Segmental organization, projection identity, and functional recruitment of ventral V3 interneurons in the adult lumbar spinal cord

Abstract

V3 neurons are a genetically defined population of excitatory Sim1-expressing spinal INs that contribute to locomotor stability, motor coordination, and spinal motor circuit function. Although ventromedial (V3VM) and ventrolateral (V3VL) V3 subpopulations have been identified with distinct functions, their segmental organization, projection-associated molecular diversity, and functional recruitment in the adult lumbar spinal cord remain poorly understood. This study investigated these characteristics in adult mice. Using Sim1CreTdTomato mice, V3VM and V3VL were mapped within lumbar segments L1-L6 based on their anatomical positions in the ventral horn. Expression of the transcription factors Zfhx3 and NFIB was examined in the rostral (L2) and caudal (L6) segments as markers of putative long- and short-range projection phenotypes. Functional recruitment was assessed using cFos immunoreactivity after in vivo stimulation of the L2 or L6 ventral roots. A reproducible rostrocaudal distribution pattern was identified. V3VM neurons predominated throughout the lumbar spinal cord, representing 80% of ventral V3 (V3V) neurons in L1-L2, 92% in L3-L5, and 62% in L6. In contrast, V3VL neurons were present in the rostral lumbar, relatively sparse within the mid-lumbar enlargement, but increased significantly in the caudal lumbar cord, comprising 20.3%, 7.7%, and 37.3% of V3V neurons in L1-L2, L3-L5, and L6, respectively (n = 3 mice). Molecular analyses demonstrated that both V3VM and V3VL populations expressed Zfhx3 and NFIB, suggesting molecular heterogeneity within V3V neurons (n = 2 mice). Functional analyses revealed segment-dependent recruitment following motor pathway activation. L2 ventral root stimulation recruited V3VM and V3VL neurons similarly (52.0% and 55.5%, respectively), whereas L6 stimulation preferentially activated V3VM neurons (74.0%) compared with V3VL neurons (43.5%) (n = 5 mice). cFos expression extended into adjacent lumbar and sacral segments and was observed bilaterally, suggesting involvement of V3V neurons in intersegmental and commissural spinal networks. These findings suggest that V3V neurons exhibit spatial, molecular, and functional specialization along the rostrocaudal axis, providing insight into the organization of spinal motor circuits.