Abstract
BackgroundIntracellular calcium is an important ion involved in the regulation and modulation of many neuronal functions. From regulating cell cycle and proliferation to initiating signaling cascades and regulating presynaptic neurotransmitter release, the concentration and timing of calcium activity governs the function and fate of neurons. Changes in calcium transients can be used in high-throughput screening applications as a basic measure of neuronal maturity, especially in developing or immature neuronal cultures derived from stem cells.ResultsUsing human induced pluripotent stem cell derived neurons and dissociated mouse cortical neurons combined with the calcium indicator Fluo-4, we demonstrate that PeakCaller reduces type I and type II error in automated peak calling when compared to the oft-used PeakFinder algorithm under both basal and pharmacologically induced conditions.ConclusionHere we describe PeakCaller, a novel MATLAB script and graphical user interface for the quantification of intracellular calcium transients in neuronal cultures. PeakCaller allows the user to set peak parameters and smoothing algorithms to best fit their data set. This new analysis script will allow for automation of calcium measurements and is a powerful software tool for researchers interested in high-throughput measurements of intracellular calcium.
Highlights
Intracellular calcium is an important ion involved in the regulation and modulation of many neuronal functions
In this study we use Fluo-4, a [Ca2+] indicator, and analyze Ca2+ signaling in commercially available hiPSCderived cortical neurons and dissociated mouse cortical neuron cultures
The PeakFinder algorithm uses an a priori approach that assumes that a peak is present, as it denotes a peak based on the highest point within a set window, and will always produce false positives in an instance where there is a background of fluorescence
Summary
In this study we use Fluo-4, a [Ca2+] indicator, and analyze Ca2+ signaling in commercially available hiPSCderived cortical neurons and dissociated mouse cortical neuron cultures. After the application of PTX to cultured neurons, cells will enter a state of heightened excitability followed by loss of excitation after exhaustion of glutamate stores This change in excitation and inhibition can be measured by looking at the activity of Ca2+ in neurons [27]. PeakCaller accurately identified windows of high Ca2+ activity in neurons, while PeakFinder consistently overestimated the number of peaks and number of active cells (Fig. 10). In this context, PeakFinder becomes less accurate and more vulnerable to type I error as the number of true peaks decreases relative to background fluorescence. PeakCaller’s smoothing functions can be set to account for global changes in fluorescence and can be set with narrower look-back and look-ahead windows to properly identify the majority of peaks in a given ROI
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