An Analysis Pipeline for Two-Photon Calcium Imaging Data to Reveal the Functional Connectivity of the Mouse Somatosensory Cortex

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Investigating functional connectivity in the primary somatosensory cortex (S1) is crucial for deciphering sensory processing mechanisms, which involves different cortical layers and multiple brain regions along its pathway. Here, we present a computational pipeline for analyzing two-photon calcium imaging data to infer cortical functional connectivity from neuronal activity evoked in the mouse S1 by whisker stimulations. A dataset of imaging acquisitions performed across 3 different cortical layers in both anesthetized and awake mice was used for the development of the pipeline. Signals were collected while delivering multi-whisker air-puff stimuli to the mouse snout. Neuronal activity was extracted using the well-established CaImAn tool, followed by Savitzky-Golay filtering and thresholdbased peak detection. Functional connectivity was then estimated by analyzing activation peak delays (APD) between neuronal pairs and relative to the single consecutive stimuli, employing statistical tests to assess non-random and directionally biased interactions. Network topology was investigated using modularity and betweenness centrality. This study highlights the utility of the pipeline in unravelling brain networks and in clarifying the importance of stimulus-driven hubs in sensory processing. The methods and preliminary findings reported here offer insights into the organizational principles of the S1 and its microcircuitry involved in the encoding of whisker-related signals.

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