Three-dimensional Interrogation of Cell Types and Instinctive Behavior in the Periaqueductal Gray

Summary The periaqueductal gray (PAG) is a critical midbrain hub that relays information from the forebrain to motor and autonomic brainstem centers to orchestrate instinctive behaviors. The current organization of the PAG into four main radial columns lacks the resolution needed to account for the vast range of PAG functions. Using spatially resolved single-cell transcriptomic measurements, we uncovered widespread transcriptional heterogeneity in the PAG with >100 excitatory and inhibitory neuronal populations, which further assemble into 19 spatial metaclusters. We explored the transcriptional and spatial logic of PAG function during instinctive behaviors and demonstrated the regional recruitment of cell types for distinct behaviors. Unexpectedly, certain behaviors trigger differential spatial activation patterns within given cell types, illustrating the complexity of PAG molecular and functional 3D organization. The newly uncovered spatial motifs and high precision cellular map of instinctive behavior in the PAG open new avenues for a mechanistic understanding of PAG function.