Yves Fregnac

Didier Aude Rauscent

L E Ray

Marie-Jeanne Cabirol-Pol

Keith Sillar

John Simmers

Dr Denis Combes
email: denis.combes@u-bordeaux2.fr

Development and neuromodulation of spinal locomotor networks in the metamorphosing frog

Keywords: development, locomotion, Xenopus laevis, metamorphosis, spinal motor networks, neuromodulation

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 on 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 [START_REF] Shi | Amphibian metamorphosis; from morphology to molecular biology[END_REF]. 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 [START_REF] Shi | Amphibian metamorphosis; from morphology to molecular biology[END_REF], the associated developmental changes that occur in actual central neural network function remain largely unknown (but see, e.g. [START_REF] Hoskins | Metamorphosis of the amphibian eye[END_REF]).

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 [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF], 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 [START_REF] Ramanathan | Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis[END_REF]. 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.

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 [START_REF] Dodd | The Biology of Metamorphosis[END_REF]; also see [START_REF] Shi | Amphibian metamorphosis; from morphology to molecular biology[END_REF]. The entire metamorphic process is orchestrated by thyroid hormones (TH), comprising tetraiodothyronine (thyroxine or T4) which is converted to a more active form, triiodothyronine (T3), by monodeiodinases in target tissues where it regulates gene expression [START_REF] Denver | Thyroid hormone-dependent gene expression program for Xenopus neural development[END_REF]. Evidence for the causal effects of TH on metamorphosis originally derived from the finding that exogenous T3 and T4 induce precocious metamorphosis [START_REF] Gudernatsch | Feeding experiments on tadpoles. I. The influence of specific organs given as food on growth and differentiation: a contribution to the knowledge of organs with internal secretion[END_REF], that the inhibition of endogenous T3 synthesis blocks metamorphosis [START_REF] Dodd | The Biology of Metamorphosis[END_REF], and the close correlation of the normal metamorphic process with elevated plasma concentrations of T3 and T4 (Fig. 1). During pro-metamorphosis (stages 54-58; [START_REF] Nieuwkoop | Normal Tables for Xenopus laevis[END_REF], the hindlimbs develop as TH levels rise until a peak is reached some two weeks later at metamorphic climax (stage 59-64). Circulating TH subsequently returns to premetamorphic levels at stage 66 when the tail has completely regressed and the animal employs solely a limb-based mode of locomotion.

A diverse range of changes in nervous system organization and function during metamorphosis has been documented. For example, the bilaterally-paired, giant Mauthner neurons of the medulla involved in larval escape responses undergo degeneration [START_REF] Kollross | Metamorphosis: A Problem in developmental biology[END_REF], as do larval-specific mechanoreceptor Rohon-Beard neurons [START_REF] Hughes | The development of the primary sensory system in Xenopus laevis[END_REF]. In contrast, postembryonic neurogenesis and proliferation of secondary motoneurons and sensory systems (dorsal root ganglion cells) is required to service the newly developing limbs [START_REF] Van Mier | The development of the motor system in the clawed toad Xenopus laevis[END_REF]. Other types of neuron already present at larval stages, such as cerebellar Purkinje cells, continue to mature and acquire dendritic complexity in accordance with their more complex adult integrative functions [START_REF] Hauser | Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis[END_REF], while still other neuron types, such as trigeminal motoneurons, are reassigned to different jaw muscle targets in the adult frog [START_REF] Omerza | Redeployment of trigeminal motor axons during metamorphosis[END_REF].

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 [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]. In intact premetamorphic larvae, as in the earlier postembryonic stages [START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF], locomotion consists of alternating left-right myotomal contractions that propagate along the body in a rostro-caudal sequence. Although the limb buds appear at stage 49, undulatory propulsion remains the only mode of locomotion during this developmental period. Consistent with this axial-based swimming behaviour, in vitro recordings from ventral motor roots of the isolated CNS (Fig. 2A1, left) during episodes of fictive locomotion show bilateral alternation of bursts within a given segment, with a brief rostro-caudal delay between segments (Fig. 2A2).

