Baranidharan; Wang, Lihong V.

    2016-03-01

    Neural scientists can benefit greatly from imaging tools that can penetrate thick brain tissue. Compared with traditional optical microscopy methods, photoacoustic imaging can beat the optical diffusion limit and achieve such deep tissue imaging with high spatial resolution. In this study, we used an optical-resolution photoacoustic microscope to image the odor-evoked neuronal activities in a drosophila model. Drosophila brain neurons stably express GCaMP5G, a calcium-sensitive fluorescent protein whose optical absorption coefficient changes with calcium influx during action potentials. We recorded an ~20% odor-evoked fractional photoacoustic signal increase at all depths of the drosophila brain in vivo, with and without removal of the brain cuticle, at a recording rate of 1 kHz. Our results were confirmed by concurrent fluorescent recordings. Furthermore, by performing fast 2D scanning, we imaged the antenna lobe region, which is of particular interest in neuroscience, at a volumetric rate of ~1 Hz with a sub-neuron resolution of 3 μm. Unlike optical imaging, which requires surgical removal of the scattering brain cuticle, our photoacoustic system can image through the cuticle and measure neuronal signals of the whole drosophila brain without invasive surgery, enabling minimal disturbance to the animal's behaviors. In conclusion, we have demonstrated photoacoustic imaging of calcium signals in drosophila brains for the first time. Utilizing the deep imaging capability of photoacoustic tomography, our methods could potentially be extended to in vivo imaging of neuronal activities from deep brains in other animal models.

  10. Multispot two-photon imaging of mice heart tissue detecting calcium waves

    NASA Astrophysics Data System (ADS)

    de Mauro, C.; Cecchetti, C. A.; Alfieri, D.; Borile, G.; Mongillo, M.; Pavone, F. S.

    2012-06-01

    High rate, full field image acquisition in multiphoton imaging is achievable by parallelization of the excitation and of the detection paths. Via a Diffractive Optical Elements (DOEs) which splits a pulsed laser, and a spatial resolved descanned detection path, a new approach to microscopy has been developed. By exploiting the three operating mode, single beam, 16 beamlets or 64 beamlets, the best experimental conditions can be found by adapting the power per beamlet. This Multiphoton Multispot system (MCube) has been characterized in thick tissue samples, and subsequently used for the first time for Ca2+ imaging of acute heart slices. A test sample with fixed mice heart slices with embedded sub-resolution fluorescent beads has been used to test the capability of optical axial resolution up to ~200 microns in depth. Radial and axial resolutions of 0.6 microns and 3 microns have been respectively obtained with a 40X water immersion objective, getting close to the theoretical limit. Then images of heart slices cardiomyocites, loaded with Fluo4-AM have been acquired. The formation of Ca2+ waves during electrostimulated beating has been observed, and the possibility of easily acquire full frame images at 15 Hz (16 beamlets) has been demonstrated, towards the in vivo study of time resolved cellular dynamics and arrhythmia trigger mechanisms in particular. A very high speed two-photon Random Access system for in vivo electrophysiological studies, towards the correlation of voltage and calcium signals in arrhythmia phenomena, is now under developing at Light4tech.

  11. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.

    PubMed

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René

    2016-01-08

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

  12. Microwave fixation and localization of calcium in synaptic terminals using x-ray microanalysis and electron energy loss spectroscopy imaging.

    PubMed

    Mizuhira, V; Hasegawa, H

    1997-01-01

    The distribution of calcium ions is demonstrated in synaptic terminals by means of a two-step chemical precipitation of calcium ions in the rat brain. K-oxalate/K-antimonate chemical replacement with simultaneous computerized microwave irradiation was used. This precipitate in nerve cell structures was investigated by computerized electron probe x-ray microanalysis (EDX) and electron energy loss spectroscopic (EELS) imaging. The values obtained by EDX agreed with those of the standard sample and theoretical values of Ca-antimonate. Typical EELS spectra of Ca:L, O:K, and Sb:M were obtained from nerve terminals in the same tissue block as that used for EDX analysis. Excellent net Ca:L and Sb:M EELS digital images were obtained after their background images were subtracted. Calcium ions were distributed in the nerve terminals, synaptic vesicles, mitochondria, and synaptic membranes.

  13. Multi-modal in vivo imaging of brain blood oxygenation, blood flow and neural calcium dynamics during acute seizures

    NASA Astrophysics Data System (ADS)

    Ringuette, Dene; Jeffrey, Melanie A.; Carlen, Peter L.; Levi, Ofer

    2016-03-01

    Dysfunction of the vascular endothelium has been implicated in the development of epilepsy. To better understand the relation between vascular function and seizure and provide a foundation for interpreting results from functional imaging in chronic disease models, we investigate the relationship between intracellular calcium dynamics and local cerebral blood flow and blood oxygen saturation during acute seizure-like events and pharmacological seizure rescue. To probe the relation between the aforementioned physiological markers in an acute model of epilepsy in rats, we integrated three different optical modalities together with electrophysiological recordings: Laser speckle contrast imaging (LSCI) was used to study changes in flow speeds, Intrinsic optical signal imaging (IOSI) was used to monitor changes in oxygenated, de-oxygenated, and total hemoglobin concentration, and Calcium-sensitive dye imaging was used to monitor intracellular calcium dynamics. We designed a dedicated cortical flow chamber to remove superficial blood and dye resulting from the injection procedure, which reduced spurious artifacts. The near infrared light used for IOSI and LSCI was delivered via a light pipe integrated with the flow chamber to minimize the effect of fluid surface movement on illumination stability. Calcium-sensitive dye was injected via a glass electrode used for recording the local field potential. Our system allowed us to observe and correlate increases in intracellular calcium, blood flow and blood volume during seizure-like events and provide a quantitative analysis of neurovascular coupling changes associated with seizure rescue via injection of an anti-convulsive agent.

  14. A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store

    PubMed Central

    Henderson, Mark J.; Baldwin, Heather A.; Werley, Christopher A.; Boccardo, Stefano; Whitaker, Leslie R.; Yan, Xiaokang; Holt, Graham T.; Schreiter, Eric R.; Looger, Loren L.; Cohen, Adam E.; Kim, Douglas S.; Harvey, Brandon K.

    2015-01-01

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states. PMID:26451944

  15. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

    PubMed

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu

    2014-06-01

    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

  16. A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.

    PubMed

    Henderson, Mark J; Baldwin, Heather A; Werley, Christopher A; Boccardo, Stefano; Whitaker, Leslie R; Yan, Xiaokang; Holt, Graham T; Schreiter, Eric R; Looger, Loren L; Cohen, Adam E; Kim, Douglas S; Harvey, Brandon K

    2015-01-01

    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  17. Calcium Imaging of Neuronal Activity in Drosophila Can Identify Anticonvulsive Compounds.

    PubMed

    Streit, Anne K; Fan, Yuen Ngan; Masullo, Laura; Baines, Richard A

    2016-01-01

    Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds

  18. Calcium Imaging of Neuronal Activity in Drosophila Can Identify Anticonvulsive Compounds

    PubMed Central

    Streit, Anne K.; Fan, Yuen Ngan; Masullo, Laura; Baines, Richard A.

    2016-01-01

    Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds

  19. Dual energy x-ray imaging and scoring of coronary calcium: physics-based digital phantom and clinical studies

    NASA Astrophysics Data System (ADS)

    Zhou, Bo; Wen, Di; Nye, Katelyn; Gilkeson, Robert C.; Wilson, David L.

    2016-03-01

    Coronary artery calcification (CAC) as assessed with CT calcium score is the best biomarker of coronary artery disease. Dual energy x-ray provides an inexpensive, low radiation-dose alternative. A two shot system (GE Revolution-XRd) is used, raw images are processed with a custom algorithm, and a coronary calcium image (DECCI) is created, similar to the bone image, but optimized for CAC visualization, not lung visualization. In this report, we developed a physicsbased, digital-phantom containing heart, lung, CAC, spine, ribs, pulmonary artery, and adipose elements, examined effects on DECCI, suggested physics-inspired algorithms to improve CAC contrast, and evaluated the correlation between CT calcium scores and a proposed DE calcium score. In simulation experiment, Beam hardening from increasing adipose thickness (2cm to 8cm) reduced Cg by 19% and 27% in 120kVp and 60kVp images, but only reduced Cg by <7% in DECCI. If a pulmonary artery moves or pulsates with blood filling between exposures, it can give rise to a significantly confounding PA signal in DECCI similar in amplitude to CAC. Observations suggest modifications to DECCI processing, which can further improve CAC contrast by a factor of 2 in clinical exams. The DE score had the best correlation with "CT mass score" among three commonly used CT scores. Results suggest that DE x-ray is a promising tool for imaging and scoring CAC, and there still remains opportunity for further DECCI processing improvements.

  20. Preparation and preclinical evaluation of 68Ga-DOTA-amlodipine for L-type calcium channel imaging

    PubMed Central

    Firuzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Shafiee-Ardestani, Mahdi; Khalaj, Ali

    2016-01-01

    Aim: In order to develop a possible tracer for L-type calcium channel imaging, we here report the development of a Ga-68 amlodipine derivative for possible PET imaging. Materials and Methods: Amlodipine DOTA conjugate was synthesized, characterized and went through calcium channel blockade, toxicity, apoptosis/necrosis tests. [68Ga] DOTA AMLO was prepared at optimized conditions followed by stability tests, partition coefficient determination and biodistribution studies using tissue counting and co incidence imaging up to 2 h. Results: [68Ga] DOTA AMLO was prepared at pH 4–5 in 7–10 min at 95°C in high radiochemical purity (>99%, radio thin layer chromatography; specific activity: 1.9–2.1 GBq/mmol) and was stable up to 4 h with a log P of −0.94. Calcium channel rich tissues including myocardium, and tissues with smooth muscle cells such as colon, intestine, and lungs demonstrated significant uptake. Co incidence images supported the biodistribution data up to 2 h. Conclusions: The complex can be a candidate for further positron emission tomography imaging for L type calcium channels. PMID:27833311

  1. The need for calcium imaging in nonhuman primates: New motor neuroscience and brain-machine interfaces.

    PubMed

    O'Shea, Daniel J; Trautmann, Eric; Chandrasekaran, Chandramouli; Stavisky, Sergey; Kao, Jonathan C; Sahani, Maneesh; Ryu, Stephen; Deisseroth, Karl; Shenoy, Krishna V

    2017-01-01

    A central goal of neuroscience is to understand how populations of neurons coordinate and cooperate in order to give rise to perception, cognition, and action. Nonhuman primates (NHPs) are an attractive model with which to understand these mechanisms in humans, primarily due to the strong homology of their brains and the cognitively sophisticated behaviors they can be trained to perform. Using electrode recordings, the activity of one to a few hundred individual neurons may be measured electrically, which has enabled many scientific findings and the development of brain-machine interfaces. Despite these successes, electrophysiology samples sparsely from neural populations and provides little information about the genetic identity and spatial micro-organization of recorded neurons. These limitations have spurred the development of all-optical methods for neural circuit interrogation. Fluorescent calcium signals serve as a reporter of neuronal responses, and when combined with post-mortem optical clearing techniques such as CLARITY, provide dense recordings of neuronal populations, spatially organized and annotated with genetic and anatomical information. Here, we advocate that this methodology, which has been of tremendous utility in smaller animal models, can and should be developed for use with NHPs. We review here several of the key opportunities and challenges for calcium-based optical imaging in NHPs. We focus on motor neuroscience and brain-machine interface design as representative domains of opportunity within the larger field of NHP neuroscience.

  2. Calcium (Ca2+) waves data calibration and analysis using image processing techniques

    PubMed Central

    2013-01-01

    Background Calcium (Ca2+) propagates within tissues serving as an important information carrier. In particular, cilia beat frequency in oviduct cells is partially regulated by Ca2+ changes. Thus, measuring the calcium density and characterizing the traveling wave plays a key role in understanding biological phenomena. However, current methods to measure propagation velocities and other wave characteristics involve several manual or time-consuming procedures. This limits the amount of information that can be extracted, and the statistical quality of the analysis. Results Our work provides a framework based on image processing procedures that enables a fast, automatic and robust characterization of data from two-filter fluorescence Ca2+ experiments. We calculate the mean velocity of the wave-front, and use theoretical models to extract meaningful parameters like wave amplitude, decay rate and time of excitation. Conclusions Measurements done by different operators showed a high degree of reproducibility. This framework is also extended to a single filter fluorescence experiments, allowing higher sampling rates, and thus an increased accuracy in velocity measurements. PMID:23679062

  3. Inferring Neuronal Dynamics from Calcium Imaging Data Using Biophysical Models and Bayesian Inference.

    PubMed

    Rahmati, Vahid; Kirmse, Knut; Marković, Dimitrije; Holthoff, Knut; Kiebel, Stefan J

    2016-02-01

    Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits.

  4. Inferring Neuronal Dynamics from Calcium Imaging Data Using Biophysical Models and Bayesian Inference

    PubMed Central

    Rahmati, Vahid; Kirmse, Knut; Marković, Dimitrije; Holthoff, Knut; Kiebel, Stefan J.

    2016-01-01

    Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits. PMID:26894748

  5. Image-based Modeling of Biofilm-induced Calcium Carbonate Precipitation

    NASA Astrophysics Data System (ADS)

    Connolly, J. M.; Rothman, A.; Jackson, B.; Klapper, I.; Cunningham, A. B.; Gerlach, R.

    2013-12-01

    Pore scale biological processes in the subsurface environment are important to understand in relation to many engineering applications including environmental contaminant remediation, geologic carbon sequestration, and petroleum production. Specifically, biofilm induced calcium carbonate precipitation has been identified as an attractive option to reduce permeability in a lasting way in the subsurface. This technology may be able to replace typical cement-based grouting in some circumstances; however, pore-scale processes must be better understood for it to be applied in a controlled manor. The work presented will focus on efforts to observe biofilm growth and ureolysis-induced mineral precipitation in micro-fabricated flow cells combined with finite element modelling as a tool to predict local chemical gradients of interest (see figure). We have been able to observe this phenomenon over time using a novel model organism that is able to hydrolyse urea and express a fluorescent protein allowing for non-invasive observation over time with confocal microscopy. The results of this study show the likely existence of a wide range of local saturation indices even in a small (1 cm length scale) experimental system. Interestingly, the locations of high predicted index do not correspond to the locations of higher precipitation density, highlighting the need for further understanding. Figure 1 - A micro-fabricated flow cell containing biofilm-induced calcium carbonate precipitation. (A) Experimental results: Active biofilm is in green and dark circles are calcium carbonate crystals. Note the channeling behavior in the top of the image, leaving a large hydraulically inactive area in the biofilm mass. (B) Finite element model: The prediction of relative saturation of calcium carbonate (as calcite). Fluid enters the system at a low saturation state (blue) but areas of high supersaturation (red) are predicted within the hydraulically inactive area in the biofilm. If only effluent

  6. Calcium Imaging of Basal Forebrain Activity during Innate and Learned Behaviors

    PubMed Central

    Harrison, Thomas C.; Pinto, Lucas; Brock, Julien R.; Dan, Yang

    2016-01-01

    The basal forebrain (BF) plays crucial roles in arousal, attention, and memory, and its impairment is associated with a variety of cognitive deficits. The BF consists of cholinergic, GABAergic, and glutamatergic neurons. Electrical or optogenetic stimulation of BF cholinergic neurons enhances cortical processing and behavioral performance, but the natural activity of these cells during behavior is only beginning to be characterized. Even less is known about GABAergic and glutamatergic neurons. Here, we performed microendoscopic calcium imaging of BF neurons as mice engaged in spontaneous behaviors in their home cages (innate) or performed a go/no-go auditory discrimination task (learned). Cholinergic neurons were consistently excited during movement, including running and licking, but GABAergic and glutamatergic neurons exhibited diverse responses. All cell types were activated by overt punishment, either inside or outside of the discrimination task. These findings reveal functional similarities and distinctions between BF cell types during both spontaneous and task-related behaviors. PMID:27242444

  7. Adaptive optics for in vivo two-photon calcium imaging of neuronal networks

    NASA Astrophysics Data System (ADS)

    Meimon, Serge; Conan, Jean-Marc; Mugnier, Laurent M.; Michau, Vincent; Cossart, Rosa; Malvache, Arnaud

    2014-03-01

    The landscape of biomedical research in neuroscience has changed dramatically in recent years as a result of spectacular progress in dynamic microscopy. However, the optical accessibility of deep brain structures or deeper regions of the surgically exposed hippocampus (a few 100 microns typically) remains limited, due to volumic aberrations created by the sample inhomogeneities. Adaptive optics can correct for these aberrations. Our goal is to realize a novel adaptive optics module dedicated to in vivo two-photon calcium imaging of the hippocampus. The key issue in adaptive optics is the ability to perform an accurate and reliable wavefront sensing. In two- photon microscopy indirect methods are required. Two families of approaches have been proposed so far, the modal sensorless technique and a method based on pupil segmentation. We present here a formal comparison of these approaches, in particular as a function of the amount of aberrations.

  8. New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging.

    PubMed

    Oheim, Martin; van 't Hoff, Marcel; Feltz, Anne; Zamaleeva, Alsu; Mallet, Jean-Maurice; Collot, Mayeul

    2014-10-01

    Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck

  9. Model-Free Reconstruction of Excitatory Neuronal Connectivity from Calcium Imaging Signals

    PubMed Central

    Stetter, Olav; Battaglia, Demian; Soriano, Jordi; Geisel, Theo

    2012-01-01

    A systematic assessment of global neural network connectivity through direct electrophysiological assays has remained technically infeasible, even in simpler systems like dissociated neuronal cultures. We introduce an improved algorithmic approach based on Transfer Entropy to reconstruct structural connectivity from network activity monitored through calcium imaging. We focus in this study on the inference of excitatory synaptic links. Based on information theory, our method requires no prior assumptions on the statistics of neuronal firing and neuronal connections. The performance of our algorithm is benchmarked on surrogate time series of calcium fluorescence generated by the simulated dynamics of a network with known ground-truth topology. We find that the functional network topology revealed by Transfer Entropy depends qualitatively on the time-dependent dynamic state of the network (bursting or non-bursting). Thus by conditioning with respect to the global mean activity, we improve the performance of our method. This allows us to focus the analysis to specific dynamical regimes of the network in which the inferred functional connectivity is shaped by monosynaptic excitatory connections, rather than by collective synchrony. Our method can discriminate between actual causal influences between neurons and spurious non-causal correlations due to light scattering artifacts, which inherently affect the quality of fluorescence imaging. Compared to other reconstruction strategies such as cross-correlation or Granger Causality methods, our method based on improved Transfer Entropy is remarkably more accurate. In particular, it provides a good estimation of the excitatory network clustering coefficient, allowing for discrimination between weakly and strongly clustered topologies. Finally, we demonstrate the applicability of our method to analyses of real recordings of in vitro disinhibited cortical cultures where we suggest that excitatory connections are characterized

  10. FRET-based calcium imaging: a tool for high-throughput/content phenotypic drug screening in Alzheimer disease.

    PubMed

    Honarnejad, Kamran; Kirsch, Achim K; Daschner, Alexander; Szybinska, Aleksandra; Kuznicki, Jacek; Herms, Jochen

    2013-12-01

    Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.

  11. Multimodal second harmonic generation and two photon fluorescence imaging of microdomain calcium contraction coupling in single cardiomyocytes

    NASA Astrophysics Data System (ADS)

    Chan, James; Awasthi, Samir; Izu, Leighton; Mao, Ziliang; Jian, Zhong; Landas, Trevor; Lerner, Aaron; Shimkunas, Rafael; Woldeyesus, Rahwa; Bossuyt, Julie; Wood, Brittani; Chen, Yi-Je; Matthews, Dennis; Lieu, Deborah; Chiamvimonvat, Nipavan; Lam, Kit; Chen-Izu, Ye

    2016-11-01

    The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.

  12. Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data.

    PubMed

    Fallani, Fabrizio De Vico; Corazzol, Martina; Sternberg, Jenna R; Wyart, Claire; Chavez, Mario

    2015-05-01

    The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network approach to calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer information propagation between the activities of different neurons. The resulting functional network was then modeled as a directed graph and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions.

  13. A finite rate of innovation algorithm for fast and accurate spike detection from two-photon calcium imaging

    NASA Astrophysics Data System (ADS)

    Oñativia, Jon; Schultz, Simon R.; Dragotti, Pier Luigi

    2013-08-01

    Objective. Inferring the times of sequences of action potentials (APs) (spike trains) from neurophysiological data is a key problem in computational neuroscience. The detection of APs from two-photon imaging of calcium signals offers certain advantages over traditional electrophysiological approaches, as up to thousands of spatially and immunohistochemically defined neurons can be recorded simultaneously. However, due to noise, dye buffering and the limited sampling rates in common microscopy configurations, accurate detection of APs from calcium time series has proved to be a difficult problem. Approach. Here we introduce a novel approach to the problem making use of finite rate of innovation (FRI) theory (Vetterli et al 2002 IEEE Trans. Signal Process. 50 1417-28). For calcium transients well fit by a single exponential, the problem is reduced to reconstructing a stream of decaying exponentials. Signals made of a combination of exponentially decaying functions with different onset times are a subclass of FRI signals, for which much theory has recently been developed by the signal processing community. Main results. We demonstrate for the first time the use of FRI theory to retrieve the timing of APs from calcium transient time series. The final algorithm is fast, non-iterative and parallelizable. Spike inference can be performed in real-time for a population of neurons and does not require any training phase or learning to initialize parameters. Significance. The algorithm has been tested with both real data (obtained by simultaneous electrophysiology and multiphoton imaging of calcium signals in cerebellar Purkinje cell dendrites), and surrogate data, and outperforms several recently proposed methods for spike train inference from calcium imaging data.

  14. SarConfoCal: simultaneous sarcomere length and cytoplasmic calcium measurements for laser scanning confocal microscopy images.

    PubMed

    Pasqualin, Côme; Gannier, François; Yu, Angèle; Malécot, Claire O; Bredeloux, Pierre; Maupoil, Véronique

    2016-12-22

    Simultaneous recordings of myocytes contractility and their cytoplasmic calcium concentration allow powerful studies, particularly on heart failure and other cardiac dysfunctions. Such studies require dedicated and expensive experimental devices that are difficult to use. Thus we propose SarConfoCal, the first and only software to simultaneously analyse both cytoplasmic calcium variations (from fluorescence signal) and myocytes contractility (from sarcomere length measurement) on laser scanning confocal microscopy images. SarConfoCal is easy to set up and use, especially by people without programming skills.

  15. Optimization of a GCaMP calcium indicator for neural activity imaging.

    PubMed

    Akerboom, Jasper; Chen, Tsai-Wen; Wardill, Trevor J; Tian, Lin; Marvin, Jonathan S; Mutlu, Sevinç; Calderón, Nicole Carreras; Esposti, Federico; Borghuis, Bart G; Sun, Xiaonan Richard; Gordus, Andrew; Orger, Michael B; Portugues, Ruben; Engert, Florian; Macklin, John J; Filosa, Alessandro; Aggarwal, Aman; Kerr, Rex A; Takagi, Ryousuke; Kracun, Sebastian; Shigetomi, Eiji; Khakh, Baljit S; Baier, Herwig; Lagnado, Leon; Wang, Samuel S-H; Bargmann, Cornelia I; Kimmel, Bruce E; Jayaraman, Vivek; Svoboda, Karel; Kim, Douglas S; Schreiter, Eric R; Looger, Loren L

    2012-10-03

    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

  16. Optimization of a GCaMP calcium indicator for neural activity imaging

    PubMed Central

    Akerboom, Jasper; Chen, Tsai-Wen; Wardill, Trevor J.; Tian, Lin; Marvin, Jonathan S.; Mutlu, Sevinç; Calderón, Nicole Carreras; Esposti, Federico; Borghuis, Bart G.; Sun, Xiaonan Richard; Gordus, Andrew; Orger, Michael B.; Portugues, Ruben; Engert, Florian; Macklin, John J.; Filosa, Alessandro; Aggarwal, Aman; Kerr, Rex; Takagi, Ryousuke; Kracun, Sebastian; Shigetomi, Eiji; Khakh, Baljit S.; Baier, Herwig; Lagnado, Leon; Wang, Samuel S.-H.; Bargmann, Cornelia I.; Kimmel, Bruce E.; Jayaraman, Vivek; Svoboda, Karel; Kim, Douglas S.; Schreiter, Eric R.; Looger, Loren L.

    2012-01-01

    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general. PMID:23035093

  17. New calcium-selective smart contrast agents for magnetic resonance imaging.

    PubMed

    Verma, Kirti Dhingra; Forgács, Attila; Uh, Hyounsoo; Beyerlein, Michael; Maier, Martin E; Petoud, Stéphane; Botta, Mauro; Logothetis, Nikos K

    2013-12-23

    Calcium plays a vital role in the human body and especially in the central nervous system. Precise maintenance of Ca(2+) levels is very crucial for normal cell physiology and health. The deregulation of calcium homeostasis can lead to neuronal cell death and brain damage. To study this functional role played by Ca(2+) in the brain noninvasively by using magnetic resonance imaging, we have synthesized a new set of Ca(2+) -sensitive smart contrast agents (CAs). The agents were found to be highly selective to Ca(2+) in the presence of other competitive anions and cations in buffer and in physiological fluids. The structure of CAs comprises Gd(3+)-DO3A (DO3A=1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane) coupled to a Ca(2+) chelator o-amino phenol-N,N,O-triacetate (APTRA). The agents are designed to sense Ca(2+) present in extracellular fluid of the brain where its concentration is relatively high, that is, 1.2-0.8 mM. The determined dissociation constant of the CAs to Ca(2+) falls in the range required to sense and report changes in extracellular Ca(2+) levels followed by an increase in neural activity. In buffer, with the addition of Ca(2+) the increase in relaxivity ranged from 100-157%, the highest ever known for any T1-based Ca(2+)-sensitive smart CA. The CAs were analyzed extensively by the measurement of luminescence lifetime measurement on Tb(3+) analogues, nuclear magnetic relaxation dispersion (NMRD), and (17)O NMR transverse relaxation and shift experiments. The results obtained confirmed that the large relaxivity enhancement observed upon Ca(2+) addition is due to the increase of the hydration state of the complexes together with the slowing down of the molecular rotation and the retention of a significant contribution of the water molecules of the second sphere of hydration.

  18. Synthesis and characterization of bioresorbable calcium phosphosilicate nanocomposite particles for fluorescence imaging and biomedical applications

    NASA Astrophysics Data System (ADS)

    Morgan, Thomas T.

    Organically doped calcium phosphosilicate nanoparticles (CPSNPs) were developed and characterized, driven by the need for non-toxic vectors for drug delivery and fluorescence biological imaging applications. In particular, advancement in drug delivery for the chemotherapeutic treatment of cancers is required to increase drug efficacy and improve patient quality of life. Additionally, brighter and more photostable fluorophores are needed to meet demands for improved sensitivity and experimental diversity, which may lead to improvements in early detection of solid tumors and advancement in understanding of biological processes. A literature survey on the state of the field for nanoparticle based biological fluorescence imaging and drug delivery is presented in Chapter 1. Chapter 2 focuses on the characterization techniques used in this work. The development and optical characterization of 20-40 nm diameter, citrate functionalized Cy3 amidite doped calcium phosphosilicate nanoparticles (Cy3 CPSNPs) for in vitro fluorescence imaging is outlined in Chapters 3 and 4, respectively. In particular, sodium citrate was used to functionalize the surface and provide electrosteric dispersion of these particles. CPSNPs stabilized with sodium citrate routinely exhibited highly negative zeta potentials greater than -25 mV in magnitude. Furthermore, the fluorescence quantum yield of the encapsulated fluorophore was improved by more than 4.5-fold when compared to the unencapsulated dye. The bioimaging and drug delivery capability of CPSNPs was explored. Cy3 CPSNPs dissolved quickly in the acidic environment experienced during endocytosis, releasing the encapsulated fluorophore. This is consistent with solution phase experiments that show the particles are dissolved at pH 5. CPSNPs loaded with fluorescein and a hydrophobic growth inhibitor, ceramide C6, proved the ability to simultaneously image and delivery of the hydrophobic drug to cells in vitro. Chapter 5 examined the colloidal

  19. Chronic imaging of movement-related Purkinje cell calcium activity in awake behaving mice

    PubMed Central

    Gaffield, Michael A.; Amat, Samantha B.; Bito, Haruhiko

    2015-01-01

    Purkinje cells (PCs) are a major site of information integration and plasticity in the cerebellum, a brain region involved in motor task refinement. Thus PCs provide an ideal location for studying the mechanisms necessary for cerebellum-dependent motor learning. Increasingly, sophisticated behavior tasks, used in combination with genetic reporters and effectors of activity, have opened up the possibility of studying cerebellar circuits during voluntary movement at an unprecedented level of quantitation. However, current methods used to monitor PC activity do not take full advantage of these advances. For example, single-unit or multiunit electrode recordings, which provide excellent temporal information regarding electrical activity, only monitor a small population of cells and can be quite invasive. Bolus loading of cell-permeant calcium (Ca2+) indicators is short-lived, requiring same-day imaging immediately after surgery and/or indicator injection. Genetically encoded Ca2+ indicators (GECIs) overcome many of these limits and have garnered considerable use in many neuron types but only limited use in PCs. Here we employed these indicators to monitor Ca2+ activity in PCs over several weeks. We could repeatedly image from the same cerebellar regions across multiple days and observed stable activity. We used chronic imaging to monitor PC activity in crus II, an area previously linked to licking behavior, and identified a region of increased activity at the onset of licking. We then monitored this same region after training tasks to initiate voluntary licking behavior in response to different sensory stimuli. In all cases, PC Ca2+ activity increased at the onset of rhythmic licking. PMID:26561609

  20. Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

    PubMed Central

    Trivedi, Vikas; Truong, Thai V.; Trinh, Le A.; Holland, Daniel B.; Liebling, Michael; Fraser, Scott E.

    2015-01-01

    We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines. PMID:26114028

  1. Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey.

    PubMed

    McClellan, A D; McPherson, D; O'Donovan, M J

    1994-11-07

    Neurons in the brainstem and spinal cord of the lamprey were retrogradely labeled with Calcium Green-dextran, an indicator dye that increases its fluorescence when intracellular calcium levels increase. Optical signals could be recorded from these labeled neurons during spinal cord stimulation, nerve stimulation, or spontaneous activity, up to 4 days after dye application and for distances of 5-14 mm away from the application site. Optical signals were enhanced by 4-AP, a potassium channel blocker, and blocked by cadmium, a calcium channel blocker. Taken together, the results suggest that the optical signals recorded from labeled neurons were due to calcium influx during electrical activity. Thus, retrograde labeling with calcium indicator dyes may provide a general purpose method for simultaneously monitoring the activity-related changes of intracellular calcium in anatomically identified groups of neurons in the lamprey nervous system.

  2. Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.

    PubMed

    Miyamoto, Akitoshi; Bannai, Hiroko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2013-05-03

    Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals.

  3. LFP-guided targeting of a cortical barrel column for in vivo two-photon calcium imaging

    PubMed Central

    Lee, Joon-Hyuk; Shin, Hee-Sup; Lee, Kwang-Hyung; Chung, Sooyoung

    2015-01-01

    Two-photon microscopy of bulk-loaded functional dyes is an outstanding physiological technique that enables simultaneous functional mapping of hundreds of brain cells in vivo at single-cell resolution. However, precise targeting of a specific cortical location is not easy due to its fine dimensionality. To enable precise targeting, intrinsic-signal optical imaging is often additionally performed. However, the intrinsic-signal optical imaging is not only time-consuming but also ineffective in ensuring precision. Here, we propose an alternative method for precise targeting based on local field potential (LFP) recording, a conventional electrophysiological method. The heart of this method lies in use of the same glass pipette to record LFPs and to eject calcium dye. After confirming the target area by LFP using a glass pipette, the calcium dye is ejected from the same pipette without a time delay or spatial adjustment. As a result, the calcium dye is loaded into the same ensemble of brain cells from which the LFP was obtained. As a validation of the proposed LFP-based method, we targeted and successfully loaded calcium dye into layer 2/3 of a mouse barrel column. PMID:26511063

  4. LFP-guided targeting of a cortical barrel column for in vivo two-photon calcium imaging.

    PubMed

    Lee, Joon-Hyuk; Shin, Hee-Sup; Lee, Kwang-Hyung; Chung, Sooyoung

    2015-10-29

    Two-photon microscopy of bulk-loaded functional dyes is an outstanding physiological technique that enables simultaneous functional mapping of hundreds of brain cells in vivo at single-cell resolution. However, precise targeting of a specific cortical location is not easy due to its fine dimensionality. To enable precise targeting, intrinsic-signal optical imaging is often additionally performed. However, the intrinsic-signal optical imaging is not only time-consuming but also ineffective in ensuring precision. Here, we propose an alternative method for precise targeting based on local field potential (LFP) recording, a conventional electrophysiological method. The heart of this method lies in use of the same glass pipette to record LFPs and to eject calcium dye. After confirming the target area by LFP using a glass pipette, the calcium dye is ejected from the same pipette without a time delay or spatial adjustment. As a result, the calcium dye is loaded into the same ensemble of brain cells from which the LFP was obtained. As a validation of the proposed LFP-based method, we targeted and successfully loaded calcium dye into layer 2/3 of a mouse barrel column.

  5. Imaging of drug loading distributions in individual microspheres of calcium silicate hydrate - an X-ray spectromicroscopy study

    NASA Astrophysics Data System (ADS)

    Guo, Xiaoxuan; Wang, Zhiqiang; Wu, Jin; Wang, Jian; Zhu, Ying-Jie; Sham, Tsun-Kong

    2015-04-01

    Imaging is one of the most direct and ideal ways to track drug loading distributions in drug carriers on the molecular level, which will facilitate the optimization of drug carriers and drug loading capacities. Herein, we report the mapping of an individual mesoporous calcium silicate hydrate (CSH) microsphere before and after the loading of ibuprofen (IBU) and the interactions between drug carriers and drug molecules simultaneously by scanning transmission X-ray microscopy (STXM). Nanoscaled X-ray absorption near edge structure (XANES) spectroscopy clearly indicates that IBU is bonded to calcium and silicate sites via carboxylic acid groups. More importantly, STXM has been successfully used to determine the absolute thickness of IBU, revealing its distribution in the CSH microsphere.Imaging is one of the most direct and ideal ways to track drug loading distributions in drug carriers on the molecular level, which will facilitate the optimization of drug carriers and drug loading capacities. Herein, we report the mapping of an individual mesoporous calcium silicate hydrate (CSH) microsphere before and after the loading of ibuprofen (IBU) and the interactions between drug carriers and drug molecules simultaneously by scanning transmission X-ray microscopy (STXM). Nanoscaled X-ray absorption near edge structure (XANES) spectroscopy clearly indicates that IBU is bonded to calcium and silicate sites via carboxylic acid groups. More importantly, STXM has been successfully used to determine the absolute thickness of IBU, revealing its distribution in the CSH microsphere. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07471h

  6. Functional evaluation of neural stem cell differentiation by single cell calcium imaging.

    PubMed

    Eiriz, Maria Francisca; Grade, Sofia; Rosa, Alexandra; Xapelli, Sara; Bernardino, Liliana; Agasse, Fabienne; Malva, João O

    2011-09-01

    Neurogenesis in the adult mammalian brain occurs in two specific brain areas, the subventricular zone (SVZ) bordering the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Although these regions are prone to produce new neurons, cultured cells from these neurogenic niches tend to be mixed cultures, containing both neurons and glial cells. Several reports highlight the potential of the self-healing capacity of the brain following injury. Even though much knowledge has been produced on the neurogenesis itself, brain repairing strategies are still far away from patients cure. Here we review general concepts in the neurogenesis field, also addressing the methods available to study neural stem cell differentiation. A major problem faced by research groups and companies dedicated to brain regenerative medicine resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. To address this issue, we developed a unique single cell calcium imaging-based method to functionally discriminate different cell types derived from SVZ neural stem cell cultures. The unique functional profile of each SVZ cell type was correlated at the single cell level with the immunodetection of specific phenotypic markers. This platform was raised on the basis of the functional response of neurons, oligodendrocytes and immature cells to depolarising agents, to thrombin and to histamine, respectively. We also outline key studies in which our new platform was extremely relevant in the context of drug discovery and development in the area of brain regenerative medicine.

