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. 2020 Jul 28;10:12568. doi: 10.1038/s41598-020-69412-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Chitosan-coated magnetic nanoparticles (NPs) impacts synchronicity in calcium signalling. (A1–A6) Image processing analysis to extract spatially resolved neuronal connectivity map based on primary cortical neurons labelled with Fluo-4. (A1) Time-lapse images show pseudo-coloured calcium activity captured with digital fluorescent microscopy. (A2) Automated image segmentation spatially selects individual regions of interest (ROIs) based on fluorescently active cell bodies. (A3) Changes in calcium activity get recorded over time and (A4) transferred into a calcium spike raster plot based on calcium influx. (A5) A cross-correlation matrix presents high (blue) and low (red) probability of temporally connected calcium spiking activity, which is used in (A6) to connect the ROIs in a connectivity map. (B) Representing fluorescent images of primary neurons incubated for 10 h in the digital imaging system. (C1–C2) Connectivity maps present changes in synchronous calcium spiking activity over an 8-h time span in cortical neurons grown for 9 days in vitro, independent of room temperature (w/o heating), physiological temperature (w/heating) and of incubation with chitosan-coated magnetic NPs (w/NPs). (D1–F2) Network analysis shows (D1–D2) number of active neurons under different temperature conditions, with and without NPs, (E1–E2) number of connections between ROIs under different temperature conditions, with and without NPs, and (F1–F2) active cell normalized count of connections under different temperature conditions, with and without NPs. These four plots indicate the activity of the network and how it changes under the different culture conditions.