GABA and Glycine Synaptic Release on Axotomized Motoneuron Cell Bodies Promotes Motor Axon Regeneration

Motor axon regeneration after traumatic nerve injuries is a slow process that adversely influences patient outcomes because muscle reinnervation delays result in irreversible muscle atrophy and suboptimal axon regeneration. This advocates for investigating methods to accelerate motor axon growth. Electrical nerve stimulation and exercise both enhance motor axon regeneration in rodents and patients, but these interventions cannot always be easily implemented. A roadblock to uncover novel therapeutic approaches based on the effects of activity is the lack of understanding of the synaptic drives responsible for activity-mediated facilitation of axon regeneration. We hypothesized that the relevant excitatory inputs facilitating axon regrowth originate in GABA/glycine synapses, which become depolarizing after downregulation of the potassium chloride cotransporter 2 in motoneurons following axotomy. To test this, we injected tetanus toxin (TeTx) into the tibialis anterior (TA) muscle of mice to block the release of GABA/glycine specifically onto TA motoneurons. Thereafter, we axotomized all sciatic motoneurons by nerve crush and analyzed the time courses of muscle reinnervation in TeTx-treated (TA) and untreated (lateral gastrocnemius [LG]) motoneurons. Muscle reinnervation was slower in TA motoneurons with blocked GABA/glycine synapses, as measured by recovery of M responses and anatomical reinnervation of neuromuscular junctions. Post hoc immunohistochemistry confirmed the removal of the vesicle-associated membrane proteins 1 and 2 by TeTx activity, specifically from inhibitory synapses. These proteins are necessary for the exocytotic release of neurotransmitters. Therefore, we conclude that GABA/glycine neurotransmission on regenerating motoneurons facilitates axon growth and muscle reinnervation, and we discuss possible interventions to modulate these inputs on regenerating motoneurons.