Spinal motor neuron plasticity after hindlimb unloading in aged mice and its modulation by exercise with TRPM8-mediated cutaneous stimulation

Age-related motor decline is influenced not only by muscle atrophy but also by deterioration of the neuromuscular system. However, the effects of aging and disuse on spinal motor neuron (MN) plasticity remain poorly understood. In this study, we examined how short-term hindlimb unloading (HU)-a disuse model-affects neuromuscular junction (NMJ) integrity and spinal motor neuron (MN) synaptic inputs in young (3 months) and aged (22 months) mice. We also assessed whether TRPM8-mediated skin cooling (SC), which activates MN via spinal interneurons, could enhance neuromuscular recovery when applied during low-intensity treadmill exercise. Two weeks of HU induced comparable muscle atrophy in both age groups; however, motor function, assessed by the beam walking test, declined only in aged mice. NMJs in aged mice showed fragmentation and partial denervation, which were not exacerbated by HU. In contrast, HU significantly reduced the number of C-boutons-cholinergic synaptic inputs on MNs-exclusively in aged mice, indicating selective vulnerability of spinal cholinergic circuits to aging and disuse. Glutamatergic (VGLUT1-positive) inputs declined with age but were unaffected by HU. Notably, exercise combined with TRPM8-mediated SC during reloading restored C-bouton density and improved motor function in aged mice, whereas exercise alone had no effect. These findings suggest that aging and disuse jointly disrupt spinal cholinergic circuits and that TRPM8-mediated cutaneous stimulation can promote recovery of spinal synaptic inputs. Our results identify C-boutons as a potential therapeutic target to mitigate age-related physical decline and highlight SC as a strategy to preserve or restore motor control in older individuals.