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BMC Neuroscience

, 16:P299 | Cite as

Computational estimation of calcium fluxes in isolated magnocellular neurons

  • S Kortus
  • G Dayanithi
  • M Zapotocky
Open Access
Poster presentation

Keywords

Clearance Rate Thapsigargin Calcium Transient Intracellular Calcium Concentration Calcium Flux 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Current optical methods based on fluorescent indicators permit to measure the intracellular calcium concentration with a high temporal resolution. To analyze the physiological mechanisms that underly the calcium dynamics, however, knowledge of the calcium fluxes into and out of the cell is needed. Here we present a method that permits to separately estimate the influx and clearance rates, based on the measurement of Ca2+ concentration during a series of depolarization-evoked calcium transients. We apply this method to investigate calcium clearance mechanisms in isolated magnocellular neurons of the rat supraoptic nucleus.

In the simplest case, we assume that the cytoplasmic Ca2+ concentration during the transient is governed by a time-dependent influx Jinflux(t) through voltage-gated Calcium channels and by an outflux Jclearance that depends only on the instantaneous calcium concentration [Ca2+](t):
d [ Ca 2 + ] / d t = J influx ( t ) J clearance ( [ Ca 2 + ] . Open image in new window
(1)

To separate the two fluxes, we first estimate the clearance function Jclearance([Ca2+]). Near the end of the transient, Jclearance dominates over Jinflux, and Jclearance may be obtained directly as the measured Ca2+ decay rate [1]. In contrast, near the peak of a transient the two fluxes are comparable, and Jclearance therefore significantly exceeds -d[Ca2+]/dt. However, in this case the clearance rate obtained from a higher transient can be used as a good estimate. If the assumption of Eq.1 is satisfied, the clearance function is obtained as the envelope of the recorded return curves in the d[Ca2+]/dt vs. [Ca2+] plot. The calcium influx rate Jinflux(t) during each transient is then estimated by substracting Jclearance([Ca2+](t)) from the measured rate d[Ca2+]/dt. We tested the adequacy of this procedure using surrogate calcium dynamics data.

For cells in which the endoplasmic reticulum (ER) noticeably contributes to the calcium transient [2], the clearance function Jclearance is not solely dependent on the cytoplasmic calcium concentration, as was assumed above. In this case, the method described above can still be applied to experiments performed in presence of Thapsigargin or cyclopiazonic acid (CPA), to avoid release or uptake of Ca2+ by the ER. Comparison of the estimated fluxes from the experiments with and without Thapsigargin/CPA can be used to investigate the ER-dependent calcium fluxes. We apply this method to freshly isolated magnocellular neurons, in which we used Fura-2AM to measure the cytoplasmic [Ca2+] during depolarization-evoked Ca2+ transients of various amplitudes and durations; depolarization was induced [3] by changing the external K+ concentration.

Our method of estimating the Ca2+ fluxes may be used also in other cell types to help characterize the contribution of individual mechanisms to calcium dynamics.

References

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Copyright information

© Kortus et al. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Institute of Physiology of the Czech Academy of SciencesPragueCzech Republic
  2. 2.Institute of Biophysics and Informatics, First Faculty of MedicineCharles University in PraguePragueCzech Republic
  3. 3.Institute of Experimental MedicineCzech Academy of SciencesPragueCzech Republic
  4. 4.INSERM U710/EPHEUniversité Montpellier 2MontpellierFrance

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