  7. Fluorescent Protein-photoprotein Fusions and Their Applications in Calcium Imaging.

    PubMed

    Bakayan, Adil; Domingo, Beatriz; Vaquero, Cecilia F; Peyriéras, Nadine; Llopis, Juan

    2017-03-01

    Calcium-activated photoproteins, such as aequorin, have been used as luminescent Ca(2+) indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of green fluorescent protein (GFP) to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca(2+) levels. GFP-aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins and their biological applications to image Ca(2+) dynamics in organelles, cells, tissue explants and in live organisms.

  8. Identification of neuronal network properties from the spectral analysis of calcium imaging signals in neuronal cultures.

    PubMed

    Tibau, Elisenda; Valencia, Miguel; Soriano, Jordi

    2013-01-01

    Neuronal networks in vitro are prominent systems to study the development of connections in living neuronal networks and the interplay between connectivity, activity and function. These cultured networks show a rich spontaneous activity that evolves concurrently with the connectivity of the underlying network. In this work we monitor the development of neuronal cultures, and record their activity using calcium fluorescence imaging. We use spectral analysis to characterize global dynamical and structural traits of the neuronal cultures. We first observe that the power spectrum can be used as a signature of the state of the network, for instance when inhibition is active or silent, as well as a measure of the network's connectivity strength. Second, the power spectrum identifies prominent developmental changes in the network such as GABAA switch. And third, the analysis of the spatial distribution of the spectral density, in experiments with a controlled disintegration of the network through CNQX, an AMPA-glutamate receptor antagonist in excitatory neurons, reveals the existence of communities of strongly connected, highly active neurons that display synchronous oscillations. Our work illustrates the interest of spectral analysis for the study of in vitro networks, and its potential use as a network-state indicator, for instance to compare healthy and diseased neuronal networks.

  9. Calcium imaging of motoneuron activity in the en-bloc spinal cord preparation of the neonatal rat.

    PubMed

    Lev-Tov, A; O'Donovan, M J

    1995-09-01

    1. This paper describes the use of calcium imaging to monitor patterns of activity in neonatal rat motoneurons retrogradely labeled with the calcium-sensitive dye, calcium green-dextran. 2. Pressure ejection of calcium green-dextran into ventral roots and into the surgically peeled ventrolateral funiculi (VLF) at the lumbar cord labeled spinal motoneurons and interneurons. The back labeled motoneurons often formed two or three discrete clusters of cells. 3. Fluorescent changes (10-20%) could be detected in labeled motoneurons after a single antidromic stimulus of the segmental ventral root. These changes progressively increased in amplitude during stimulus trains (1-5 s) at frequencies from 5 to 50 Hz, presumably reflecting a frequency-dependent increase in free intracellular calcium. 4. Stimulation of the ipsilateral VLF at the caudal lumbar level (L6), elicited frequency-dependent, synaptically induced motoneuronal discharge. Frequency-dependent fluorescent changes could be detected in calcium green-labeled motoneurons during the VLF-induced synaptic activation. 5. The spatial spread of synaptic activity among calcium green-labeled clusters of motoneurons could be resolved after dorsal root stimulation. Low-intensity stimulation of the roots produced fluorescence changes restricted to the lateral clusters of motoneurons. With increasing stimulation intensity the fluorescence change increased in the lateral cells and could spread into the medial motoneuronal group. After a single supramaximal stimulus a similar pattern was observed with activity beginning laterally and spreading medially. 6. Substantial changes in fluorescence of calcium green-labeled motoneurons were also observed during motoneuron bursting induced by bath application of the glycine receptor antagonist strychnine or the potassium channel blocker 4-aminopyridine (4-AP). 7. Our results show that membrane-impermeant fluorescent calcium indicators can be used as a tool to study the activity of

  10. Feasibility of real time dual-energy imaging based on a flat panel detector for coronary artery calcium quantification.

    PubMed

    Xu, Tong; Ducote, Justin L; Wong, Jerry T; Molloi, Sabee

    2006-06-01

    The feasibility of a real-time dual-energy imaging technique with dynamic filtration using a flat panel detector for quantifying coronary arterial calcium was evaluated. In this technique, the x-ray beam was switched at 15 Hz between 60 kVp and 120 kVp with the 120 kVp beam having an additional 0.8 mm silver filter. The performance of the dynamic filtration technique was compared with a static filtration technique (4 mm Al+0.2 mm Cu for both beams). The ability to quantify calcium mass was evaluated using calcified arterial vessel phantoms with 20-230 mg of hydroxylapatite. The vessel phantoms were imaged over a Lucite phantom and then an anthropomorphic chest phantom. The total thickness of Lucite phantom ranges from 13.5-26.5 cm to simulate patient thickness of 16-32 cm. The calcium mass was measured using a densitometric technique. The effective dose to patient was estimated from the measured entrance exposure. The effects of patient thickness on contrast-to-noise ratio (CNR), effective dose, and the precision of calcium mass quantification (i.e., the frame to frame variability) were studied. The effects of misregistration artifacts were also measured by shifting the vessel phantoms manually between low- and high-energy images. The results show that, with the same detector signal level, the dynamic filtration technique produced 70% higher calcium contrast-to-noise ratio with only 4% increase in patient dose as compared to the static filtration technique. At the same time, x-ray tube loading increased by 30% with dynamic filtration. The minimum detectability of calcium with anatomical background was measured to be 34 mg of hydroxyapatite. The precision in calcium mass measurement, determined from 16 repeated dual-energy images, ranges from 13 mg to 41 mg when the patient thickness increased from 16 to 32 cm. The CNR was found to decrease with the patient thickness linearly at a rate of (-7%/cm). The anatomic background produced measurement root-mean-square (RMS

  11. Feasibility of real time dual-energy imaging based on a flat panel detector for coronary artery calcium quantification

    SciTech Connect

    Xu Tong; Ducote, Justin L.; Wong, Jerry T.; Molloi, Sabee

    2006-06-15

    The feasibility of a real-time dual-energy imaging technique with dynamic filtration using a flat panel detector for quantifying coronary arterial calcium was evaluated. In this technique, the x-ray beam was switched at 15 Hz between 60 kVp and 120 kVp with the 120 kVp beam having an additional 0.8 mm silver filter. The performance of the dynamic filtration technique was compared with a static filtration technique (4 mm Al+0.2 mm Cu for both beams). The ability to quantify calcium mass was evaluated using calcified arterial vessel phantoms with 20-230 mg of hydroxylapatite. The vessel phantoms were imaged over a Lucite phantom and then an anthropomorphic chest phantom. The total thickness of Lucite phantom ranges from 13.5-26.5 cm to simulate patient thickness of 16-32 cm. The calcium mass was measured using a densitometric technique. The effective dose to patient was estimated from the measured entrance exposure. The effects of patient thickness on contrast-to-noise ratio (CNR), effective dose, and the precision of calcium mass quantification (i.e., the frame to frame variability) were studied. The effects of misregistration artifacts were also measured by shifting the vessel phantoms manually between low- and high-energy images. The results show that, with the same detector signal level, the dynamic filtration technique produced 70% higher calcium contrast-to-noise ratio with only 4% increase in patient dose as compared to the static filtration technique. At the same time, x-ray tube loading increased by 30% with dynamic filtration. The minimum detectability of calcium with anatomical background was measured to be 34 mg of hydroxyapatite. The precision in calcium mass measurement, determined from 16 repeated dual-energy images, ranges from 13 mg to 41 mg when the patient thickness increased from 16 to 32 cm. The CNR was found to decrease with the patient thickness linearly at a rate of (-7%/cm). The anatomic background produced measurement root-mean-square (RMS

  12. Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium

    PubMed Central

    1991-01-01

    A new microscope technique, termed "W" (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized. PMID:1918140

  13. Population calcium imaging of spontaneous respiratory and novel motor activity in the facial nucleus and ventral brainstem in newborn mice

    PubMed Central

    Persson, Karin; Rekling, Jens C

    2011-01-01

    Abstract The brainstem contains rhythm and pattern forming circuits, which drive cranial and spinal motor pools to produce respiratory and other motor patterns. Here we used calcium imaging combined with nerve recordings in newborn mice to reveal spontaneous population activity in the ventral brainstem and in the facial nucleus. In Fluo-8 AM loaded brainstem–spinal cord preparations, respiratory activity on cervical nerves was synchronized with calcium signals at the ventrolateral brainstem surface. Individual ventrolateral neurons at the level of the parafacial respiratory group showed perfect or partial synchrony with respiratory nerve bursts. In brainstem–spinal cord preparations, cut at the level of the mid-facial nucleus, calcium signals were recorded in the dorsal, lateral and medial facial subnuclei during respiratory activity. Strong activity initiated in the dorsal subnucleus, followed by activity in lateral and medial subnuclei. Whole-cell recordings from facial motoneurons showed weak respiratory drives, and electrical field potential recordings confirmed respiratory drive to particularly the dorsal and lateral subnuclei. Putative facial premotoneurons showed respiratory-related calcium signals, and were predominantly located dorsomedial to the facial nucleus. A novel motor activity on facial, cervical and thoracic nerves was synchronized with calcium signals at the ventromedial brainstem extending from the level of the facial nucleus to the medulla–spinal cord border. Cervical dorsal root stimulation induced similar ventromedial activity. The medial facial subnucleus showed calcium signals synchronized with this novel motor activity on cervical nerves, and cervical dorsal root stimulation induced similar medial facial subnucleus activity. In conclusion, the dorsal and lateral facial subnuclei are strongly respiratory-modulated, and the brainstem contains a novel pattern forming circuit that drives the medial facial subnucleus and cervical motor

  14. Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Yuan, Tao; Gao, Simon S.; Saggau, Peter; Oghalai, John S.

    2010-01-01

    Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing, loss are commonly available. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes study of their hair cells difficult. We develop a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae are harvested, left intact within their otic capsule bone, and fixed in a recording chamber. The bone overlying the cochlear epithelium is opened and Reissner's membrane is incised. A fluorescent calcium indicator is applied to the preparation. A custom-built upright two-photon microscope was used to image the preparation using 3-D scanning. We are able to image about one third of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we find that outer hair cells demonstrate increased fluorescence compared with surrounding supporting cells. This methodology is then used to visualize hair cell calcium changes during mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are expected to be reduced.

  15. Forward-filling of dextran-conjugated indicators for calcium imaging at the Drosophila larval neuromuscular junction.

    PubMed

    Macleod, Gregory T

    2012-07-01

    Calcium imaging is a technique in which Ca(2+)-binding molecules are loaded into live cells and as they bind Ca(2+) they "indicate" the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. This article describes the forward-filling of dextran-conjugated indicators at the Drosophila larval neuromuscular junction (NMJ). This technique is particularly well suited for imaging changes in cytosolic Ca(2+) as dextran conjugation prevents compartmentalization of the Ca(2+) indicator. The major drawback is that the nerves must be severed at the start of the loading process, several hours before nerve terminals are ready to examine.

  16. Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy.

    PubMed

    Yuan, Tao; Gao, Simon S; Saggau, Peter; Oghalai, John S

    2010-01-01

    Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing, loss are commonly available. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes study of their hair cells difficult. We develop a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae are harvested, left intact within their otic capsule bone, and fixed in a recording chamber. The bone overlying the cochlear epithelium is opened and Reissner's membrane is incised. A fluorescent calcium indicator is applied to the preparation. A custom-built upright two-photon microscope was used to image the preparation using 3-D scanning. We are able to image about one third of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we find that outer hair cells demonstrate increased fluorescence compared with surrounding supporting cells. This methodology is then used to visualize hair cell calcium changes during mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are expected to be reduced.

  17. An automated multi-modal object analysis approach to coronary calcium scoring of adaptive heart isolated MSCT images

    NASA Astrophysics Data System (ADS)

    Wu, Jing; Ferns, Gordon; Giles, John; Lewis, Emma

    2012-02-01

    Inter- and intra- observer variability is a problem often faced when an expert or observer is tasked with assessing the severity of a disease. This issue is keenly felt in coronary calcium scoring of patients suffering from atherosclerosis where in clinical practice, the observer must identify firstly the presence, followed by the location of candidate calcified plaques found within the coronary arteries that may prevent oxygenated blood flow to the heart muscle. This can be challenging for a human observer as it is difficult to differentiate calcified plaques that are located in the coronary arteries from those found in surrounding anatomy such as the mitral valve or pericardium. The inclusion or exclusion of false positive or true positive calcified plaques respectively will alter the patient calcium score incorrectly, thus leading to the possibility of incorrect treatment prescription. In addition to the benefits to scoring accuracy, the use of fast, low dose multi-slice CT imaging to perform the cardiac scan is capable of acquiring the entire heart within a single breath hold. Thus exposing the patient to lower radiation dose, which for a progressive disease such as atherosclerosis where multiple scans may be required, is beneficial to their health. Presented here is a fully automated method for calcium scoring using both the traditional Agatston method, as well as the Volume scoring method. Elimination of the unwanted regions of the cardiac image slices such as lungs, ribs, and vertebrae is carried out using adaptive heart isolation. Such regions cannot contain calcified plaques but can be of a similar intensity and their removal will aid detection. Removal of both the ascending and descending aortas, as they contain clinical insignificant plaques, is necessary before the final calcium scores are calculated and examined against ground truth scores of three averaged expert observer results. The results presented here are intended to show the requirement and

  18. FRET imaging of calcium signaling in live cells in the microenvironment.

    PubMed

    Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei; Fang, Jing; Zhong, Wenxuan; Wang, Yingxiao

    2013-02-01

    The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

  19. A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

    PubMed Central

    Kirkby, Paul A.; Naga Srinivas, N.K.M.; Silver, R. Angus

    2010-01-01

    We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 μm; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space PMID:20588506

  20. Molecular imaging of in vivo calcium ion expression in area postrema of total sleep deprived rats: Implications for cardiovascular regulation by TOF-SIMS analysis

    NASA Astrophysics Data System (ADS)

    Mai, Fu-Der; Chen, Li-You; Ling, Yong-Chien; Chen, Bo-Jung; Wu, Un-In; Chang, Hung-Ming

    2010-05-01

    Excessive calcium influx in chemosensitive neurons of area postrema (AP) is detrimental for sympathetic activation and participates in the disruption of cardiovascular activities. Since total sleep deprivation (TSD) is a stressful condition known to harm the cardiovascular function, the present study is aimed to determine whether the in vivo calcium expression in AP would significantly alter following TSD by the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and calretinin (a specific calcium sensor protein in AP neurons) immunohistochemistry. The results indicated that in normal rats, the calcium intensity was estimated to be 0.5 × 10 5 at m/ z 40.08. However, following TSD, the intensity for calcium ions was greatly increased to 1.2 × 10 5. Molecular imaging revealed that after TSD, various strongly expressed calcium signals were distributed throughout AP with clear identified profiles instead of randomly scattered within this region in normal rats. Immunohistochemical staining corresponded well with ionic image in which a majority of calcium-enriched gathering co-localized with calretinin positive neurons. The functional significance of TSD-induced calcium augmentation was demonstrated by increased heart rate and mean arterial pressure, clinical markers for cardiovascular dysfunction. Considering AP-mediated sympathetic activation is important for cardiovascular regulation, exaggerated calcium influx in AP would render this neurocircuitry more vulnerable to over-excitation, which might serve as the underlying mechanism for the development of TSD-relevant cardiovascular deficiency.

  1. Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators.

    PubMed

    Kettunen, Petronella; Demas, Jay; Lohmann, Christian; Kasthuri, Narayanan; Gong, Yandao; Wong, Rachel O L; Gan, Wen-Biao

    2002-09-15

    The use of fluorescence-based calcium indicators has, over the years, unraveled important calcium-dependent mechanisms underlying neuronal function and development. However, difficulties associated with the loading of calcium indicators have limited their widespread use, particularly for the study of neuronal processing in the adult nervous system. Here, we show that in the central and peripheral nervous systems, populations of neurons and their processes, including dendritic spines and filopodia, can be labeled rapidly and efficiently by delivering calcium indicator-coated particles using a 'gene gun'. Importantly, neuronal labeling occurred both in vitro and in vivo, and across a wide range of ages and preparations. The labeled cells demonstrate spontaneous and evoked calcium transients, indicating that particle-mediated delivery is not deleterious to neuronal function. Furthermore, unlike loading with patch pipettes, cytoplasmic content is preserved following ballistic loading. This enables the study of calcium-dependent second messenger pathways without loss of signaling components. The ballistic delivery of calcium indicators thus opens up many new avenues for further exploration of the structure and function of the nervous system from single spines to neuronal networks.

  2. Sleep- and wake-dependent changes in neuronal activity and reactivity demonstrated in fly neurons using in vivo calcium imaging.

    PubMed

    Bushey, Daniel; Tononi, Giulio; Cirelli, Chiara

    2015-04-14

    Sleep in Drosophila shares many features with mammalian sleep, but it remains unknown whether spontaneous and evoked activity of individual neurons change with the sleep/wake cycle in flies as they do in mammals. Here we used calcium imaging to assess how the Kenyon cells in the fly mushroom bodies change their activity and reactivity to stimuli during sleep, wake, and after short or long sleep deprivation. As before, sleep was defined as a period of immobility of >5 min associated with a reduced behavioral response to a stimulus. We found that calcium levels in Kenyon cells decline when flies fall asleep and increase when they wake up. Moreover, calcium transients in response to two different stimuli are larger in awake flies than in sleeping flies. The activity of Kenyon cells is also affected by sleep/wake history: in awake flies, more cells are spontaneously active and responding to stimuli if the last several hours (5-8 h) before imaging were spent awake rather than asleep. By contrast, long wake (≥29 h) reduces both baseline and evoked neural activity and decreases the ability of neurons to respond consistently to the same repeated stimulus. The latter finding may underlie some of the negative effects of sleep deprivation on cognitive performance and is consistent with the occurrence of local sleep during wake as described in behaving rats. Thus, calcium imaging uncovers new similarities between fly and mammalian sleep: fly neurons are more active and reactive in wake than in sleep, and their activity tracks sleep/wake history.

  3. Chronic calcium imaging of neurons in the mouse visual cortex using a troponin C-based indicator.

    PubMed

    Santos, Alexandre Ferrão; Hübener, Mark

    2014-05-01

    This protocol describes the use of the genetically encoded troponin C-based calcium indicator TN-XXL to chronically monitor the functional properties of single neocortical neurons in the mouse visual cortex. A cranial window is implanted over the brain of a mouse expressing TN-XXL in pyramidal neurons of the cerebral cortex. Several days later, the visual cortex is mapped and photographed to facilitate repeated imaging of the same region using two-photon microscopy. Initial two-photon imaging may be done ∼2 wk after the window is implanted. We show the application of this technique for long-term in vivo imaging of stimulus response properties. Beyond providing functional information, long-term imaging of TN-XXL-labeled neurons also enables the simultaneous monitoring of structural properties down to the level of single dendritic spines.

  4. Calcium and voltage imaging in arrhythmia models by high-speed microscopy

    NASA Astrophysics Data System (ADS)

    de Mauro, C.; Cecchetti, C. A.; Alfieri, D.; Borile, G.; Urbani, A.; Mongillo, M.; Pavone, F. S.

    2014-03-01

    Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

  5. Imaging intracellular Ca²⁺ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators.

    PubMed

    Jiang, Ruotian; Haustein, Martin D; Sofroniew, Michael V; Khakh, Baljit S

    2014-11-19

    Astrocytes display spontaneous intracellular Ca(2+) concentration fluctuations ([Ca(2+)]i) and in several settings respond to neuronal excitation with enhanced [Ca(2+)]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca(2+)]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca(2+)]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca(2+)]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca(2+)]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca(2+)]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca(2+)]i signals in the striatal microcircuitry.

  6. Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

    PubMed Central

    Jiang, Ruotian; Haustein, Martin D.; Sofroniew, Michael V.; Khakh, Baljit S.

    2014-01-01

    Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry. PMID:25490346

  7. A fully automated multi-modal computer aided diagnosis approach to coronary calcium scoring of MSCT images

    NASA Astrophysics Data System (ADS)

    Wu, Jing; Ferns, Gordon; Giles, John; Lewis, Emma

    2012-03-01

    Inter- and intra- observer variability is a problem often faced when an expert or observer is tasked with assessing the severity of a disease. This issue is keenly felt in coronary calcium scoring of patients suffering from atherosclerosis where in clinical practice, the observer must identify firstly the presence, followed by the location of candidate calcified plaques found within the coronary arteries that may prevent oxygenated blood flow to the heart muscle. However, it can be difficult for a human observer to differentiate calcified plaques that are located in the coronary arteries from those found in surrounding anatomy such as the mitral valve or pericardium. In addition to the benefits to scoring accuracy, the use of fast, low dose multi-slice CT imaging to perform the cardiac scan is capable of acquiring the entire heart within a single breath hold. Thus exposing the patient to lower radiation dose, which for a progressive disease such as atherosclerosis where multiple scans may be required, is beneficial to their health. Presented here is a fully automated method for calcium scoring using both the traditional Agatston method, as well as the volume scoring method. Elimination of the unwanted regions of the cardiac image slices such as lungs, ribs, and vertebrae is carried out using adaptive heart isolation. Such regions cannot contain calcified plaques but can be of a similar intensity and their removal will aid detection. Removal of both the ascending and descending aortas, as they contain clinical insignificant plaques, is necessary before the final calcium scores are calculated and examined against ground truth scores of three averaged expert observer results. The results presented here are intended to show the feasibility and requirement for an automated scoring method to reduce the subjectivity and reproducibility error inherent with manual clinical calcium scoring.

  8. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

    PubMed Central

    Gee, J. Michael; Gibbons, Meredith B.; Taheri, Marsa; Palumbos, Sierra; Morris, S. Craig; Smeal, Roy M.; Flynn, Katherine F.; Economo, Michael N.; Cizek, Christian G.; Capecchi, Mario R.; Tvrdik, Petr; Wilcox, Karen S.; White, John A.

    2015-01-01

    Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5–14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal

  9. Combined system for high-time-resolution dual-excitation fluorescence photometry and fluorescence imaging of calcium transients in single normal and diseased skeletal muscle fibers

    NASA Astrophysics Data System (ADS)

    Uttenweiler, Dietmar; Wojciechowski, Reinhold; Makabe, Makoto; Veigel, Claudia; Fink, Rainer H.

    1994-12-01

    Fast photometric measurements and video-imaging of fluorescent indicators both are powerful tools in measuring the intracellular free calcium concentration of muscle and many other cells. as photometric systems yield a high temporal resolution, calcium imaging systems have high spatial but significantly reduced temporal resolution. Therefore we have developed an integrated system combining both methods and based mostly on standard components. As a common, sensitive Ca2+- indicator we used the fluorescent probe Fura-2, which is alternatingly excited for ratio measurements at 340/380 nm. We used a commercially available dual excitation photometric system (OSP-3; Olympus) for attaching a CCD-camera and a frame grabber board. To achieve the synchronization we had to design circuitries for external triggering, synchronization and accurate control of the filter changer, which we added to the system. Additionally, the software for a triggered image acquisition was developed. With this integrated setup one can easily switch between the fast photometric mode (ratio frequency 100 Hz) and the imaging mode (ratio frequency 4.17 Hz). The calcium images are correlated with the 25 times faster spot measurements and are analyzed by means of image processing. With this combined system we study release and uptake of calcium ions of normal and diseased skeletal muscle from mdx mice. Such a system will also be important for other cellular studies in which fluorescence indicators are used to monitor similar time dependent alterations as well as changes in cellular distributions of calcium.

  10. Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.

    PubMed

    Jayasinghe, Isuru D; Munro, Michelle; Baddeley, David; Launikonis, Bradley S; Soeller, Christian

    2014-10-06

    Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

  11. Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging

    PubMed Central

    Jayasinghe, Isuru D.; Munro, Michelle; Baddeley, David; Launikonis, Bradley S.; Soeller, Christian

    2014-01-01

    Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle. PMID:25100314

  12. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  13. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli.

    PubMed

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-06-27

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca(2+) concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

  14. Two-photon calcium imaging from motion-sensitive neurons in head-fixed Drosophila during optomotor walking behavior

    PubMed Central

    Seelig, Johannes D.; Chiappe, M. Eugenia; Lott, Gus K.; Dutta, Anirban; Osborne, Jason E.; Reiser, Michael B.; Jayaraman, Vivek

    2010-01-01

    Drosophila melanogster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a novel technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball’s motion is tracked at high resolution and can be treated as a proxy for the fly’s own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system (HS) lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in HS-neurons correlated with robust optomotor behavior during walking. Our technique allows an entirely new set of questions to be addressed by monitoring behavior and physiology in identified neurons in a powerful genetic model organism with an extensive repertoire of walking behaviors. PMID:20526346

  15. Development of an in vitro model using calcium imaging to investigate antidotal efficacy against saxitoxin. Report September 1994-September 1996

    SciTech Connect

    Doebler, J.A.; Dant, B.C.; Chang, F.C.T.

    1997-05-01

    Studies were conducted to investigate the utility of a novel in vitro system to monitor the efficacy of potential antidotes against sodium channel neurotoxins. Intracellular free calcium levels were measured in PC-12 (rat adrenal pheochromocytoma) cells utilizing a calcium imaging system with Fura-2 as the ratiometric calcium-sensitive indicator. Elevations in Ca(++) induced by high extracellular potassium (K(+)) and 4-aminopyridine (4-AP) were demonstrated, thus confirming the responsiveness of the system. Saxitoxin (STX), however, did not produce any alteration in Ca(++), perhaps due to a relatively low level of neuronal activity, i.e., impulse generation, in the in vitro state. Therefore, we attempted to activate PC-12 cells using the sodium channel agonist veratridine (VER) to determine whether STX could reduce enhanced Ca(++) levels. Although VER generally increased Ca(++), this response was somewhat variable and thus could not be used to demonstrate STX-induced toxic effects. Such variability may be inherent to cell lines; thus, the use of primary cultures is recommended to develop an in vitro system using Ca(++) levels as an endpoint to evaluate the efficacy of antidotes against sodium channel toxins.

  16. Migration of calcium deposits into subacromial-subdeltoid bursa and into humeral head as a rare complication of calcifying tendinitis: sonography and imaging.

    PubMed

    Della Valle, Valeria; Bassi, Emilio Maria; Calliada, Fabrizio

    2015-09-01

    Calcifying tendinitis of the shoulder is a common condition characterized by the deposition of calcium, predominantly hydroxyapatite crystals, in the rotator cuff. A rare complication of this condition is the migration of calcium deposits from tendons, usually the supraspinatus, into the subacromial-subdeltoid bursa or into the humeral greater tuberosity. These complications are responsible for intense acute shoulder pain and functional disability. Patient anamnesis and clinical symptoms must be considered to make the diagnosis, but imaging, particularly sonography, is often necessary, showing a typical presentation related to the locations of calcium deposits. We present sonographic and other imaging features of subacromial-subdeltoid bursitis and humeral osteitis related to the migration of calcium.

  17. Calcium supplements

    MedlinePlus

    ... TYPES OF CALCIUM SUPPLEMENTS Forms of calcium include: Calcium carbonate: Over-the-counter (OTC) antacid products, such as Tums and Rolaids, contain calcium carbonate. These sources of calcium do not cost much. ...

  18. Multifunctional calcium phosphate nano-contrast agent for combined nuclear, magnetic and near-infrared in vivo imaging.

    PubMed

    Ashokan, Anusha; Gowd, Genekehal S; Somasundaram, Vijay H; Bhupathi, Arun; Peethambaran, Reshmi; Unni, A K K; Palaniswamy, Shanmugasundaram; Nair, Shantikumar V; Koyakutty, Manzoor

    2013-09-01

    Combination of three imaging techniques such as nuclear, magnetic and near-infrared fluorescence can aid in improved diagnosis of disease by synergizing specific advantages of each of these techniques such as deep tissue penetration of radiation signals, anatomical and functional details provided by magnetic contrast and better spatial resolution of optical signals. In the present work, we report the development of a multimodal contrast agent based on calcium phosphate nanoparticles (nCP), doped with both indocyanine green (ICG) and Gadolinium (Gd(3+)), and labeled with 99m-Technetium-methylene diphosphonate ((99m)Tc-MDP) for combined optical, magnetic and nuclear imaging. In order to obtain the desired tri-modal contrast properties, the concentrations of ICG, Gd(3+) and (99m)Tc were optimized at ∼0.15wt%, 3.38at% and ∼0.002ng/mg of nCP, respectively. The leaching-out of ICG was protected by an additional coating of polyethyleneimine (PEI). Toxicological evaluation of the final construct carried out on healthy human mononuclear cells, red-blood cells and platelets, showed excellent hemocompatibility. In vivo multimodal imaging using mice models revealed the ability to provide near-infrared, magnetic and nuclear contrast simultaneously. The nanoparticles also showed the potential for improved MR based angio-imaging of liver. Retention of intravenously administrated nanoparticles in the liver was reduced with PEGylation and the clearance was observed within 48h without causing any major histological changes in vital organs. Thus, we developed a non-toxic tri-modal nano-contrast agent using calcium phosphate nanoparticles and demonstrated its potential for combined nuclear, magnetic and near-infrared imaging in vivo.

  19. Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

    PubMed

    Collot, Mayeul; Loukou, Christina; Yakovlev, Aleksey V; Wilms, Christian D; Li, Dongdong; Evrard, Alexis; Zamaleeva, Alsu; Bourdieu, Laurent; Léger, Jean-François; Ropert, Nicole; Eilers, Jens; Oheim, Martin; Feltz, Anne; Mallet, Jean-Maurice

    2012-09-12

    We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

  20. Label-Free Imaging of Dynamic and Transient Calcium Signaling in Single Cells.

    PubMed

    Lu, Jin; Li, Jinghong

    2015-11-09

    Cell signaling consists of diverse events that occur at various temporal and spatial scales, ranging from milliseconds to hours and from single biomolecules to cell populations. The pathway complexities require the development of new techniques that detect the overall signaling activities and are not limited to quantifying a single event. A plasmonic-based electrochemical impedance microscope (P-EIM) that can provide such data with excellent temporal and spatial resolution and does not require the addition of any labels for detection has now been developed. The highly dynamic and transient calcium signaling activities at the early stage of G-protein-coupled receptor (GPCR) stimulation were thus studied. It could be shown that a subpopulation of cells is more responsive towards agonist stimulation, and the heterogeneity of the local distributions and the transient activities of the ion channels during agonist-activated calcium flux in single HeLa cells were investigated.

  1. Simultaneous calcium fluorescence imaging and MR of ex vivo organotypic cortical cultures: a new test bed for functional MRI.

    PubMed

    Bai, Ruiliang; Klaus, Andreas; Bellay, Tim; Stewart, Craig; Pajevic, Sinisa; Nevo, Uri; Merkle, Hellmut; Plenz, Dietmar; Basser, Peter J

    2015-12-01

    Recently, several new functional (f)MRI contrast mechanisms including diffusion, phase imaging, proton density, etc. have been proposed to measure neuronal activity more directly and accurately than blood-oxygen-level dependent (BOLD) fMRI. However, these approaches have proved difficult to reproduce, mainly because of the dearth of reliable and robust test systems to vet and validate them. Here we describe the development and testing of such a test bed for non-BOLD fMRI. Organotypic cortical cultures were used as a stable and reproducible biological model of neuronal activity that shows spontaneous activity similar to that of in vivo brain cortex without any hemodynamic confounds. An open-access, single-sided magnetic resonance (MR) "profiler" consisting of four permanent magnets with magnetic field of 0.32 T was used in this study to perform MR acquisition. A fluorescence microscope with long working distance objective was mounted on the top of a custom-designed chamber that keeps the organotypic culture vital, and the MR system was mounted on the bottom of the chamber to achieve real-time simultaneous calcium fluorescence optical imaging and MR acquisition on the same specimen. In this study, the reliability and performance of the proposed test bed were demonstrated by a conventional CPMG MR sequence acquired simultaneously with calcium imaging, which is a well-characterized measurement of neuronal activity. This experimental design will make it possible to correlate directly the other candidate functional MR signals to the optical indicia of neuronal activity in the future.

  2. An integrative approach for analyzing hundreds of neurons in task performing mice using wide-field calcium imaging

    PubMed Central

    Mohammed, Ali I.; Gritton, Howard J.; Tseng, Hua-an; Bucklin, Mark E.; Yao, Zhaojie; Han, Xue

    2016-01-01

    Advances in neurotechnology have been integral to the investigation of neural circuit function in systems neuroscience. Recent improvements in high performance fluorescent sensors and scientific CMOS cameras enables optical imaging of neural networks at a much larger scale. While exciting technical advances demonstrate the potential of this technique, further improvement in data acquisition and analysis, especially those that allow effective processing of increasingly larger datasets, would greatly promote the application of optical imaging in systems neuroscience. Here we demonstrate the ability of wide-field imaging to capture the concurrent dynamic activity from hundreds to thousands of neurons over millimeters of brain tissue in behaving mice. This system allows the visualization of morphological details at a higher spatial resolution than has been previously achieved using similar functional imaging modalities. To analyze the expansive data sets, we developed software to facilitate rapid downstream data processing. Using this system, we show that a large fraction of anatomically distinct hippocampal neurons respond to discrete environmental stimuli associated with classical conditioning, and that the observed temporal dynamics of transient calcium signals are sufficient for exploring certain spatiotemporal features of large neural networks. PMID:26854041

  3. An integrative approach for analyzing hundreds of neurons in task performing mice using wide-field calcium imaging.

    PubMed

    Mohammed, Ali I; Gritton, Howard J; Tseng, Hua-an; Bucklin, Mark E; Yao, Zhaojie; Han, Xue

    2016-02-08

    Advances in neurotechnology have been integral to the investigation of neural circuit function in systems neuroscience. Recent improvements in high performance fluorescent sensors and scientific CMOS cameras enables optical imaging of neural networks at a much larger scale. While exciting technical advances demonstrate the potential of this technique, further improvement in data acquisition and analysis, especially those that allow effective processing of increasingly larger datasets, would greatly promote the application of optical imaging in systems neuroscience. Here we demonstrate the ability of wide-field imaging to capture the concurrent dynamic activity from hundreds to thousands of neurons over millimeters of brain tissue in behaving mice. This system allows the visualization of morphological details at a higher spatial resolution than has been previously achieved using similar functional imaging modalities. To analyze the expansive data sets, we developed software to facilitate rapid downstream data processing. Using this system, we show that a large fraction of anatomically distinct hippocampal neurons respond to discrete environmental stimuli associated with classical conditioning, and that the observed temporal dynamics of transient calcium signals are sufficient for exploring certain spatiotemporal features of large neural networks.

