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BACKGROUND AND AIMS: The effects of low-threshold stimulation of muscle afferents (Ia) on glutamatergic and cholinergic innervation of α-motoneurons (Mns) were tested. Two types of synaptic terminals were analyzed: (1) glutamatergic Ia carrying VGLUT1, contacting monosynaptically Mns; (2) cholinergic C-terminals, carrying VAChT, originating from V0c-interneurons of lamina X, which might receive indirect input from sensory afferents of unknown origin. Our aim wasto clarify whether enhancement of proprioceptive input to ankle extensor Mns, via direct electrical stimulation of Ia afferents in the tibial nerve of awake rats, will affect excitatory innervation of lateral gastrocnemius (LG) Mns. METHODS: LG Mns were identified with True Blue (TB) tracer injected intramuscularly. Tibial nerve was stimulated for 7 days with bursts of 3 pulses (pulse width 200 µs, 4 ms of inter-pulse interval, 25 ms inter-burst interval) in four 20 min sessions daily. The Hoffmann reflex recorded from the soleus muscle, LG synergist, allowed controlling low-threshold stimulation. Proprioceptive Ia glutamatergic and cholinergic C-terminals abutting TB-labeled Mns were detected immunohistochemically on transverse spinalsections, using input-specific anti- VGLUT1 and anti-VAChT antibodies. RESULTS: Confocal analysis revealed that the number of both VGLUT1 and VAChT immunoreactive terminals, contacting the soma and proximal dendrites of LG Mns, increased after stimulation by about 35% and 20%, respectively, comparing to sham-stimulated side (P<0.03, Wilcoxon test). CONCLUSIONS: One week of repetitive low-threshold stimulation of proprioceptive fibers in the tibial nerve enriched glutamatergic and cholinergic innervation of LG Mns indicating that this method might be useful for enhancing an activity of selected group of Mns which are the most vulnerable to the spinal cord injuries (Skup et al. 2012, EJN 36: 2679). Supported by grants: NCN 2013/09/B/NZ4/03306, statutory for the Nencki Institute.