The cerebellum is increasingly recognized as a key node in non-motor circuits, yet the behavioral significance of its projections to the midbrain raphe nuclei remains unexplored. We hypothesized that the deep cerebellar nuclei (DCN) regulate affective and cognitive states through a direct monosynaptic pathway to the raphe.

In male C57BL/6J mice, monosynaptic retrograde tracing revealed that the dentate nucleus preferentially targets the dorsal raphe nucleus (DRN) and caudal linear nucleus, whereas the fastigial and interposed nuclei innervate the median raphe nucleus, indicating target-specific topographic organization. We then combined chemogenetic silencing, fiber photometry, and optogenetic terminal activation to probe the pathway’s function.

Chemogenetic silencing increased anxiety-like avoidance in the elevated plus maze and selectively impaired social novelty discrimination in the three-chamber test, without affecting motor coordination, general locomotion, or basic sociability. Fiber photometry showed elevated DCN-raphe calcium activity durin open-arm occupancy, but not during consummatory, social, or conditioned aversive events. Optogenetic activation of DCN terminals in the DRN produced context-dependent darting behavior in the open arms and increased exploratory approach frequency.

These converging findings identify the DCN-raphe pathway as a context-dependent modulator of anxiety-like exploratory behavior and social novelty processing, broadening the functional map of cerebellar non-motor circuits.