21Understanding how neural networks generate activity patterns and communicate with each 22 other requires monitoring the electrical activity from many neurons simultaneously. Perfectly 23 suited tools for addressing this challenge are genetically encoded voltage indicators (GEVIs) 24 because they can be targeted to specific cell types and optically report the electrical activity 25 of individual, or populations of neurons. However, analyzing and interpreting the data from 26 voltage imaging experiments is challenging because high recording speeds and properties of 27 current GEVIs yield only low signal-to-noise ratios, making it necessary to apply specific 28 analytical tools. Here, we present NOSA (Neuro-Optical Signal Analysis), a novel open 29 source software designed for analyzing voltage imaging data and identifying temporal 30 interactions between electrical activity patterns of different origin. 31In this manuscript we explain the challenges that arise during voltage imaging 32 experiments and provide hands-on analytical solutions. We demonstrate how NOSA's 33 baseline fitting, filtering algorithms and movement correction can compensate for shifts in 34 baseline fluorescence and extract electrical patterns from low signal-to-noise recordings. 35 Moreover, NOSA contains powerful features to identify oscillatory frequencies in electrical 36 patterns and extract neuronal firing characteristics. NOSA is the first open-access software to 37 provide an option for analyzing simultaneously recorded optical and electrical data derived 38 from patch-clamp or other electrode-based recordings. To identify temporal relations 39 between electrical activity patterns we implemented different options to perform cross 40 correlation analysis, demonstrating their utility during voltage imaging in Drosophila and 41mice. All features combined, NOSA will facilitate the first steps into using GEVIs and help to 42 realize their full potential for revealing cell-type specific connectivity and functional 43interactions. If you would like to test NOSA, please send an email to the lead contact. 44 45 3
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