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Abstract

Introduction. The pupil size can inform about arousal or attention. Animal studies have combined pupillometry with electrophysiology and behavior to infer arousal linked to neuronal firing patterns from specific locations. Whole-brain functional magnetic resonance imaging (fMRI) allows tracking the connectivity patterns that emerge upon distinct neurological states. However, fMRI is not compatible with behavior tests. In this work, we merge fMRI with pupillometry and cingulate cortex (Cg) calcium recordings (Fig. 1A) to investigate the missing link between the brain state and the whole-brain neural activity. Methods. GCaMP was expressed in Cg on 4-week old SD rats. After 3-4 weeks, an optical fiber was implanted on Cg and the anesthetized animals (alpha-chloralose) were transferred to a 14T MRI scanner. An MRI-compatible camera was used to track pupil size changes. fMRI (n=71 trials, 10 animals) was acquired using GE-EPI (TR=1s, TE=12.5ms, resolution=0.4x0.4x0.6mm). Biopac was used to record neuronal activity from Cg. The pupil size was extracted from each video frame using Matlab. A pupil-fMRI correlation map was obtained from the voxel-wise correlation between pupil dilations and the fMRI signal. Results. fMRI revealed negative correlations between the pupil dynamics and most of the brain. The momentary spatial correlation between the pupil-fMRI map and each fMRI volume allowed creating an fMRI arousal time course (fAtc), indicative of the varying arousal level during fMRI (Fig. 1B). A region in the brainstem (A5) was positively correlated with dilations, but only under a particular electrophysiological state (during uncoupling of the calcium oscillations) (Fig. 1C). The pupil dynamics and the fAtc were negatively correlated with the neuronal activity in the 2-3Hz band (i.e. dilations were associated to less synchronized states) (Fig. 1D). Conclusion. This new platform (concurrent fMRI + pupillometry + GCaMP) constitutes a powerful tool to track brain state changes from fMRI, which is critical in whole-brain studies investigating consciousness from animal models.