From the beginning of prometamorphosis at stage 53 until stage 58, the emerging hindlimbs gradually acquire motility, although this consists of alternate abductions/adductions that are often unilateral, apparently to compensate for rolling body movements of the animal. Otherwise, normal locomotion is still exclusively axial, with the hindlimbs that are still insufficiently developed to participate in active propulsion normally being held extended against the body (Fig. 2B1). However, in vitro recordings have revealed that these apparent limb adductions during axial swimming are more complex than a simple tonic activation of the motoneurons innervating hindlimb extensor muscles. Rather, recordings from motor nerve branches to the left and right anterior tibialis (ankle flexor: Flex) and gastrocnemius (ankle extensor: Ext) muscles (see Fig. 2B1 lower) showed that both flexor and extensor motoneurons to a given limb displayed strong rhythmic bursting during fictive axial swimming. It is important to note, however, that this early appendicular rhythmicity, which occurs in strict 1:1 coordination with the axial motor pattern (see boxed cycle in Fig. 2B2), consists of a co-activation of flexor and extensor motoneurons to the same limb, and an alternation between motor bursts in opposite limbs.

From stage 59, at the onset of the metamorphic climax, the limbs are fully functional and they now participate actively in body propulsion (Fig 3A1). Until the tail is resorbed, the animal is still able to employ axial swimming, either alone or in combination with limb-based swimming. In the latter condition, moreover, the two patterns may be expressed independently and with completely different cycle frequencies (Fig. 3A2; see dispersed phase values in circular plot at right) or limb activity may remain co-ordinated 1:1 with the axial rhythm (Fig. 3A3; see tightly concentrated phase values at right). In both cases, and in contrast to the earlier combined pattern during pro-metamorphosis (see Fig. 2B2), the appendicular pattern is now coordinated appropriately for generating independent hindlimb kick cycles, with homolateral flexor and extensor motoneurons bursting in alternation, and homologous bilateral motoneurons bursting synchronously. These in vitro results therefore indicate that by metamorphic climax, the hindlimb network is fully functional and generates adultspecific patterns of locomotor output. However, during this critical transitional period, the temporal relationship between the primary axial and secondary limb networks can change spontaneously from decoupled (Fig. 3A2) to coupled (Fig. 3A3) modes of coordination, and vice versa, presumably in response to changing behavioural demands.

Finally, from stage 62, resorption of the tail begins and locomotion becomes exclusively appendicular. As seen in the in vitro recordings of Fig. 3B2 obtained from a now tail-less stage 64 froglet (Fig. 3B1), bouts of rhythmicity expressed in limb motor nerves corresponded to bilaterally-synchronous hindlimb extensions (power-stroke phase of each cycle) that alternate with bipedal flexions (return-stroke phase).

The progressive changes in locomotor strategy that accompany the emergence of the hindlimbs during Xenopus metamorphosis are schematised in Fig. 4. Within a single limb (Fig. 4A), the most striking developmental change in the underlying motor command is the switch from precocious synchronous bursting in homolateral flexor and extensor motoneurons during pro-metamorphosis (Fig. 4A1, see ellipses) to the typical adult pattern at metamorphic climax when these same motoneurons to antagonistic muscles now burst in strict alternation (Fig. 4A2). In terms of the coordination between functionally homologous motoneurons in opposing hindlimbs (Fig. 4B), the opposite transition occurs in keeping with a switch in bilateral limb movement from left-right alternation (Fig. 4B1) to synchronous hindlimb thrusting (Fig. 4B2).

These developmental changes in limb motor activity occur in parallel with a progressive functional separation of limb circuitry from the primary axial network (Fig. 4C1) until a point (around the metamorphic climax) when the two co-existing circuits can be conjointly active but in rhythms with two completely different frequencies (Fig. 4C2).

