Regenerating supernumerary axons are cholinergic and emerge from both autonomic and motor neurons in the rat spinal cord.

Multipolar neurons in the mammalian nervous system normally exhibit one axon and several dendrites. However, in response to an axonal injury, adult motoneurons may regenerate supernumerary axons. Supernumerary axons emerge from the cell body or dendritic trees in addition to the stem motor axon. It is not known whether these regenerating axons contain neurotransmitters for synaptic transmission at their terminals. Here, using immunohistochemistry for choline acetyltransferase, an enzyme that synthesizes acetylcholine, we demonstrate the emergence of cholinergic supernumerary axons at 6 weeks after a unilateral L5-S2 ventral root avulsion and acute implantation of the avulsed L6 ventral root into the adult rat spinal cord. Light microscopic serial reconstruction of choline acetyltransferase immunoreactive arbors shows that these supernumerary axons originate from both autonomic and motor neurons. The supernumerary axons emerge from the cell body or dendrites, exhibit an abnormal projection pattern within the intramedullary gray and white matters, make frequent abrupt turns in direction, and form bouton-like swellings as well as growth cone-like terminals. Double labeling immunohistochemistry studies show that the choline acetyltransferase immunoreactive supernumerary axons co-localized with two proteins associated with axonal growth and elongation, growth-associated protein 43 and p75, the low affinity neurotrophic factor receptor. Our findings suggest that regenerating supernumerary axons selectively transport and store choline acetyltransferase, supporting the notion that supernumerary axons may develop functional and active synaptic transmission. Therefore, regenerating supernumerary axons may contribute to the plasticity in neural circuits following injury in the adult nervous system.