Elsevier

Journal of Physiology-Paris

Volume 100, Issues 5–6, November–December 2006, Pages 317-327
Journal of Physiology-Paris

Development and neuromodulation of spinal locomotor networks in the metamorphosing frog

https://doi.org/10.1016/j.jphysparis.2007.05.009Get rights and content

Abstract

Metamorphosis in the anuran frog, Xenopus laevis, involves profound structural and functional transformations in most of the organism’s physiological systems as it encounters a complete alteration in body plan, habitat, mode of respiration and diet. The metamorphic process also involves a transition in locomotory strategy from axial-based undulatory swimming using alternating contractions of left and right trunk muscles, to bilaterally-synchronous kicking of the newly developed hindlimbs in the young adult. At critical stages during this behavioural switch, functional larval and adult locomotor systems co-exist in the same animal, implying a progressive and dynamic reconfiguration of underlying spinal circuitry and neuronal properties as limbs are added and the tail regresses. To elucidate the neurobiological basis of this developmental process, we use electrophysiological, pharmacological and neuroanatomical approaches to study isolated in vitro brain stem/spinal cord preparations at different metamorphic stages. Our data show that the emergence of secondary limb motor circuitry, as it supersedes the primary larval network, spans a developmental period when limb circuitry is present but not functional, functional but co-opted into the axial network, functionally separable from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, recent experiments on spontaneously active in vitro preparations from intermediate metamorphic stage animals have revealed that the biogenic amines serotonin (5-HT) and noradrenaline (NA) exert short-term adaptive control over circuit activity and inter-network coordination: whereas bath-applied 5-HT couples axial and appendicular rhythms into a single unified pattern, NA has an opposite decoupling effect. Moreover, the progressive and region-specific appearance of spinal cord neurons that contain another neuromodulator, nitric oxide (NO), suggests it plays a role in the maturation of limb locomotor circuitry. In summary, during Xenopus metamorphosis the network responsible for limb movements is progressively segregated from an axial precursor, and supra- and intra-spinal modulatory inputs are likely to play crucial roles in both its functional flexibility and maturation.

Introduction

To survive development and reach maturity, an animal’s nervous system must be capable of adapting to continually changing biomechanical and environmental circumstances. Such plasticity in nervous system operation not only requires the assembly of new neuronal circuitry to accommodate additional behavioural demands, but also a dynamic reconfiguration of pre-existing connectivity within maturing networks as well as changes in the integrative electrical properties of constituent neurons. Neuronal networks that generate programmed behaviours such as respiration, locomotion and feeding are useful models for studying developmental correlates of nervous system plasticity since such behaviours can be clearly defined at the neuroethological level, and thus the functional consequences of any developmental alteration in the underlying motor network can be charted with relative accuracy.

The metamorphosis of tadpole to frog in anuran amphibians constitutes one of the most dramatic developmental transformations in biology, involving massive alterations in body structure, a transfer from aquatic to aerial respiration, a switch from herbivorous to carnivorous diet, and biochemical, physiological and morphological changes in virtually all of the animal’s organs (Shi, 2000). With respect to locomotion, the alteration in body plan from tadpole to adult is accompanied by the growth of limbs and the regression of the tail as the organism switches its locomotory strategy from the employment of undulatory, tail-based swimming in larvae to limb-based kick propulsion in the young adult. These biomechanical modifications to the locomotory system during metamorphosis require a dynamic anatomical and functional restructuring of underlying neural circuitry within the central nervous system (CNS). Such a remodelling must include the appearance of new sensory and motor pathways to service the emerging limbs, in direct contrast to the concurrent loss of larval spinal circuitry that accompanies the regression and eventual elimination of the tail. Therefore, the gradual replacement of the tail-based swim network by adult limb-kick circuitry during the metamorphic process implies that addition, deletion and functional reassignment of spinal neuronal elements are occurring simultaneously. However, although Xenopus metamorphosis has been widely studied from endocrinological and molecular biological perspectives (Shi, 2000), the associated developmental changes that occur in actual central neural network function remain largely unknown (but see, e.g. Hoskins, 1990).

