Reduction of the current of injury leaving the amputation inhibits limb regeneration in the red spotted newt

Abstract

Immediately following amputation of the limb in salamanders, a strong, steady, and polarized flow of ionic current is produced by the injury. Current flows in a proximodistal direction within the Limb stump and is associated with a fall in electrical potential of about 50 mV/mm near the stump's end. This current is electrogenically driven by the Na+-dependent, internally positive transcutaneous voltage of the intact skin of the limb stump. Reduction of this LIME; the skin's battery, by topical application of Na+ blocking agents leads to inhibition or disruption of normal limb regeneration. This suggests electrical factors are a critical control of limb regeneration. Here we test another means to reduce the injury current and its associated electrical field within the forelimb stump of red spotted newts. A fine (40 gauge) insulated, multistrand wire was inserted beneath the skin of the animal's back, with the uninsulated portion terminating either at the shoulder region or at the base of the tail. When this cathodal (negative) electrode is connected to a regulated current source, sufficient current was pulled into the stump end from an external anode (placed in the water the animal was immersed in) to markedly reduce or null the endogenous current for the first 8 days following amputation. The extent of Limb regeneration in sham-treated and experimentally treated animals was determined 1 month following amputation at the elbow. Sham-treated animals regenerated normally, with most producing digits within this time. Limb regeneration was completely arrested, or caused to be strikingly hypomorphic, in half of the experimentally treated animals. This effect was independent of where the subcutaneous electrode was placed and suggests that electrical (physiological) factors are indeed a critical control of limb regeneration in urodeles.