  4. Comparison of the amyloid pore forming properties of rat and human Alzheimer's beta-amyloid peptide 1-42: Calcium imaging data.

    PubMed

    Di Scala, Coralie; Yahi, Nouara; Flores, Alessandra; Boutemeur, Sonia; Kourdougli, Nazim; Chahinian, Henri; Fantini, Jacques

    2016-03-01

    The data here consists of calcium imaging of human neuroblastoma SH-SY5Y cells treated with the calcium-sensitive dye Fluo-4AM and then incubated with nanomolar concentrations of either human or rat Alzheimer's β-amyloid peptide Aβ1-42. These data are both of a qualitative (fluorescence micrographs) and semi-quantitative nature (estimation of intracellular calcium concentrations of cells probed by Aβ1-42 peptides vs. control untreated cells). Since rat Aβ1-42 differs from its human counterpart at only three amino acid positions, this comparative study is a good assessment of the specificity of the amyloid pore forming assay. The interpretation of this dataset is presented in the accompanying study "Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides" [1].

  5. Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis

    PubMed Central

    Sanchez, Susana; Barry, Nicholas P.; Kirschner, Nina; Meyer, Wilfried; Mauro, Theodora M.; Moll, Ingrid; Gratton, Enrico

    2015-01-01

    Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca2+ concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca2+ concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca2+ distribution in skin were based on Ca2+ precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca2+ concentrations, also assessing organelle Ca2+ concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca2+ in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca2+ at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca2+ shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca2+ gradient towards the stratum granulosum. Ca2+ FLIM overcomes prior limitations in the observation of epidermal Ca2+ dynamics, and will allow further insights into basic epidermal physiology. PMID:21193994

  6. Oxygen spectroscopy and polarization-dependent imaging contrast (PIC)-mapping of calcium carbonate minerals and biominerals.

    PubMed

    DeVol, Ross T; Metzler, Rebecca A; Kabalah-Amitai, Lee; Pokroy, Boaz; Politi, Yael; Gal, Assaf; Addadi, Lia; Weiner, Steve; Fernandez-Martinez, Alejandro; Demichelis, Raffaella; Gale, Julian D; Ihli, Johannes; Meldrum, Fiona C; Blonsky, Adam Z; Killian, Christopher E; Salling, C B; Young, Anthony T; Marcus, Matthew A; Scholl, Andreas; Doran, Andrew; Jenkins, Catherine; Bechtel, Hans A; Gilbert, Pupa U P A

    2014-07-17

    X-ray absorption near-edge structure (XANES) spectroscopy and spectromicroscopy have been extensively used to characterize biominerals. Using either Ca or C spectra, unique information has been obtained regarding amorphous biominerals and nanocrystal orientations. Building on these results, we demonstrate that recording XANES spectra of calcium carbonate at the oxygen K-edge enables polarization-dependent imaging contrast (PIC) mapping with unprecedented contrast, signal-to-noise ratio, and magnification. O and Ca spectra are presented for six calcium carbonate minerals: aragonite, calcite, vaterite, monohydrocalcite, and both hydrated and anhydrous amorphous calcium carbonate. The crystalline minerals reveal excellent agreement of the extent and direction of polarization dependences in simulated and experimental XANES spectra due to X-ray linear dichroism. This effect is particularly strong for aragonite, calcite, and vaterite. In natural biominerals, oxygen PIC-mapping generated high-magnification maps of unprecedented clarity from nacre and prismatic structures and their interface in Mytilus californianus shells. These maps revealed blocky aragonite crystals at the nacre-prismatic boundary and the narrowest calcite needle-prisms. In the tunic spicules of Herdmania momus, O PIC-mapping revealed the size and arrangement of some of the largest vaterite single crystals known. O spectroscopy therefore enables the simultaneous measurement of chemical and orientational information in CaCO3 biominerals and is thus a powerful means for analyzing these and other complex materials. As described here, PIC-mapping and spectroscopy at the O K-edge are methods for gathering valuable data that can be carried out using spectromicroscopy beamlines at most synchrotrons without the expense of additional equipment.

  7. Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.

    PubMed

    Ford, Kevin J; Davis, Graeme W

    2014-10-29

    The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.

  8. Archaerhodopsin Voltage Imaging: Synaptic Calcium and BK Channels Stabilize Action Potential Repolarization at the Drosophila Neuromuscular Junction

    PubMed Central

    Ford, Kevin J.

    2014-01-01

    The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2–0.5 mm) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mm), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse. PMID:25355206

  9. Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging.

    PubMed

    Broillet, M; Randin, O; Chatton, J

    2001-03-02

    The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.

  10. Imaging spinal neuron ensembles active during locomotion with genetically encoded calcium indicators.

    PubMed

    Hinckley, Christopher A; Pfaff, Samuel L

    2013-03-01

    Advances in molecular-genetic tools for labeling neuronal subtypes, and the emerging development of robust genetic probes for neural activity, are likely to revolutionize our understanding of the functional organization of neural circuits. In principle, these tools should be able to detect activity at cellular resolution for large ensembles of identified neuron types as they participate in specific behaviors. This report describes the use of genetically encoded calcium indicators (GECIs), combined with two-photon microscopy, to characterize V1 interneurons, known to be critical for setting the duration of the step cycle. All V1 interneurons arise from a common precursor population and express engrailed-1 (En1). Our data show that although neighboring interneurons that arise from the same developmental lineage and share many features, such as projection patterns and neurotransmitter profiles, they are not irrevocably committed to having the same pattern of activity.

  11. Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology.

    PubMed

    Russell, James T

    2011-08-01

    The design and engineering of organic fluorescent Ca(2+) indicators approximately 30 years ago opened the door for imaging cellular Ca(2+) signals with a high degree of temporal and spatial resolution. Over this time, Ca(2+) imaging has revolutionized our approaches for tissue-level spatiotemporal analysis of functional organization and has matured into a powerful tool for in situ imaging of cellular activity in the living animal. In vivo Ca(2+) imaging with temporal resolution at the millisecond range and spatial resolution at micrometer range has been achieved through novel designs of Ca(2+) sensors, development of modern microscopes and powerful imaging techniques such as two-photon microscopy. Imaging Ca(2+) signals in ensembles of cells within tissue in 3D allows for analysis of integrated cellular function, which, in the case of the brain, enables recording activity patterns in local circuits. The recent development of miniaturized compact, fibre-optic-based, mechanically flexible microendoscopes capable of two-photon microscopy opens the door for imaging activity in awake, behaving animals. This development is poised to open a new chapter in physiological experiments and for pharmacological approaches in the development of novel therapies.

  12. Detecting cells using non-negative matrix factorization on calcium imaging data.

    PubMed

    Maruyama, Ryuichi; Maeda, Kazuma; Moroda, Hajime; Kato, Ichiro; Inoue, Masashi; Miyakawa, Hiroyoshi; Aonishi, Toru

    2014-07-01

    We propose a cell detection algorithm using non-negative matrix factorization (NMF) on Ca2+ imaging data. To apply NMF to Ca2+ imaging data, we use the bleaching line of the background fluorescence intensity as an a priori background constraint to make the NMF uniquely dissociate the background component from the image data. This constraint helps us to incorporate the effect of dye-bleaching and reduce the non-uniqueness of the solution. We demonstrate that in the case of noisy data, the NMF algorithm can detect cells more accurately than Mukamel's independent component analysis algorithm, a state-of-art method. We then apply the NMF algorithm to Ca2+ imaging data recorded on the local activities of subcellular structures of multiple cells in a wide area. We show that our method can decompose rapid transient components corresponding to somas and dendrites of many neurons, and furthermore, that it can decompose slow transient components probably corresponding to glial cells.

  13. Imaging and analysis of nonratiometric calcium indicators at the Drosophila larval neuromuscular junction.

    PubMed

    Macleod, Gregory T

    2012-07-01

    Ca(2+) indicators can be loaded into a Drosophila larval neuromuscular junction (NMJ) preparation using several methods, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. This article describes how such an NMJ preparation loaded with Ca(2+) indicator is set up for imaging of the muscle fiber during stimulation of its innervating nerve cell. A simple protocol is provided for collecting and analyzing a set of imaging data, together with the sequence of calculations involved in image analysis. The change in the intensity of the Ca(2+) indicator must be quantified to obtain an estimate of the change in the concentration of free Ca(2+) (Δ[Ca(2+)]). The change in intensity is conventionally represented as the expression "ΔF/F." Simply put, this is the change in fluorescence intensity relative to the resting fluorescence intensity. If the K(D) of the Ca(2+) indicator is in excess of the maximum value of [Ca(2+)] during the response, then ΔF/F is considered to be linearly related to Δ[Ca(2+)]. In practice, ΔF/F is calculated for each image using a simple algorithm ([F(stim) - F(rest)]/F(rest)), where F(stim) is the intensity of the Ca(2+) indicator in each image, and F(rest) is the intensity before nerve stimulation. Finally, various options for building a Ca(2+)-imaging rig are considered.

  14. Evolving Role of Molecular Imaging with (18)F-Sodium Fluoride PET as a Biomarker for Calcium Metabolism.

    PubMed

    Raynor, William; Houshmand, Sina; Gholami, Saeid; Emamzadehfard, Sahra; Rajapakse, Chamith S; Blomberg, Björn Alexander; Werner, Thomas J; Høilund-Carlsen, Poul F; Baker, Joshua F; Alavi, Abass

    2016-08-01

    (18)F-sodium fluoride (NaF) as an imaging tracer portrays calcium metabolic activity either in the osseous structures or in soft tissue. Currently, clinical use of NaF-PET is confined to detecting metastasis to the bone, but this approach reveals indirect evidence for disease activity and will have limited use in the future in favor of more direct approaches that visualize cancer cells in the read marrow where they reside. This has proven to be the case with FDG-PET imaging in most cancers. However, a variety of studies support the application of NaF-PET to assess benign osseous diseases. In particular, bone turnover can be measured from NaF uptake to diagnose osteoporosis. Several studies have evaluated the efficacy of bisphosphonates and their lasting effects as treatment for osteoporosis using bone turnover measured by NaF-PET. Additionally, NaF uptake in vessels tracks calcification in the plaques at the molecular level, which is relevant to coronary artery disease. Also, NaF-PET imaging of diseased joints is able to project disease progression in osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. Further studies suggest potential use of NaF-PET in domains such as back pain, osteosarcoma, stress-related fracture, and bisphosphonate-induced osteonecrosis of the jaw. The critical role of NaF-PET in disease detection and characterization of many musculoskeletal disorders has been clearly demonstrated in the literature, and these methods will become more widespread in the future. The data from PET imaging are quantitative in nature, and as such, it adds a major dimension to assessing disease activity.

  15. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter.

    PubMed

    Xiong, Tou Cheu; Ronzier, Elsa; Sanchez, Frédéric; Corratgé-Faillie, Claire; Mazars, Christian; Thibaud, Jean-Baptiste

    2014-01-01

    Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

  16. Long-Term Two-Photon Calcium Imaging of Neuronal Populations with Subcellular Resolution in Adult Non-human Primates.

    PubMed

    Sadakane, Osamu; Masamizu, Yoshito; Watakabe, Akiya; Terada, Shin-Ichiro; Ohtsuka, Masanari; Takaji, Masafumi; Mizukami, Hiroaki; Ozawa, Keiya; Kawasaki, Hiroshi; Matsuzaki, Masanori; Yamamori, Tetsuo

    2015-12-01

    Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity in vivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up- and downregulate it for more than 100 days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons in layer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates.

  17. Three-dimensional distribution of sensory stimulation-evoked neuronal activity of spinal dorsal horn neurons analyzed by in vivo calcium imaging.

    PubMed

    Nishida, Kazuhiko; Matsumura, Shinji; Taniguchi, Wataru; Uta, Daisuke; Furue, Hidemasa; Ito, Seiji

    2014-01-01

    The spinal dorsal horn comprises heterogeneous populations of interneurons and projection neurons, which form neuronal circuits crucial for processing of primary sensory information. Although electrophysiological analyses have uncovered sensory stimulation-evoked neuronal activity of various spinal dorsal horn neurons, monitoring these activities from large ensembles of neurons is needed to obtain a comprehensive view of the spinal dorsal horn circuitry. In the present study, we established in vivo calcium imaging of multiple spinal dorsal horn neurons by using a two-photon microscope and extracted three-dimensional neuronal activity maps of these neurons in response to cutaneous sensory stimulation. For calcium imaging, a fluorescence resonance energy transfer (FRET)-based calcium indicator protein, Yellow Cameleon, which is insensitive to motion artifacts of living animals was introduced into spinal dorsal horn neurons by in utero electroporation. In vivo calcium imaging following pinch, brush, and heat stimulation suggests that laminar distribution of sensory stimulation-evoked neuronal activity in the spinal dorsal horn largely corresponds to that of primary afferent inputs. In addition, cutaneous pinch stimulation elicited activities of neurons in the spinal cord at least until 2 spinal segments away from the central projection field of primary sensory neurons responsible for the stimulated skin point. These results provide a clue to understand neuronal processing of sensory information in the spinal dorsal horn.

  18. Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

    PubMed Central

    Buchmann, Peter; Argast, Paul; Hübener, Mark; Bonhoeffer, Tobias; Keller, Georg B.

    2014-01-01

    In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice. PMID:24637961

  19. Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140

    PubMed Central

    Tsukamoto, Seiichi; Fujii, Teruyuki; Oyama, Kotaro; Shintani, Seine A.; Ishiwata, Shin’ichi

    2016-01-01

    In cardiac muscle, contraction is triggered by sarcolemmal depolarization, resulting in an intracellular Ca2+ transient, binding of Ca2+ to troponin, and subsequent cross-bridge formation (excitation–contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca2+ dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)–based Ca2+ sensor yellow Cameleon–Nano (YC-Nano) fused to α-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, α-actinin–YC-Nano140 is best suited for high-precision analysis of EC coupling and α-actinin–YC-Nano140 enables quantitative analyses of intracellular calcium transients and sarcomere dynamics at low and high temperatures, during spontaneous beating and with electrical stimulation. We use this tool to show that calcium transients are synchronized along the length of a myofibril. However, the averaging of SL along myofibrils causes a marked underestimate (∼50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via β-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that β-adrenergic stimulation with 50 nM isoproterenol accelerated Ca2+ dynamics, in association with an approximately twofold increase in sarcomere lengthening velocity. We conclude that our experimental system has a broad range of potential applications for the unveiling molecular mechanisms of EC coupling in cardiomyocytes at the single sarcomere level. PMID:27670899

  20. Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx.

    PubMed

    Jaffe, Lionel F

    2007-03-01

    For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.

  1. Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: A sup 44 Ca stable isotope study

    SciTech Connect

    Chandra, S.; Fullmer, C.S.; Smith, C.A.; Wasserman, R.H.; Morrison, G.H. )

    1990-08-01

    The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.

  2. Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free calcium ions.

    PubMed

    Si, Di; Epstein, Tamir; Lee, Yong-Eun Koo; Kopelman, Raoul

    2012-01-17

    Ca(2+) is a universal second messenger and plays a major role in intracellular signaling, metabolism, and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca(2+) measurement involves the introduction of optically sensitive Ca(2+) indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca(2+) indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most-sensitive indicators, cytotoxicity of the indicators, interference from nonspecific binding caused by cellular biomacromolecules, challenging calibration, and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca(2+) indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine-based Ca(2+) fluorescence indicators. The rhod-2-containing PEBBLEs presented here show a stable sensing range at near-neutral pH (pH 6-9). Because of the protection of the PEBBLE matrix, the interference of protein-nonspecific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (K(d) = 478 nM) and intracellular (K(d) = 293 nM) measurements. These nanosensors are useful quantitative tools for the measurement and imaging of the cytosolic nanomolar free Ca(2+) levels.

  3. Calcium imaging of sleep-wake related neuronal activity in the dorsal pons.

    PubMed

    Cox, Julia; Pinto, Lucas; Dan, Yang

    2016-02-25

    The dorsal pons has long been implicated in the generation of rapid eye movement (REM) sleep, but the underlying circuit mechanisms remain poorly understood. Using cell-type-specific microendoscopic Ca(2+) imaging in and near the laterodorsal tegmental nucleus, we found that many glutamatergic neurons are maximally active during REM sleep (REM-max), while the majority of GABAergic neurons are maximally active during wakefulness (wake-max). Furthermore, the activity of glutamatergic neurons exhibits a medio-lateral spatial gradient, with medially located neurons more selectively active during REM sleep.

  4. Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.

    PubMed

    Zhong, Chongbo; Talmage, David A; Role, Lorna W

    2015-06-24

    Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)-accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

  5. Calcium imaging in the ant Camponotus fellah reveals a conserved odour-similarity space in insects and mammals

    PubMed Central

    2010-01-01

    Background Olfactory systems create representations of the chemical world in the animal brain. Recordings of odour-evoked activity in the primary olfactory centres of vertebrates and insects have suggested similar rules for odour processing, in particular through spatial organization of chemical information in their functional units, the glomeruli. Similarity between odour representations can be extracted from across-glomerulus patterns in a wide range of species, from insects to vertebrates, but comparison of odour similarity in such diverse taxa has not been addressed. In the present study, we asked how 11 aliphatic odorants previously tested in honeybees and rats are represented in the antennal lobe of the ant Camponotus fellah, a social insect that relies on olfaction for food search and social communication. Results Using calcium imaging of specifically-stained second-order neurons, we show that these odours induce specific activity patterns in the ant antennal lobe. Using multidimensional analysis, we show that clustering of odours is similar in ants, bees and rats. Moreover, odour similarity is highly correlated in all three species. Conclusion This suggests the existence of similar coding rules in the neural olfactory spaces of species among which evolutionary divergence happened hundreds of million years ago. PMID:20187931

  6. High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

    PubMed

    Pezzati, R; Bossi, M; Podini, P; Meldolesi, J; Grohovaz, F

    1997-08-01

    The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the variations organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

  7. Functional Microarchitecture of the Mouse Dorsal Inferior Colliculus Revealed through In Vivo Two-Photon Calcium Imaging

    PubMed Central

    Barnstedt, Oliver; Keating, Peter; Weissenberger, Yves

    2015-01-01

    The inferior colliculus (IC) is an obligatory relay for ascending auditory inputs from the brainstem and receives descending input from the auditory cortex. The IC comprises a central nucleus (CNIC), surrounded by several shell regions, but the internal organization of this midbrain nucleus remains incompletely understood. We used two-photon calcium imaging to study the functional microarchitecture of both neurons in the mouse dorsal IC and corticocollicular axons that terminate there. In contrast to previous electrophysiological studies, our approach revealed a clear functional distinction between the CNIC and the dorsal cortex of the IC (DCIC), suggesting that the mouse midbrain is more similar to that of other mammals than previously thought. We found that the DCIC comprises a thin sheet of neurons, sometimes extending barely 100 μm below the pial surface. The sound frequency representation in the DCIC approximated the mouse's full hearing range, whereas dorsal CNIC neurons almost exclusively preferred low frequencies. The response properties of neurons in these two regions were otherwise surprisingly similar, and the frequency tuning of DCIC neurons was only slightly broader than that of CNIC neurons. In several animals, frequency gradients were observed in the DCIC, and a comparable tonotopic arrangement was observed across the boutons of the corticocollicular axons, which form a dense mesh beneath the dorsal surface of the IC. Nevertheless, acoustically responsive corticocollicular boutons were sparse, produced unreliable responses, and were more broadly tuned than DCIC neurons, suggesting that they have a largely modulatory rather than driving influence on auditory midbrain neurons. SIGNIFICANCE STATEMENT Due to its genetic tractability, the mouse is fast becoming the most popular animal model for sensory neuroscience. Nevertheless, many aspects of its neural architecture are still poorly understood. Here, we image the dorsal auditory midbrain and its

  8. In Vivo Two-Photon Imaging of Axonal Dieback, Blood Flow, and Calcium Influx with Methylprednisolone Therapy after Spinal Cord Injury

    PubMed Central

    Tang, Peifu; Zhang, Yiling; Chen, Chao; Ji, Xinran; Ju, Furong; Liu, Xingyu; Gan, Wen-Biao; He, Zhigang; Zhang, Shengxiang; Li, Wei; Zhang, Lihai

    2015-01-01

    Severe spinal cord injury (SCI) can cause neurological dysfunction and paralysis. However, the early dynamic changes of neurons and their surrounding environment after SCI are poorly understood. Although methylprednisolone (MP) is currently the standard therapeutic agent for treating SCI, its efficacy remains controversial. The purpose of this project was to investigate the early dynamic changes and MP's efficacy on axonal damage, blood flow, and calcium influx into axons in a mouse SCI model. YFP H-line and Thy1-GCaMP transgenic mice were used in this study. Two-photon microscopy was used for imaging of axonal dieback, blood flow, and calcium influx post-injury. We found that MP treatment attenuated progressive damage of axons, increased blood flow, and reduced calcium influx post-injury. Furthermore, microglia/macrophages accumulated in the lesion site after SCI and expressed the proinflammatory mediators iNOS, MCP-1 and IL-1β. MP treatment markedly inhibited the accumulation of microglia/macrophages and reduced the expression of the proinflammatory mediators. MP treatment also improved the recovery of behavioral function post-injury. These findings suggest that MP exerts a neuroprotective effect on SCI treatment by attenuating progressive damage of axons, increasing blood flow, reducing calcium influx, and inhibiting the accumulation of microglia/macrophages after SCI. PMID:25989524

  9. In vivo two-photon imaging of axonal dieback, blood flow, and calcium influx with methylprednisolone therapy after spinal cord injury.

    PubMed

    Tang, Peifu; Zhang, Yiling; Chen, Chao; Ji, Xinran; Ju, Furong; Liu, Xingyu; Gan, Wen-Biao; He, Zhigang; Zhang, Shengxiang; Li, Wei; Zhang, Lihai

    2015-05-19

    Severe spinal cord injury (SCI) can cause neurological dysfunction and paralysis. However, the early dynamic changes of neurons and their surrounding environment after SCI are poorly understood. Although methylprednisolone (MP) is currently the standard therapeutic agent for treating SCI, its efficacy remains controversial. The purpose of this project was to investigate the early dynamic changes and MP's efficacy on axonal damage, blood flow, and calcium influx into axons in a mouse SCI model. YFP H-line and Thy1-GCaMP transgenic mice were used in this study. Two-photon microscopy was used for imaging of axonal dieback, blood flow, and calcium influx post-injury. We found that MP treatment attenuated progressive damage of axons, increased blood flow, and reduced calcium influx post-injury. Furthermore, microglia/macrophages accumulated in the lesion site after SCI and expressed the proinflammatory mediators iNOS, MCP-1 and IL-1β. MP treatment markedly inhibited the accumulation of microglia/macrophages and reduced the expression of the proinflammatory mediators. MP treatment also improved the recovery of behavioral function post-injury. These findings suggest that MP exerts a neuroprotective effect on SCI treatment by attenuating progressive damage of axons, increasing blood flow, reducing calcium influx, and inhibiting the accumulation of microglia/macrophages after SCI.

  10. Peripheral inflammation upregulates P2X receptor expression in satellite glial cells of mouse trigeminal ganglia: a calcium imaging study.

    PubMed

    Kushnir, Raya; Cherkas, Pavel S; Hanani, Menachem

    2011-09-01

    Satellite glial cells (SGCs) in sensory ganglia are altered structurally and biochemically as a result of nerve injury. Whereas there is ample evidence that P2 purinergic receptors in central glial cells are altered after injury, there is very little information on similar changes in SGCs, although it is well established that SGCs are endowed with P2 receptors. Using calcium imaging, we characterized changes in P2 receptors in SGCs from mouse trigeminal ganglia in short-term cultures. Seven days after the induction of submandibular inflammation with complete Freund's adjuvant, there was a marked increase in the sensitivity of SGCs to ATP, with the threshold of activation decreasing from 5 μM to 10 nM. A similar observation was made in the intact trigeminal ganglion after infra-orbital nerve axotomy. Using pharmacological tools, we investigated the receptor mechanisms underlying these changes in cultured SGCs. We found that in control tissues response to ATP was mediated by P2Y (metabotropic) receptors, whereas after inflammation the response was mediated predominantly by P2X (ionotropic) receptors. As the contribution of P2X1,3,6 receptors was excluded, and the sensitivity to a P2X7 agonist did not change after inflammation, it appears that after inflammation the responses to ATP are largely due to P2X2 and/or 5 receptors, with a possible contribution of P2X4 receptors. We conclude that inflammation induced a large increase in the sensitivity of SGCs to ATP, which involved a switch from P2Y to P2X receptors. We propose that the over 100-fold augmented sensitivity of SGCs to ATP after injury may contribute to chronic pain states.

  11. An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.

    PubMed

    Cerveira, Joana; Begum, Julfa; Di Marco Barros, Rafael; van der Veen, Annemarthe G; Filby, Andrew

    2015-08-01

    Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to

  12. SPECT myocardial perfusion imaging as an adjunct to coronary calcium score for the detection of hemodynamically significant coronary artery stenosis

    PubMed Central

    2012-01-01

    Background Coronary artery calcifications (CAC) are markers of coronary atherosclerosis, but do not correlate well with stenosis severity. This study intended to evaluate clinical situations where a combined approach of coronary calcium scoring (CS) and nuclear stress test (SPECT-MPI) is useful for the detection of relevant CAD. Methods Patients with clinical indication for invasive coronary angiography (ICA) were included into our study during 08/2005-09/2008. At first all patients underwent CS procedure as part of the study protocol performed by either using a multidetector computed tomography (CT) scanner or a dual-source CT imager. CAC were automatically defined by dedicated software and the Agatston score was semi-automatically calculated. A stress-rest SPECT-MPI study was performed afterwards and scintigraphic images were evaluated quantitatively. Then all patients underwent ICA. Thereby significant CAD was defined as luminal stenosis ≥75% in quantitative coronary analysis (QCA) in ≥1 epicardial vessel. To compare data lacking Gaussian distribution an unpaired Wilcoxon-Test (Mann–Whitney) was used. Otherwise a Students t-test for unpaired samples was applied. Calculations were considered to be significant at a p-value of <0.05. Results We consecutively included 351 symptomatic patients (mean age: 61.2±12.3 years; range: 18–94 years; male: n=240) with a mean Agatston score of 258.5±512.2 (range: 0–4214). ICA verified exclusion of significant CAD in 66/67 (98.5%) patients without CAC. CAC was detected in remaining 284 patients. In 132/284 patients (46.5%) with CS>0 significant CAD was confirmed by ICA, and excluded in 152/284 (53.5%) patients. Sensitivity for CAD detection by CS alone was calculated as 99.2%, specificity was 30.3%, and negative predictive value was 98.5%. An additional SPECT in patients with CS>0 increased specificity to 80.9% while reducing sensitivity to 87.9%. Diagnostic accuracy was 84.2%. Conclusions In patients without CS=0

  13. A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.

    PubMed

    Andjelic, Sofija; Lumi, Xhevat; Veréb, Zoltán; Josifovska, Natasha; Facskó, Andrea; Hawlina, Marko; Petrovski, Goran

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

  14. A Simple Method for Establishing Adherent Ex Vivo Explant Cultures from Human Eye Pathologies for Use in Subsequent Calcium Imaging and Inflammatory Studies

    PubMed Central

    Veréb, Zoltán; Facskó, Andrea; Hawlina, Marko

    2014-01-01

    A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment. PMID:25276840

  15. Optical electrocorticogram (OECoG) using wide-field calcium imaging reveals the divergence of neuronal and glial activity during acute rodent seizures.

    PubMed

    Daniel, Andy G S; Laffont, Philippe; Zhao, Mingrui; Ma, Hongtao; Schwartz, Theodore H

    2015-08-01

    The role of glia in epilepsy has been widely debated. Using in vivo bulk loading of calcium dyes, we imaged neuronal and glial activity in an acute pharmacologic rodent model of neocortical seizures. Optical calcium-based ECoG maps revealed that neuronal waves propagated rapidly and remained mostly confined to the seizure focus. Glial waves were triggered by ictal onset but propagated slowly in a stereotypical fashion far beyond the seizure focus. Although related at their onset, the divergence of these two phenomena during seizure evolution calls into question their interdependence and the criticality of the role of glia in seizure onset and neurovascular coupling. This article is part of a Special Issue entitled "Status Epilepticus".

  16. Calcium - urine

    MedlinePlus

    ... into the urine, which causes calcium kidney stones Sarcoidosis Taking too much calcium Too much production of ... Milk-alkali syndrome Proximal renal tubular acidosis Rickets Sarcoidosis Vitamin D Review Date 5/3/2015 Updated ...

  17. Indocyanine green-encapsulating calcium phosphosilicate nanoparticles: Bifunctional theranostic vectors for near infrared diagnostic imaging and photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Altinoglu, Erhan I.

    The synthesis, laundering, and properties of calcium phosphosilicate nanoparticles (CPSNPs) that encapsulate the NIR fluorophore indocyanine green (ICG) related to multifunctional fluorescent photosensitization is presented. Imaging with transmission electron microscopy (TEM) revealed the well dispersed state of the nanoparticles, the spherical morphology, and the log normal mean particle diameter of 16 nm. Electron energy loss spectroscopy (EELS) mapping identified a Ca:P:Si ratio of 1:1.72:0.41 and a homogeneous composition without evidence of an element rich or deficient architecture. Zeta potential of the as-synthesized, citrate-functionalized CPSNPs was -29 +/-3 mV. A theoretical solids loading of 1.9 x 1013 CPSNP/mL was calculated for a standard suspension. The mean ICG content per suspension is 2 x 10 -6 M, which equates to approximately 63 fluorophore molecules encapsulated per CPSNP. For imaging and diagnostic considerations, the doped CPSNPs exhibited significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore. The quantum efficiency of the fluorescent agent is 200% greater at 0.053+/-0.003 over the free fluorophore in PBS. Also, photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. These performance enhancements are attributed to the matrix shielding effect of the NP around the internalized fluorophore molecules. The in vivo emission signal stability from ICG-CPSNPs was compared to the free fluorophore by whole animal NIR imaging. The duration of fluorescent signal from the ICG-CPSPNPs was extended to up to four days post-injection, highlighting the potential for long-term imaging and sensitive tracking applications using ICG when encapsulated within the protective matrix of CPSNPs. The surfaces of the ICG-CPSNPs were covalently bound with polyethylene glycol (PEG). The pharmacokinetic behavior of the

  18. Reversibly phototunable TiO{sub 2} photonic crystal modulated by Ag nanoparticles' oxidation/reduction

    SciTech Connect

    Liu Jian; Zhou Jinming; Ye Changqing; Li Mingzhu; Wang Jingxia; Jiang Lei; Song Yanlin

    2011-01-10

    We report a reversibly phototunable photonic crystal system whose reflectance at the stop band position can be modulated by alternating UV/visible (UV/Vis) irradiation. The phototunable system consists of Ag nanoparticles and TiO{sub 2} photonic crystal. The stop bands intensity of Ag loaded TiO{sub 2} photonic crystals were found to be dependent on the redox states of Ag nanoparticles. The quasi 'on' and 'off' states of the stop band were reversibly modulated by the Ag nanoparticles' oxidation/reduction through alternating UV/Vis light irradiation.

  19. FocusStack and StimServer: a new open source MATLAB toolchain for visual stimulation and analysis of two-photon calcium neuronal imaging data.

    PubMed

    Muir, Dylan R; Kampa, Björn M

    2014-01-01

    Two-photon calcium imaging of neuronal responses is an increasingly accessible technology for probing population responses in cortex at single cell resolution, and with reasonable and improving temporal resolution. However, analysis of two-photon data is usually performed using ad-hoc solutions. To date, no publicly available software exists for straightforward analysis of stimulus-triggered two-photon imaging experiments. In addition, the increasing data rates of two-photon acquisition systems imply increasing cost of computing hardware required for in-memory analysis. Here we present a Matlab toolbox, FocusStack, for simple and efficient analysis of two-photon calcium imaging stacks on consumer-level hardware, with minimal memory footprint. We also present a Matlab toolbox, StimServer, for generation and sequencing of visual stimuli, designed to be triggered over a network link from a two-photon acquisition system. FocusStack is compatible out of the box with several existing two-photon acquisition systems, and is simple to adapt to arbitrary binary file formats. Analysis tools such as stack alignment for movement correction, automated cell detection and peri-stimulus time histograms are already provided, and further tools can be easily incorporated. Both packages are available as publicly-accessible source-code repositories.

  20. Ligand and electrically induced acitivation patterns in myenteric neuronal networks. Confocal calcium imaging as a bridge between basic and human physiology.

    PubMed

    Bisschops, R

    2008-01-01

    Confocal imaging in combination with fluorescent calcium indicators provides the possibility to study neuronal activation in entire neuronal networks. The experiments presented in this essay aimed at applying confocal calcium imaging to study activation patterns in neuronal networks of myenteric ganglia in situ. First we studied the response to electrical train stimulation (ETS). ETS induced Ca2+ transients in 52.2% and 65.4% of the neurons when applied orally and aborally respectively. We observed more responses during aboral ETS which is not in line with the hypothesis of neuronal polarity, suggesting complex neuronal activation patterns and neuronal interaction in ETS-induced activation in myenteric ganglia. We demonstrated that ghrelin has a direct excitatory effect on myenteric neurons in situ via ghrelin receptor activation. Ghrelin induced Ca2+ transients in one third of the myenteric neurons, involving release of Ca2+ from intracellular stores and direct GHS-receptor activation. We found that CRF activates one fifth of the myenteric neurons, via CRF1 receptor activation. These CRF induced Ca2+ signals involved somatic influx through (mainly R-type) voltage operated Ca2+ channels. Finally we set up human studies in healthy volunteers and dyspeptic patients to test the effect of ghrelin on gastrointestinal motility. Intravenous administration of ghrelin induced a premature phase 3 activity front that originated in the stomach and an increase in gastric tone. Ghrelin decreased gastric emptying time for fluids and reduced symptom scores for fullness and pain. These studies provide further evidence for a role of ghrelin in the regulation of gastrointestinal motility, and possibly provide new therapeutic approaches. Our studies show that confocal calcium imaging allows to assess neuronal activation of myenteric neurons. The influence of new hormones or new pharmaceutical compounds on the myenteric plexus can hereby be easily assessed.

  1. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

    PubMed

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2015-04-24

    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

  2. Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system.

    PubMed

    Nakamura, Saki; Nakanishi, Ayumi; Takazawa, Minami; Okihiro, Shunsuke; Urano, Shiro; Fukui, Koji

    2016-01-01

    Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOX(TM) increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

  3. Zolpidem reduces hippocampal neuronal activity in freely behaving mice: a large scale calcium imaging study with miniaturized fluorescence microscope.

    PubMed

    Berdyyeva, Tamara; Otte, Stephani; Aluisio, Leah; Ziv, Yaniv; Burns, Laurie D; Dugovic, Christine; Yun, Sujin; Ghosh, Kunal K; Schnitzer, Mark J; Lovenberg, Timothy; Bonaventure, Pascal

    2014-01-01

    Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal's state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (∼ 65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders.

  4. Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

    PubMed

    Lin, Juqiang; Xu, Han; Wu, Yangzhe; Tang, Mingjie; McEwen, Gerald D; Liu, Pin; Hansen, Dane R; Gilbertson, Timothy A; Zhou, Anhong

    2013-02-05

    G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

  5. Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy.