Here, we review recent evidence from our exploration of in vitro CNS preparations of the clawed toad Xenopus laevis, which continue to produce motor rhythms that share basic features with the patterns that drive real locomotion in vivo. Comparisons of the “fictive” locomotor patterns generated by in vitro preparations at different metamorphic stages have begun to unravel how the developmental remodelling of spinal locomotor circuitry is achieved (Combes et al., 2004), with the eventual aim of determining the neurobiological substrates of such plasticity at the cellular and systems levels. We have also reported that during metamorphosis, neurons that produce the gaseous neuromodulator, nitric oxide (NO), are distributed in a spatial and temporal pattern appropriate for a developmental role in the assembly of the limb motor circuitry (Ramanathan et al., 2006). Preliminary results have also suggested that NO exerts modulatory actions on the expression of spinal locomotor output throughout this period. More recent evidence has shown that the monoamines serotonin and noradrenaline also exert powerful, but opposing, modulatory actions on the expression of spinal locomotor output during metamorphosis, indicating that these signalling molecules may dynamically regulate the short-term operation of spinal locomotor circuitry, in parallel with long-term network development. Finally, comparison is drawn between the metamorphic changes in Xenopus and the different strategies utilized by other animals in the ontogeny of their locomotory systems.

Section snippets

Frog metamorphosis

Anuran metamorphosis involves three major types of developmental process: firstly, a resorption of larval-specific organs such as the gills and tail; second, a de novo formation of adult-specific organs such as the lungs and limbs; and third, a remodelling of pre-existing structures, such as the liver and intestine, into their adult formats (Dodd and Dodd, 1976; also see Shi, 2000). The entire metamorphic process is orchestrated by thyroid hormones (TH), comprising tetraiodothyronine (thyroxine

Developmental segregation of spinal locomotor circuitry

What general developmental rules allow the metamorphosing frog to first construct and then disengage an adult locomotor network from a functionally distinct and pre-existing tadpole precursor? To address the neural basis of this developmental plasticity, we have established in vitro preparations of the spinal cord and brain stem at different metamorphic stages. Such isolated preparations continue to generate fictive locomotor rhythms spontaneously throughout 2–3 days in vitro (Combes et al.,

Comparison with rodent locomotor network development

Striking parallels and differences exist between the developmental changes in limb motor coordination in metamorphosing Xenopus and alterations in appendicular output that occur during the maturation of other vertebrate locomotor systems. In the rat, for example, the acquisition of the adult pattern of locomotion relies on a progressive maturation of spinal circuitry that extends through the pre- and peri-natal period. Five days prior to birth (embryonic day 16, E16), the lumbar region of the

Neuromodulation and locomotor network development

As for higher vertebrates, descending projections from the brainstem play important roles in both the modulation and maturation of amphibian spinal locomotor networks (for review, see McLean et al., 2000). To date, the best studied supraspinal control pathways in Xenopus are the serotonergic and noradrenergic systems which are present at the time of hatching and originate in two brainstem cell populations, the raphe nucleus (Sillar et al., 1995) and the isthmic region (the amphibian equivalent

Comparison with other metamorphosing locomotor systems

It is also instructive to compare the metamorphic changes in locomotory behaviour in anuran amphibians like Xenopus with the developmental transformations that occur in other amphibian species as well as in certain insects (Fig. 5). It is important to remember that the transition from the anuran tadpole to froglet involves a switch between two completely different locomotor strategies (primary axial-based swimming and secondary limb-based propulsion) while the organism continues to behave in

Conclusion

The establishment of new in vitro preparations of the Xenopus CNS which are capable of generating motor rhythms in the limb and/or tail ventral roots appropriate to drive locomotor movements of the host organism’s developmental stage (Combes et al., 2004), has thus permitted initial insights into how the complex metamorphic transition in locomotor strategy is accomplished. In addition, these preparations offer numerous avenues for further studies to address key facets of the neural plasticity

Acknowledgments

This work is supported by a doctoral studentship from the Conseil Régional d’Aquitaine to A. Rauscent, the CNRS (“ATIPE jeune chercheur”, France), and a research interchange grant from the Leverhulme Trust (UK).

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