Connectivity of neuropeptide Y-Cre spinal interneurons reveal monosynaptic inputs from pathways involved in pain and itch in mice

Neuropeptide Y (NPY) is an inhibitory neuropeptide expressed in the spinal dorsal horn and has been implicated in the inhibition of pain, chemical and mechanical itch. Although its functional role has been studied extensively, the presynaptic inputs to spinal NPY-expressing interneurons remain poorly characterized, leaving their precise position within the somatosensory-regulatory network unresolved. Here, we combined monosynaptic retrograde rabies tracing with immunohistochemistry and RNAscope to map the upstream connectivity of Npy-Cre neurons in the lumbar spinal cord in mice. Tracing experiments revealed that Npy-Cre neurons receive input from multiple sources, including local and distal spinal divisions, dorsal root ganglia (DRG), and supraspinal regions. Lumbar inputs comprised spinal populations associated with cool-sensing, mechanical pain transmission and itch regulation. DRG inputs included both myelinated A-fibers and non-myelinated nociceptive fibers, indicating a heterogeneous sensory profile. Supraspinal inputs originated from motor and sensory cortices, midbrain structures such as the periaqueductal gray and red nucleus, and brainstem nuclei. Together, these findings indicated that Npy-Cre interneurons integrate signals from diverse sensory and descending pathways, supporting a more central role in somatosensory modulation than previously recognized. PERSPECTIVE: This study maps monosynaptic inputs to spinal neuropeptide Y (NPY) interneurons from spinal, peripheral and supraspinal regions involved in pain, itch and motor regulation. These findings position NPY circuits as modulators of descending and sensory signaling, suggesting potential targets for therapeutic strategies in persistent pain and itch conditions.