The lumbosacral spinal cord contains neural circuits crucial for locomotion, organized into rostrocaudal levels with distinct somatosensory and motor neuron pools that project to and from the muscles of the lower limbs. However, the specific spinal levels that innervate each muscle and the locations of neuron pools vary significantly between individuals, presenting challenges for targeted therapies and neurosurgical interventions aimed at restoring locomotion. Non-invasive approaches to functionally map the segmental distribution of muscle innervation — or projectome— are therefore essential. Here, we developed a pipeline dedicated to record blood oxygenation level dependent (BOLD) signals in the lumbosacral spinal cord using functional magnetic resonance imaging (fMRI). We assessed spinal activity across different conditions targeting the extensor/flexor muscles of the right leg (ankle, knee, and hip) in 12 healthy participants. To enhance clinical relevance, we included not only active movements but also two conditions that did not rely on participants’ performance: passive stretches and muscle-specific tendon vibration, which activates proprioceptive afferents of the vibrated muscle. BOLD activity patterns were primarily located on the side ipsilateral to the movement, stretch, or vibration, both at the group and participant levels, indicating the BOLD activity being associated with the projectome. The fMRI-derived rostrocaudal BOLD activity patterns exhibited mixed alignment with expected innervation maps from invasive studies, varying by muscle and condition. While some muscles and conditions matched well across studies, others did not. Significant variability among individual participants underscores the need for personalized mapping of projections for targeted therapies and neurosurgical interventions. To support the interpretation of BOLD activity patterns, we developed a decision tree-based framework that combines reconstruction of neural structures from high-resolution anatomical MRI datasets and muscle-specific fMRI activity to produce personalized projectomes. Our findings provide a valuable proof-of-concept for the potential of fMRI to map the lumbosacral spinal cord’s functional organization, while shedding light on challenges associated with this endeavor.

In the past decade, exploration of spontaneous blood-oxygen-level-dependent (BOLD) signal fluctuations has expanded beyond the brain to include the spinal cord. While most studies have predominantly focused on the cervical region, the lumbosacral segments play a crucial role in motor control and sensory processing of the lower limbs. Addressing this gap, the aims of the current study were twofold: first, confirming the presence and nature of organized spontaneous BOLD signals in the human lumbosacral spinal cord; second, systematically assessing the impact of various denoising strategies on signal quality and functional connectivity (FC) patterns. Given the susceptibility of spinal cord functional magnetic resonance imaging (fMRI) to noise, this step is pivotal to ensure the robustness of intrinsic FC. Our findings uncovered bilateral FC between the ventral and dorsal horns. Importantly, these patterns were consistently observed across denoising methods and demonstrating fair to excellent split-half temporal stability. Importantly, the evaluation of diverse denoising strategies highlighted the efficacy of physiological noise modeling (PNM)-based pipelines in cleaning the signal while preserving the strength of connectivity estimates. Together, our results provide evidence of robust FC patterns in the lumbosacral spinal cord, thereby paving the way for future studies probing caudal spinal activity.