    PubMed

    Huang, Alex Y; Myers, Jay T; Barkauskas, Deborah; Howell, Scott J; Oleinick, Nancy L; McCormick, Thomas S; Cooper, Kevin D; Baron, Elma D; Lam, Minh

    2012-09-01

    The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT.

  6. Calcium Chloride in Neonatal Parenteral Nutrition Solutions with and without Added Cysteine: Compatibility Studies Using Laser and Micro-Flow Imaging Methodology

    PubMed Central

    Huston, Robert K.; Christensen, J. Mark; Alshahrani, Sultan M.; Mohamed, Sumeia M.; Clark, Sara M.; Nason, Jeffrey A.; Wu, Ying Xing

    2015-01-01

    Background Previous studies of compatibility of calcium chloride (CaCl2) and phosphates have not included particle counts in the range specified by the United States Pharmacopeia. Micro-flow imaging techniques have been shown to be comparable to light obscuration when determining particle count and size in pharmaceutical solutions. Objective The purpose of this study was to do compatibility testing for parenteral nutrition (PN) solutions containing CaCl2 using dynamic light scattering and micro-flow imaging techniques. Methods Solutions containing TrophAmine (Braun Medical Inc, Irvine, CA), CaCl2, and sodium phosphate (NaPhos) were compounded with and without cysteine. All solutions contained standard additives to neonatal PN solutions including dextrose, trace metals, and electrolytes. Control solutions contained no calcium or phosphate. Solutions were analyzed for particle size and particle count. Means of Z-average particle size and particle counts of controls were determined. Study solutions were compared to controls and United States Pharmacopeia (USP) Chapter 788 guidelines. The maximum amount of Phos that was compatible in solutions that contained at least 10 mmol/L of Ca in 2.5% amino acids (AA) was determined. Compatibility of these solutions was verified by performing analyses of 5 repeats of these solutions. Microscopic analyses of the repeats were also performed. Results Amounts of CaCl2 and NaPhos that were compatible in solutions containing 1.5%, 2%, 2.5%, and 3% AA were determined. The maximum amount of NaPhos that could be added to TrophAmine solutions of > = 2.5% AA containing at least 10 mmol/L of CaCl2 was 7.5 mmol/L. Adding 50 mg/dL of cysteine increased the amount of NaPhos that could be added to solutions containing 10 mmol/L of CaCl2 to 10 mmol/L. Conclusion Calcium chloride can be added to neonatal PN solutions containing NaPhos in concentrations that can potentially provide an intravenous intake of adequate amounts of calcium and phosphorus

  7. Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation.

    PubMed

    Peters, P C; Thoni, C; Harry, E J

    2006-01-01

    A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.

  8. Detection of 2-Photon Oxidation from a NIR Laser Using Confocal Microscopy

    DTIC Science & Technology

    2006-01-01

    cells were then pre-loaded with antioxidants, ascorbic acid or N- acetyl -L- cysteine ( NAC ), they inhibit nonlinear oxidation with different...Scientific) or N- acetyl -L- cysteine ( NAC ) (cat A9165, Sigma-Aldrich) were co-loaded with the dye and acted to inhibit fluorescence; cells (preloaded...Effects on 2-Photon Excitation Oxidation RPE cells preloaded with CM-H;DCFDA and varying concentrations of inhibitors (AA and NAC ) were exposed to

  9. A Computational Model Based on Multi-Regional Calcium Imaging Represents the Spatio-Temporal Dynamics in a Caenorhabditis elegans Sensory Neuron

    PubMed Central

    Kuramochi, Masahiro; Doi, Motomichi

    2017-01-01

    Due to the huge number of neuronal cells in the brain and their complex circuit formation, computer simulation of neuronal activity is indispensable to understanding whole brain dynamics. Recently, various computational models have been developed based on whole-brain calcium imaging data. However, these analyses monitor only the activity of neuronal cell bodies and treat the cells as point unit. This point-neuron model is inexpensive in computational costs, but the model is unrealistically simplistic at representing intact neural activities in the brain. Here, we describe a novel three-unit Ordinary Differential Equation (ODE) model based on the neuronal responses derived from a Caenorhabditis elegans salt-sensing neuron. We recorded calcium responses in three regions of the ASER neuron using a simple downstep of NaCl concentration. Our simple ODE model generated from a single recording can adequately reproduce and predict the temporal responses of each part of the neuron to various types of NaCl concentration changes. Our strategy which combines a simple recording data and an ODE mathematical model may be extended to realistically understand whole brain dynamics by computational simulation. PMID:28072834

  10. Calcium in diet

    MedlinePlus

    ... of calcium dietary supplements include calcium citrate and calcium carbonate. Calcium citrate is the more expensive form of ... the body on a full or empty stomach. Calcium carbonate is less expensive. It is absorbed better by ...

  11. Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging.

    PubMed

    Murata, Kaori; Takahashi, Ayumi; Morita, Takao; Nezu, Akihiro; Fukumoto, Satoshi; Saitoh, Masato; Tanimura, Akihiro

    2016-01-01

    Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca(2+) release from intracellular stores and subsequent Ca(2+) entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca(2+) responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca(2+) concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca(2+) responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca(2+) responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca(2+) responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

  12. Monitoring Brain Activity with Protein Voltage and Calcium Sensors

    PubMed Central

    Storace, Douglas A.; Braubach, Oliver R.; Jin, Lei; Cohen, Lawrence B.; Sung, Uhna

    2015-01-01

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo. PMID:25970202

  13. Calcium - ionized

    MedlinePlus

    ... 245. Read More Acute kidney failure Albumin - blood (serum) test Bone tumor Calcium blood test Hyperparathyroidism Hypoparathyroidism Malabsorption Milk-alkali syndrome Multiple myeloma Osteomalacia Paget disease of the bone Rickets Sarcoidosis Vitamin D Review ...

  14. Calcium Carbonate.

    PubMed

    Al Omari, M M H; Rashid, I S; Qinna, N A; Jaber, A M; Badwan, A A

    2016-01-01

    Calcium carbonate is a chemical compound with the formula CaCO3 formed by three main elements: carbon, oxygen, and calcium. It is a common substance found in rocks in all parts of the world (most notably as limestone), and is the main component of shells of marine organisms, snails, coal balls, pearls, and eggshells. CaCO3 exists in different polymorphs, each with specific stability that depends on a diversity of variables.

  15. Calcium Hydroxylapatite

    PubMed Central

    Yutskovskaya, Yana Alexandrovna; Philip Werschler, WM.

    2015-01-01

    Background: Calcium hydroxylapatite is one of the most well-studied dermal fillers worldwide and has been extensively used for the correction of moderate-to-severe facial lines and folds and to replenish lost volume. Objectives: To mark the milestone of 10 years of use in the aesthetic field, this review will consider the evolution of calcium hydroxylapatite in aesthetic medicine, provide a detailed injection protocol for a global facial approach, and examine how the unique properties of calcium hydroxylapatite provide it with an important place in today’s market. Methods: This article is an up-to-date review of calcium hydroxylapatite in aesthetic medicine along with procedures for its use, including a detailed injection protocol for a global facial approach by three expert injectors. Conclusion: Calcium hydroxylapatite is a very effective agent for many areas of facial soft tissue augmentation and is associated with a high and well-established safety profile. Calcium hydroxylapatite combines high elasticity and viscosity with an ability to induce long-term collagen formation making it an ideal agent for a global facial approach. PMID:25610523

  16. Calcium orthophosphates

    PubMed Central

    Dorozhkin, Sergey V.

    2011-01-01

    The present overview is intended to point the readers’ attention to the important subject of calcium orthophosphates. This type of materials is of special significance for human beings, because they represent the inorganic part of major normal (bones, teeth and antlers) and pathological (i.e., those appearing due to various diseases) calcified tissues of mammals. For example, atherosclerosis results in blood vessel blockage caused by a solid composite of cholesterol with calcium orthophosphates, while dental caries and osteoporosis mean a partial decalcification of teeth and bones, respectively, that results in replacement of a less soluble and harder biological apatite by more soluble and softer calcium hydrogenphosphates. Therefore, the processes of both normal and pathological calcifications are just an in vivo crystallization of calcium orthophosphates. Similarly, dental caries and osteoporosis might be considered an in vivo dissolution of calcium orthophosphates. Thus, calcium orthophosphates hold a great significance for humankind, and in this paper, an overview on the current knowledge on this subject is provided. PMID:23507744

  17. Characterization of calcium and zinc spatial distributions at the fibrocartilage zone of bone-tendon junction by synchrotron radiation-based micro X-ray fluorescence analysis combined with backscattered electron imaging

    NASA Astrophysics Data System (ADS)

    Lu, Hongbin; Chen, Can; Wang, Zhanwen; Qu, Jin; Xu, Daqi; Wu, Tianding; Cao, Yong; Zhou, Jingyong; Zheng, Cheng; Hu, Jianzhong

    2015-09-01

    Tendon attaches to bone through a functionally graded fibrocartilage zone, including uncalcified fibrocartilage (UF), tidemark (TM) and calcified fibrocartilage (CF). This transition zone plays a pivotal role in relaxing load transfer between tendon and bone, and serves as a boundary between otherwise structurally and functionally distinct tissue types. Calcium and zinc are believed to play important roles in the normal growth, mineralization, and repair of the fibrocartilage zone of bone-tendon junction (BTJ). However, spatial distributions of calcium and zinc at the fibrocartilage zone of BTJ and their distribution-function relationship are not totally understood. Thus, synchrotron radiation-based micro X-ray fluorescence analysis (SR-μXRF) in combination with backscattered electron imaging (BEI) was employed to characterize the distributions of calcium and zinc at the fibrocartilage zone of rabbit patella-patellar tendon complex (PPTC). For the first time, the unique distributions of calcium and zinc at the fibrocartilage zone of the PPTC were clearly mapped by this method. The distributions of calcium and zinc at the fibrocartilage zone of the PPTC were inhomogeneous. A significant accumulation of zinc was exhibited in the transition region between UF and CF. The highest zinc content (3.17 times of that of patellar tendon) was found in the TM of fibrocartilage zone. The calcium content began to increase near the TM and increased exponentially across the calcified fibrocartilage region towards the patella. The highest calcium content (43.14 times of that of patellar tendon) was in the transitional zone of calcified fibrocartilage region and the patella, approximately 69 μm from the location with the highest zinc content. This study indicated, for the first time, that there is a differential distribution of calcium and zinc at the fibrocartilage zone of PPTC. These observations reveal new insights into region-dependent changes across the fibrocartilage zone of

  18. Comparison of glomerular activity patterns by fMRI and wide-field calcium imaging: Implications for principles underlying odor mapping.

    PubMed

    Sanganahalli, Basavaraju G; Rebello, Michelle R; Herman, Peter; Papademetris, Xenophon; Shepherd, Gordon M; Verhagen, Justus V; Hyder, Fahmeed

    2016-02-01

    Functional imaging signals arise from distinct metabolic and hemodynamic events at the neuropil, but how these processes are influenced by pre- and post-synaptic activities need to be understood for quantitative interpretation of stimulus-evoked mapping data. The olfactory bulb (OB) glomeruli, spherical neuropil regions with well-defined neuronal circuitry, can provide insights into this issue. Optical calcium-sensitive fluorescent dye imaging (OICa(2+)) reflects dynamics of pre-synaptic input to glomeruli, whereas high-resolution functional magnetic resonance imaging (fMRI) using deoxyhemoglobin contrast reveals neuropil function within the glomerular layer where both pre- and post-synaptic activities contribute. We imaged odor-specific activity patterns of the dorsal OB in the same anesthetized rats with fMRI and OICa(2+) and then co-registered the respective maps to compare patterns in the same space. Maps by each modality were very reproducible as trial-to-trial patterns for a given odor, overlapping by ~80%. Maps evoked by ethyl butyrate and methyl valerate for a given modality overlapped by ~80%, suggesting activation of similar dorsal glomerular networks by these odors. Comparison of maps generated by both methods for a given odor showed ~70% overlap, indicating similar odor-specific maps by each method. These results suggest that odor-specific glomerular patterns by high-resolution fMRI primarily tracks pre-synaptic input to the OB. Thus combining OICa(2+) and fMRI lays the framework for studies of OB processing over a range of spatiotemporal scales, where OICa(2+) can feature the fast dynamics of dorsal glomerular clusters and fMRI can map the entire glomerular sheet in the OB.

  19. Characterization of semi-solid Self-Emulsifying Drug Delivery Systems (SEDDS) of atorvastatin calcium by Raman image spectroscopy and chemometrics.

    PubMed

    Breitkreitz, Márcia C; Sabin, Guilherme P; Polla, Griselda; Poppi, Ronei J

    2013-01-25

    A methodology based on Raman image spectroscopy and chemometrics for homogeneity evaluation of formulations containing atorvastatin calcium in Gelucire(®) 44/14 is presented. In the first part of the work, formulations with high amounts of Gelucire(®) 44/14 (80%) and solvents of different polarities (diethylene glycol monoethyl ether, propyleneglycol, propylene glycol monocaprylate and glyceryl mono/dicaprylate/caprate) were prepared for miscibility screening evaluation by classical least squares (CLS). It was observed that Gelucire(®) 44/14 presented higher affinity for the lipophilic solvents glyceryl mono/dicaprylate/caprate and propylene glycol monocaprylate, whose samples were observed to be homogeneous, and lower affinity for the hydrophilic solvents diethylene glycol monoethyl ether and propyleneglycol, whose samples were heterogeneous. In the second part of the work, the ratio of glyceryl mono/dicaprylate/caprate and Gelucire(®) 44/14 was determined based on studies in water and allowed the selection of the proportions of these two excipients in the preconcentrate that provided supersaturation of atorvastatin upon dilution. The preconcentrate was then evaluated for homogeneity by partial least squares (PLS) and an excellent miscibility was observed in this proportion as well. Therefore, it was possible to select a formulation that presented simultaneously homogeneous preconcentrate and solubility enhancement in water by Raman image spectroscopy and chemometrics.

  20. Accurate spike estimation from noisy calcium signals for ultrafast three-dimensional imaging of large neuronal populations in vivo

    PubMed Central

    Deneux, Thomas; Kaszas, Attila; Szalay, Gergely; Katona, Gergely; Lakner, Tamás; Grinvald, Amiram; Rózsa, Balázs; Vanzetta, Ivo

    2016-01-01

    Extracting neuronal spiking activity from large-scale two-photon recordings remains challenging, especially in mammals in vivo, where large noises often contaminate the signals. We propose a method, MLspike, which returns the most likely spike train underlying the measured calcium fluorescence. It relies on a physiological model including baseline fluctuations and distinct nonlinearities for synthetic and genetically encoded indicators. Model parameters can be either provided by the user or estimated from the data themselves. MLspike is computationally efficient thanks to its original discretization of probability representations; moreover, it can also return spike probabilities or samples. Benchmarked on extensive simulations and real data from seven different preparations, it outperformed state-of-the-art algorithms. Combined with the finding obtained from systematic data investigation (noise level, spiking rate and so on) that photonic noise is not necessarily the main limiting factor, our method allows spike extraction from large-scale recordings, as demonstrated on acousto-optical three-dimensional recordings of over 1,000 neurons in vivo. PMID:27432255

  1. In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans

    PubMed Central

    Zhan, Hong; Stanciauskas, Ramunas; Stigloher, Christian; Dizon, Kevin K.; Jospin, Maelle; Bessereau, Jean-Louis; Pinaud, Fabien

    2014-01-01

    Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca2+ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. PMID:25232639

  2. An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.

    PubMed

    Ellefsen, Kyle L; Settle, Brett; Parker, Ian; Smith, Ian F

    2014-09-01

    Local Ca(2+) transients such as puffs and sparks form the building blocks of cellular Ca(2+) signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500 frames s(-1)) imaging of subcellular Ca(2+) signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1 Gb min(-1)) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca(2+) events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca(2+) release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events.

  3. Fast Kinetics of Calcium Signaling and Sensor Design

    PubMed Central

    Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

    2015-01-01

    Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

  4. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.

    PubMed

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K

    2005-07-01

    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  5. Calcium Test

    MedlinePlus

    ... if a person has symptoms of a parathyroid disorder , malabsorption , or an overactive thyroid. A total calcium level is often measured as part of a routine health screening. It is included in the comprehensive metabolic panel (CMP) and the basic metabolic panel (BMP) , ...

  6. Calcium cyanide

    Integrated Risk Information System (IRIS)

    Jump to main content . Integrated Risk Information System Recent Additions | Contact Us Search : All EPA IRIS • You are here : EPA Home • Research • Environmental Assessment • IRIS • IRIS Summaries Redirect Page As of September 28 , 2010 , the assessment summary for calcium cyanide is included in th

  7. Imaging of a glucose analog, calcium and NADH in neurons and astrocytes: dynamic responses to depolarization and sensitivity to pioglitazone

    PubMed Central

    Pancani, Tristano; Anderson, Katie L.; Porter, Nada M.; Thibault, Olivier

    2011-01-01

    Neuronal Ca2+ dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca2+ sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca2+ signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca2+ dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including FURA-2 and NADH imaging, provides results that are consistent with the idea that Ca2+ levels may rapidly alter glycolytic activity, and that downstream events beyond Ca2+ dysregulation with aging, may alter cellular metabolism in the brain. PMID:21978418

  8. Imaging of a glucose analog, calcium and NADH in neurons and astrocytes: dynamic responses to depolarization and sensitivity to pioglitazone.

    PubMed

    Pancani, Tristano; Anderson, Katie L; Porter, Nada M; Thibault, Olivier

    2011-12-01

    Neuronal Ca(2+) dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca(2+) sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca(2+) signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca(2+) dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including Fura-2 and NADH imaging, provides results that are consistent with the idea that Ca(2+) levels may rapidly alter glycolytic activity, and that downstream events beyond Ca(2+) dysregulation with aging, may alter cellular metabolism in the brain.

  9. Determining auditory-evoked activities from multiple cells in layer 1 of the dorsal cortex of the inferior colliculus of mice by in vivo calcium imaging.

    PubMed

    Ito, Tetsufumi; Hirose, Junichi; Murase, Kazuyuki; Ikeda, Hiroshi

    2014-11-24

    Layer 1 of the dorsal cortex of the inferior colliculus (DCIC) is distinguished from other layers by its cytoarchitecture and fiber connections. However, the information of the sound types represented in layer 1 of the DCIC remains unclear because placing electrodes on such thin structures is challenging. In this study, we utilized in vivo calcium imaging to assess auditory-evoked activities in multiple cells in layer 1 of DCIC and to characterize sound stimuli producing strong activity. Most cells examined showed strong responses to broad-band noise and low-frequency tone bursts of high sound intensity. In some cases, we successfully obtained frequency response areas, which are receptive fields to tone frequencies and intensities, and ~30% of these showed V-shape tunings. This is the first systematic study to record auditory responses of cells in layer 1 of DCIC. These results indicate that cells in this area are selective to tones with low frequency, implying the importance of such auditory information in the neural circuitry of layer 1 of DCIC.

  10. ToF-SIMS images and spectra of biomimetic calcium silicate-based cements after storage in solutions simulating the effects of human biological fluids

    NASA Astrophysics Data System (ADS)

    Torrisi, A.; Torrisi, V.; Tuccitto, N.; Gandolfi, M. G.; Prati, C.; Licciardello, A.

    2010-01-01

    ToF-SIMS images were obtained from a section of a tooth, obturated by means of a new calcium-silicate based cement (wTCF) after storage for 1 month in a saline solutions (DPBS), in order to simulate the body fluid effects on the obturation. Afterwards, ToF-SIMS spectra were obtained from model samples, prepared by using the same cement paste, after storage for 1 month and 8 months in two different saline solutions (DPBS and HBSS). ToF-SIMS spectra were also obtained from fluorine-free cement (wTC) samples after storage in HBSS for 1 month and 8 months and used for comparison. It was found that the composition of both the saline solution and the cement influenced the composition of the surface of disks and that longer is the storage greater are the differences. Segregation phenomena occur both on the cement obturation of the tooth and on the surface of the disks prepared by using the same cement. Indirect evidences of formation of new crystalline phases are supplied.

  11. Coronary artery calcium screening: current status and recommendations from the European Society of Cardiac Radiology and North American Society for Cardiovascular Imaging.

    PubMed

    Oudkerk, Matthijs; Stillman, Arthur E; Halliburton, Sandra S; Kalender, Willi A; Möhlenkamp, Stefan; McCollough, Cynthia H; Vliegenthart, Rozemarijn; Shaw, Leslee J; Stanford, William; Taylor, Allen J; van Ooijen, Peter M A; Wexler, Lewis; Raggi, Paolo

    2008-08-01

    Current guidelines and literature on screening for coronary artery calcium for cardiac risk assessment are reviewed for both general and special populations. It is shown that for both general and special populations a zero score excludes most clinically relevant coronary artery disease. The importance of standardization of coronary artery calcium measurements by multi-detector CT is discussed.

  12. Coronary artery calcium screening: current status and recommendations from the European Society of Cardiac Radiology and North American Society for Cardiovascular Imaging.

    PubMed

    Oudkerk, Matthijs; Stillman, Arthur E; Halliburton, Sandra S; Kalender, Willi A; Möhlenkamp, Stefan; McCollough, Cynthia H; Vliegenthart, Rozemarijn; Shaw, Leslee J; Stanford, William; Taylor, Allen J; van Ooijen, Peter M A; Wexler, Lewis; Raggi, Paolo

    2008-12-01

    Current guidelines and literature on screening for coronary artery calcium for cardiac risk assessment are reviewed for both general and special populations. It is shown that for both general and special populations a zero score excludes most clinically relevant coronary artery disease. The importance of standardization of coronary artery calcium measurements by multidetector CT is discussed.

  13. Visualizing sensory transmission between dorsal root ganglion and dorsal horn neurons in co-culture with calcium imaging.

    PubMed

    Ohshiro, Hiroyuki; Ogawa, Shinji; Shinjo, Katsuhiro

    2007-09-15

    Sensory information is conveyed to the central nervous system by primary afferent neurons within dorsal root ganglia (DRG), which synapse onto neurons of the dorsal horn of the spinal cord. This synaptic connection is central to the processing of both sensory and pain stimuli. Here, we describe a model system to monitor synaptic transmission between DRG neurons and dorsal horn neurons that is compatible with high-throughput screening. This co-culture preparation comprises DRG and dorsal horn neurons and utilizes Ca(2+) imaging with the indicator dye Fura-2 to visualize synaptic transmission. Addition of capsaicin to co-cultures stimulated DRG neurons and led to activation of dorsal horn neurons as well as increased intracellular Ca(2+) concentrations. This effect was dose-dependent and absent when DRG neurons were omitted from the culture. NMDA receptors are a critical component of synapses between DRG and dorsal horn neurons as MK-801, a use-dependent non-competitive antagonist, prevented activation of dorsal horn neurons following capsaicin treatment. This model system allows for rapid and efficient analysis of noxious stimulus-evoked Ca(2+) signal transmission and provides a new approach both for investigating synaptic transmission in the spinal cord and for screening potential analgesic compounds.

  14. Sodium–calcium exchangers contribute to the regulation of cytosolic calcium levels in mouse taste cells

    PubMed Central

    Laskowski, Agnieszka I; Medler, Kathryn F

    2009-01-01

    Taste cells use multiple signalling mechanisms to generate unique calcium responses to distinct taste stimuli. Some taste stimuli activate G-protein coupled receptors (GPCRs) that cause calcium release from intracellular stores while other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). We recently demonstrated that a constitutive calcium influx exists in taste cells that is regulated by mitochondrial calcium transport and that the magnitude of this calcium influx correlates with the signalling mechanisms used by the taste cells. In this study, we used calcium imaging to determine that sodium–calcium exchangers (NCXs) also routinely contribute to the regulation of basal cytosolic calcium and that their relative role correlates with the signalling mechanisms used by the taste cells. RT-PCR analysis revealed that multiple NCXs and sodium–calcium–potassium exchangers (NCKXs) are expressed in taste cells. Thus, a dynamic relationship exists between calcium leak channels and calcium regulatory mechanisms in taste cells that functions to keep cytosolic calcium levels in the appropriate range for cell function. PMID:19581381

  15. Calcium and bone disease

    PubMed Central

    Blair, Harry C.; Robinson, Lisa J.; Huang, Christopher L.-H.; Sun, Li; Friedman, Peter A.; Schlesinger, Paul H.; Zaidi, Mone

    2013-01-01

    Calcium transport and calcium signaling are of basic importance in bone cells. Bone is the major store of calcium and a key regulatory organ for calcium homeostasis. Bone, in major part, responds to calcium-dependent signals from the parathyroids and via vitamin D metabolites, although bone retains direct response to extracellular calcium if parathyroid regulation is lost. Improved understanding of calcium transporters and calcium-regulated cellular processes has resulted from analysis of genetic defects, including several defects with low or high bone mass. Osteoblasts deposit calcium by mechanisms including phosphate and calcium transport with alkalinization to absorb acid created by mineral deposition; cartilage calcium mineralization occurs by passive diffusion and phosphate production. Calcium mobilization by osteoclasts is mediated by acid secretion. Both bone forming and bone resorbing cells use calcium signals as regulators of differentiation and activity. This has been studied in more detail in osteoclasts, where both osteoclast differentiation and motility are regulated by calcium. PMID:21674636

  16. On the solvability of the quantum Rabi model and its 2-photon and two-mode generalizations

    SciTech Connect

    Zhang, Yao-Zhong

    2013-10-15

    We study the solvability of the time-independent matrix Schrödinger differential equations of the quantum Rabi model and its 2-photon and two-mode generalizations in Bargmann Hilbert spaces of entire functions. We show that the Rabi model and its 2-photon and two-mode analogs are quasi-exactly solvable. We derive the exact, closed-form expressions for the energies and the allowed model parameters for all the three cases in the solvable subspaces. Up to a normalization factor, the eigenfunctions for these models are given by polynomials whose roots are determined by systems of algebraic equations.

  17. Activation of muscarinic receptors increases the activity of the granule neurones of the rat dorsal cochlear nucleus--a calcium imaging study.

    PubMed

    Kőszeghy, Áron; Vincze, János; Rusznák, Zoltán; Fu, Yuhong; Paxinos, George; Csernoch, László; Szücs, Géza

    2012-06-01

    Acetylcholine modulates the function of the cochlear nucleus via several pathways. In this study, the effects of cholinergic stimulation were studied on the cytoplasmic Ca(2+) concentration of granule neurones of the rat dorsal cochlear nucleus (DCN). Ca(2+) transients were recorded in Oregon-Green-BAPTA 1-loaded brain slices using a calcium imaging technique. For the detection, identification and characterisation of the Ca(2+) transients, a wavelet analysis-based method was developed. Granule cells were identified on the basis of their size and localisation. The action potential-coupled character of the Ca(2+) transients of the granule cells was established by recording fluorescence changes and electrical activity simultaneously. Application of the cholinergic agonist carbamyl-choline (CCh) significantly increased the frequency of the Ca(2+) transients (from 0.37 to 6.31 min(-1), corresponding to a 17.1-fold increase; n = 89). This effect was antagonised by atropine, whereas CCh could still evoke an 8.3-fold increase of the frequency of the Ca(2+) transients when hexamethonium was present. Using immunolabelling, the expression of both type 1 and type 3 muscarinic receptors (M1 and M3 receptors, respectively) was demonstrated in the granule cells. Application of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (an M3-specific antagonist) prevented the onset of the CCh effect, whereas an M1-specific antagonist (pirenzepine) was less effective. We conclude that cholinergic stimulation increases the activity of granule cells, mainly by acting on their M3 receptors. The modulation of the firing activity of the granule cells, in turn, may modify the firing of projection neurones and may adjust signal processing in the entire DCN.

  18. Calcium and Vitamin D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium is required for the bone formation phase of bone remodeling. Typically about 5 nmol (200 mg) of calcium is removed from the adult skeleton and replaced each day. To supply this amount, one would need to consume about 600 mg of calcium, since calcium is not very efficiently absorbed. Calcium ...

  19. The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.

    PubMed

    Neumann, Sebastian; Kovtun, Anna; Dietzel, Irmgard D; Epple, Matthias; Heumann, Rolf

    2009-12-01

    Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.

  20. Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials.

    PubMed

    Weiss, P; Obadia, L; Magne, D; Bourges, X; Rau, C; Weitkamp, T; Khairoun, I; Bouler, J M; Chappard, D; Gauthier, O; Daculsi, G

    2003-11-01

    This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation. Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 microm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.

  1. Broadband thermo-optic switch based on a W2 photonic crystal waveguide

    NASA Astrophysics Data System (ADS)

    Cui, Kaiyu; Feng, Xue; Huang, Yidong; Zhao, Qiang; Huang, Zhilei; Zhang, Wei

    2013-02-01

    Broadband thermo-optic switch based on an ultra-compact W2 photonic crystal waveguide (PCW) is demonstrated with an integrated titanium/aluminum microheater on its surface. The operating principle relies on shifting a transmission-dip caused by the enhanced coupling between the defect modes in W2 PCW. As a result, broadband switching functionality with larger extinction ratio can be attained. Moreover, microheaters with different width are evaluated by the power consumptions and heating transfer efficiency, and an optimized slab microheater is utilized. Finally, switching functionality with bandwidth up to 24 nm (1557~1581 nm) is measured by the PCW with footprint of only 8μm×17.6 μm, while the extinction ratio is in excess of 15 dB over the entire bandwidth. What's more, the switching speed is obtained by the measurement of alternating current modulation. Response time for this thermo-optic switch is 11.0+/-3.0 μs for rise time and 40.3+/-5.3 μs for fall time, respectively.

  2. Calcium and Vitamin D

    MedlinePlus

    ... A calcium-rich diet (including dairy, nuts, leafy greens and fish) helps to build and protect your ... yogurt and cheese are high in calcium. Certain green vegetables and other foods contain calcium in smaller ...

  3. Coronary Calcium Scan

    MedlinePlus

    ... Scan Coronary Calcium Scan Related Topics Angina Atherosclerosis Coronary Heart Disease Electrocardiogram Heart Attack Send a link to NHLBI ... calcium, or calcifications, are a sign of atherosclerosis, coronary heart disease, or coronary microvascular disease. A coronary calcium scan ...

  4. Calcium uptake and bioelectrical activity of denervated and myotonic muscle

    PubMed Central

    Radu, H.; Gödri, I.; Albu, E.; Radu, A.; Robu, R.

    1970-01-01

    Calcium uptake on muscle microsomal fraction has been investigated in connection with bioelectrical activity in some muscle diseases. The findings showed a significant increase of calcium uptake in denervated muscle, which exhibited spontaneous bioelectrical activity (fibrillations). In myotonias, a low calcium uptake was peculiar to Steinert's disease but not to myotonia congenita. In other muscle diseases, such as progressive muscular dystrophy (Duchenne's type) or Charcot-Marie-Tooth's disease, the ability of muscle microsomal fraction to bind calcium was not changed. Starting with the key role of calcium in excitation-contraction coupling, the implications of calcium uptake disturbances in muscle electrogenesis are discussed. Images PMID:5431720

  5. Store-operated calcium entry is essential for glial calcium signalling in CNS white matter.

    PubMed

    Papanikolaou, M; Lewis, A; Butt, A M

    2017-02-28

    'Calcium signalling' is the ubiquitous response of glial cells to multiple extracellular stimuli. The primary mechanism of glial calcium signalling is by release of calcium from intracellular stores of the endoplasmic reticulum (ER). Replenishment of ER Ca(2+) stores relies on store-operated calcium entry (SOCE). However, despite the importance of calcium signalling in glial cells, little is known about their mechanisms of SOCE. Here, we investigated SOCE in glia of the mouse optic nerve, a typical CNS white matter tract that comprises bundles of myelinated axons and the oligodendrocytes and astrocytes that support them. Using quantitative RT-PCR, we identified Orai1 channels, both Stim1 and Stim2, and the transient receptor potential M3 channel (TRPM3) as the primary channels for SOCE in the optic nerve, and their expression in both astrocytes and oligodendrocytes was demonstrated by immunolabelling of optic nerve sections and cultures. The functional importance of SOCE was demonstrated by fluo-4 calcium imaging on isolated intact optic nerves and optic nerve cultures. Removal of extracellular calcium ([Ca(2+)]o) resulted in a marked depletion of glial cytosolic calcium ([Ca(2+)]i), which recovered rapidly on restoration of [Ca(2+)]o via SOCE. 2-aminoethoxydiphenylborane (2APB) significantly decreased SOCE and severely attenuated ATP-mediated calcium signalling. The results provide evidence that Orai/Stim and TRPM3 are important components of the 'calcium toolkit' that underpins SOCE and the sustainability of calcium signalling in white matter glia.

  6. Presynaptic Calcium Signalling in Cerebellar Mossy Fibres

    PubMed Central

    Thomsen, Louiza B.; Jörntell, Henrik; Midtgaard, Jens

    2009-01-01

    Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX)-sensitive fast Na+ spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette. PMID:20162034

  7. Calcium wave of tubuloglomerular feedback.

    PubMed

    Peti-Peterdi, János

    2006-08-01

    ATP release from macula densa (MD) cells into the interstitium of the juxtaglomerular (JG) apparatus (JGA) is an integral component of the tubuloglomerular feedback (TGF) mechanism that controls the glomerular filtration rate. Because the cells of the JGA express a number of calcium-coupled purinergic receptors, these studies tested the hypothesis that TGF activation triggers a calcium wave that spreads from the MD toward distant cells of the JGA and glomerulus. Ratiometric calcium imaging of in vitro microperfused isolated JGA-glomerulus complex dissected from rabbits was performed with fluo-4/fura red and confocal fluorescence microscopy. Activation of TGF by increasing tubular flow rate at the MD rapidly produced a significant elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) in extraglomerular mesangial cells (by 187.6 +/- 45.1 nM) and JG renin granular cells (by 281.4 +/- 66.6 nM). Subsequently, cell-to-cell propagation of the calcium signal at a rate of 12.6 +/- 1.1 microm/s was observed upstream toward proximal segments of the afferent arteriole and adjacent glomeruli, as well as toward intraglomerular elements including the most distant podocytes (5.9 +/- 0.4 microm/s). The same calcium wave was observed in nonperfusing glomeruli, causing vasoconstriction and contractions of the glomerular tuft. Gap junction uncoupling, an ATP scavenger enzyme cocktail, and pharmacological inhibition of P(2) purinergic receptors, but not adenosine A(1) receptor blockade, abolished the changes in [Ca(2+)](i) and propagation of the calcium wave. These studies provided evidence that both gap junctional communication and extracellular ATP are integral components of the TGF calcium wave.

  8. Estrogen evokes a rapid effect on intracellular calcium in neurons characterized by calcium oscillations in the arcuate nucleus.