However, occasionally at this mixed developmental stage, a return to co-ordinated axialand limb-based activity can occur (Fig. 4C3; see experimental recordings in Fig. 3A2,A3), and our recent data suggest that this switch between coupled and decoupled axial and limb motor rhythms may be subject to neuromodulatory control (see below).

Thus at early pro-metamorphic stages, an appendicular motor command appears to exist already, but in an immature form that remains subordinate to the axial network in terms of cycle frequency, homolateral burst synergies, and left/right alternation. This suggests that initially during metamorphosis, the newly-differentiated limb motoneurons and pre-existing axial motoneurons share common synaptic inputs from tail-swimming circuitry. As metamorphosis proceeds, however, the de novo limb-kick circuitry progressively disengages from the faster tail network, becoming increasingly free to operate at its own slower cycle frequency and with a different pattern of burst timing that is appropriate for adult hindlimb control. Thus at later metamorphic stages, the ability of axial-and limb-based motor patterns to be expressed conjointly at very different frequencies (and occasionally independently) is clearly indicative of two co-existing spinal rhythmogenic networks that are approaching a complete functional segregation.

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 in vitro rat spinal cord generates a motor pattern that consists of synchronized activity in flexor/extensor motoneurons and on both sides of the cord [START_REF] Nishimaru | Formation of the central pattern generator for locomotion in the rat and mouse[END_REF] for a review see [START_REF] Vinay | Development of posture and locomotion: an interplay of endogenously generated activities and neurotrophic actions by descending pathways[END_REF]. Two days later (at E18.5), however, the hindlimb motor command now displays bilateral alternation, although synchronous bursting still occurs in homolateral flexor and extensor motoneurons. This intermediate developmental coordination therefore strongly resembles the transitional phase in prometamorphic Xenopus (Stage 57-58), where limb motor output during fictive axial swimming also consists of left-right alternation, with flexor and extensor motoneurons on the same side bursting in synchrony (see Fig. 2B2; Fig. 4A1,B1). As the rat approaches birth (at E22), however, the adult-like pattern of motor coordination becomes established in which, unlike in late pro-metamorphic Xenopus where a shift to bilateral synchrony occurs, the newborn rat now expresses both left-right and homolateral flexor-extensor alternation.

Although the mechanisms underlying the developmental changes in Xenopus locomotory network coordination remain to be elucidated, some testable hypotheses can also be formulated from comparison with the early maturation of the rodent locomotory system. In the latter, for example, the transition from in-phase bursting on the two sides of the cord at E16 to left-right alternation in lumbar root bursts at birth [START_REF] Nishimaru | Formation of the central pattern generator for locomotion in the rat and mouse[END_REF] is thought to derive from a combination of maturational changes, including a decline in widespread electrical coupling and a maturational shift in the equilibrium potential for chloride ions toward more hyperpolarized values. This in turn accounts for a switch in GABA-and glycine-evoked potentials from functional excitation to inhibition (Ben-Ari, 2001) with a resultant transformation of the discharge of cross-cord coupled neurons from synchrony to alternation [START_REF] Nishimaru | Formation of the central pattern generator for locomotion in the rat and mouse[END_REF][START_REF] Clarac | The maturation of locomotor networks[END_REF][START_REF] Kudo | Developmental changes in rhythmic spinal neuronal activity in the rat fetus[END_REF].

