A deep learning pipeline for three-dimensional brain-wide mapping of local neuronal ensembles in teravoxel light-sheet microscopy

Teravoxel-scale, cellular-resolution images of cleared rodent brains acquired with light-sheet fluorescence microscopy have transformed the way we study the brain. Realizing the potential of this technology requires computational pipelines that generalize across experimental protocols and map neuronal activity at the laminar and subpopulation-specific levels, beyond atlas-defined regions. Here, we present artficial intelligence-based cartography of ensembles (ACE), an end-to-end pipeline that employs three-dimensional deep learning segmentation models and advanced cluster-wise statistical algorithms, to enable unbiased mapping of local neuronal activity and connectivity. Validation against state-of-the-art segmentation and detection methods on unseen datasets demonstrated ACE's high generalizability and performance. Applying ACE in two distinct neurobiological contexts, we discovered subregional effects missed by existing atlas-based analyses and showcase ACE's ability to reveal localized or laminar neuronal activity brain-wide. Our open-source pipeline enables whole-brain mapping of neuronal ensembles at a high level of precision across a wide range of neuroscientific applications.