Functional roles of periaqueductal gray preproenkephalin cells in defensive behavior

In order to survive, animals must precisely regulate their approach and defensive behaviors towards threats. The periaqueductal gray (PAG), particularly its dorsal division, is a key hub in the innate defensive system, transforming sensory information into coordinated defensive motor responses. Neurons within the dorsal PAG exhibit distinct functional activity in relation to certain behaviors, and cells tuned to approach and escape from various threats have been reported. Despite its significance, the cellular and neurochemical identity underlying the functional heterogeneity of PAG neurons is not understood. The endogenous opioid system, including endogenous neuropeptides and their receptors, is widely expressed in the PAG, and has been mainly investigated in the context of PAG-mediated pain modulation. In this study, we explored the specific contribution of this system to defensive behavior by focusing on the functional roles of preproenkephalin-positive (PENK) cells within the PAG. Using spatially resolved transcriptomics, we analyzed the expression profile of PAG PENK+ neurons and found two major cell clusters located in the dorsal and ventrolateral PAG and expressing glutamatergic and GABAergic markers respectively. Additionally, we employed one-photon calcium imaging recordings in freely moving mice and characterized the functional responses of PENK+ cells to a diverse array of threats, including exposure to predators, prey and aggressive conspecifics. Thus, our study shed light into the cellular heterogeneity of a brainstem structure that modulates innate defensive behaviors.