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Elucidating regulators of astrocytic Ca 2+ signaling via multi-threshold event detection (MTED)
Ist Teil von
Glia, 2021-12, Vol.69 (12), p.2798-2811
Ort / Verlag
United States
Erscheinungsjahr
2021
Quelle
Wiley Online Library All Journals
Beschreibungen/Notizen
Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca
activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca
activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca
activity recordings, hindering global comparisons. Here, we present a biophysically-based analytical concept for deciphering the complex spatio-temporal changes of Ca
biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel-based multi-threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca
activity across Ca
indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. We identified extended Ca
activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long-lasting astrocytic Ca
activity. Our MTED strategy, as a parameter-free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.