Nerve sprouting induced by a piece of peripheral nerve placed over a normally innervated frog muscle

1. The effects of a segment of peripheral nerve on the innervation of a skeletal muscle were investigated in the cutaneous pectoris muscle of the frog Rana esculenta. An explant of the sciatic nerve was placed in an aneural region of the muscle at a distance of 1-3 mm from the zone of neuromuscular junctions. The muscles were examined morphologically and electrophysiologically at different post-operative times. 2. After the first month nerve sprouts were seen arising from both nerve terminals and intramuscular nodes of Ranvier. Both the number of sites of sprouting and the relative distance to the explant tip increased with time (up to 5 months), suggesting spread of a nerve sprouting-promoting stimulus. 3. Most neurites resulting from sprouting were seen growing towards the nerve explant, in the vicinity of which active neurite proliferation occurred. Some of them entered the explant as observed in semi-thin and thin sections which revealed the presence of both myelinated and unmyelinated nerve fibres. 4. Electrical stimulation of the nerve explant in preparations autografted for more than 2 months resulted in the contraction of bundles of muscle fibres. Endplate potentials of similar amplitudes were recorded intracellularly in some muscle fibres of such preparations when stimulating either the nerve explant or the cutaneous pectoris nerve. When two stimuli were paired by gradually reducing the interval of time between them, the second response was gradually facilitated. This confirmed that nerve fibres stimulated through the explant corresponded to new neuritic processes resulting from motor nerve sprouting. 5. Pieces of perineural tissue and segments of peripheral nerve killed by alcohol treatment or by repeated freezing and thawing were used as controls. They did not induce any nerve sprouting. 6. The results indicate that cells surviving in a segment of peripheral nerve trunk actively induce intact axons to sprout both from their terminals and intramuscular nodes of Ranvier; moreover these cells promote attraction and proliferation of growing neurites. The possibility of release of a diffusible factor by glial cells is discussed.