Developmental segregation of spinal networks driving axial- and hindlimb-based locomotion in metamorphosing Xenopus laevis

Abstract

Amphibian metamorphosis includes a complete reorganization of an organism's locomotory system from axial-based swimming in larvae to limbed propulsion in the young adult. At critical stages during this behavioural switch, larval and adult motor systems operate in the same animal, commensurate with a gradual and dynamic reconfiguration of spinal locomotor circuitry. To study this plasticity, we have developed isolated preparations of the spinal cord and brainstem from pre- to post-metamorphic stages of the amphibian Xenopus laevis, in which spinal motor output patterns expressed spontaneously or in the presence of NMDA correlate with locomotor behaviour in the freely swimming animal. Extracellular ventral root recordings along the spinal cord of pre-metamorphic tadpoles revealed motor output corresponding to larval axial swimming, whereas postmetamorphic animals expressed motor patterns appropriate for bilaterally synchronous hindlimb flexion-extension kicks. However, in vitro recordings from metamorphic climax stages, with the tail and the limbs both functional, revealed two distinct motor patterns that could occur either independently or simultaneously, albeit at very different frequencies. Activity at 0.5-1 Hz in lumbar ventral roots corresponded to bipedal extension-flexion cycles, while the second, faster pattern (2-5 Hz) recorded from tail ventral roots corresponded to larval-like swimming. These data indicate that at intermediate stages during metamorphosis separate networks, one responsible for segmentally organized axial locomotion and another for more localized appendicular rhythm generation, coexist in the spinal cord and remain functional after isolation in vitro. These preparations now afford the opportunity to explore the cellular basis of locomotor network plasticity and reconfiguration necessary for behavioural changes during development.

Figure 1. Axial-based swimming in pre-metamorphic Xenopus larvae

In a Stage 50 tadpole (A), undulatory swimming movements and forward propulsion are generated by alternate bilateral contractions of axial myotomes with a characteristic rostro-caudal delay along the body (B). When the brainstem and spinal cord are isolated in vitro (C), extracellular recordings from selected spinal ventral roots (D; recorded roots indicated in C) reveal spontaneous bursting in axial motorneurones which, as with swimming in vivo, alternates across the cord and propagates rostro-caudally (dotted lines indicate delay between VR2 and VR17). Note that the horizontal bar in B indicates a complete cycle.

Figure 2. Limb-based swimming in post-metamorphic Xenopus froglets

In a stage 64 juvenile (A), the tail has resorbed and swimming is now produced exclusively by bilaterally synchronous cycles of hindlimb extensions and flexions (B; cycle indicated by bar). The fictive correlate of this behaviour is generated by the isolated spinal cord and brainstem (C), as seen in D, where several cycles beginning with intense coordinately active bursts in left and right extensor lumbar motorneurones alternate with bilateral hindlimb flexor bursts. In this example, 10 µm NMDA was present in the bath. Note that a fictive swim episode could begin with either extensor (as illustrated) or flexor bursting. Note that the rhythm frequency is an order of magnitude slower than the pre-metamorphic tadpole (Fig. 1D).

Figure 3. Combined axial- and hindlimb-based locomotion early in metamorphosis

In a stage 58 tadpole (A), the hindlimbs are present but not yet fully functional. During swimming, they are maintained against the tail which then generates undulatory movements (B; cycle indicated by bar). After isolation (C), the spinal cord can spontaneously generate motor activity (D and E) which, in this example, begins with simultaneous brief flexor and more prolonged extensor discharge, followed ∼500 ms later by rapid bursting in caudal tail segments (VR15). Lumbar activity then becomes coordinated with axial bursting (Db), with ipsilateral extensor and flexor motorneurones firing simultaneously, and in phase-opposition with contralateral partners. Hindlimb motor discharge can wax and wane within a single axial swimming episode (E), the latter invariably outlasting the limb root activity (see also D).

Figure 4. Segregated axial- and hindlimb-based locomotion in intermediate metamorphic climax tadpoles

At stage 61 (A) the hindlimbs are now fully functional and a combination of rhythmic bilateral limb kicks and tail undulations are used to propel the animal (B). The isolated cord (C) can generate spontaneous motor patterns (D) appropriate for both locomotor modes: higher frequency bursting in tail spinal segments and slower, bilaterally synchronous bursting in lumbar hindlimb motorneurones corresponding to fictive kicking. As in vivo, the two motor programmes may be co-ordinately active (as in D) or can operate independently, with bursting expressed solely in limb motorneurones (E), or more frequently, activity occurring only in axial motorneurones (F). Note that the horizontal bar in B indicates a limb-kick cycle.