Peptidergic and functional delineation of the Edinger-Westphal nucleus

Many neuronal populations that release fast-acting excitatory and inhibitory neurotransmitters in the brain also contain slower-acting neuropeptides. These facultative peptidergic cell types are common, but it remains uncertain whether neurons that solely release peptides exist. Our fluorescence in situ hybridization, genetically targeted electron microscopy, and electrophysiological characterization suggest that most neurons of the non-cholinergic, centrally projecting Edinger-Westphal nucleus in mice are obligately peptidergic. We further show, using anterograde projection mapping, monosynaptic retrograde tracing, angled-tip fiber photometry, and chemogenetic modulation and genetically targeted ablation in conjunction with canonical assays for anxiety, that this peptidergic population activates in response to loss of motor control and promotes anxiety responses. Together, these findings elucidate an integrative, ethologically relevant role for the Edinger-Westphal nucleus and functionally align the nucleus with the periaqueductal gray, where it resides. This work advances our understanding of peptidergic modulation of anxiety and provides a framework for future investigations of peptidergic systems.