Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Beerman RW

PROVIDER: S-EPMC4684902 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Beerman Rebecca W RW Matty Molly A MA Au Gina G GG Looger Loren L LL Choudhury Kingshuk Roy KR Keller Philipp J PJ Tobin David M DM

Cell reports 20151207 10

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 ...[more]

PMID: 26673320

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Project description:Calcium imaging is commonly used to record dynamic changes in excitability from axons or cell bodies in the nervous system of vertebrates. These recordings often reveal discrete calcium transients that have variable amplitudes, durations, and rates of rise and decay, all of which can arise from an unstable or "noisy" baseline. This often leads to considerable ambiguity about how to discriminate and quantify calcium transients. We describe an analytical methodology that objectively identifies multiple calcium transients from multiple recording sites and quantifies the degree of temporal synchrony between each event. The methodology consists of multiple steps. The first step involves baselining, to either preserve the underlying shape of calcium transients or remove unwanted frequency components and transform the peaks of calcium transients into more easily detectable patterns. The second step is the application of at least one of two different spike detection algorithms, one based on a gradient estimate and the other on template matching. The third step is the quantification of synchrony between pairs of recordings using at least one of two time lag correlation measures. The fourth step is the identification of statistically significant coincident firing patterns. This allows discrimination of neuronal firing patterns between different sites that appear to occur simultaneously and that statistically could not be attributed to chance. The analytical methods we have demonstrated can be applied not only to calcium imaging but also to many other physiological recordings, where discrimination and temporal correlation of biological signals from multiple sites is required, particularly when arising from unstable baselines, with variable signal-to-noise ratios.NEW & NOTEWORTHY Dynamic imaging of intracellular calcium is commonly used to record changes in excitability in central and peripheral neurons. We describe a novel analytical methodology that objectively discriminates calcium transients from low signal-to-noise recordings from multiple sites and quantifies the degree of temporal synchrony between events. These new methods can be applied not only to calcium imaging but also to many other physiological recordings where discrimination and temporal correlation of biological signals from multiple sites is required.

Dataset Information

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

Publications

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

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