The left-right alternation of locomotor circuitry in the spinal cord of premetamorphic Xenopus larvae is mediated by inhibitory glycinergic cross-cord connections [START_REF] Soffe | Ionic and pharmacological properties of reciprocal inhibition in Xenopus embryo motoneurones[END_REF], as it is in the rat [START_REF] Cowley | Effects of inhibitory amino acid antagonists on reciprocal inhibitory interactions during rhythmic motor activity in the in vitro neonatal rat spinal cord[END_REF][START_REF] Kjaerulff | Crossed rhythmic synaptic input to motoneurons during selective activation of the contralateral spinal locomotor network[END_REF]. A reasonable hypothesis therefore is that a weakening (rather than a strengthening, as is likely the case in rodents) of cross-cord glycinergic inhibition occurs in the developing limb circuitry of Xenopus, and in conjunction with an increase (and/or an emergence) of cross-cord excitation, enables network activity to switch from left-right alternation appropriate for axial swimming, to bilateral co-activation necessary for synchronous limb-kicking. This situation is in direct contrast to the requirement for a switch from synchrony to alternation in the activity of homolateral limb flexor and extensor motoneurons. Here the most likely developmental solution is that any electrical coupling between adjacent ipsilateral motoneuron pools becomes weakened in parallel with the de novo formation of intersegmental inhibitory connections to ensure alternating flexor and extensor bursting (see also [START_REF] Kudo | Developmental changes in rhythmic spinal neuronal activity in the rat fetus[END_REF].

Finally it has to be remembered that in contrast to the foetal rodent, the limbbased locomotory circuitry of metamorphosing Xenopus is assembled under the dominance of a pre-existing functional system (for tail-based swimming) which initially appropriates the still immature limb network to its own pattern of activity. Thus whereas locomotion in adult mammals derives from the progressive acquisition of functional spinal circuitry from an embryonic (and therefore non-functional) precursor, the emergence of limb-based locomotion in Xenopus is fundamentally different. Here, an already functional locomotor system (for axial swimming) is replaced by a second completely different one (for limb-based swimming), with the two systems having to coexist and operate within the same organism while the transition is taking place.

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 [START_REF] Sillar | Involvement of brainstem serotonergic interneurons in the development of a vertebrate spinal locomotor circuit[END_REF] and the isthmic region (the amphibian equivalent of the locus coeruleus [START_REF] Marin | Do amphibians have a true locus coeruleus?[END_REF]), respectively. The biogenic amines serotonin (5HT) and noradrenaline (NA) have very different short-term modulatory actions on the spinal locomotory circuitry of the hatchling tadpole. Whereas exogenously-applied 5HT increases the duration and intensity of axial motoneuron bursts with little effect on swimming frequency [START_REF] Sillar | Phase-dependent modulation of a cutaneous sensory pathway by glicinergic inhibition from the locomotor rhythm generator in Xenopus embryos[END_REF], NA slows fictive swimming and reduces burst durations relative to cycle periods [START_REF] Mcdearmid | Aminergic modulation of glycine release in a spinal network controlling swimming in Xenopus laevis[END_REF]. An important common target of this aminergic modulation is the strength of inhibitory synaptic connections within locomotory spinal circuitry, and in particular, the glycinergic inhibition of motoneurons by cross-cord commissural interneurons responsible for reciprocal midcycle inhibition and the left-right alternation necessary for undulatory swimming. Again 5HT and NA exert opposing modulatory effects, with 5HT decreasing the amplitude of mid-cycle glycernergic IPSPs during fictive swimming, whereas NA enhances them [START_REF] Mcdearmid | Aminergic modulation of glycine release in a spinal network controlling swimming in Xenopus laevis[END_REF].

5HT has also been implicated in the maturation of the larval locomotory system, and in particular, the developmental acquisition of more efficient and flexible axial swimming behaviour. This conclusion derived from previous findings that the early maturation of postembryonic swimming rhythmicity between late embryonic and early larval stages [START_REF] Sillar | Segregation of NMDA AND NON-NMDA receptors at separate synaptic contacts: evidence from spontaneous EPSPs in Xenopus embryo spinal neurons[END_REF] is strikingly similar to the effects of exogenous 5HT described above (Sillar et al., 1992), and that these effects occurred in an agedependent, rostro-caudal fashion in temporal correlation with the progressive ingrowth of serotonergic axons to the spinal cord [START_REF] Sillar | Control of locomotor movements during vertebrate development[END_REF][START_REF] Sillar | Involvement of brainstem serotonergic interneurons in the development of a vertebrate spinal locomotor circuit[END_REF][START_REF] Van Mier | The development of the motor system in the clawed toad Xenopus laevis[END_REF].

