Activity and Functional Connectivity of Lumbar Spinal Interneurons at Rest: Irregular, Sparse Yet Structured

Spinal interneurons are central to sensorimotor integration. Even in the absence of overt sensory inputs or motor output, spinal interneurons remain active, yet their functional organization across spatial and temporal scales remains poorly understood. We characterized the discharge properties and functional connectivity of 2,847 lumbar spinal interneurons recorded in vivo from quiescent decerebrate cats (3 males, 5 females) using microelectrode arrays inserted into rostral (L3-4) and caudal (L5-7) spinal segments at superficial (0-1500 µm) and deep (1500-3000 µm) depths. Superficial neurons generally discharged at higher and less variable rates than deeper neurons, forming a consistent depth-dependent gradient at both segments. Functional connectivity was sparse yet structured, with distinct depth profiles across timescales. Short-timescale correlations (±10 ms) occurred in ∼2% of neuron pairs, which were predominantly excitatory and prominent caudally at ∼1500 µm depth. Longer-timescale correlations (∼400 ms) were similarly rare but showed equal proportions of excitation and inhibitory and were more pronounced at deeper depths. Graph-theoretic network analysis showed that timescale-specific correlations are architecturally distinct and non-randomly organized. Short-timescale networks exhibited depth-specific hubs near 1500 µm and lower clustering, whereas longer-timescale networks showed more clustering. At the ensemble level, neurons shared low-frequency oscillatory drive, particularly in rostral recordings. Separately, a subset of ensembles with dense short-timescale connectivity were consistently observed near 1500 µm in the caudal array, linking slow and fast interactions. Together, these findings show that spinal interneuron circuitry exhibits spatially and temporally structured organization even at rest, establishing a critical baseline for studies of spinal injury and disease.Significance Statement Spinal interneurons are essential for sensorimotor processing, yet their functional organization remains poorly defined. By recording the resting discharge behavior of 2,847 lumbar interneurons from in vivo cats, we show that spinal interneuron networks are sparse but systematically organized both spatially and temporally. Neurons exhibited depth-dependent discharge properties and formed distinct short- and longer-timescale functional networks with different architectures and hub locations. Shared low-frequency activity links distributed ensembles, particularly in rostral segments, while highly-connected ensembles emerge from specific layers in caudal segments. These findings reveal a multilayered, temporally distinct organization of spinal interneuron circuitry and provide a foundational reference for understanding how spinal networks adapt following injury or disease.