Computational estimation of calcium fluxes in isolated magnocellular neurons
KeywordsClearance Rate Thapsigargin Calcium Transient Intracellular Calcium Concentration Calcium Flux
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.
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 . 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 , 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  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.
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