Effects of locomotor training on hindlimb regeneration in the urodele amphibian Pleurodeles waltlii

Abstract

1. The effects of locomotor training on hindlimb regeneration were studied in the urodele amphibian Pleurodeles waltlii. 2. After amputation of one hindlimb at mid-femur, adult animals were subjected to regular training sessions (1 h daily, 5 days a week, over 8 months) of terrestrial stepping. 3. Eight months post-amputation, trained animals exhibited regenerated limbs of reduced size as compared to animals kept in their aquaria. Histological data showed an abnormal regeneration of both the femur and distal structures (e.g. digit muscles, metatarsi and phalanges) while medial structures (e.g. tibia and fibula) were totally re-formed. The study of the electromyographical activity in regenerated limbs during stepping and that of their reflex responsiveness to electrical stimulation showed that both motor and sensory innervations were functional in the limb stump of trained animals. 4. The regenerative capacity of the abnormal stumps was preserved since following a second amputation a quite normal hindlimb was regenerated in 3 months, provided the re-amputated animals were not trained to terrestrial stepping. 5. The stress due to handling, change in locomotor medium (aquatic vs. terrestrial) and the friction of the wound epidermis with the ground were not involved in the disruption of limb regeneration. 6. The locomotor pattern, the reflex responsiveness and the muscle fibre composition were similar in supernumerary forelimbs grafted on the back and in normal forelimbs. However, the supernumerary forelimbs regenerated normally even in animals subjected to locomotor training while the hindlimb did not. It is concluded that the disrupting effects of locomotor training on limb regeneration were localized to the the limb directly involved in locomotion. 7. The mechanisms underlying abnormal limb regeneration in animals subjected to locomotor training are discussed.

Figure 1. Longitudinal sections of regenerates in control (A-C) and in trained animals with (D and E) and without (F and G) digits

Sections were stained with eosin and haematoxylin. f, femur; fi, fibula; m, metatarsis; p, phalanges; t, tibia; ta, tarsis.

Figure 2. EMG pattern and electrophoretic pattern of myosin in normal and supernumerary forelimbs

A, EMG pattern of regenerates during overground stepping in a control animal (left panel) and in a locomotor trained animal (right panel). For each channel, EMG activity was full-wave rectified and filtered. Calibrations apply to each channel. i, ipsilateral; co, contralateral; regen, regenerate; pifi, puboischiofemoralis internus. B, EMG pattern in a supernumerary forelimb during overground stepping. Conventions as in A. su, supernumerary; bic, biceps brachii. Data are from the same individual. C, electrophoretic pattern of myosin isoforms contained in muscles of contralateral forelimb (left) and supernumerary forelimb (right). Arrowheads: slow myosin isoform; thick line: intermediate myosin isoform; thin lines: fast myosin isoforms.

Figure 3. Regeneration in supernumerary animal

Regeneration of the supernumerary forelimb (arrowed) in a non-trained (A) and in a trained (B) supernumerary animal. Regeneration of the right hindlimb in a non-trained (C) and in a trained (D) supernumerary animal.