    PubMed

    Fricke, Oliver; Kow, Lee-Ming; Bogun, Magda; Pfaff, Donald W

    2007-06-01

    Rapid estrogen effects became an interesting topic to explain estrogen effects not associated with the classical nuclear pathway. The rapid estrogen effect on intracellular calcium oscillations was characterized in neurons of the arcuate nucleus. Ratiometric calcium imaging (fura-2AM) was used to measure intracellular calcium in brain slices of female Swiss Webster mice (median of age 27 days p.n.). Calcium oscillations were dependent on intracellular calcium and also on calcium influx from the extracellular space. The perfusion of slices with calcium-free solution inhibited spontaneous calcium oscillations. The metabotropic glutamate receptor agonist t-ACPD (5 microM) and low concentrated ryanodine (100 nM) induced intracellular calcium release when slices were perfused with calcium-free solution. 17beta-estradiol (10 nM) also induced intracellular calcium release in calcium-free ACSF. This effect was inhibited by the preceding administration of thapsigargin (2 microM) indicating the association of the rapid estrogen effect with intracellular calcium stores. The administration of the non-selective phospholipase C-inhibitor ET-18 (30 microM), but not U73122 (10 microM), and the inhibition of protein kinase A by H-89 (0.25 microM) suppressed the rapid estrogen effect. Analyses indicated a qualitative, but not quantitatively significant effect of 17beta-estradiol on calcium oscillations.

  9. Plant Calcium Content: Ready to Remodel

    PubMed Central

    Yang, Jian; Punshon, Tracy; Guerinot, Mary Lou; Hirschi, Kendal D.

    2012-01-01

    By identifying the relationship between calcium location in the plant cell and nutrient bioavailability, the plant characteristics leading to maximal calcium absorption by humans can be identified. Knowledge of plant cellular and molecular targets controlling calcium location in plants is emerging. These insights should allow for better strategies for increasing the nutritional content of foods. In particular, the use of preparation-free elemental imaging technologies such as synchrotron X-ray fluorescence (SXRF) microscopy in plant biology may allow researchers to understand the relationship between subcellular location and nutrient bioavailability. These approaches may lead to better strategies for altering the location of calcium within the plant to maximize its absorption from fruits and vegetables. These modified foods could be part of a diet for children and adults identified as at-risk for low calcium intake or absorption with the ultimate goal of decreasing the incidence and severity of inadequate bone mineralization. PMID:23016135

  10. Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices

    PubMed Central

    Sasaki, Takuya; Ishikawa, Tomoe; Abe, Reimi; Nakayama, Ryota; Asada, Akiko; Matsuki, Norio; Ikegaya, Yuji

    2014-01-01

    Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level. PMID:24710057

  11. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING IN CENTER, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT WITH SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  12. [Do cows drink calcium?].

    PubMed

    Geishauser, T; Lechner, S; Plate, I; Heidemann, B

    2008-03-01

    The objective of this study was to investigate how well cows drink the Propeller calcium drink, and it's effect on blood calcium concentration. Drinking was tested in 120 cows right after calving, before cows drank anything else. 60 cows each were offered 20 liters of Propeller calcium drink or 20 liters of water. Cows drank the Propeller as good as water. 72% of all cows drank all 20 liters, 18% drank on average 8.2 liters and 10% drank less than 1 liter. Blood calcium concentration was studied in 16 cows right after calving. Eight cows each were offered 20 liters of Propeller calcium drink or no calcium drink. Blood calcium significantly increased ten minutes after Propeller intake and stayed significantly elevated for 24 hours. Without calcium drink blood calcium levels decreased significantly. Advantages of the new Propeller calcium drink over calcium gels or boli could be that cows now drink calcium themselves and that the Propeller increases blood calcium concentration rapidly and long lasting.

  13. Characteristics of two calcium pectinates prepared from citrus pectin using either calcium chloride or calcium hydroxide.

    PubMed

    Guo, Xiujun; Duan, Hanying; Wang, Chao; Huang, Xuesong

    2014-07-09

    Calcium pectinate (CaP) was prepared from citrus pectin using either calcium chloride (C-CaP) or calcium hydroxide (HO-CaP) as the source of calcium for the reaction. The production yields and the rates of decalcification for the two calcium pectinates were compared and both found to be lower for C-CaP than for HO-CaP. In an attempt to explain these differences, certain chemical and structural characteristics of the two products, including functional groups (-CH3, C═O, COO-), rheological properties, morphology, and egg-box junction zones, were investigated by Fourier transformation infrared (FTIR) spectroscopy, rheology, scanning electron microscopy (SEM), and X-ray diffraction (XRD). The results from FTIR showed that, with an increase in calcium content, the wavenumber values and peak areas of FTIR for -CH3, C═O, and COO- groups all changed dramatically for C-CaP, while they were virtually unchanged for HO-CaP. Rheological analysis of the CaP gel showed that C-CaP had a stronger cross-linked network structure and a greater range of elastic behavior as compared to HO-CaP. SEM images of two CaP gels showed irregular membranes. C-CaP maintained a tight structure and a smooth surface, whereas HO-CaP was loose and rough. The results from XRD revealed a higher degree of crystallinity within C-CaP than within HO-CaP, which indicated that C-CaP possessed compact, ordered, and stable egg-box junction zones while the junction zones in HO-CaP were metastable and loose.

  14. Calcium and Mitosis

    NASA Technical Reports Server (NTRS)

    Hepler, P.

    1983-01-01

    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  15. Calcium and bones

    MedlinePlus

    ... very bad at absorbing calcium. Most people absorb only 15% to 20% of the calcium they eat in their diet. Vitamin D is the hormone that helps the gut absorb more calcium. Many older adults have common risks that make bone health worse. ...

  16. Get Enough Calcium

    MedlinePlus

    ... a food with 45% DV of calcium. Check food labels. The Daily Value (DV) on a food label tells you the amount of a nutrient (like ... serving, or 60% DV. Learn how to check food labels for calcium information. Use this calcium shopping list ...

  17. Measuring calcium dynamics in living cells with genetically encodable calcium indicators.

    PubMed

    McCombs, Janet E; Palmer, Amy E

    2008-11-01

    Genetically encoded calcium indicators (GECIs) allow researchers to measure calcium dynamics in specific targeted locations within living cells. Such indicators enable dissection of the spatial and temporal control of calcium signaling processes. Here we review recent progress in the development of GECIs, highlighting which indicators are most appropriate for measuring calcium in specific organelles and localized domains in mammalian tissue culture cells. An overview of recent approaches that have been undertaken to ensure that the GECIs are minimally perturbed by the cellular environment is provided. Additionally, the procedures for introducing GECIs into mammalian cells, conducting calcium imaging experiments, and analyzing data are discussed. Because organelle-targeted indicators often pose an additional challenge, we underscore strategies for calibrating GECIs in these locations.

  18. Two-Photon Processor and SeNeCA: a freely available software package to process data from two-photon calcium imaging at speeds down to several milliseconds per frame.

    PubMed

    Tomek, Jakub; Novak, Ondrej; Syka, Josef

    2013-07-01

    Two-Photon Processor (TPP) is a versatile, ready-to-use, and freely available software package in MATLAB to process data from in vivo two-photon calcium imaging. TPP includes routines to search for cell bodies in full-frame (Search for Neural Cells Accelerated; SeNeCA) and line-scan acquisition, routines for calcium signal calculations, filtering, spike-mining, and routines to construct parametric fields. Searching for somata in artificial in vivo data, our algorithm achieved better performance than human annotators. SeNeCA copes well with uneven background brightness and in-plane motion artifacts, the major problems in simple segmentation methods. In the fast mode, artificial in vivo images with a resolution of 256 × 256 pixels containing ≈ 100 neurons can be processed at a rate up to 175 frames per second (tested on Intel i7, 8 threads, magnetic hard disk drive). This speed of a segmentation algorithm could bring new possibilities into the field of in vivo optophysiology. With such a short latency (down to 5-6 ms on an ordinary personal computer) and using some contemporary optogenetic tools, it will allow experiments in which a control program can continuously evaluate the occurrence of a particular spatial pattern of activity (a possible correlate of memory or cognition) and subsequently inhibit/stimulate the entire area of the circuit or inhibit/stimulate a different part of the neuronal system. TPP will be freely available on our public web site. Similar all-in-one and freely available software has not yet been published.

  19. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging

    PubMed Central

    Sofroniew, Nicholas James; Flickinger, Daniel; King, Jonathan; Svoboda, Karel

    2016-01-01

    Imaging is used to map activity across populations of neurons. Microscopes with cellular resolution have small (<1 millimeter) fields of view and cannot simultaneously image activity distributed across multiple brain areas. Typical large field of view microscopes do not resolve single cells, especially in the axial dimension. We developed a 2-photon random access mesoscope (2p-RAM) that allows high-resolution imaging anywhere within a volume spanning multiple brain areas (∅ 5 mm x 1 mm cylinder). 2p-RAM resolution is near diffraction limited (lateral, 0.66 μm, axial 4.09 μm at the center; excitation wavelength = 970 nm; numerical aperture = 0.6) over a large range of excitation wavelengths. A fast three-dimensional scanning system allows efficient sampling of neural activity in arbitrary regions of interest across the entire imaging volume. We illustrate the use of the 2p-RAM by imaging neural activity in multiple, non-contiguous brain areas in transgenic mice expressing protein calcium sensors. DOI: http://dx.doi.org/10.7554/eLife.14472.001 PMID:27300105

  20. Calcium hydroxide poisoning

    MedlinePlus

    These products contain calcium hydroxide: Cement Limewater Many industrial solvents and cleaners (hundreds to thousands of construction products, flooring strippers, brick cleaners, cement thickening products, and many ...

  1. Calcium: total or ionized?

    PubMed

    Schenck, Patricia A; Chew, Dennis J

    2008-05-01

    Measurement of serum total calcium (tCa) has been relied on for assessment of calcium status, despite the fact that it is the ionized calcium (iCa) fraction that has biologic activity. Serum tCa does not accurately predict iCa status in many clinical conditions. For accurate assessment of iCa status, iCa should be directly measured. Anaerobic measurement of serum iCa under controlled conditions provides the most reliable assessment of calcium status; aerobic measurement of iCa with species-specific pH correction is highly correlated with anaerobic measurements.

  2. Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism

    PubMed Central

    Petrou, Terry; Olsen, Hervør L.; Thrasivoulou, Christopher; Masters, John R.; Ashmore, Jonathan F.

    2017-01-01

    Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i. We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds. PMID:27980039

  3. Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements.

    PubMed

    Kopp, Richard F; Leech, Colin A; Roe, Michael W

    2014-03-01

    Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol's mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10 μM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100 μM resveratrol, excitation at 340 nm resulted in a large intensity increase at 510 nm, but the expected concurrent decline with 380 nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.

  4. Calcium fluoride window mounting

    NASA Astrophysics Data System (ADS)

    Berger, D. Douglas

    1982-10-01

    A technique has been developed for joining a large calcium fluoride crystal to a stainless-steel flange by means of a silver transition ring. The process involves both vacuum brazing using a copper-silver alloy and air brazing using silver chloride. This paper describes the procedure used in fabricating a high-vacuum leak-tight calcium fluoride window assembly.

  5. Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling

    PubMed Central

    Li, Ruizhen; Leblanc, Julie; He, Kevin; Liu, X. Johné

    2016-01-01

    Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results—most important, the different sensitivities of the spindle to BAPTA and EGTA—suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling. PMID:27582389

  6. Kidney and calcium homeostasis.

    PubMed

    Jeon, Un Sil

    2008-12-01

    Plasma calcium concentration is maintained within a narrow range (8.5-10.5 mg/dL) by the coordinated action of parathyroid hormone (PTH), 1,25(OH)2D3, calcitonin, and ionized calcium (iCa(2+)) itself. The kidney plays a key role in this process by the fine regulation of calcium excretion. More than 95% of filtered calcium is reabsorbed along the renal tubules. In the proximal tubules, 60% of filtered calcium is reabsorbed by passive mechanisms. In the thick ascending limb, 15% of calcium is reabsorbed by paracellular diffusion through paracellin-1 (claudin-16). The calcium sensing receptor (CaSR) in the basolateral membrane of the thick ascending limb senses the change in iCa(2+) and inhibits calcium reabsorption independent to PTH and 1,25(OH)2D3. The fine regulation of calcium excretion occurs in the distal convoluted tubules and connecting tubules despite the fact that only 10-15% of filtered calcium is reabsorbed there. Transient receptor potential vanilloid 5 (TRPV5) and 6 (TRPV6) in the apical membrane act as the main portal of entry, calbindin-D28K delivers Ca(2+) in the cytoplasm, and then Na(2+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase in the basolateral membrane serve as an exit. In the cortical collecting duct, TRPV6 is expressed, but the role might be negligible. In addition to PTH and 1,25(OH)2D3, acid-base disturbance, diuretics, and estrogen affect on these calcium channels. Recently, klotho and fibroblast growth factor 23 (FGF23) are suggested as new players in the calcium metabolism. Klotho is exclusively expressed in the kidney and co-localized with TRPV5, NCX1, and calbindin-D28K. Klotho increases calcium reabsorption through trafficking of TRPV5 to the plasma membrane, and also converts FGF receptor to the specific FGF23 receptor. FGF23:klotho complex bound to FGF receptor inhibits 1α-hydroxylase of vitamin D, and contributes to calcium reabsorption and phosphate excretion in the kidney.

  7. Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion

    PubMed Central

    1996-01-01

    Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of

  8. The CamKKβ Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells.

    PubMed

    Jurcsisn, Jennifer G; Pye, Richard L; Ali, Jon; Barr, Barbara L; Wyatt, Christopher N

    2015-01-01

    It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO609 and Ca(2+) imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca(2+) ([Ca(2+)](I)). Furthermore, STO609 (100 μM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK(Ca) inhibitor paxilline.Taken together these data suggest that ST0609 should be used with caution during Ca(2+) imaging studies as it can directly interact with Ca(2+) binding dyes. The rapid inhibitory effect of STO609 on BK(Ca) was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK(Ca), inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BK(Ca). Future work will determine if the inhibition of BK(Ca) is via CamKKβ or via an off target action of STO609 on the channel itself.

  9. Light Sheet Fluorescence Microscopy Quantifies Calcium Oscillations in Root Hairs of Arabidopsis thaliana.

    PubMed

    Candeo, Alessia; Doccula, Fabrizio G; Valentini, Gianluca; Bassi, Andrea; Costa, Alex

    2017-03-31

    Calcium oscillations play a role in the regulation of the development of tip-growing plant cells. Using optical microscopy, calcium oscillations have been observed in a few systems (e.g. pollen tubes, fungal hyphae, algal rhizoids). High resolution, non-phototoxic and rapid imaging methods are required to study the calcium oscillation in root hairs. We show that Light Sheet Fluorescence Microscopy is optimal to image growing root hairs of Arabidopsis thaliana and to follow their oscillatory tip-focused calcium gradient. We describe a protocol for performing live imaging of root hairs in seedlings expressing the cytosolic localised ratiometric calcium indicator Yellow Cameleon 3.6. Using this protocol, we measured the calcium gradient in a large number of root hairs. We characterised their calcium oscillations and correlated them with the rate of hair growth. The method was then used to screen the effect of auxin on the properties of the growing root hairs.

  10. Calcium wave of Brain Astrocytes

    NASA Astrophysics Data System (ADS)

    Cornell Bell, A. H.

    1997-03-01

    Time lapse confocal scanning laser microscopy was used to study hippocampal astrocyte cultures loaded with a calcium indicator, Fluo3-AM (4 uM). kThe neurotransmitter kainate (100uM) overwhelms the Na+-buffering capacity of astrocytes within 100 sec resulting in reversal of the Na+/Ca2+ exchanger. This results in a subcellular site where Ca2+ entering the cytoplasm contributes to a long-distance Ca2+ wave which travels at 20 um/sec without decrement. Image analysis has shown calcium waves not only at a high Kainate dose, but also at a low Kainate dose, e.g. 10uM. These are, however, shortlived and burried in an extremely noisy background and only detectable by analyzing the calcium waves images for spatio-temporal coherence. As the kainate dose increases, more large scale coherent structures with visible geometric features (spiral waves and target waves) can be observed. Multiple spiral waves are produced when the Kainate dose increases to 100 uM. These waves travel at a constant velocity across entire microscope fields for long time periods (>30 mins). Na+ channels have no effect on the Kainate wave. Voltage-gated Ca2+ channels are not involved and Ca2+ enters through reversal of the exchanger. Ca2+ release from stores does not contribute to the kainate wave. Removal of Na+ or Ca2+ from outside and the specific Na+/Ca2+ exchange inhibitor benzamil (10 uM) inhibit the kainate wave. A functional antibody to alpha6-Integrin which is localized to membrane regions between cells inhibits the spread of the kainate wave in a dose and time-dependent manner. Fluorescence Recovery after Photobleach (FRAP) techniques indicate that gap junctions remain open between cells. This would imply that Ca2+ or IP3 need not pass through the gap junction, but reversal of the exchanger would propel the Ca2+ wave at the cell surface.

  11. Presynaptic calcium currents in squid giant synapse.

    PubMed Central

    Llinás, R; Steinberg, I Z; Walton, K

    1981-01-01

    A voltage clamp study has been performed in the presynaptic terminal of the squid stellate ganglion. After blockage of the voltage-dependent sodium and potassium conductances, an inward calcium current is demonstrated. Given a step-depolarization pulse, this voltage- and time-dependent conductance has an S-shaped onset. At the "break" of the voltage step, a rapid tail current is observed. From these results a kinetic model is generated which accounts for the experimental results and predicts for the time course and amplitude a possible calcium entry during presynaptic action potentials. Images FIGURE 1 PMID:7225510

  12. [Calcium and health].

    PubMed

    Ortega Anta, Rosa M; Jiménez Ortega, Ana I; López-Sobaler, Ana M

    2015-04-07

    An adequate intake of calcium is only not limited to avoid the risk of osteoporosis and its benefits in longterm bone health, but also it has been linked to protection against various major diseases, such as hypertension, cancer, kidney stones, insulin resistance, diabetes... and several investigations suggest its importance in preventing and controlling obesity. Studies conducted in Spanish representative samples show that a high percentage of adults and children (> 75%) don't achieve the recommended intake of calcium. Moreover, are growing trends among the population suggesting that calcium intake and dairy consumption (main food source of the mineral) are high, and even excessive, in many individuals. This misconception results in that the calcium intake is increasingly far from the recommended one. The maximum tolerable intake of the mineral is fixed at 2.500 mg/day, but this intake is unusual, and it's more disturbing and frequent, to find intakes below the recommended calcium intakes (1.000 and 1.200 mg/day in adults, men and women, respectively). Data from different studies highlight the risk of an inadequate calcium intake and the damages that may affect the health in a long term. It is not about transmitting indiscriminate guidelines in order to increase the intake of calcium / dairy, but the recommended intakes must be met to achieve both the nutritional and health benefits. Also activities for demystification of misconceptions are need, increasingly frequent, that may impair health population.

  13. Calcium Signaling and Neurodegeneration

    PubMed Central

    2010-01-01

    Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so–called “calcium hypothesis of neurodegenerative diseases.” The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early–stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy. PMID:22649630

  14. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  15. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  16. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  17. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation of calcium carbonate from calcium chloride in the “Calcium...

  18. CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    CALCIUM CHLORIDE PLANT LOOKING EAST. CALCIUM CHLORIDE BUILDING ON LEFT, CALCIUM CHLORIDE STORAGE BUILDING ON RIGHT OF CENTER WITH TOP OF SA (SODA ASH) BUILDING IN RIGHT BACKGROUND. - Solvay Process Company, Calcium Chloride Plant, Between Willis & Milton Avenues, Solvay, Onondaga County, NY

  19. Broadband transient absorption spectroscopy with 1- and 2-photon excitations: Relaxation paths and cross sections of a triphenylamine dye in solution

    SciTech Connect

    Moreno, J.; Dobryakov, A. L.; Hecht, S. E-mail: skovale@chemie.hu-berlin.de; Kovalenko, S. A. E-mail: skovale@chemie.hu-berlin.de; Ioffe, I. N.; Granovsky, A. A.

    2015-07-14

    1-photon (382 nm) and 2-photon (752 nm) excitations to the S{sub 1} state are applied to record and compare transient absorption spectra of a push-pull triphenylamine (TrP) dye in solution. After 1-photon excitation, ultrafast vibrational and structural molecular relaxations are detected on a 0.1 ps time scale in nonpolar hexane, while in polar acetonitrile, the spectral evolution is dominated by dipolar solvation. Upon 2-photon excitation, transient spectra in hexane reveal an unexpected growth of stimulated emission (SE) and excited-state absorption (ESA) bands. The behavior is explained by strong population transfer S{sub 1} → S{sub n} due to resonant absorption of a third pump photon. Subsequent S{sub n} → S{sub 1} internal conversion (with τ{sub 1} = 1 ps) prepares a very hot S{sub 1} state which cools down with τ{sub 2} = 13 ps. The pump pulse energy dependence proves the 2-photon origin of the bleach signal. At the same time, SE and ESA are strongly affected by higher-order pump absorptions that should be taken into account in nonlinear fluorescence applications. The 2-photon excitation cross sections σ{sup (2)} = 32 ⋅ 10{sup −50} cm{sup 4} s at 752 nm are evaluated from the bleach signal.

  20. Broadband transient absorption spectroscopy with 1- and 2-photon excitations: Relaxation paths and cross sections of a triphenylamine dye in solution.

    PubMed

    Moreno, J; Dobryakov, A L; Ioffe, I N; Granovsky, A A; Hecht, S; Kovalenko, S A

    2015-07-14

    1-photon (382 nm) and 2-photon (752 nm) excitations to the S1 state are applied to record and compare transient absorption spectra of a push-pull triphenylamine (TrP) dye in solution. After 1-photon excitation, ultrafast vibrational and structural molecular relaxations are detected on a 0.1 ps time scale in nonpolar hexane, while in polar acetonitrile, the spectral evolution is dominated by dipolar solvation. Upon 2-photon excitation, transient spectra in hexane reveal an unexpected growth of stimulated emission (SE) and excited-state absorption (ESA) bands. The behavior is explained by strong population transfer S1 → Sn due to resonant absorption of a third pump photon. Subsequent Sn → S1 internal conversion (with τ1 = 1 ps) prepares a very hot S1 state which cools down with τ2 = 13 ps. The pump pulse energy dependence proves the 2-photon origin of the bleach signal. At the same time, SE and ESA are strongly affected by higher-order pump absorptions that should be taken into account in nonlinear fluorescence applications. The 2-photon excitation cross sections σ(2) = 32 ⋅ 10(-50) cm(4) s at 752 nm are evaluated from the bleach signal.

  1. Calcium signaling and cytotoxicity.

    PubMed Central

    Kass, G E; Orrenius, S

    1999-01-01

    The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery. Images Figure 1 Figure 2 Figure 3 PMID:10229704

  2. High power broadband mid-infrared supercontinuum fiber laser using a novel chalcogenide AsSe2 photonic crystal fiber

    NASA Astrophysics Data System (ADS)

    Diouf, Mbaye; Ben Salem, Amine; Cherif, Rim; Wague, Ahmadou; Zghal, Mourad

    2016-05-01

    A high power supercontinuum (SC) based on a new type of chalcogenide AsSe2 material for broadband mid-infrared light source is numerically reported. Ultra-broadband coherent mid-IR SC generation with more than 3 octave-spanning from 1.7 to 14 μm in a novel design of chalcogenide AsSe2 photonic crystal fiber (PCF) is demonstrated. To the best of our knowledge and aiming to properly model the nonlinear propagation, an accurate fit of the Raman response function and the corresponding Raman gain of the novel AsSe2 chalcogenide glass are proposed numerically for the first time. The obtained SC is generated by pumping at 3.9 μm in the anomalous dispersion regime in only 8 mm long fiber. Our study shows that the initially generated SC from 150 fs pulse duration with 8.8 kW peak power exhibits high power proportion of more than 80% for wavelengths beyond 3 μm which is very promising for designing high power SC fiber laser sources in the mid-IR atmospheric windows and the molecular fingerprint region.

  3. A search for 2-photon emission from the 662 keV state in ^137Ba using Gammasphere

    NASA Astrophysics Data System (ADS)

    Lister, C. J.; McCutchan, E. A.; Moran, K.; Zhu, S.; Carpenter, M. P.; Greene, J. P.; Millener, J. D.; Sutter, R. J.; Alburger, D. E.

    2012-10-01

    Two photon decays from excited nuclear states provide an interesting test both of QED and nuclear structure. It has been extensively studied for cases where one photon decay is forbidden [1]. Two photon decay in direct competition with the first order process has never been convincingly demonstrated. Nonetheless, observation of this decay will provide additional challenging tests for experiment and theory. The ^137Ba case is particularly interesting as the decay has high multipolarity, M4, so the 2-photon process can have contributions from both quadrupole-quadrupole and dipole-octupole multipolarities. Gammasphere is the perfect tool for this investigation, having good energy resolution, good efficiency, good coverage of angles, and sufficient granularity to minimize pile-up and count-rate difficulties. A short test experiment showed the power of Gammasphere and the dauntingly high Compton scattering background that need suppression. However, new calculations and new measurements from Brookhaven suggest that the two photon branch is ˜2 x 10-6 and should be measurable. This work was supported by DOE contracts, DE-FG02-94ER40848, DE-AC02-06CH11357 and DE-AC02-98CH10946.[4pt] [1] J. Kramp, et. al, Nucl. Phys. A474 (1987) 412

  4. Calcium Channel Blockers

    MedlinePlus

    ... such as high blood pressure, chest pain and Raynaud's disease. Find out more about this class of medication. ... Irregular heartbeats (arrhythmia) Some circulatory conditions, such as Raynaud's disease For black people and older people, calcium channel ...

  5. Stoichiometry of Calcium Medicines

    ERIC Educational Resources Information Center

    Pinto, Gabriel

    2005-01-01

    The topic of calcium supplement and its effects on human lives is presented in the way of questions to the students. It enables the students to realize the relevance of chemistry outside the classroom surrounding.

  6. Calcium-D-glucarate.

    PubMed

    2002-08-01

    Calcium-D-glucarate is the calcium salt of D-glucaric acid, a substance produced naturally in small amounts by mammals, including humans. Glucaric acid is also found in many fruits and vegetables with the highest concentrations to be found in oranges, apples, grapefruit, and cruciferous vegetables. Oral supplementation of calcium-D-glucarate has been shown to inhibit beta-glucuronidase, an enzyme produced by colonic microflora and involved in Phase II liver detoxification. Elevated beta-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as breast, prostate, and colon cancers. Other potential clinical applications of oral calcium-D-glucarate include regulation of estrogen metabolism and as a lipid-lowering agent.

  7. Calcium-sensing receptors.

    PubMed

    Goodman, William G

    2004-01-01

    It is now known that variations in extracellular calcium concentration exert diverse physiologic effects in a variety of tissues that are mediated by a calcium-sensing receptor (CaSRs). In parathyroid tissue, the CaSR represents the molecular mechanism by which parathyroid cells detect changes in blood ionized calcium concentration, modulate parathyroid hormone (PTH) secretion accordingly, and thus maintain serum calcium levels within a narrow physiologic range. In the kidney, the CaSR regulates renal calcium excretion and influences the transepithelial movement of water and other electrolytes. More generally, activation of the CaSR represents an important signal transduction pathway in intestine, placenta, brain, and perhaps bone. Some of these actions involve cell cycle regulation, changes that may be relevant to understanding the pathogenesis of parathyroid gland hyperplasia in secondary hyperparathyroidism caused by chronic kidney disease. The CaSR represents an appealing target for therapeutic agents designed to modify parathyroid gland function in vivo, offering the prospect of novel therapies for selected disorders of bone and mineral metabolism. Other receptors capable of responding to extracellular calcium ions also have been identified, but the functional importance of these interactions remains to be determined.

  8. Calcium/Vitamin D Supplementation and Coronary Artery Calcification

    PubMed Central

    Manson, JoAnn E.; Allison, Matthew A.; Carr, J. Jeffrey; Langer, Robert D.; Cochrane, Barbara B.; Hendrix, Susan L.; Hsia, Judith; Hunt, Julie R.; Lewis, Cora E.; Margolis, Karen L.; Robinson, Jennifer G.; Rodabough, Rebecca J.; Thomas, Asha M.

    2010-01-01

    Objectives Coronary artery calcified plaque is a marker for atheromatous plaque burden and predicts future risk of cardiovascular events. The relationship between calcium plus vitamin D supplementation and coronary artery calcium (CAC) has not been previously assessed in a randomized trial setting. We compared coronary artery calcium scores among women randomized to calcium/vitamin D supplementation versus placebo following trial completion. Methods In an ancillary substudy of women randomized to calcium carbonate (1000 mg of elemental calcium daily) plus vitamin D3 (400 IU daily) versus placebo, nested within the Women’s Health Initiative trial of estrogen among women with hysterectomy, we measured CAC with cardiac computed tomography in 754 women aged 50–59 years at randomization. Imaging for CAC was performed at 28 of 40 centers following a mean of 7 years of treatment and scans were read centrally. Coronary artery calcium scores were measured by a central reading center with masking to randomization assignments. Results Post-trial CAC measurements were similar in women randomized to calcium/vitamin D supplementation (calcium/D) and those receiving placebo. The mean CAC score was 91.6 for calcium/D and 100.5 for placebo (rank test p-value=0.74). After adjustment for coronary risk factors, multivariate odds ratios for increasing CAC score cutpoints (CAC >0, ≥10, and ≥100) for calcium/D vs placebo were 0.92 (95% confidence interval, 0.64–1.34), 1.29 (0.88–1.87), and 0.90 (0.56–1.44), respectively. Corresponding odds ratios among women with >50% adherence to study pills and for higher levels of CAC (>300), were similar. Conclusions Treatment with moderate doses of calcium plus vitamin D3 did not appear to alter coronary artery calcified plaque burden among postmenopausal women. PMID:20551849

  9. Oxidative calcium release from catechol.

    PubMed

    Riley, Patrick A; Stratford, Michael R L

    2015-04-01

    Oxidation of 4-methylcatechol previously exposed to aqueous calcium chloride was shown by ion chromatography to be associated with release of calcium ions. The catechol was oxidised to the corresponding orthoquinone by the use of tyrosinase from Agaricus bisporus. The oxidative release of calcium from the catechol is ascribed to the diminution of the available hydroxyl functions able to act as chelating groups. Our results suggest that the redox status of melanin may regulate calcium binding and influence calcium levels in pigmented cells.

  10. Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study

    PubMed Central

    Canepari, Marco; Djurisic, Maja; Zecevic, Dejan

    2007-01-01

    The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca2+ imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca2+]i changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca2+]i signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP–AP pairing evoked supra-linear summation of [Ca2+]i transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca2+ influx through NMDA receptors (0 to −15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals. PMID:17272348

  11. Comprehensive coronary risk determination in primary prevention: an imaging and clinical based definition combining computed tomographic coronary artery calcium score and national cholesterol education program risk score.

    PubMed

    Nasir, Khurram; Vasamreddy, Chandra; Blumenthal, Roger S; Rumberger, John A

    2006-06-16

    Cardiovascular disease (CVD) is the leading cause of mortality and a major cause of morbidity. Coronary heart disease (CHD) accounts for nearly half of all CVD deaths. Currently estimation of risk in primary prevention is based on the Framingham risk equations, which inputs traditional risk factors and is helpful in predicting the development of CHD in asymptomatic individuals. However many individuals suffer events in the absence of established risk factors for atherosclerosis and broad based population risk estimations may have little precision when applied to a given individual. To meet the challenge of CHD risk assessment, several tools have been developed to identify atherosclerotic disease in its preclinical stages. This paper aims to incorporate information from coronary artery calcification (CAC) scoring from a computed tomographic "heartscan" (using Electron Beam Tomography (EBT) as the validated prototype) along with current Framingham risk profiling in order to refine risk on an absolute scale by combining imaging and clinical data to affect a more comprehensive calculation of absolute risk in a given individual. For CAC scores above the 75th percentile but <90th percentile, 10 years is added to chronological age, and for CAC scores above the 90th percentile, 20 years is added to current chronological age. Among those in whom a positive CAC score is the norm such as older individuals (men> or =55 years, women> or =65 years) a CAC = 0 will result in an age point score corresponding to the age-group whose median CAC score is zero i.e., 40-44 years for men and 55-59 years for women. The utilization of CAC scores allows the inclusion of sub-clinical disease definition into the context of modifiable risk factors as well as identifies high-risk individuals requiring aggressive treatment.

  12. Spectral Properties of Single Gold Nanoparticles in Close Proximity to Biological Fluorophores Excited by 2-Photon Excitation

    PubMed Central

    Anzalone, Andrea; Gabriel, Manuela; Estrada, Laura C.; Gratton, Enrico

    2015-01-01

    Metallic nanoparticles (NPs) are able to modify the excitation and emission rates (plasmonic enhancement) of fluorescent molecules in their close proximity. In this work, we measured the emission spectra of 20 nm Gold Nanoparticles (AuNPs) fixed on a glass surface submerged in a solution of different fluorophores using a spectral camera and 2-photon excitation. While on the glass surface, we observed the presence in the emission at least 3 components: i) second harmonic signal (SHG), ii) a broad emission from AuNPS and iii) fluorescence arising from fluorophores nearby. When on the glass surface, we found that the 3 spectral components have different relative intensities when the incident direction of linear polarization was changed indicating different physical origins for these components. Then we measured by fluctuation correlation spectroscopy (FCS) the scattering and fluorescence signal of the particles alone and in a solution of 100 nM EGFP using the spectral camera or measuring the scattering and fluorescence from the particles. We observed occasional fluorescence bursts when in the suspension we added fluorescent proteins. The spectrum of these burst was devoid of the SHG and of the broad emission in contrast to the signal collected from the gold nanoparticles on the glass surface. Instead we found that the spectrum during the burst corresponded closely to the spectrum of the fluorescent protein. An additional control was obtained by measuring the cross-correlation between the reflection from the particles and the fluorescence arising from EGFP both excited at 488 nm. We found a very weak cross-correlation between the AuNPs and the fluorescence confirming that the burst originate from a few particles with a fluorescence signal. PMID:25909648

  13. Calcium transients during early development in single starfish (Asterias forbesi) oocytes

    PubMed Central

    1984-01-01

    Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. We have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. We used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approximately 1 microM at the point of insemination and the region of elevated free calcium expands across the oocyte in approximately 20 s (17-19 degrees C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes. PMID:6490725

  14. [Microbial geochemical calcium cycle].

    PubMed

    Zavarzin, G A

    2002-01-01

    The participation of microorganisms in the geochemical calcium cycle is the most important factor maintaining neutral conditions on the Earth. This cycle has profound influence on the fate of inorganic carbon, and, thereby, on the removal of CO2 from the atmosphere. The major part of calcium deposits was formed in the Precambrian, when prokaryotic biosphere predominated. After that, calcium recycling based on biogenic deposition by skeletal organisms became the main process. Among prokaryotes, only a few representatives, e.g., cyanobacteria, exhibit a special calcium function. The geochemical calcium cycle is made possible by the universal features of bacteria involved in biologically mediated reactions and is determined by the activities of microbial communities. In the prokaryotic system, the calcium cycle begins with the leaching of igneous rock predominantly through the action of the community of organotrophic organisms. The release of carbon dioxide to the soil air by organotrophic aerobes leads to leaching with carbonic acid and soda salinization. Under anoxic conditions, of major importance is the organic acid production by primary anaerobes (fermentative microorganisms). Calcium carbonate is precipitated by secondary anaerobes (sulfate reducers) and to a smaller degree by methanogens. The role of the cyanobacterial community in carbonate deposition is exposed by stromatolites, which are the most common organo-sedimentary Precambrian structures. Deposition of carbonates in cyanobacterial mats as a consequence of photoassimilation of CO2 does not appear to be a significant process. It is argued that carbonates were deposited at the boundary between the "soda continent", which emerged as a result of subaerial leaching with carbonic acid, and the ocean containing Ca2+. Such ecotones provided favorable conditions for the development of the benthic cyanobacterial community, which was a precursor of stromatolites.

