Cortical depth-dependent functional magnetic resonance image (fMRI), also known as layer-fMRI, has the potential to capture directional neural information flow of brain computations within and across large-scale cortical brain networks. For example, layer-fMRI can differentiate feedforward and feedback cortical input in hierarchically organized brain networks. Recent advancements in 3D-echo-planar imaging (EPI) sampling approaches and MR-contrast generation strategies have allowed proof-of-principle studies showing that layer-fMRI can provide sufficient data quality for capturing laminar changes in functional connectivity. These studies have, however, not shown how reliable the signal is and how repeatable the respective results are. It is especially unclear whether whole-brain layer-fMRI functional connectivity protocols are widely applicable across common neuroscience-driven analysis approaches. Moreover, there are no established preprocessing fMRI methods that are optimized to work for whole-brain layer-fMRI datasets. In this work, we aimed to serve the field of layer-fMRI and build tools for future routine whole-brain layer-fMRI in application-based neuroscience research. We have developed publicly available sequences, acquisition protocols, and processing pipelines for whole-brain layer-fMRI. These protocols are validated across 60 hours of scanning in nine participants. Specifically, we identified and exploited methodological advancements for maximizing tSNR efficiency and test-retest reliability. We are sharing an extensive multi-modal whole-brain layer-fMRI dataset (20 scan hours of movie watching in a single participant) for benchmarking future method developments: The Kenshu dataset. With this dataset, we are also exemplifying the usefulness of whole-brain layer-fMRI in conjunction with commonly applied analysis approaches in modern cognitive neuroscience fMRI studies. This includes connectivity analyses, representational similarity matrix estimations, general linear model analyses, principal component analysis clustering, and so on. We believe that this work paves the road for future routine measurements of directional functional connectivity across the entire brain.
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