Summary Up and down states are among the most prominent features of the thalamo-cortical system during non-rapid eye movement (NREM) sleep and many forms of anesthesia. Cortical interneurons, including parvalbumin (PV) cells, display firing activity during cortical down states, and this GABAergic signaling is associated with prolonged down-state durations. However, what drives PV interneurons to fire during down states remains unclear. We here tested the hypothesis that background thalamic activity may lead to suprathreshold activation of PV cells during down states. To this aim, we performed two-photon guided juxtasomal recordings from PV interneurons in the barrel field of the somatosensory cortex (S1bf) of anesthetized mice, while simultaneously collecting the local field potential (LFP) in S1bf and the multi-unit activity (MUA) in the ventral posteromedial (VPM) thalamic nucleus. We found that activity in the VPM was associated with longer down-state duration in S1bf and that down states displaying PV cell firing were associated with increased VPM activity. Moreover, thalamic inhibition through application of muscimol reduced the fraction of spikes discharged by PV cells during cortical down states. Finally, we inhibited PV interneurons using optogenetics during down states while monitoring cortical LFP under control conditions and after thalamic muscimol injection. We found increased latency of the optogenetically triggered down-to-up transitions upon thalamic pharmacological blockade compared to controls. These findings demonstrate that spontaneous thalamic activity inhibits cortex during down states through the activation of PV interneurons.

In vivo two-photon calcium imaging is a powerful approach in neuroscience. However, processing two-photon calcium imaging data is computationally intensive and time-consuming, making online frame-by-frame analysis challenging. This is especially true for large field-of-view (FOV) imaging. Here, we present CITE-On (Cell Identification and Trace Extraction Online), a convolutional neural network-based algorithm for fast automatic cell identification, segmentation, identity tracking, and trace extraction in two-photon calcium imaging data. CITE-On processes thousands of cells online, including during mesoscopic two-photon imaging, and extracts functional measurements from most neurons in the FOV. Applied to publicly available datasets, the offline version of CITE-On achieves performance similar to that of state-of-the-art methods for offline analysis. Moreover, CITE-On generalizes across calcium indicators, brain regions, and acquisition parameters in anesthetized and awake head-fixed mice. CITE-On represents a powerful tool to speed up image analysis and facilitate closed-loop approaches, for example in combined all-optical imaging and manipulation experiments.

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