  15. Calcium dynamics during NMDA-induced membrane potential oscillations in lamprey spinal neurons--contribution of L-type calcium channels (CaV1.3).

    PubMed

    Wang, Di; Grillner, Sten; Wallén, Peter

    2013-05-15

      NMDA receptor-dependent, intrinsic membrane potential oscillations are an important element in the operation of the lamprey locomotor network. They involve a cyclic influx of calcium, leading to an activation of calcium-activated potassium (KCa) channels that in turn contributes to the termination of the depolarized plateau and membrane repolarization. In this study, we have investigated the calcium dynamics in different regions of lamprey spinal neurons during membrane potential oscillations, using confocal calcium imaging in combination with intracellular recordings. Calcium fluctuations were observed in both soma and dendrites, timed to the oscillations. The calcium level increased sharply at the onset of membrane depolarization, to reach its maximum by the end of the plateau. The calcium peak in distal dendrites typically occurred earlier than in the soma during the oscillatory cycle. The L-type calcium channel blocker nimodipine increased the duration of the depolarized plateau phase in most cells tested, whereas the agonist Bay K 8644 decreased plateau duration. Bay K 8644 increased the amplitude of calcium fluctuations, particularly in distal dendrites, whereas nimodipine caused a decrease, suggesting that L-type low-voltage-activated calcium channels are mainly localized in these regions. Our results thus indicate that dendritic CaV1.3-like calcium channels are activated during NMDA-mediated membrane potential oscillations. This calcium influx activates KCa channels involved in plateau termination.

  16. Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

    PubMed Central

    Helassa, Nordine; Podor, Borbala; Fine, Alan; Török, Katalin

    2016-01-01

    Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators. PMID:27922063

  17. Guideline for minimizing radiation exposure during acquisition of coronary artery calcium scans with the use of multidetector computed tomography: a report by the Society for Atherosclerosis Imaging and Prevention Tomographic Imaging and Prevention Councils in collaboration with the Society of Cardiovascular Computed Tomography.

    PubMed

    Voros, Szilard; Rivera, Juan J; Berman, Daniel S; Blankstein, Ron; Budoff, Matthew J; Cury, Ricardo C; Desai, Milind Y; Dey, Damini; Halliburton, Sandra S; Hecht, Harvey S; Nasir, Khurram; Santos, Raul D; Shapiro, Michael D; Taylor, Allen J; Valeti, Uma S; Young, Phillip M; Weissman, Gaby

    2011-01-01

    Coronary artery calcium (CAC) scanning is an important tool for risk stratification in intermediate-risk, asymptomatic subjects without previous coronary disease. However, the clinical benefit of improved risk prediction needs to be balanced against the risk of the use of ionizing radiation. Although there is increasing emphasis on the need to obtain CAC scans at low-radiation exposure to the patient, very few practical documents exist to aid laboratories and health care professionals on how to obtain such low-radiation scans. The Tomographic Imaging Council of the Society for Atherosclerosis Imaging and Prevention, in collaboration with the Prevention Council and the Society of Cardiovascular Computed Tomography, created a task force and writing group to generate a practical document to address parameters that can be influenced by careful attention to image acquisition. Patient selection for CAC scanning should be based on national guidelines. It is recommended that laboratories performing CAC examinations monitor radiation exposure (dose-length-product [DLP]) and effective radiation dose (E) in all patients. DLP should be <200 mGy × cm; E should average 1.0-1.5 mSv and should be <3.0 mSv. On most scanner platforms, CAC imaging should be performed in an axial mode with prospective electrocardiographic triggering, using tube voltage of 120 kVp. Tube current should be carefully selected on the basis of patient size, potentially using chest lateral width measured on the topogram. Scan length should be limited for the coverage of the heart only. When patients and imaging parameters are selected appropriately, CAC scanning can be performed with low levels of radiation exposure.

  18. Calcium Apatite Deposition Disease: Diagnosis and Treatment

    PubMed Central

    2016-01-01

    Calcium apatite deposition disease (CADD) is a common entity characterized by deposition of calcium apatite crystals within and around connective tissues, usually in a periarticular location. CADD most frequently involves the rotator cuff. However, it can theoretically occur in almost any location in the musculoskeletal system, and many different locations of CADD have been described. When CADD presents in an unexpected location it can pose a diagnostic challenge, particularly when associated with pain or swelling, and can be confused with other pathologic processes, such as infection or malignancy. However, CADD has typical imaging characteristics that usually allows for a correct diagnosis to be made without additional imaging or laboratory workup, even when presenting in unusual locations. This is a review of the common and uncommon presentations of CADD in the appendicular and axial skeleton as well as an updated review of pathophysiology of CADD and current treatments. PMID:28042481

  19. Nonequilibrium Calcium Dynamics Regulate the Autonomous Firing Pattern of Rat Striatal Cholinergic Interneurons

    PubMed Central

    Goldberg, Joshua A.; Teagarden, Mark A.; Foehring, Robert C.; Wilson, Charles J.

    2009-01-01

    Striatal cholinergic interneurons discharge rhythmically in two patterns associated with different afterhyperpolarization timescales, each dictated by a different calcium-dependent potassium current. Single spiking depends on a medium-duration afterhyperpolarization (mAHP) generated by rapid SK currents that are associated with N-type calcium channels. Periodic bursting is driven by a delayed and slowly decaying afterhyperpolarization (sAHP) current associated with L-type channels. Using calcium imaging we show that the calcium transients underlying these currents exhibit two corresponding timescales throughout the somatodendritic tree. This result is not consistent with spatial compartmentalization of calcium entering through the two calcium channels and acting on the two potassium currents, or with differences in channel gating kinetics of the calcium dependent potassium currents. Instead, we show that nonequilibrium dynamics of calcium redistribution among cytoplasmic binding sites with different calcium binding kinetics can give rise to multiple timescales within the same cytoplasmic volume. The resulting independence of mAHP and sAHP currents allows cytoplasmic calcium to control two different and incompatible firing patterns (single spiking or bursting and pausing), depending on whether calcium influx is pulsatile or sustained. During irregular firing, calcium entry at both timescales can be detected, suggesting that an interaction between the medium and slow calcium-dependent afterhyperpolarizations may underlie this firing pattern. PMID:19571130

  20. Nonequilibrium calcium dynamics regulate the autonomous firing pattern of rat striatal cholinergic interneurons.

    PubMed

    Goldberg, Joshua A; Teagarden, Mark A; Foehring, Robert C; Wilson, Charles J

    2009-07-01

    Striatal cholinergic interneurons discharge rhythmically in two patterns associated with different afterhyperpolarization timescales, each dictated by a different calcium-dependent potassium current. Single spiking depends on a medium-duration afterhyperpolarization (mAHP) generated by rapid SK currents that are associated with N-type calcium channels. Periodic bursting is driven by a delayed and slowly decaying afterhyperpolarization (sAHP) current associated with L-type channels. Using calcium imaging we show that the calcium transients underlying these currents exhibit two corresponding timescales throughout the somatodendritic tree. This result is not consistent with spatial compartmentalization of calcium entering through the two calcium channels and acting on the two potassium currents, or with differences in channel gating kinetics of the calcium dependent potassium currents. Instead, we show that nonequilibrium dynamics of calcium redistribution among cytoplasmic binding sites with different calcium binding kinetics can give rise to multiple timescales within the same cytoplasmic volume. The resulting independence of mAHP and sAHP currents allows cytoplasmic calcium to control two different and incompatible firing patterns (single spiking or bursting and pausing), depending on whether calcium influx is pulsatile or sustained. During irregular firing, calcium entry at both timescales can be detected, suggesting that an interaction between the medium and slow calcium-dependent afterhyperpolarizations may underlie this firing pattern.

  1. Calcium metabolism and correcting calcium deficiencies.

    PubMed

    Emkey, Ronald D; Emkey, Gregory R

    2012-09-01

    Calcium is the most abundant cation in the human body, of which approximately 99% occurs in bone, contributing to its rigidity and strength. Bone also functions as a reservoir of Ca for its role in multiple physiologic and biochemical processes. This article aims to provide a thorough understanding of the absorptive mechanisms and factors affecting these processes to enable one to better appreciate an individual's Ca needs, and to provide a rationale for correcting Ca deficiencies. An overview of Ca requirements and suggested dosing regimens is presented, with discussion of various Ca preparations and potential toxicities of Ca treatment.

  2. Inositol trisphosphate and calcium signalling

    NASA Astrophysics Data System (ADS)

    Berridge, Michael J.

    1993-01-01

    Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

  3. Location matters: somatic and dendritic SK channels answer to distinct calcium signals.

    PubMed

    Rudolph, Stephanie; Thanawala, Monica S

    2015-07-01

    Voltage-dependent calcium channels (VDCCs) couple neuronal activity to diverse intracellular signals with exquisite spatiotemporal specificity. Using calcium imaging and electrophysiology, Jones and Stuart (J Neurosci 33: 19396-19405, 2013) examined the intimate relationship between distinct types of VDCCs and small-conductance calcium-activated potassium (SK) channels that contribute to the compartmentalized control of excitability in the soma and dendrites of cortical pyramidal neurons. Here we discuss the importance of calcium domains for signal specificity, explore the possible functions and mechanisms for local control of SK channels, and highlight technical considerations for the optical detection of calcium signals.

  4. Gravimetric Determination of Calcium as Calcium Carbonate Hydrate.

    ERIC Educational Resources Information Center

    Henrickson, Charles H.; Robinson, Paul R.

    1979-01-01

    The gravimetric determination of calcium as calcium carbonate is described. This experiment is suitable for undergraduate quantitative analysis laboratories. It is less expensive than determination of chloride as silver chloride. (BB)

  5. Preparation and properties of calcium oxide from eggshells via calcination

    NASA Astrophysics Data System (ADS)

    Tangboriboon, N.; Kunanuruksapong, R.; Sirivat, A.

    2012-12-01

    Duck eggs are one of the most versatile cooking ingredients in which residue eggshells are discarded. Raw duck eggshells were calcined at temperatures between 300 to 900 °C, for 1, 3, and 5 h. Both the raw and calcined duck eggshells were characterized by FTIR, STA, XRD, XRF, TEM, BET, a particle size analyzer, and an impedance analyzer. The proper calcination conditions are: 900 °C and 1 h, yielding calcium oxide with a purity of 99.06 % w/w. The calcium carbonate of the rhombohedral form (CaCO3) transforms completely into the calcium oxide or lime of the face centered cubic form (CaO) at 900 °C, as shown by XRD diffraction patterns. The transmission electron microscopy (TEM) images of the calcium oxide reveal a moderately good dispersion of nearly uniform particles. The calcium oxide has a white color, a spherical shape, high porosity, and narrow particles size distribution. The percentage of ceramic yield of the calcium oxide is 53.53, as measured by STA (TG-DTA-DTG). The calcium oxide has a N2 adsorption-desorption isotherm indicating the meso-porosity range. The dielectric constant and the electrical conductivity of the calcined calcium oxide are 35 and 1:0×10-6(Ω·m)-1, respectively, at the frequency of 500 Hz.

  6. Mechanically Induced Intercellular Calcium Communication in Confined Endothelial Structures

    PubMed Central

    Junkin, Michael; Lu, Yi; Long, Juexuan; Deymier, Pierre A.; Hoying, James B.; Wong, Pak Kin

    2012-01-01

    Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures. PMID:23267827

  7. Calcium Handling and Arrhythmogenesis.

    PubMed

    Bompotis, Georgios C; Pappas, Loukas K; Angelidis, Christos; Kossyvakis, Charalampos; Giannopoulos, Georgios; Deftereos, Spyridon

    2016-01-01

    Intracellular calcium homeostasis plays a fundamental role in the electric and mechanical function of the heart by modulating action potential pattern and duration, by linking cell membrane depolarization to myocardial contraction and by regulating cardiac automaticity. Abnormalities of intracellular calcium regulation disrupt the electrophysiological properties of the heart and create an arrhythmogenic milieu, which promotes atrial and ventricular arrhythmogenesis and impairs cardiac automaticity and atrioventricular conduction. In this brief review, we summarize the basic genetic, molecular and electrophysiological mechanisms linking inherited or acquired intracellular Ca(2+) dysregulation to arrhythmogenesis.

  8. Calcium metabolism in microgravity.

    PubMed

    Heer, M; Kamps, N; Biener, C; Korr, C; Boerger, A; Zittermann, A; Stehle, P; Drummer, C

    1999-09-09

    Unloading of weight bearing bones as induced by microgravity or immobilization has significant impacts on the calcium and bone metabolism and is the most likely cause for space osteoporosis. During a 4.5 to 6 month stay in space most of the astronauts develop a reduction in bone mineral density in spine, femoral neck, trochanter, and pelvis of 1%-1.6% measured by Dual Energy X-ray Absorption (DEXA). Dependent on the mission length and the individual turnover rates of the astronauts it can even reach individual losses of up to 14% in the femoral neck. Osteoporosis itself is defined as the deterioration of bone tissue leading to enhanced bone fragility and to a consequent increase in fracture risk. Thinking of long-term missions to Mars or interplanetary missions for years, space osteoporosis is one of the major concerns for manned spaceflight. However, decrease in bone density can be initiated differently. It either can be caused by increases in bone formation and bone resorption resulting in a net bone loss, as obtained in fast looser postmenopausal osteoporosis. On the other hand decrease in bone formation and increase in bone resorption also leads to bone losses as obtained in slow looser postmenopausal osteoporosis or in Anorexia Nervosa patients. Biomarkers of bone turnover measured during several missions indicated that the pattern of space osteoporosis is very similar to the pattern of Anorexia Nervosa patients or slow looser postmenopausal osteoporosis. However, beside unloading, other risk factors for space osteoporosis exist such as stress, nutrition, fluid shifts, dehydration and bone perfusion. Especially nutritional factors may contribute considerably to the development of osteoporosis. From earthbound studies it is known that calcium supplementation in women and men can prevent bone loss of 1% bone per year. Based on these results we studied the calcium intake during several European missions and performed an experiment during the German MIR 97 mission

  9. Analysis of spontaneous and nerve-evoked calcium transients in intact extraocular muscles in vitro.

    PubMed

    Feng, Cheng-Yuan; Hennig, Grant W; Corrigan, Robert D; Smith, Terence K; von Bartheld, Christopher S

    2012-07-01

    Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2(max) duration of 2-12 s, velocity of 25-50 μm/s) and two fast "flash-like" types (Type 1, 30-90 ms; Type 2, 90-150 ms 1/2(max) duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs.

  10. Analysis of Spontaneous and Nerve-Evoked Calcium Transients in Intact Extraocular Muscles in Vitro

    PubMed Central

    Feng, Cheng-Yuan; Hennig, Grant W.; Corrigan, Robert D.; Smith, Terence K.; von Bartheld, Christopher S.

    2012-01-01

    Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2max duration of 2–12 s, velocity of 25–50 μm/s) and two fast “flash-like” types (Type 1, 30–90 ms; Type 2, 90–150 ms 1/2max duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs. PMID:22579493

  11. Calcium channels, external calcium concentration and cell proliferation.

    PubMed

    Borowiec, Anne-Sophie; Bidaux, Gabriel; Pigat, Natascha; Goffin, Vincent; Bernichtein, Sophie; Capiod, Thierry

    2014-09-15

    Evidence for a role for calcium channel proteins in cell proliferation is numerous suggesting that calcium influx is essential in this physiological process. Several studies in the past thirty years have demonstrated that calcium channel expression levels are determinant in cell proliferation. Voltage-gated, store-operated, second messengers and receptor-operated calcium channels have been associated to cell proliferation. However, the relationship between calcium influx and cell proliferation can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Thus, protein expression could be more important than channel function to trigger cell proliferation suggesting that additional channel functions may be responsible to reconcile calcium channel expression and cell proliferation. When needed, external calcium concentration is obviously important for calcium channel function but it also regulates calcium sensing receptor (CaSR) activity. CaSR can up- or down-regulate cell proliferation depending on physiological conditions. CaSR sensitivity to external calcium is within the 0.5 to 5 mM range and therefore, the role of these receptors in cell proliferation must be taken into account. We therefore suggest here that cell proliferation rates could depend on the relative balance between calcium influx and CaSR activation.

  12. Calcium bioaccessibility and uptake by human intestinal like cells following in vitro digestion of casein phosphopeptide-calcium aggregates.

    PubMed

    Perego, Silvia; Del Favero, Elena; De Luca, Paola; Dal Piaz, Fabrizio; Fiorilli, Amelia; Cantu', Laura; Ferraretto, Anita

    2015-06-01

    Casein phosphopeptides (CPPs), derived by casein proteolysis, can bind calcium ions and keep them in solution. In vitro studies have demonstrated CPP-induced cell calcium uptake, depending on the formation of (CPP + calcium) complexes and on the degree of differentiation of the intestinal cells. With the present study, we address the persistence of the complexes and of the CPP-induced calcium uptake in intestinal like cells after the digestion process, thus examining their eligibility to serve as nutraceuticals. A calcium-preloaded CPP preparation of commercial origin (Ca-CPPs) was subjected to in vitro digestion. The evolution of the supramolecular structure of the Ca-CPP complexes was studied using laser-light and X-ray scattering. The bioactivity of the pre- and post-digestion Ca-CPPs was determined in differentiated Caco2 and HT-29 cells by video imaging experiments using Fura-2. We found that Ca-CPP aggregates keep a complex supramolecular organization upon digestion, despite getting smaller in size and increasing internal calcium dispersion. Concomitantly and most interestingly, digested Ca-CPPs clearly enhance the uptake of calcium ions, especially in Caco2 cells. In contrast, digestion depletes the ability of post-loaded decalcified-CPPs (Ca-dekCPPs), with a weaker internal structure, to induce calcium uptake. The enhanced bioactivity reached upon digestion strongly suggests a recognized role of Ca-CPPs, in the form used here, as nutraceuticals.

  13. Diet and calcium stones.

    PubMed Central

    Hughes, J; Norman, R W

    1992-01-01

    OBJECTIVE: To review the current literature on the dietary modification of urinary risk factors as a means of reducing the likelihood of recurrent stone formation and to develop practical dietary recommendations that might be useful to this end. DATA SOURCES: MEDLINE was searched for English-language articles published from 1983 to 1990. Additional references were selected from the bibliographies of identified articles. STUDY SELECTION: Nonrandomized trials and retrospective reviews were included because of a paucity of randomized controlled trials. DATA SYNTHESIS: Information on the dietary intake of calcium, oxalate, protein, sodium and fibre and on alcohol and fluid intake was used to develop practical guidelines on dietary modification. CONCLUSION: Dietary modification plays an important role in the reduction of urinary risk factors in patients with calcium stone disease of the urinary tract. As an initial form of prevention attention should be directed toward moderating the intake of calcium, oxalate, protein, sodium and alcohol and increasing the intake of fibre and water. Future research should include an assessment of the long-term reduction of dietary and urinary risk factors and the rates of recurrence of calcium stones. PMID:1310430

  14. Calcium silicate insulation structure

    DOEpatents

    Kollie, Thomas G.; Lauf, Robert J.

    1995-01-01

    An insulative structure including a powder-filled evacuated casing utilizes a quantity of finely divided synthetic calcium silicate having a relatively high surface area. The resultant structure-provides superior thermal insulating characteristics over a broad temperature range and is particularly well-suited as a panel for a refrigerator or freezer or the insulative barrier for a cooler or a insulated bottle.

  15. Calcium biofortification of crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than half of the world's population is deficient in calcium (Ca), iron (Fe), iodine (I), magnesium (Mg), selenium (Se), or zinc (Zn). The consumption of plants, directly or via livestock, containing inadequate concentrations of particular minerals causes these deficiencies. Agronomic and geneti...

  16. High Blood Calcium (Hypercalcemia)

    MedlinePlus

    ... as sarcoidosis • Hormone disorders, such as overactive thyroid (hyperthyroidism) • A genetic condition called familial hypocalciuric hypercalcemia • Kidney ... topics: www.hormone.org (search for PHPT, calcium, hyperthyroidism, or osteoporosis) • MedlinePlus (National Institutes of Health-NIH): ...

  17. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  18. The Plasma Membrane Calcium Pump

    NASA Technical Reports Server (NTRS)

    Rasmussen, H.

    1983-01-01

    Three aspect of cellular calcium metabolism in animal cells was discussed including the importance of the plasma membrane in calcium homeostasis, experiments dealing with the actual mechanism of the calcium pump, and the function of the pump in relationship to the mitochondria and to the function of calmodulin in the intact cell.

  19. Impregnating Coal With Calcium Carbonate

    NASA Technical Reports Server (NTRS)

    Sharma, Pramod K.; Voecks, Gerald E.; Gavalas, George R.

    1991-01-01

    Relatively inexpensive process proposed for impregnating coal with calcium carbonate to increase rates of gasification and combustion of coal and to reduce emission of sulfur by trapping sulfur in calcium sulfide. Process involves aqueous-phase reactions between carbon dioxide (contained within pore network of coal) and calcium acetate. Coal impregnated with CO2 by exposing it to CO2 at high pressure.

  20. Bioceramics of calcium orthophosphates.

    PubMed

    Dorozhkin, Sergey V

    2010-03-01

    A strong interest in use of ceramics for biomedical applications appeared in the late 1960's. Used initially as alternatives to metals in order to increase a biocompatibility of implants, bioceramics have become a diverse class of biomaterials, presently including three basic types: relatively bioinert ceramics, bioactive (or surface reactive) and bioresorbable ones. Furthermore, any type of bioceramics could be porous to provide tissue ingrowth. This review is devoted to bioceramics prepared from calcium orthophosphates, which belong to the categories of bioresorbable and bioactive compounds. During the past 30-40 years, there have been a number of major advances in this field. Namely, after the initial work on development of bioceramics that was tolerated in the physiological environment, emphasis was shifted towards the use of bioceramics that interacted with bones by forming a direct chemical bond. By the structural and compositional control, it became possible to choose whether the bioceramics of calcium orthophosphates was biologically stable once incorporated within the skeletal structure or whether it was resorbed over time. At the turn of the millennium, a new concept of calcium orthophosphate bioceramics, which is able to regenerate bone tissues, has been developed. Current biomedical applications of calcium orthophosphate bioceramics include replacements for hips, knees, teeth, tendons and ligaments, as well as repair for periodontal disease, maxillofacial reconstruction, augmentation and stabilization of the jawbone, spinal fusion and bone fillers after tumor surgery. Potential future applications of calcium orthophosphate bioceramics will include drug-delivery systems, as well as they will become effective carriers of growth factors, bioactive peptides and/or various types of cells for tissue engineering purposes.

  1. Imaging calcium waves in cerebellar Bergmann glia.

    PubMed

    Beierlein, Michael

    2013-01-01

    This protocol describes methods for recording synaptically evoked Ca(2+) waves from individual Bergmann glia (BG) in slices of cerebellar cortex. Unlike protoplasmic, star-shaped astrocytes, whose thin processes pose a serious challenge to stable Ca(2+) measurements, BG are large radial cells, with several main processes that run over distances of several hundred micrometers toward the pia and ensheathe thousands of parallel fiber (PF) synapses. Stimulation of PF synapses with brief bursts can trigger long-lasting Ca(2+) responses in BG processes, which can be reliably recorded using a cooled charge-coupled device (CCD) camera. This protocol was developed to enable measurements of Ca(2+) waves in individual BG loaded with a high-affinity Ca(2+) indicator such as Fura-2 for up to 2 h. Because BG recorded in slices rarely display spontaneous (i.e., tetrodotoxin [TTX]-sensitive) or intrinsic Ca(2+) transients, Ca(2+) waves can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Fluorescence measurements obtained using CCD technology offer a straightforward means of characterizing the mechanisms and potential functional consequences of widespread and long-lasting, store-mediated Ca(2+) increases in astrocytes.

  2. Cellular-resolution population imaging reveals robust sparse coding in the Drosophila Mushroom Body

    PubMed Central

    Honegger, Kyle S.; Campbell, Robert A. A.; Turner, Glenn C.

    2011-01-01

    Sensory stimuli are represented in the brain by the activity of populations of neurons. In most biological systems, studying population coding is challenging since only a tiny proportion of cells can be recorded simultaneously. Here we used 2-photon imaging to record neural activity in the relatively simple Drosophila mushroom body (MB), an area involved in olfactory learning and memory. Using the highly sensitive calcium indicator, GCaMP3, we simultaneously monitored the activity of >100 MB neurons in vivo (about 5% of the total population). The MB is thought to encode odors in sparse patterns of activity, but the code has yet to be explored either on a population level or with a wide variety of stimuli. We therefore imaged responses to odors chosen to evaluate the robustness of sparse representations. Different odors activated distinct patterns of MB neurons, however we found no evidence for spatial organization of neurons by either response probability or odor tuning within the cell body layer. The degree of sparseness was consistent across a wide range of stimuli, from monomolecular odors to artificial blends and even complex natural smells. Sparseness was mainly invariant across concentrations, largely because of the influence of recent odor experience. Finally, in contrast to sensory processing in other systems, no response features distinguished natural stimuli from monomolecular odors. Our results indicate that the fundamental feature of odor processing in the MB is to create sparse stimulus representations in a format that facilitates arbitrary associations between odor and punishment or reward. PMID:21849538

  3. Calcium Phosphates and Human Beings

    NASA Astrophysics Data System (ADS)

    Dorozhkin, Sergey V.

    2006-05-01

    This article describes the general importance of calcium phosphates for human beings. The basic information on the structure and chemical properties of the biologically relevant calcium phosphates is summarized. Basic facts on the natural occurrence and the industrial use of natural calcium phosphates are discussed. Fundamental details on the presence of calcium phosphates in major calcified tissues (bones and teeth) of humans and mammals, as well as on biomaterials made of calcium phosphates are discussed. The article will be of value for chemistry teachers for expansion of their general background and point the students' attention to the rapidly growing topic of bone-substituting biomaterials.

  4. Modulation of calcium signalling by the endoplasmic reticulum in Carassius neurons.

    PubMed

    Lukyanets, Igor A; Lukyanetz, Elena A

    2013-04-19

    It is known that endoplasmic reticulum (ER), being a calcium store participates in the regulation of intracellular calcium concentration. Ca-ATPase of the ER is one of the crucial agents providing the calcium-accumulating function of this intracellular structure. We studied the role of the ER in modulation of calcium signalling in Carassius neurons using a Ca2+-imaging technique. We tested the role of the ER in the maintenance of a steady state calcium level in the cytoplasm and in modulation of Ca2+ transients evoked by cell depolarizations. The ER calcium stores were depleted using inhibitors of ER Ca-ATPase, which provided blocking of Ca2+ uptake by the ER. Our experiments firstly showed that the ER can significantly modulate the characteristics of intracellular calcium signals in Carassius neurons during their activity. These findings also indicate that the ER modulates the shape of Ca2+ signals rather than the basal level of intracellular Ca2+ in these neurons.

  5. Role of magnesium and calcium in alcohol-induced hypertension and strokes as probed by in vivo television microscopy, digital image microscopy, optical spectroscopy, 31P-NMR, spectroscopy and a unique magnesium ion-selective electrode.

    PubMed

    Altura, B M; Altura, B T

    1994-10-01

    It is not known why alcohol ingestion poses a risk for development of hypertension, stroke and sudden death. Of all drugs, which result in body depletion of magnesium (Mg), alcohol is now known to be the most notorious cause of Mg-wasting. Recent data obtained through the use of biophysical (and noninvasive) technology suggest that alcohol may induce hypertension, stroke, and sudden death via its effects on intracellular free Mg2+ ([Mg2+]i), which in turn alter cellular and subcellular bioenergetics and promote calcium ion (Ca2+) overload. Evidence is reviewed that demonstrates that the dietary intake of Mg modulates the hypertensive actions of alcohol. Experiments with intact rats indicates that chronic ethanol ingestion results in both structural and hemodynamic alterations in the microcirculation, which, in themselves, could account for increased vascular resistance. Chronic ethanol increases the reactivity of intact microvessels to vasoconstrictors and results in decreased reactivity to vasodilators. Chronic ethanol ingestion clearly results in vascular smooth muscle cells that exhibit a progressive increase in exchangeable and cellular Ca2+ concomitant with a progressive reduction in Mg content. Use of 31P-NMR spectroscopy coupled with optical-backscatter reflectance spectroscopy revealed that acute ethanol administration to rats results in dose-dependent deficits in phosphocreatine (PCr), the [PCr]/[ATP] ratio, intracellular pH (pHi), oxyhemoglobin, and the mitochondrial level of oxidized cytochrome oxidase aa3 concomitant with a rise in brain-blood volume and inorganic phosphate. Temporal studies performed in vivo, on the intact brain, indicate that [Mg2+]i is depleted before any of the bioenergetic changes. Pretreatment of animals with Mg2+ prevents ethanol from inducing stroke and prevents all of the adverse bioenergetic changes from taking place. Use of quantitative digital imaging microscopy, and mag-fura-2, on single-cultured canine cerebral vascular

  6. Calcium signaling in taste cells.

    PubMed

    Medler, Kathryn F

    2015-09-01

    The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

  7. Probing Deep Brain Circuitry: New Advances in in Vivo Calcium Measurement Strategies.

    PubMed

    Girven, Kasey S; Sparta, Dennis R

    2017-02-15

    The study of neuronal ensembles in awake and behaving animals is a critical question in contemporary neuroscience research. Through the examination of calcium fluctuations, which are correlated with neuronal activity, we are able to better understand complex neural circuits. Recently, the development of technologies including two-photon microscopy, miniature microscopes, and fiber photometry has allowed us to examine calcium activity in behaving subjects over time. Visualizing changes in intracellular calcium in vivo has been accomplished utilizing GCaMP, a genetically encoded calcium indicator. GCaMP allows researchers to tag cell-type specific neurons with engineered fluorescent proteins that alter their levels of fluorescence in response to changes in intracellular calcium concentration. Even with the evolution of GCaMP, in vivo calcium imaging had yet to overcome the limitation of light scattering, which occurs when imaging from neural tissue in deep brain regions. Currently, researchers have created in vivo methods to bypass this problem; this Review will delve into three of these state of the art techniques: (1) two-photon calcium imaging, (2) single photon calcium imaging, and (3) fiber photometry. Here we discuss the advantages and disadvantages of the three techniques. Continued advances in these imaging techniques will provide researchers with unparalleled access to the inner workings of the brain.

  8. Particle size and shape of calcium hydroxide

    PubMed Central

    Komabayashi, Takashi; D’souza, Rena N; Dechow, Paul C; Safavi, Kamran E.; Spångberg, Larz S.W.

    2009-01-01

    The aim of this study was to examine the particle length, width, perimeter, and aspect ratio of calcium hydroxide powder using a flow particle image analyzer (FPIA). Five sample groups each with 10mg calcium hydroxide were mixed with 15mL of alcohol and sonicated. Digital images of the particle samples were taken using the FPIA and analyzed with a one-way ANOVA. The overall averages±S.D. among the five groups for particle length (μm), width (μm), perimeter (μm), and aspect ratio were 2.255±1.994, 1.620±1.464, 6.699±5.598, and 0.737±0.149, respectively. No statistical significance was observed among the groups for all parameters. When the total of 46,818 particles from all five groups were classified into the five length categories of 0.5μm increments, there were significant differences in width, perimeter, and aspect ratio (all p-values<0.0001). In conclusion, calcium hydroxide particles have a size and shape that may allow direct penetration into open dentin tubules. PMID:19166791

  9. Breast Microcalcification Detection Using Super-Resolution Ultrasound Image Reconstruction

    DTIC Science & Technology

    2010-09-01

    microcalcifications often occur as one of two types: calcium oxalate dihydrate or calcium hydroxyapatite. Their sizes range approximately from 0.1 mm to 0.5 mm...super-resolution imaging, ultrasound imaging, wave equation. 1. INTRODUCTION Microcalcifications, tiny specks of mineral deposits ( calcium ), are the

  10. Fruit Calcium: Transport and Physiology

    PubMed Central

    Hocking, Bradleigh; Tyerman, Stephen D.; Burton, Rachel A.; Gilliham, Matthew

    2016-01-01

    Calcium has well-documented roles in plant signaling, water relations and cell wall interactions. Significant research into how calcium impacts these individual processes in various tissues has been carried out; however, the influence of calcium on fruit ripening has not been thoroughly explored. Here, we review the current state of knowledge on how calcium may impact the development, physical traits and disease susceptibility of fruit through facilitating developmental and stress response signaling, stabilizing membranes, influencing water relations and modifying cell wall properties through cross-linking of de-esterified pectins. We explore the involvement of calcium in hormone signaling integral to the physiological mechanisms behind common disorders that have been associated with fruit calcium deficiency (e.g., blossom end rot in tomatoes or bitter pit in apples). This review works toward an improved understanding of how the many roles of calcium interact to influence fruit ripening, and proposes future research directions to fill knowledge gaps. Specifically, we focus mostly on grapes and present a model that integrates existing knowledge around these various functions of calcium in fruit, which provides a basis for understanding the physiological impacts of sub-optimal calcium nutrition in grapes. Calcium accumulation and distribution in fruit is shown to be highly dependent on water delivery and cell wall interactions in the apoplasm. Localized calcium deficiencies observed in particular species or varieties can result from differences in xylem morphology, fruit water relations and pectin composition, and can cause leaky membranes, irregular cell wall softening, impaired hormonal signaling and aberrant fruit development. We propose that the role of apoplasmic calcium-pectin crosslinking, particularly in the xylem, is an understudied area that may have a key influence on fruit water relations. Furthermore, we believe that improved knowledge of the calcium

  11. DISTILLATION OF CALCIUM

    DOEpatents

    Barton, J.

    1954-07-27

    This invention relates to an improvement in the process for the purification of caicium or magnesium containing an alkali metal as impurity, which comprises distiiling a batch of the mixture in two stages, the first stage distillation being carried out in the presence of an inert gas at an absolute pressure substantially greater than the vapor pressure of calcium or maguesium at the temperature of distillation, but less than the vaper pressure at that temperature of the alkali metal impurity so that only the alkali metal is vaporized and condensed on a condensing surface. A second stage distilso that substantially only the calcium or magnesium distills under its own vapor pressure only and condenses in solid form on a lower condensing surface.

  12. Nutrition in calcium nephrolithiasis

    PubMed Central

    2013-01-01

    Idiopathic calcium nephrolithiasis is a multifactorial disease with a complex pathogenesis due to genetic and environmental factors. The importance of social and health effects of nephrolithiasis is further highlighted by the strong tendency to relapse of the disease. Long-term prospective studies show a peak of disease recurrence within 2–3 years since onset, 40-50% of patients have a recurrence after 5 years and more than 50-60% after 10 years. International nutritional studies demonstrated that nutritional habits are relevant in therapy and prevention approaches of nephrolithiasis. Water, right intake of calcium, low intake of sodium, high levels of urinary citrate are certainly important for the primary and secondary prevention of nephrolithiasis. In this review is discussed how the correction of nutritional mistakes can reduce the incidence of recurrent nephrolithiasis. PMID:23634702

  13. Complexometric Determination of Calcium

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    Ethylenediaminetetraacetate (EDTA) complexes with numerous mineral ions, including calcium and magnesium. This reaction can be used to determine the amount of these minerals in a sample by a complexometric titration. Endpoints in the titration are detected using indicators that change color when they complex with mineral ions. Calmagite and eriochrome black T (EBT) are such indicators that change from blue to pink when they complex with calcium and magnesium. In the titration of a mineral-containing solution with EDTA, the solution turns from pink to blue at the endpoint with either indicator. The pH affects a complexometric EDTA titration in several ways, and must be carefully controlled. A major application of EDTA titration is testing the hardness of water, for which the method described is an official one (Standard Methods for the Examination of Water and Wastewater, Method 2340C; AOAC Method 920.196).

  14. Synthesis of calcium superoxide

    NASA Technical Reports Server (NTRS)

    Rewick, R. T.; Blucher, W. G.; Estacio, P. L.

    1972-01-01

    Efforts to prepare Ca(O2) sub 2 from reactions of calcium compounds with 100% O3 and with O(D-1) atoms generated by photolysis of O3 at 2537 A are described. Samples of Ca(OH) sub 2, CaO, CaO2, Ca metal, and mixtures containing suspected impurities to promote reaction have been treated with excess O3 under static and flow conditions in the presence and absence of UV irradiation. Studies with KO2 suggest that the superoxide anion is stable to radiation at 2537 A but reacts with oxygen atoms generated by the photolysis of O3 to form KO3. Calcium superoxide is expected to behave in an analogous.

  15. Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

    NASA Technical Reports Server (NTRS)

    Allen, G. J.; Kwak, J. M.; Chu, S. P.; Llopis, J.; Tsien, R. Y.; Harper, J. F.; Schroeder, J. I.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca

  16. Availability of calcium from skim milk, calcium sulfate and calcium carbonate for bone mineralization in pigs.

    PubMed

    Pointillart, A; Coxam, V; Sève, B; Colin, C; Lacroix, C H; Guéguen, L

    2000-01-01

    Dairy products provide abundant, accessible calcium for humans, while some calcium sulfate-rich mineral waters could provide appreciable amounts of calcium. But there is little evidence that this calcium is as available as milk calcium for making bone. The availability of calcium was studied by monitoring bone parameters in 2-month-old pigs fed restricted amounts of calcium (70% RDA) for 2.5 months. The 3 main (> or = 50% Ca intake) Ca sources were either CaCO3 or CaSO4 or skim milk powder (29% of the diet). The bones of the pigs fed the "milk" diet had higher (P < 0.01) ash contents, breaking strength and density (DEXA) than those of the two others groups, in which the bone values were similar. Thus, the calcium provided by a diet containing milk appears to ensure better bone mineralization than do calcium salts included in a non-milk diet. The calcium restriction may have enhanced some milk properties to stimulate calcium absorption in these young, rapidly growing pigs.

  17. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells

    NASA Astrophysics Data System (ADS)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel

    1988-06-01

    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  18. A model of propagating calcium-induced calcium release mediated by calcium diffusion

    PubMed Central

    1989-01-01

    The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading. PMID:2738577

  19. Calcium bioavailability and kinetics of calcium ascorbate and calcium acetate in rats.

    PubMed

    Cai, Jianwei; Zhang, Qinmin; Wastney, Meryl E; Weaver, Connie M

    2004-01-01

    The objective was to investigate the bioavailability and mechanism of calcium absorption of calcium ascorbate (ASC) and calcium acetate (AC). A series of studies was performed in adult Sprague-Dawley male rats. In the first study, each group of rats (n = 10/group) was assigned to one of the five test meals labeled with (45)Ca: (i) 25 mg calcium as heated ASC or (ii) unheated ASC, (iii) 25 mg calcium as unheated AC, (iv) 3.6 mg Ca as unheated ASC, or (v) unheated AC. Femur uptake indicated better calcium bioavailability from ASC than AC at both calcium loads. A 5-min heat treatment partly reduced bioavailability of ASC. Kinetic studies were performed to further investigate the mechanism of superior calcium bioavailability from ASC. Two groups of rats (n = 10/group) received oral doses of 25 mg Ca as ASC or AC. Each dose contained 20 micro Ci (45)Ca. Two additional groups of rats (n = 10/group) received an intravenous injection (iv) of 10 micro Ci (45)Ca after receiving an unlabeled oral dose of 25 mg calcium as ASC or AC. Sequential blood samples were collected over 48 hrs. Urine and fecal samples were collected every 12 hrs for 48 hrs and were analyzed for total calcium and (45)Ca content. Total calcium and (45)Ca from serum, urine, and feces were fitted by a compartment kinetics model with saturable and nonsaturable absorption pathways by WinSAAM (Windows-based Simulation Analysis and Modeling). The difference in calcium bioavailability between the two salts was due to differences in saturable rather than passive intestinal absorption and not to endogenous secretion or calcium deposition rate. The higher bioavailability of calcium ascorbate was due to a longer transit time in the small intestine compared with ASC.

  20. Calcium signalling and calcium channels: evolution and general principles.

    PubMed

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-09-15

    Calcium as a divalent cation was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules handling calcium. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, neurotransmitters, second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms.

  1. Elemental calcium intake associated with calcium acetate/calcium carbonate in the treatment of hyperphosphatemia

    PubMed Central

    Wilson, Rosamund J; Copley, J Brian

    2017-01-01

    Background Calcium-based and non-calcium-based phosphate binders have similar efficacy in the treatment of hyperphosphatemia; however, calcium-based binders may be associated with hypercalcemia, vascular calcification, and adynamic bone disease. Scope A post hoc analysis was carried out of data from a 16-week, Phase IV study of patients with end-stage renal disease (ESRD) who switched to lanthanum carbonate monotherapy from baseline calcium acetate/calcium carbonate monotherapy. Of the intent-to-treat population (N=2520), 752 patients with recorded dose data for calcium acetate (n=551)/calcium carbonate (n=201) at baseline and lanthanum carbonate at week 16 were studied. Elemental calcium intake, serum phosphate, corrected serum calcium, and serum intact parathyroid hormone levels were analyzed. Findings Of the 551 patients with calcium acetate dose data, 271 (49.2%) had an elemental calcium intake of at least 1.5 g/day at baseline, and 142 (25.8%) had an intake of at least 2.0 g/day. Mean (95% confidence interval [CI]) serum phosphate levels were 6.1 (5.89, 6.21) mg/dL at baseline and 6.2 (6.04, 6.38) mg/dL at 16 weeks; mean (95% CI) corrected serum calcium levels were 9.3 (9.16, 9.44) mg/dL and 9.2 (9.06, 9.34) mg/dL, respectively. Of the 201 patients with calcium carbonate dose data, 117 (58.2%) had an elemental calcium intake of at least 1.5 g/day, and 76 (37.8%) had an intake of at least 2.0 g/day. Mean (95% CI) serum phosphate levels were 5.8 (5.52, 6.06) mg/dL at baseline and 5.8 (5.53, 6.05) mg/dL at week 16; mean (95% CI) corrected serum calcium levels were 9.7 (9.15, 10.25) mg/dL and 9.2 (9.06, 9.34) mg/dL, respectively. Conclusion Calcium acetate/calcium carbonate phosphate binders, taken to control serum phosphate levels, may result in high levels of elemental calcium intake. This may lead to complications related to calcium balance. PMID:28182142

  2. Theoretical aspects of calcium signaling

    NASA Astrophysics Data System (ADS)

    Pencea, Corneliu Stefan

    2001-08-01

    Experiments investigating intracellular calcium dynamics have revealed that calcium signals differentially affect a variety of intracellular processes, from fertilization and cell development and differentiation to subsequent cellular activity, ending with cell death. As an intracellular messenger, calcium transmits information within and between cells, thus regulating their activity. To control such a variety of processes, calcium signals have to be very flexible and also precisely regulated. The cell uses a calcium signaling ``toolkit'', where calcium ions can act in different contexts of space, amplitude and time. For different tasks, the cell selects the particular signal, or combination of signals, that triggers the appropriate physiological response. The physical foundations of such a versatile cellular signaling toolkit involving calcium are not completely understood, despite important experimental and theoretical progress made recently. The declared goal of this work is to investigate physical mechanisms on which the propagation of differential signals can be based. The dynamics of calcium near a cluster of inositol trisphosphate (IP3) activated calcium channels has been investigated analytically and numerically. Our work has demonstrated that clusters of different IP3 receptors can show similar bistable behavior, but differ in both the transient and long term dynamics. We have also investigated the conditions under which a calcium signal propagates between a pair of localized stores. We have shown that the propagation of the signal across a random distribution of such stores shows a percolation transition manifested in the shape of the wave front. More importantly, our work indicates that specific distribution of stores can be interpreted as calcium circuits that can perform important signal analyzing task, from unidirectional propagation and coincidence detection to a complete set of logic gates. We believe that phenomena like the ones described are

  3. Revisiting intracellular calcium signaling semantics.

    PubMed

    Haiech, Jacques; Audran, Emilie; Fève, Marie; Ranjeva, Raoul; Kilhoffer, Marie-Claude

    2011-12-01

    Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.

  4. Cardiovascular effects of calcium supplements.

    PubMed

    Reid, Ian R

    2013-07-05

    Calcium supplements reduce bone turnover and slow the rate of bone loss. However, few studies have demonstrated reduced fracture incidence with calcium supplements, and meta-analyses show only a 10% decrease in fractures, which is of borderline statistical and clinical significance. Trials in normal older women and in patients with renal impairment suggest that calcium supplements increase the risk of cardiovascular disease. To further assess their safety, we recently conducted a meta-analysis of trials of calcium supplements, and found a 27%-31% increase in risk of myocardial infarction, and a 12%-20% increase in risk of stroke. These findings are robust because they are based on pre-specified analyses of randomized, placebo-controlled trials and are consistent across the trials. Co-administration of vitamin D with calcium does not lessen these adverse effects. The increased cardiovascular risk with calcium supplements is consistent with epidemiological data relating higher circulating calcium concentrations to cardiovascular disease in normal populations. There are several possible pathophysiological mechanisms for these effects, including effects on vascular calcification, vascular cells, blood coagulation and calcium-sensing receptors. Thus, the non-skeletal risks of calcium supplements appear to outweigh any skeletal benefits, and are they appear to be unnecessary for the efficacy of other osteoporosis treatments.

  5. 21 CFR 184.1195 - Calcium citrate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... acid with calcium hydroxide or calcium carbonate. It occurs as a fine white, odorless powder and... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium citrate. 184.1195 Section 184.1195 Food... Specific Substances Affirmed as GRAS § 184.1195 Calcium citrate. (a) Calcium citrate...

  6. 21 CFR 184.1195 - Calcium citrate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... acid with calcium hydroxide or calcium carbonate. It occurs as a fine white, odorless powder and... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium citrate. 184.1195 Section 184.1195 Food... Specific Substances Affirmed as GRAS § 184.1195 Calcium citrate. (a) Calcium citrate...

  7. 21 CFR 573.260 - Calcium silicate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium silicate. 573.260 Section 573.260 Food and... Listing § 573.260 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used as an anticaking agent in animal feed, provided that the amount of calcium silicate does...

  8. 21 CFR 573.260 - Calcium silicate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium silicate. 573.260 Section 573.260 Food and... Listing § 573.260 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used as an anticaking agent in animal feed, provided that the amount of calcium silicate does...

  9. 21 CFR 573.260 - Calcium silicate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium silicate. 573.260 Section 573.260 Food and... Listing § 573.260 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used as an anticaking agent in animal feed, provided that the amount of calcium silicate does...

  10. 21 CFR 573.260 - Calcium silicate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium silicate. 573.260 Section 573.260 Food and... Listing § 573.260 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used as an anticaking agent in animal feed, provided that the amount of calcium silicate does...

  11. 21 CFR 573.260 - Calcium silicate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium silicate. 573.260 Section 573.260 Food and... Listing § 573.260 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used as an anticaking agent in animal feed, provided that the amount of calcium silicate does...

  12. 21 CFR 184.1195 - Calcium citrate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Specific Substances Affirmed as GRAS § 184.1195 Calcium citrate. (a) Calcium citrate (Ca3(C6H5O7)2·4H2O, CAS Reg. No. 813-0994-095) is the calcium salt of citric acid. It is prepared by neutralizing citric acid with calcium hydroxide or calcium carbonate. It occurs as a fine white, odorless powder...

  13. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    , localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.

  14. New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

    PubMed Central

    Vorndran, C; Minta, A; Poenie, M

    1995-01-01

    A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol. Images FIGURE 6 FIGURE 8 PMID:8580355

  15. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium pantothenate, calcium chloride double salt..., calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may... information required by the Act, the following: (1) The name of the additive “calcium chloride double salt...

  16. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium pantothenate, calcium chloride double salt..., calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may... information required by the Act, the following: (1) The name of the additive “calcium chloride double salt...

  17. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium pantothenate, calcium chloride double salt..., calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may... information required by the Act, the following: (1) The name of the additive “calcium chloride double salt...

  18. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium pantothenate, calcium chloride double salt..., calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may... information required by the Act, the following: (1) The name of the additive “calcium chloride double salt...

  19. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium pantothenate, calcium chloride double salt... Dietary and Nutritional Additives § 172.330 Calcium pantothenate, calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may be safely used in foods for...

  20. Calcium channels and migraine.

    PubMed

    Pietrobon, Daniela

    2013-07-01

    Missense mutations in CACNA1A, the gene that encodes the pore-forming α1 subunit of human voltage-gated Ca(V)2.1 (P/Q-type) calcium channels, cause a rare form of migraine with aura (familial hemiplegic migraine type 1: FHM1). Migraine is a common disabling brain disorder whose key manifestations are recurrent attacks of unilateral headache that may be preceded by transient neurological aura symptoms. This review, first, briefly summarizes current understanding of the pathophysiological mechanisms that are believed to underlie migraine headache, migraine aura and the onset of a migraine attack, and briefly describes the localization and function of neuronal Ca(V)2.1 channels in the brain regions that have been implicated in migraine pathogenesis. Then, the review describes and discusses i) the functional consequences of FHM1 mutations on the biophysical properties of recombinant human Ca(V)2.1 channels and native Ca(V)2.1 channels in neurons of knockin mouse models carrying the mild R192Q or severe S218L mutations in the orthologous gene, and ii) the functional consequences of these mutations on neurophysiological processes in the cerebral cortex and trigeminovascular system thought to be involved in the pathophysiology of migraine, and the insights into migraine mechanisms obtained from the functional analysis of these processes in FHM1 knockin mice. This article is part of a Special Issue entitled: Calcium channels.

  1. Differential Dendritic Integration of Synaptic Potentials and Calcium in Cerebellar Interneurons.

    PubMed

    Tran-Van-Minh, Alexandra; Abrahamsson, Therése; Cathala, Laurence; DiGregorio, David A

    2016-08-17

    Dendritic voltage integration determines the transformation of synaptic inputs into output firing, while synaptic calcium integration drives plasticity mechanisms thought to underlie memory storage. Dendritic calcium integration has been shown to follow the same synaptic input-output relationship as dendritic voltage, but whether similar operations apply to neurons exhibiting sublinear voltage integration is unknown. We examined the properties and cellular mechanisms of these dendritic operations in cerebellar molecular layer interneurons using dendritic voltage and calcium imaging, in combination with synaptic stimulation or glutamate uncaging. We show that, while synaptic potentials summate sublinearly, concomitant dendritic calcium signals summate either linearly or supralinearly depending on the number of synapses activated. The supralinear dendritic calcium triggers a branch-specific, short-term suppression of neurotransmitter release that alters the pattern of synaptic activation. Thus, differential voltage and calcium integration permits dynamic regulation of neuronal input-output transformations without altering intrinsic nonlinear integration mechanisms.

  2. Calcium transporters: From fields to the table

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium transporters regulate calcium fluxes within cells. Plants, like all organisms, contain channels, pumps, and exchangers to carefully modulate intracellular calcium levels. This review presents a summary of the recent advances in cloning and characterizing of these transporters and highlight...

  3. Vitamin D, Calcium, and Bone Health

    MedlinePlus

    ... in Balance › Vitamin D, Calcium, and Bone Health Vitamin D, Calcium, and Bone Health March 2012 Download ... also helps keep your bones strong. Why are vitamin D and calcium important to bone health? Vitamin ...

  4. Major Minerals - Calcium, Magnesium, Phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium, magnesium and phosphorus are essential elements critically important for the function of the musculoskeletal system, including the formation and transduction of energy and the maintenance of healthy bone. The major calcium concern for physically active healthy middle-aged adults is to consu...

  5. Electrochemical cell with calcium anode

    DOEpatents

    Cooper, John F.; Hosmer, Pamela K.; Kelly, Benjamin E.

    1979-01-01

    An electrochemical cell comprising a calcium anode and a suitable cathode in an alkaline electrolyte consisting essentially of an aqueous solution of an hydroxide and a chloride. Specifically disclosed is a mechanically rechargeable calcium/air fuel cell with an aqueous NaOH/NaCl electrolyte.

  6. Calcium Intake: A Lifelong Proposition.

    ERIC Educational Resources Information Center

    Amschler, Denise H.

    1985-01-01

    This article reviews the current problem of low calcium intake in the United States among all age groups, the role of calcium in the formation and maintenance of bone mass, and major factors influencing absorption. Osteoporosis is discussed, and current recommendations for Recommended Dietary allowance are provided. (Author/MT)

  7. Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons.

    PubMed

    Luo, Fen-Lan; Yang, Nian; He, Chao; Li, Hong-Li; Li, Chao; Chen, Fang; Xiong, Jia-Xiang; Hu, Zhi-An; Zhang, Jun

    2014-11-01

    Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K(+)-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca(2+) ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.

  8. Imaging nervous system activity.

    PubMed

    Fields, R D; O'Donovan, M J

    2001-05-01

    Optical imaging methods rely upon visualization of three types of signals: (1) intrinsic optical signals, including light scattering and reflectance, birefringence, and spectroscopic changes of intrinsic molecules, such as NADH or oxyhemoglobin; (2) changes in fluorescence or absorbance of voltage-sensitive membrane dyes; and (3) changes in fluorescence or absorbance of calcium-sensitive indicator dyes. Of these, the most widely used approach is fluorescent microscopy of calcium-sensitive dyes. This unit describes protocols for the use of calcium-sensitive dyes and voltage-dependent dyes for studies of neuronal activity in culture, tissue slices, and en-bloc preparations of the central nervous system.

  9. The bioavailability of calcium in spinach and calcium-oxalate to calcium-deficient rats.

    PubMed

    Kikunaga, S; Arimori, M; Takahashi, M

    1988-04-01

    We estimated the utilization of calcium in spinach and calcium-oxalate to calcium-deficient rats, and the effect of oxalic acid on absorption of dietary calcium by using calcium-deficient rats. The body weight gain of the calcium-deficient rats for 8 days receiving a calcium-deficient diet supplemented with raw-powdered spinach (R-sp), boiled-powdered spinach (B-sp), or calcium-oxalate (Ca-ox), and a control diet supplemented with oxalic acid (OX-C) were 4.8, 2.8, 4.9, and 5.1 g, respectively. The calcium content in the liver and kidney of the rats receiving R-sp, B-sp, Ca-ox, and OX-C diets significantly increased as compared with the calcium-deficient rats. Significant differences in the liver calcium levels were not observed among the rats receiving various additional diets, though the content in the kidneys of the rats receiving R-sp, B-sp, Ca-ox, and OX-C diets were 28.0, 21.5, 0.11, and 0.59 mg, respectively. An especially large amount of calcium was accumulated in the kidneys of the rats receiving R-sp and B-sp diets. The calcium concentration in the serum of the rats receiving Ca-ox and OX-C diets was higher than the calcium concentration in the serum of the R-sp, B-sp, and calcium-deficient rats. The calcium content in the left tibiae of the rats receiving Ca-ox and OX-C diets was higher than that of the rats receiving R-sp and B-sp diets. The breaking force of the right tibiae of the rats was highest in the OX-C group, and higher in the R-sp and Ca-ox groups than the breaking force of the right tibiae of the rats fed on B-sp diet. The alkaline phosphatase activity in the small intestines of the rats rose in the order of the R-sp, B-sp, and Ca-ox groups, although significant differences of the activity were not observed between the Ca-ox and the OX-C groups. The calcium retention of the rats receiving the calcium-deficient, R-sp, B-sp, Ca-ox, and OX-C diets was -18.5, 35.2, 25.6, 41.6, and 45.8%, respectively. About 35% of the calcium in the spinach was

  10. Calcium in Plants

    PubMed Central

    WHITE, PHILIP J.; BROADLEY, MARTIN R.

    2003-01-01

    Calcium is an essential plant nutrient. It is required for various structural roles in the cell wall and membranes, it is a counter‐cation for inorganic and organic anions in the vacuole, and the cytosolic Ca2+ concentration ([Ca2+]cyt) is an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges. This article provides an overview of the nutritional requirements of different plants for Ca, and how this impacts on natural flora and the Ca content of crops. It also reviews recent work on (a) the mechanisms of Ca2+ transport across cellular membranes, (b) understanding the origins and specificity of [Ca2+]cyt signals and (c) characterizing the cellular [Ca2+]cyt‐sensors (such as calmodulin, calcineurin B‐like proteins and calcium‐dependent protein kinases) that allow plant cells to respond appropriately to [Ca2+]cyt signals. PMID:12933363

  11. Limestone reaction in calcium aluminate cement–calcium sulfate systems

    SciTech Connect

    Bizzozero, Julien Scrivener, Karen L.

    2015-10-15

    This paper reports a study of ternary blends composed of calcium aluminate cement, calcium sulfate hemihydrate and limestone. Compressive strength tests and hydration kinetics were studied as a function of limestone and calcium sulfate content. The phase evolution and the total porosity were followed and compared to thermodynamic simulation to understand the reactions involved and the effect of limestone on these binders. The reaction of limestone leads to the formation of hemicarboaluminate and monocarboaluminate. Increasing the ratio between sulfate and aluminate decreases the extent of limestone reaction.

  12. Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

    PubMed Central

    Asmat, Tauseef M.; Tenenbaum, Tobias; Jonsson, Ann-Beth

    2014-01-01

    The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells. PMID:25464500

  13. Privileged crosstalk between TRPV1 channels and mitochondrial calcium shuttling machinery controls nociception.

    PubMed

    Nita, Iulia I; Caspi, Yaki; Gudes, Sagi; Fishman, Dimitri; Lev, Shaya; Hersfinkel, Michal; Sekler, Israel; Binshtok, Alexander M

    2016-12-01

    The nociceptive noxious heat-activated receptor - TRPV1, conducts calcium and sodium, thus producing a depolarizing receptor potential, leading to activation of nociceptive neurons. TRPV1-mediated calcium and sodium influx is negatively modulated by calcium, via calcium-dependent desensitization of TRPV1 channels. A mitochondrial Ca(2+) uniporter - MCU, controls mitochondrial Ca(2+) entry while a sodium/calcium transporter - NCLX shapes calcium and sodium transients by mediating sodium entry into and removing calcium from the mitochondria. The functional interplay between TRPV1, MCU and NCLX, in controlling the cytosolic and mitochondrial calcium and sodium transients and subsequently the nociceptive excitability, is poorly understood. Here, we used cytosolic and mitochondrial fluorescent calcium and sodium imaging together with electrophysiological recordings of TRPV1-induced currents in HEK293T cells and nociceptor-like dissociated rat dorsal root ganglion neurons, while modulating NCLX or MCU expression using specific small interfering RNA (siNCLX). We show that the propagation of the TRPV1-induced cytosolic calcium and sodium fluxes into mitochondria is dependent on coordinated activity of NCLX and MCU. Thus, knocking-down of NCLX triggers down regulation of MCU dependent mitochondrial Ca(2+) uptake. This in turn decreases rate and amplitude of TRPV1-mediated cytosolic calcium, which inhibits capsaicin-induced inward current and neuronal firing. TRPV1-mediated currents were fully rescued by intracellular inclusion of the fast calcium chelator BAPTA. Finally, NCLX controls capsaicin-induced cell death, by supporting massive mitochondrial Ca(2+) shuttling. Altogether, our results suggest that NCLX, by regulating cytosolic and mitochondrial ionic transients, modulates calcium-dependent desensitization of TRPV1 channels, thereby, controlling nociceptive signaling.

  14. Defoaming effect of calcium soap.

    PubMed

    Zhang, Hui; Miller, Clarence A; Garrett, Peter R; Raney, Kirk H

    2004-11-15

    The effect of calcium oleate on foam stability was studied for aqueous solutions of two commonly used surfactants (anionic and nonionic) under alkaline conditions in the absence of oil. For the anionic surfactant, defoaming by calcium oleate appears to involve two mechanisms. One is that oleate and calcium ions are presumably incorporated into the surfactant monolayers with a resulting decrease in the maximum of the disjoining pressure curve and therefore produces less stable thin films. The other is bridging of the films by calcium oleate particles. The latter mechanism was especially important in freshly made solutions where precipitation in the aqueous phase was still occurring when the foam was generated. Foams generated after aging (hours) when precipitation was nearly complete were more stable even though solution turbidities were greater. Foams of the nonionic surfactant were less stable than those of the anionic surfactant but were also destabilized by sufficient amounts of calcium oleate and exhibited a similar aging effect. A simplified model was developed for estimating the sodium oleate concentration at which precipitation commences in solutions of the anionic surfactant containing dissolved calcium. It includes enhancement of calcium content in the electrical double layers of the surfactant micelles. Predictions of the model were in agreement with experiment.

  15. Calcium homeostasis in diabetes mellitus.

    PubMed

    Heath, H; Lambert, P W; Service, F J; Arnaud, S B

    1979-09-01

    Experimentally diabetic rats have low serum 1,25-dihydroxyvitamin D, intestinal malabsorption of calcium, secondary hyperparathyroidism, and bone loss. To examine the hypothesis that abnormalities similar to those in the diabetic rat might explain human diabetic osteopenia, we studied calcium metabolism in 40 healthy control and 82 diabetic patients aged 18--75 yr [47 untreated: fasting plasma glucose (mean +/- SE), 267 +/- 8 mg/dl; 19 treated but hyperglycemic: glucose 305 +/- 24 mg/dl; 16 treated and in better control: glucose, 146 +/- 8 mg/dl]. Serum total calcium, ionic calcium, immunoreactive parathyroid hormone (Arnaud method, GP-1M and CH-12M antisera), 25-hydroxyvitamin D (Haddad method), and 1,25-dihydroxyvitamin D (Lambert method) concentrations were normal in all 3 groups of diabetics and were not significantly different from values in the control group. We determined absorption of calcium from the intestine by a double isotope method (100 mg Ca carrier; normal range, 40--80%) in 11 control and 13 untreated, uncontrolled diabetics (mean plasma glucose, 285 +/- 17 mg/dl). Absorption of calcium in controls was 60 +/- 3% and in diabetics was 56 +/- 3% (not significantly different). We have found no derangement of calcium metabolism in adults with insulin-requiring juvenile- and adult-onset diabetes regardless of treatment status. The experimental diabetic rat model does not appear to be useful for determining the pathogenesis of adult human diabetic osteopenia.

  16. Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

    DTIC Science & Technology

    2007-03-01

    non-linear imaging modality( 2 ), which combines current state of the art imaging techniques (fluorescence spectroscopy and fluorescence lifetime...calibrated and helped us to confirm the fluorescence source from living cells under our imaging conditions (789nm 2 -photon excitation) is indeed NADH... Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.” Exp Cell Res 309( 2 ): 390-6. DeMali, K. A. and Burridge, K. (2003

  17. Coronary artery calcium measurement with multi-detector row CT: in vitro assessment of effect of radiation dose.

    PubMed

    Hong, Cheng; Bae, Kyongtae T; Pilgram, Thomas K; Suh, Jongdae; Bradley, David

    2002-12-01

    The authors assessed in vitro the effect of radiation dose on coronary artery calcium quantification with multi-detector row computed tomography. A cardiac phantom with calcified cylinders was scanned at various milliampere second settings (20-160 mAs). A clear tendency was found for image noise to decrease as tube current increased (P <.001). No tendency was found for the Agatson score or calcium volume and mass errors to vary with tube current. Calcium measurements were not significantly affected by the choice of tube current. Calcium mass error was strongly correlated with calcium volume error (P <.001). The calcium mass measurement was more accurate and less variable than the calcium volume measurement.

  18. Calcium signals in olfactory neurons.

    PubMed

    Tareilus, E; Noé, J; Breer, H

    1995-11-09

    Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

  19. Stochastic Modeling of Calcium in 3D Geometry

    PubMed Central

    Mazel, Tomáš; Raymond, Rebecca; Raymond-Stintz, Mary; Jett, Stephen; Wilson, Bridget S.

    2009-01-01

    Release of inflammatory mediators by mast cells in type 1 immediate-hypersensitivity allergic reactions relies on antigen-dependent increases in cytosolic calcium. Here, we used a series of electron microscopy images to build a 3D reconstruction representing a slice through a rat tumor mast cell, which then served as a basis for stochastic modeling of inositol-trisphosphate-mediated calcium responses. The stochastic approach was verified by reaction-diffusion modeling within the same geometry. Local proximity of the endoplasmic reticulum to either the plasma membrane or mitochondria is predicted to differentially impact local inositol trisphosphate receptor transport. The explicit consideration of organelle spatial relationships represents an important step toward building a comprehensive, realistic model of cellular calcium dynamics. PMID:19254531

  20. 21 CFR 184.1187 - Calcium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005.... Calcium alginate is prepared by the neutralization of purified alginic acid with appropriate pH...

  1. 21 CFR 172.410 - Calcium silicate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium silicate. 172.410 Section 172.410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents § 172.410 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be...

  2. 21 CFR 172.410 - Calcium silicate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium silicate. 172.410 Section 172.410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents § 172.410 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be...

  3. 21 CFR 172.410 - Calcium silicate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium silicate. 172.410 Section 172.410 Food and... PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Anticaking Agents § 172.410 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be safely used in food in accordance with...

  4. 21 CFR 172.410 - Calcium silicate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium silicate. 172.410 Section 172.410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents § 172.410 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be...

  5. 21 CFR 172.410 - Calcium silicate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium silicate. 172.410 Section 172.410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Agents § 172.410 Calcium silicate. Calcium silicate, including synthetic calcium silicate, may be...

  6. 21 CFR 184.1201 - Calcium glycerophosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium glycerophosphate. 184.1201 Section 184... as GRAS § 184.1201 Calcium glycerophosphate. (a) Calcium glycerophosphate (C3H7CaO6P, CAS Reg. No... mixture of calcium β-, and D-, and L-α-glycerophosphate. (b) The ingredient meets the specifications...

  7. Children's Bone Health and Calcium

    MedlinePlus

    ... Research Information Clinical Trials Resources and Publications Children's Bone Health and Calcium: Condition Information Skip sharing on ... media links Share this: Page Content What is bone health and how do you build strong bones? ...

  8. Calcium and Arrhythmogenesis

    PubMed Central

    Ter Keurs, Henk E. D. J.; Boyden, Penelope A.

    2010-01-01

    Triggered activity in cardiac muscle and intracellular Ca2+ have been linked in the past. However, today not only are there a number of cellular proteins that show clear Ca2+ dependence but also there are a number of arrhythmias whose mechanism appears to be linked to Ca2+-dependent processes. Thus we present a systematic review of the mechanisms of Ca2+ transport (forward excitation-contraction coupling) in the ventricular cell as well as what is known for other cardiac cell types. Second, we review the molecular nature of the proteins that are involved in this process as well as the functional consequences of both normal and abnormal Ca2+ cycling (e.g., Ca2+ waves). Finally, we review what we understand to be the role of Ca2+ cycling in various forms of arrhythmias, that is, those associated with inherited mutations and those that are acquired and resulting from reentrant excitation and/or abnormal impulse generation (e.g., triggered activity). Further solving the nature of these intricate and dynamic interactions promises to be an important area of research for a better recognition and understanding of the nature of Ca2+ and arrhythmias. Our solutions will provide a more complete understanding of the molecular basis for the targeted control of cellular calcium in the treatment and prevention of such. PMID:17429038

  9. Calcium transport in turtle bladder

    SciTech Connect

    Sabatini, S.; Kurtzman, N.A. )

    1987-12-01

    Unidirectional {sup 45}Ca fluxes were measured in the turtle bladder under open-circuit and short-circuit conditions. In the open-circuited state net calcium flux (J{sup net}{sub Ca}) was secretory (serosa to mucosa). Ouabain reversed J{sup net}{sub Ca} to an absorptive flux. Amiloride reduced both fluxes such that J{sup net}{sub Ca} was not significantly different from zero. Removal of mucosal sodium caused net calcium absorption; removal of serosal sodium caused calcium secretion. When bladders were short circuited, J{sup net}{sub Ca} decreased to approximately one-third of control value but remained secretory. When ouabain was added under short-circuit conditions, J{sup net}{sub Ca} was similar in magnitude and direction to ouabain under open-circuited conditions (i.e., absorptive). Tissue {sup 45}Ca content was {approx equal}30-fold lower when the isotope was placed in the mucosal bath, suggesting that the apical membrane is the resistance barrier to calcium transport. The results obtained in this study are best explained by postulating a Ca{sup 2+}-ATPase on the serosa of the turtle bladder epithelium and a sodium-calcium antiporter on the mucosa. In this model, the energy for calcium movement would be supplied, in large part, by the Na{sup +}-K{sup +}-ATPase. By increasing cell sodium, ouabain would decrease the activity of the mucosal sodium-calcium exchanger (or reverse it), uncovering active calcium transport across the serosa.

  10. Medical therapy, calcium oxalate urolithiasis

    NASA Technical Reports Server (NTRS)

    Ruml, L. A.; Pearle, M. S.; Pak, C. Y.

    1997-01-01

    The development of diagnostic protocols that identify specific risk factors for calcium oxalate nephrolithiasis has led to the formulation of directed medical regimens that are aimed at correcting the underlying metabolic disturbances. Initiation of these treatment programs has reduced markedly the rate of stone formation in the majority of patients who form stones. This article discusses the rationale that underlies the choice of medical therapy for the various pathophysiologic causes of calcium oxalate nephrolithiasis and the appropriate use of available medications.

  11. Using MRI to detect and differentiate calcium oxalate and calcium hydroxyapatite crystals in air-bubble-free phantom.

    PubMed

    Mustafi, Devkumar; Fan, Xiaobing; Peng, Bo; Foxley, Sean; Palgen, Jeremy; Newstead, Gillian M

    2015-12-01

    Calcium oxalate (CaOX) crystals and calcium hydroxyapatite (CaHA) crystals were commonly associated with breast benign and malignant lesions, respectively. In this research, CaOX (n = 6) and CaHA (n = 6) crystals in air-bubble-free agarose phantom were studied and characterized by using MRI at 9.4 T scanner. Calcium micro-crystals, with sizes that ranged from 200 to 500 µm, were made with either 99% pure CaOX or CaHA powder and embedded in agar to mimic the dimensions and calcium content of breast microcalcifications in vivo. MRI data were acquired with high spatial resolution T2-weighted (T2W) images and gradient echo images with five different echo times (TEs). The crystal areas were determined by setting the threshold relative to agarose signal. The ratio of crystal areas was calculated by the measurements from gradient echo images divided by T2W images. Then the ratios as a function of TE were fitted with the radical function. The results showed that the blooming artifacts due to magnetic susceptibility between agar and CaHA crystals were more than twice as large as the susceptibility in CaOX crystals (p < 0.05). In addition, larger bright rings were observed on gradient echo images around CaHA crystals compared to CaOX crystals. Our results suggest that MRI may provide useful information regarding breast microcalcifications by evaluating the apparent area of crystal ratios obtained between gradient echo and T2W images.

  12. Calcium in plant defence-signalling pathways.

    PubMed

    Lecourieux, David; Ranjeva, Raoul; Pugin, Alain

    2006-01-01

    In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

  13. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  14. Calcium metabolism and cardiovascular function after spaceflight

    NASA Technical Reports Server (NTRS)

    Hatton, Daniel C.; Yue, Qi; Dierickx, Jacqueline; Roullet, Chantal; Otsuka, Keiichi; Watanabe, Mitsuaki; Coste, Sarah; Roullet, Jean Baptiste; Phanouvang, Thongchan; Orwoll, Eric; Orwoll, Shiela; McCarron, David A.

    2002-01-01

    To determine the influence of dietary calcium on spaceflight-induced alterations in calcium metabolism and blood pressure (BP), 9-wk-old spontaneously hypertensive rats, fed either high- (2%) or low-calcium (0.02%) diets, were flown on an 18-day shuttle flight. On landing, flight animals had increased ionized calcium (P < 0.001), elevated parathyroid hormone levels (P < 0.001), reduced calcitonin levels (P < 0.05), unchanged 1,25(OH)(2)D(3) levels, and elevated skull (P < 0.01) and reduced femur bone mineral density. Basal and thrombin-stimulated platelet free calcium (intracellular calcium concentration) were also reduced (P < 0.05). There was a tendency for indirect systolic BP to be reduced in conscious flight animals (P = 0.057). However, mean arterial pressure was elevated (P < 0.001) after anesthesia. Dietary calcium altered all aspects of calcium metabolism (P < 0.001), as well as BP (P < 0.001), but the only interaction with flight was a relatively greater increase in ionized calcium in flight animals fed low- compared with high-calcium diets (P < 0.05). The results indicate that 1) flight-induced disruptions of calcium metabolism are relatively impervious to dietary calcium in the short term, 2) increased ionized calcium did not normalize low-calcium-induced elevations of BP, and 3) parathyroid hormone was paradoxically increased in the high-calcium-fed flight animals after landing.

  15. Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

    PubMed

    Ludwar, Bjoern Ch; Evans, Colin G; Cropper, Elizabeth C

    2012-07-15

    It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record

  16. Calcium signalling and calcium channels: Evolution and general principles

    PubMed Central

    Verkhratsky, Alexei; Parpura, Vladimir

    2014-01-01

    Calcium as a divalent ion was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, by neurotransmitters, by second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms. PMID:24291103

  17. Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical ...

  18. A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga

    PubMed Central

    Sviben, Sanja; Gal, Assaf; Hood, Matthew A.; Bertinetti, Luca; Politi, Yael; Bennet, Mathieu; Krishnamoorthy, Praveen; Schertel, Andreas; Wirth, Richard; Sorrentino, Andrea; Pereiro, Eva; Faivre, Damien; Scheffel, André

    2016-01-01

    Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism. PMID:27075521

  19. Calcium-binding sites on sensory processes in vertebrate hair cells.

    PubMed Central

    Moran, D T; Rowley, J C; Asher, D L

    1981-01-01

    Vertebrate lateral line and vestibular systems center their function on highly mechanosensitive hair cells. Each hair cell is equipped with one kinocilium (which resembles a motile cilium) and 50-100 actin-containing stereocilia (which resemble microvilli) at the site of stimulus reception. This report describes electron-microscopic localization of calcium-binding sites on the sensory processes of vertebrate hair cells. Using the Oschman-Wall technique for calcium localization [Oschman, J. L. & Wall, B. J. (1972) J. Cell Biol. 55, 58-73] together with electron-probe x-ray microanalysis of thin sections, we observed: (i) calcium- and iron-containing deposits in the region of the ciliary necklace in goldfish lateral line hair cells, (ii) calcium deposits upon the surface of stereocilia of hair cells of the bullfrog inner ear, and (iii) calcium deposits upon stereocilia of hair cells of the guinea pig vestibular system. Images PMID:6973762

  20. [Calcium metabolism characteristics in microgravity].

    PubMed

    Grigor'ev, A I; Larina, I M; Morukov, B V

    1999-06-01

    The results of research of calcium exchange parameters at cosmonauts taken part in long space flights (SF) onboard of orbital stations "SALUT" and "MIR" within 1978-1998 were generalized. The analysis of data received during observation of 44 cosmonauts (18 of them have taken part in long SF twice) was done. The observation was carried out before and after SF by duration 30-438 days. The content of a total calcium in blood serum was increased basically by the increase of its ionized fraction after flights of moderate (3-6 months) and large duration (6-14 months) along with the significant increase of PTH and decrease of calcitonin levels. The content of osteocalcin after SF was increased. Three cosmonauts participated in research of calcium kinetics using stable isotopes before, in time and after a 115-day SF. Reduction of intestinal absorption, excretion through a gastrointestinal tract, and increase of calcium excretion with urine were marked in time of SF. In early postflight period a level of intestinal absorption, on the average, was much lower than in SF, and the calcium removal through intestine was increased. Both renal and intestinal excretion of calcium were not normalized in 3.5-4.5 months after end of SF. Increase of resorbtive processes in bone tissues which induced negative bone balance during flight was observed in all test subjects, proceeding from estimations of speed of the basic calcium flows made on the basis of mathematical modeling. The conclusion about decrease in speed of bone tissue remodeling and strengthening of its resorption proves to be true by data of research of biochemical and endocrine markers.

  1. Calcium supplement: humanity's double-edged sword.

    PubMed

    Bunyaratavej, Narong; Buranasinsup, Shutipen

    2011-10-01

    The principle aim of the present study is to investigate the dark side of calcium, pollutions in calcium preparation especially lead (Pb), mercury (Hg) and cadmium (Cd). The collected samples were the different calcium salts in the market and 18 preparations which were classified into 3 groups: Calcium carbonate salts, Chelated calcium and natural-raw calcium. All samples were analyzed for lead, cadmium and mercury by inductively Coupled Plasma Mass Spectrometry (ICP-MS) technique, in house method based on AOAC (2005) 999.10 by ICP-MS. The calcium carbonate and the natural-raw calcium in every sample contained lead at 0.023-0.407 mg/kg of calcium powder. Meanwhile, the natural-raw calcium such as oyster, coral and animal bone showed amount of lead at 0.106-0.384 mg/kg with small amounts of mercury and cadmium. The chelated calcium such as calcium gluconate, calcium lactate and calcium citrate are free of lead.

  2. Calcium bioavailability and its relation to osteoporosis.

    PubMed

    Weaver, C M

    1992-06-01

    The balance of data suggests that calcium intake has a positive influence on bone mass in premenopausal women and has a preventive effect on the rate of bone loss in postmenopausal women. Even small advantages in bone mass provide great reductions in fracture rates. However, the majority of studies have tested the relationship of calcium intake and bone mass using calcium supplements. Few intervention studies have manipulated calcium intake through foods. Calcium is only useful to the skeleton once it is absorbed. Therefore, the bioavailability of dietary calcium becomes important in the prevention and treatment of osteoporosis. Isotopic tracer techniques have only recently been employed in the labeling of foods with calcium isotopes for evaluation of calcium absorption. Milk calcium is usually the referent food which is typically absorbed at 20-40% depending on the calcium status of the subject. The absorptive efficiency of most vegetable sources is as good or better than for dairy foods, unless they have high concentrations of oxalic acid (spinach, for example) or phytic acid (wheat bran cereal, for example). Few vegetable sources are concentrated sources of calcium. Therefore, it would be difficult to obtain adequate intakes of calcium to protect against osteoporosis without liberal use of dairy products in the diet. Alternately, calcium supplements provide concentrated amounts of absorbable calcium, but they do not provide other nutrients necessary for skeletal growth and maintenance.

  3. Neurovascular Network Explorer 1.0: a database of 2-photon single-vessel diameter measurements with MATLAB(®) graphical user interface.

    PubMed

    Sridhar, Vishnu B; Tian, Peifang; Dale, Anders M; Devor, Anna; Saisan, Payam A

    2014-01-01

    We present a database client software-Neurovascular Network Explorer 1.0 (NNE 1.0)-that uses MATLAB(®) based Graphical User Interface (GUI) for interaction with a database of 2-photon single-vessel diameter measurements from our previous publication (Tian et al., 2010). These data are of particular interest for modeling the hemodynamic response. NNE 1.0 is downloaded by the user and then runs either as a MATLAB script or as a standalone program on a Windows platform. The GUI allows browsing the database according to parameters specified by the user, simple manipulation and visualization of the retrieved records (such as averaging and peak-normalization), and export of the results. Further, we provide NNE 1.0 source code. With this source code, the user can database their own experimental results, given the appropriate data structure and naming conventions, and thus share their data in a user-friendly format with other investigators. NNE 1.0 provides an example of seamless and low-cost solution for sharing of experimental data by a regular size neuroscience laboratory and may serve as a general template, facilitating dissemination of biological results and accelerating data-driven modeling approaches.

  4. Influence of phosvitin and calcium gluconate concentration on permeation and intestinal absorption of calcium ions.

    PubMed

    Dolińska, Barbara; Łopata, Katarzyna; Mikulska, Agnieszka; Leszczyńska, Lucyna; Ryszka, Florian

    2012-06-01

    The effect of egg yolk phosvitin on the permeation and absorption of calcium was investigated in vitro in relation to calcium gluconate concentration. Obtained results indicate that phosvitin significantly reduces the intestinal calcium absorption from 1 and 10 mM of calcium gluconate solution. It is associated with the formation of the complex of Ca (II) ions with phosvitin. The process of calcium permeation increases under phosvitin influence when calcium gluconate concentrations rise up to 10 mM. At a higher concentration of calcium gluconate (20 mM), no effect of phosvitin was seen on permeation of calcium ions.

  5. Pathogenesis and treatment of calcium stones.

    PubMed

    Parks, J H; Coe, F L

    1996-09-01

    Calcium stones arise from imbalances between urinary excretions of insoluble salts and water. Idiopathic hypercalciuria and hyperparathyroidism are the calcium disorders usually associated with elevated levels of calcium in the urine. Renal tubular acidosis is associated with a disordered acid-base status that results in low urine citrate. Hypocitraturia itself is a cause of calcium stones because it leaves urine calcium free to complex with either oxalate or phosphate. Elevated urine oxalate is commonly associated with dietary excesses, bowel disease, and, rarely, primary hyperoxaluria. Hyperuricosuria, usually of dietary origin, when reversed can cause a fall in new calcium stones.

  6. Calcium scoring with dual-energy CT in men and women: an anthropomorphic phantom study

    NASA Astrophysics Data System (ADS)

    Li, Qin; Liu, Songtao; Myers, Kyle; Gavrielides, Marios A.; Zeng, Rongping; Sahiner, Berkman; Petrick, Nicholas

    2016-03-01

    This work aimed to quantify and compare the potential impact of gender differences on coronary artery calcium scoring with dual-energy CT. An anthropomorphic thorax phantom with four synthetic heart vessels (diameter 3-4.5 mm: female/male left main and left circumflex artery) were scanned with and without female breast plates. Ten repeat scans were acquired in both single- and dual-energy modes and reconstructed at six reconstruction settings: two slice thicknesses (3 mm, 0.6 mm) and three reconstruction algorithms (FBP, IR3, IR5). Agatston and calcium volume scores were estimated from the reconstructed data using a segmentation-based approach. Total calcium score (summation of four vessels), and male/female calcium scores (summation of male/female vessels scanned in phantom without/with breast plates) were calculated accordingly. Both Agatston and calcium volume scores were found comparable between single- and dual-energy scans (Pearson r= 0.99, p<0.05). The total calcium scores were larger for the thinner slice thickness. Among the scores obtained from the three reconstruction algorithms, FBP yielded the highest and IR5 yielded the lowest scores. The total calcium scores from the phantom without breast plates were significantly larger than those from the phantom with breast plates, and the difference increased with the stronger denoising in iterative algorithm and with thicker slices. Both gender-based anatomical differences and vessel size impacted the calcium scores. The calcium volume scores tended to be underestimated when the vessels were smaller. These findings are valuable for understanding inconsistencies between women and men in calcium scoring, and for standardizing imaging protocols for improved gender-specific calcium scoring.

  7. Sound-evoked network calcium transients in mouse auditory cortex in vivo.

    PubMed

    Grienberger, Christine; Adelsberger, Helmuth; Stroh, Albrecht; Milos, Ruxandra-Iulia; Garaschuk, Olga; Schierloh, Anja; Nelken, Israel; Konnerth, Arthur

    2012-02-15

    Population calcium signals generated by the action potential activity of local clusters of neurons have been recorded in the auditory cortex of mice using an optical fibre-based approach. These network calcium transients (NCaTs) occurred spontaneously as well as in response to sound stimulation. Two-photon calcium imaging experiments suggest that neurons and neuropil contribute about equally to the NCaT. Sound-evoked calcium signals had two components: an early, fast increase in calcium concentration, which corresponds to the short-latency spiking responses observed in electrophysiological experiments, and a late, slow calcium transient which lasted for at least 1 s. The slow calcium transients evoked by sound were essentially identical to spontaneous NCaTs. Their sizes were dependent on the spontaneous activity level at sound onset, suggesting that spontaneous and sensory-evoked NCaTs excluded each other. When using pure tones as stimulus, the early evoked calcium transients were more narrowly tuned than the slow NCaTs. The slow NCaTs were correlated with global ‘up states' recorded with epidural potentials, and sound presented during an epidural ‘down state' triggered a calcium transient that was associated with an epidural ‘up state'. Essentially indistinguishable calcium transients were evoked by optogenetic activation of local clusters of layer 5 pyramidal neurons in the auditory cortex, indicating that these neurons play an important role in the generation of the calcium signal. Taken together, our results identify sound-evoked slow NCaTs as an integral component of neuronal signalling in the mouse auditory cortex, reflecting the prolonged neuronal activity of local clusters of neurons that can be activated even by brief stimuli.

  8. In vitro atherosclerotic plaque and calcium quantitation by intravascular ultrasound and electron-beam computed tomography.

    PubMed

    Gutfinger, D E; Leung, C Y; Hiro, T; Maheswaran, B; Nakamura, S; Detrano, R; Kang, X; Tang, W; Tobis, J M

    1996-05-01

    The purpose of this investigation was to compare the accuracy of intravascular ultrasound (IVUS) and electron-beam computed tomography (EBCT) in quantitating human atherosclerotic plaque and calcium. In experiment 1, 12 human atherosclerotic arterial segments were obtained at autopsy and imaged by using IVUS and EBCT. The plaque from each arterial segment was dissected and a volume measurement of the dissected plaque was obtained by water displacement. The plaque from each arterial segment was ashed at 700 degrees F, and the weight of the remaining ashes was used as an estimate of the calcium mass. In experiment II, 11 calcified arterial segments were obtained at autopsy and imaged by using IVUS at one site along the artery. A corresponding histologic cross section stained with Masson's trichrome was prepared. In experiment I, the mean plaque volume measured by water displacement was 165.3 +/- 118.4 microliters. The mean plaque volume calculated by IVUS was 166.1 +/- 114.4 microliters and correlated closely with that by water displacement (r = 0.98, p < 0.0001). The mean calcium mass measured by ashing was 19.4 +/- 15.8 mg. The mean calculated calcium mass by EBCT was 19.9 mg and correlated closely with that by ashing (r=0.98, p<0.001). The mean calculated calcium volume by IVUS was 18.6 +/- 11.2 microliters and correlated linearly with the calcium mass by ashing (r = 0.87, p < 0.0003). In experiment II, the mean cross-sectional area of the calcified matrix was 1.71 +/- 0.66 mm2 by histologic examination compared with 1.44 +/- 0.66 mm2 by IVUS. There was a good correlation between the calcified cross-sectional area by histologic examination and IVUS (r = 0.76, p < 0.007); however, IVUS may underestimate the amount of calcium present depending on the intralesional calcium morphologic characteristics. In conclusion, IVUS accurately quantitates atherosclerotic plaque volume as well as the cross-sectional area and volume of intralesional calcium, especially if the

  9. The effect of variable calcium and very low calcium diets on human calcium metabolism. Ph.D. Thesis. Final Report

    NASA Technical Reports Server (NTRS)

    Chu, J.

    1971-01-01

    The effects of a very low calcium diet, with variable high and low protein intake, on the dynamics of calcium metabolism and the mechanism of calciuretics, are examined. The experiment, using male subjects, was designed to study the role of intestinal calcium absorption on urinary calcium excretion, and the rate of production of endogeneously secreted calcium in the gastrointestinal tract. The study showed an average of 70% fractional absorption rate during very low calcium intake, and that a decrease in renal tubular reabsorption of calcium is responsible for calciuretic effects of high protein intake. The study also indicates that there is a tendency to develop osteoporosis after long periods of low calcium intake, especially with a concurrent high protein intake.

  10. Chemical calcium indicators.

    PubMed

    Paredes, R Madelaine; Etzler, Julie C; Watts, Lora Talley; Zheng, Wei; Lechleiter, James D

    2008-11-01

    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 are widely used for the investigation of intracellular Ca2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators now available allow for intracellular Ca2+ detection over a very large range (<50 nM to >50 microM). High affinity indicators can be used to quantify Ca2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca2+ detection when more than one fluorophore is being imaged. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca2+ indicators including uneven dye loading, leakage, photobleaching, and changes in cell volume. Recent technical advances that permit in vivo Ca2+ measurements will also be discussed.

  11. Coronary artery calcium in breast cancer survivors after radiation therapy.

    PubMed

    Takx, Richard A P; Vliegenthart, Rozemarijn; Schoepf, U Joseph; Pilz, Lothar R; Schoenberg, Stefan O; Morris, Pamela B; Henzler, Thomas; Apfaltrer, Paul

    2017-03-24

    The purpose of the current study is to investigate whether breast cancer survivors after radiation therapy have a higher burden of coronary artery calcium as a potential surrogate of radiation-induced accelerated coronary artery disease. 333 patients were included. 54 patients underwent chest CT ≥ 6 months after the start of radiation therapy (radiation therapy group), while 279 patients had a CT scan either prior to or without undergoing radiation therapy (RT). Coronary artery calcium was quantified from CT by applying a threshold-based automated algorithm. Mean age at diagnosis was similar (p = 0.771) between RT (57.4 ± 13.1 years) and NoRT (58.0 ± 11.9 years). Median time between radiation therapy and CT was 2 years. The groups showed no significant differences in race, smoking history, cancer laterality, or cancer stage. 39 (72.2%) of RT patients had a coronary artery calcium score of 0, compared to 201 (72.0%) in patients without radiation therapy. Median coronary artery calcium burden for both groups was not significantly different (p = 0.982), nor when comparing patients who underwent left- versus right-sided radiation therapy (p = 0.453). When adjusting for the time between diagnosis and CT, radiation therapy patients had a significantly lower risk of a positive coronary artery calcium score. In conclusion, breast cancer survivors after radiation therapy are not more likely to show coronary artery calcium on follow-up CT imaging. Our results thus do not support radiation-induced accelerated coronary artery disease as an explanation for higher rates of heart disease in this group.

  12. Calcium regulation of muscle contraction.

    PubMed Central

    Szent-Györgyi, A G

    1975-01-01

    Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain. PMID:806311

  13. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  14. Vitamin D and intestinal calcium absorption.

    PubMed

    Christakos, Sylvia; Dhawan, Puneet; Porta, Angela; Mady, Leila J; Seth, Tanya

    2011-12-05

    The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.

  15. Synchrotron X-ray microanalysis and imaging of synthetic biological calcium carbonate in comparison with archaeological samples originating from the Large cave of Arcy-sur-Cure (28000-24500 BP, Yonne, France).

    PubMed

    Chalmin, Emilie; Reiche, Ina

    2013-12-01

    Biosynthetic calcite samples were investigated using combined synchrotron X-ray microspectroscopy mapping. These samples were prepared with bacteria isolated from the Large cave of Arcy-sur-Cure in which prehistoric figures are masked by an opaque calcite layer. The biotic or abiotic origin of this layer is the issue of the present work. As previously known, a large community of bacteria may be involved in the CaCO3 formation in caves. A mixture of calcite/vaterite was obtained from bacteria isolated from the cave. Therefore, we can offer conclusions on their calcifying capability. The rare presence of vaterite in cave environments may be treated as a marker of biotic carbonate formations. Moreover, an amorphous calcium phosphate phase was present in the form of a calcite/vaterite mixture in the biotic model samples. This mixture of phases could be used as a tracer of the biotic process of CaCO3 formation. These biotic tracer phases were not identified using the applied analytical methods in the natural samples taken from the opaque calcite layers that covered the prehistoric figures of the Large cave. In this case, based on the obtained results, the biotic calcite formation process is likely to be considered as an undetectable effect at minimum.

  16. Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

    NASA Astrophysics Data System (ADS)

    Restrepo, Simon; Basler, Konrad

    2016-08-01

    Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

  17. Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

    PubMed Central

    Restrepo, Simon; Basler, Konrad

    2016-01-01

    Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies. PMID:27503836

  18. Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses.

    PubMed

    Jackson, Rachel E; Burrone, Juan

    2016-01-01

    Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses.

  19. Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses

    PubMed Central

    Jackson, Rachel E.; Burrone, Juan

    2016-01-01

    Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs) that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses. PMID:27507942

  20. Extra-intestinal calcium handling contributes to normal serum calcium levels when intestinal calcium absorption is suboptimal.

    PubMed

    Lieben, Liesbet; Verlinden, Lieve; Masuyama, Ritsuko; Torrekens, Sophie; Moermans, Karen; Schoonjans, Luc; Carmeliet, Peter; Carmeliet, Geert

    2015-12-01

    The active form of vitamin D, 1,25(OH)2D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)2D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)2D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.

  1. The effect of calcium gluconate and other calcium supplements as a dietary calcium source on magnesium absorption in rats.

    PubMed

    Chonan, O; Takahashi, R; Yasui, H; Watanuki, M

    1997-01-01

    The effects of commercially available calcium supplements (calcium carbonate, calcium gluconate, oyster shell preparation and bovine bone preparation) and gluconic acid on the absorption of calcium and magnesium were evaluated for 30 days in male Wistar rats. There were no differences in the apparent absorption ratio of calcium among rats fed each calcium supplement; however, the rats fed the calcium gluconate diet had a higher apparent absorption ratio of magnesium than the rats fed the other calcium supplements. Dietary gluconic acid also more markedly stimulated magnesium absorption than the calcium carbonate diet, and the bone (femur and tibia) magnesium contents of rats fed the gluconic acid diet were significantly higher than those of the rats fed the calcium carbonate diet. Furthermore, the weight of cecal tissue and the concentrations of acetic acid and butyric acid in cecal digesta of rats fed the calcium gluconate diet or the gluconic acid diet were significantly increased. We speculate that the stimulation of magnesium absorption in rats fed the calcium gluconate diet is a result of the gluconic acid component and the effect of gluconic acid on magnesium absorption probably results from cecal hypertrophy, magnesium solubility in the large intestine and the effects of volatile fatty acids on magnesium absorption.

  2. Bidentate iminodiacetate modified dendrimer for bone imaging.

    PubMed

    Pes, Lara; Kim, Young; Tung, Ching-Hsuan

    2017-03-01

    A new dendrimer probe was designed for bone imaging. Bidentate iminodiacetate groups were introduced to the probe to obtain strong bind to bones. The assembled dendrimeric probe, with four iminodiacetate moieties and a fluorescent tag, displayed good selectivity to hydroxyapatite, calcium oxalate and calcium phosphate salts. In mice, the probe offered vivid skeletal details after intravenous delivery.

  3. Calcium Activation of Mougeotia Potassium Channels 1

    PubMed Central

    Lew, Roger R.; Serlin, Bruce S.; Schauf, Charles L.; Stockton, Marsha E.

    1990-01-01

    Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca2+] as low as 20 micromolar. However, external [Ca2+] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID:16667356

  4. Decalcification of calcium polycarbophil in rats.

    PubMed

    Yamada, T; Saito, T; Takahara, E; Nagata, O; Tamai, I; Tsuji, A

    1997-03-01

    The in vivo decalcification of calcium polycarbophil was examined. The decalcification ratio of [45Ca]calcium polycarbophil in the stomach after oral dosing to rats was more than 70% at each designated time and quite closely followed in the in vitro decalcification curve, indicating that the greater part of the calcium ion is released from calcium polycarbophil under normal gastric acidic conditions. The residual radioactivity in rat gastrointestine was nearly equal to that after oral administration of either [45Ca]calcium chloride + polycarbophil. The serum level of radioactivity was nearly equal to that after oral dosing of [45Ca]calcium lactate. These results indicate that the greater part of orally administered calcium polycarbophil released calcium ions to produce polycarbophil in vivo.

  5. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the calcium hydroxide neutralization of acetic acid. (b) The ingredient meets...

  6. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the calcium hydroxide neutralization of acetic acid. (b) The ingredient meets...

  7. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... No. 62-54-4), also known as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the calcium hydroxide neutralization of acetic acid. (b) The ingredient meets...

  8. 21 CFR 184.1185 - Calcium acetate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... as acetate of lime or vinegar salts, is the calcium salt of acetic acid. It may be produced by the calcium hydroxide neutralization of acetic acid. (b) The ingredient meets the specifications of the...

  9. Magnesium/Calcium Competition at Excitable Membranes.

    ERIC Educational Resources Information Center

    Belzer, Bill; Fry, Panni

    1998-01-01

    Considers some consequences of altering intracellular calcium supply by magnesium concentration changes. Focuses on using this procedure as an exercise with allied health students as they witness therapeutic uses of magnesium and other calcium entry inhibitors. (DDR)

  10. Calcium, vitamin D, and your bones

    MedlinePlus

    ... not exceed 4000 IU per day Calcium and Dairy Products Milk and dairy products are the best sources of calcium. They ... 2% or 1%) milk, and other lower fat dairy products. Removing some of the fat does not ...

  11. Calcium release from experimental dental materials.

    PubMed

    Okulus, Zuzanna; Buchwald, Tomasz; Voelkel, Adam

    2016-11-01

    The calcium release from calcium phosphate-containing experimental dental restorative materials was examined. The possible correlation of ion release with initial calcium content, solubility and degree of curing (degree of conversion) of examined materials was also investigated. Calcium release was measured with the use of an ion-selective electrode in an aqueous solution. Solubility was established by the weighing method. Raman spectroscopy was applied for the determination of the degree of conversion, while initial calcium content was examined with the use of energy-dispersive spectroscopy. For examined materials, the amount of calcium released was found to be positively correlated with solubility and initial calcium content. It was also found that the degree of conversion does not affect the ability of these experimental composites to release calcium ions.

  12. The ins and outs of mitochondrial calcium.

    PubMed

    Finkel, Toren; Menazza, Sara; Holmström, Kira M; Parks, Randi J; Liu, Julia; Sun, Junhui; Liu, Jie; Pan, Xin; Murphy, Elizabeth

    2015-05-22

    Calcium is thought to play an important role in regulating mitochondrial function. Evidence suggests that an increase in mitochondrial calcium can augment ATP production by altering the activity of calcium-sensitive mitochondrial matrix enzymes. In contrast, the entry of large amounts of mitochondrial calcium in the setting of ischemia-reperfusion injury is thought to be a critical event in triggering cellular necrosis. For many decades, the details of how calcium entered the mitochondria remained a biological mystery. In the past few years, significant progress has been made in identifying the molecular components of the mitochondrial calcium uniporter complex. Here, we review how calcium enters and leaves the mitochondria, the growing insight into the topology, stoichiometry and function of the uniporter complex, and the early lessons learned from some initial mouse models that genetically perturb mitochondrial calcium homeostasis.

  13. 21 CFR 184.1191 - Calcium carbonate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... three common methods of manufacture: (1) As a byproduct in the “Lime soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the “Carbonation process”; or (3) By precipitation...

  14. Calcium Supplements: Do They Interfere with Blood Pressure Drugs?

    MedlinePlus

    ... have your blood pressure and calcium levels checked. Calcium channel blockers. When given through an intravenous (IV) line, calcium may decrease the effects of calcium channel blockers, such as nifedipine (Adalat CC, Afeditab CR, ...

  15. 21 CFR 184.1195 - Calcium citrate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... calcium carbonate. It occurs as a fine white, odorless powder and usually contains four moles of water per... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium citrate. 184.1195 Section 184.1195 Food... GRAS § 184.1195 Calcium citrate. (a) Calcium citrate (Ca3(C6H5O7)2·4H2O, CAS Reg. No. 813-0994-095)...

  16. Oxalic acid decreases calcium absorption in rats

    SciTech Connect

    Weaver, C.M.; Martin, B.R.; Ebner, J.S.; Krueger, C.A.

    1987-11-01

    Calcium absorption from salts and foods intrinsically labeled with /sup 45/Ca was determined in the rat model. Calcium bioavailability was nearly 10 times greater for low oxalate kale, CaCO/sub 3/ and CaCl/sub 2/ than from CaC/sub 2/O/sub 4/ (calcium oxalate) and spinach (high in oxalates). Extrinsic and intrinsic labeling techniques gave a similar assessment of calcium bioavailability from kale but not from spinach.

  17. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  18. Oxalic acid decreases calcium absorption in rats.

    PubMed

    Weaver, C M; Martin, B R; Ebner, J S; Krueger, C A

    1987-11-01

    Calcium absorption from salts and foods intrinsically labeled with 45Ca was determined in the rat model. Calcium bioavailability was nearly 10 times greater for low oxalate kale, CaCO3 and CaCl2 than from CaC2O4 (calcium oxalate) and spinach (high in oxalates). Extrinsic and intrinsic labeling techniques gave a similar assessment of calcium bioavailability from kale but not from spinach.

  19. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato

    ERIC Educational Resources Information Center

    Liang, Willmann

    2008-01-01

    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  20. Calcium orthophosphates and human beings

    PubMed Central

    Dorozhkin, Sergey V.

    2012-01-01

    The historical development of a scientific knowledge on calcium orthophosphates from the 1770s until 1940 is described. Many forgotten and poorly known historical facts and approaches have been extracted from old publications and then they have been analyzed, systematized and reconsidered from the modern point of view. The chosen time scale starts with the earliest available studies of 1770s (to the best of my findings, calcium orthophosphates had been unknown before), passes through the entire 19th century and finishes in 1940, because since then the amount of publications on calcium orthophosphates rapidly increases and the subject becomes too broad. Furthermore, since publications of the second half of the 20th century are easily accessible, a substantial amount of them have already been reviewed by other researchers. The reported historical findings clearly demonstrate that the substantial amount of the scientific facts and experimental approaches have been known for very many decades and, in fact, the considerable quantity of relatively recent investigations on calcium orthophosphates is just either a further development of the earlier studies or a rediscovery of the already forgotten knowledge. PMID:23507803

  1. Acute calcium pyrophosphate deposition arthropathy.

    PubMed

    Rosen, Thomas; Furman, Janet

    2016-06-01

    Acute calcium pyrophosphate deposition (CPPD) arthropathy, also called pseudogout, is common, and becomes more prevalent as patients age. The presenting symptoms are similar to both gout and septic arthritis but may be treated differently. This article describes a typical patient presentation and management from an emergency medicine and orthopedic surgery standpoint.

  2. Calcium Kinetics During Space Flight

    NASA Technical Reports Server (NTRS)

    Smith, Scott M.; OBrien, K. O.; Abrams, S. A.; Wastney, M. E.

    2005-01-01

    Bone loss during space flight is one of the most critical challenges to astronaut health on space exploration missions. Defining the time course and mechanism of these changes will aid in developing means to counteract bone loss during space flight, and will have relevance for other clinical situations that impair weight-bearing activity. Bone health is a product of the balance between bone formation and bone resorption. Early space research could not clearly identify which of these was the main process altered in bone loss, but identification of the collagen crosslinks in the 1990s made possible a clear understanding that the impact of space flight was greater on bone resorption, with bone formation being unchanged or only slightly decreased. Calcium kinetics data showed that bone resorption was greater during flight than before flight (668 plus or minus 130 vs. 427 plus or minus 153 mg/d, p less than 0.001), and clearly documented that true intestinal calcium absorption was lower during flight than before flight (233 plus or minus 87 vs. 460 plus or minus 47 mg/d, p less than 0.01). Weightlessness had a detrimental effect on the balance in bone turnover: the difference between daily calcium balance during flight (-234 plus or minus 102 mg/d) and calcium balance before flight (63 plus or minus 75 mg/d) approached 300 mg/d (p less than 0.01). These data demonstrate that the bone loss that occurs during space flight is a consequence of increased bone resorption and decreased intestinal calcium absorption. Examining the changes in bone and calcium homeostasis in the initial days and weeks of space flight, as well as at later times on missions longer than 6 months, is critical to understanding the nature of bone adaptation to weightlessness. To increase knowledge of these changes, we studied bone adaptation to space flight on the 16-day Space Shuttle Columbia (STS-107) mission. When the brave and talented crew of Columbia were lost during reentry on the tragic morning

  3. 21 CFR 582.7187 - Calcium alginate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is...

  4. 21 CFR 182.8217 - Calcium phosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium phosphate. 182.8217 Section 182.8217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8217 Calcium phosphate. (a) Product. Calcium phosphate...

  5. 21 CFR 182.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium ascorbate. 182.3189 Section 182.3189 Food... GENERALLY RECOGNIZED AS SAFE Chemical Preservatives § 182.3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is generally recognized as safe when used in...

  6. 21 CFR 182.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium ascorbate. 182.3189 Section 182.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is...

  7. 21 CFR 582.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  8. 21 CFR 582.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  9. 21 CFR 182.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium ascorbate. 182.3189 Section 182.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is...

  10. 21 CFR 182.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium ascorbate. 182.3189 Section 182.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is...

  11. 21 CFR 582.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  12. 21 CFR 182.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium ascorbate. 182.3189 Section 182.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is...

  13. 21 CFR 582.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  14. 21 CFR 582.3189 - Calcium ascorbate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  15. 21 CFR 582.1217 - Calcium phosphate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  16. 21 CFR 182.1217 - Calcium phosphate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  17. 21 CFR 582.1217 - Calcium phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  18. 21 CFR 582.1217 - Calcium phosphate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  19. 21 CFR 582.1217 - Calcium phosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  20. 21 CFR 182.1217 - Calcium phosphate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium phosphate. 182.1217 Section 182.1217 Food... GENERALLY RECOGNIZED AS SAFE Multiple Purpose GRAS Food Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic). (b) Conditions of use. This substance is...

  1. 21 CFR 182.1217 - Calcium phosphate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  2. 21 CFR 182.1217 - Calcium phosphate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  3. 21 CFR 182.1217 - Calcium phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  4. 21 CFR 582.1217 - Calcium phosphate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  5. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  6. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  7. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  8. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  9. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  10. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  11. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  12. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  13. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  14. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  15. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  16. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  17. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  18. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  19. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  20. 21 CFR 73.1070 - Calcium carbonate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... COLOR ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1070 Calcium carbonate. (a) Identity. (1) The color additive calcium carbonate is a fine, white, synthetically prepared powder consisting essentially of... 21 Food and Drugs 1 2013-04-01 2013-04-01 false Calcium carbonate. 73.1070 Section 73.1070...