Our most recent findings also indicate that monoamines continue to exert important and different regulatory influences on spinal locomotor circuitry during the later metamorphic development of Xenopus. A particularly interesting example is the ability of 5-HT and NA to modulate, again in an opposing manner, the functional coupling between axial-and limb-based locomotor rhythms in animals at metamorphic climax (see Fig. 4C2, 3; Rauscent et al., unpublished observations). In stage 61 in vitro preparations that are spontaneously expressing independent axial and limb rhythms (Fig. 4C2), bath-applied 5-HT (5-10µM) causes an acceleration of appendicular rhythmicity, a slowing down of axial activity, and a 1:1 coupling of the two otherwise separate patterns into a single, combined rhythm (Fig. 4C3). In contrast, the presence of NA (10µM) has the opposite effect, in that during already spontaneously-coupled locomotor rhythmicity (as in Fig. 4C3), this amine's action is to dissociate the two rhythms by causing a differential increase and decrease, respectively, in the frequency of axial and limb activity. It seems likely that these opposing network-specific effects on cycle rate are accompanied by an amine-dependent increase (for 5-HT) and decrease (for NA) in the strength of synaptic interactions between the two circuits. Presumably such short-term plasticity in the functional relationship between co-existing locomotor networks is designed to satisfy the immediate propulsive requirements of the animal, and it now remains to be seen whether 5-HT and/or NA are additionally engaged in the longer-term developmental changes in Xenopus locomotor circuitry during metamorphosis.

Another source of supraspinal influence on Xenopus tadpole locomotor circuitry is the gaseous signalling molecule nitric oxide (NO) which has been implicated in numerous developmental and physiological processes throughout the animal kingdom.

NO has also been found to be involved in developmental mechanisms as diverse as neurogenesis, cell proliferation [START_REF] Kuzin | Nitric oxide regulates cell proliferation during Drosophila development[END_REF], differentiation [START_REF] Contestabile | Role of nitric oxide in the regulation of neuronal proliferation, survival and differentiation[END_REF] and apoptosis [START_REF] Pinsky | Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture[END_REF][START_REF] Zhang | Nitric oxide synthase inhibition prevents neuronal death in the developing visual cortex[END_REF]. Our recent findings are consistent with an equally multi-faceted role of NO in the metamorphic development of Xenopus locomotory spinal circuitry [START_REF] Ramanathan | Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis[END_REF]. This has been suggested by the progressive pattern of appearance of NO-synthase (NOS) containing neurons within the spinal cord during metamorphosis. Whereas in pre-metamorphic tadpoles, NOS expression is restricted to the brainstem (McLean andSillar, 2000, 2002), at early stages of pro-metamorphosis (coincident with the emergence of the foreand hindlimb buds), two distinct clusters of spinal NOS-positive neurons interspersed with areas devoid of stained somata are observed. Interestingly, motoneurons innervating the fore-and hindlimb buds (revealed after retrograde labelling with horseradish peroxidase (HRP)) are located exclusively in areas where NOS staining is absent, indicating that during early stages of metamorphosis, nitrergic neurons are excluded from the very cord regions where the new limb circuits are forming. Therefore it is possible that, as reported elsewhere in the Xenopus CNS [START_REF] Peunova | Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain[END_REF], the rat brain [START_REF] Moreno-Lopez | Nitric oxide is a physiological inhibitor of neurogenesis in the adult mouse subventricular zone and olfactory bulb[END_REF] and Drosophila [START_REF] Kuzin | Nitric oxide regulates cell proliferation during Drosophila development[END_REF], NO may have an early negative influence on network development, so that its absence from the cervical and lumbar spinal regions in early pro-metamorphic stages could allow the neuronal proliferation necessary for the initial assembly of limb locomotor circuitry.

However, as Xenopus reaches late pro-metamorphosis, NOS expression is found to be evenly distributed along the entire length of the spinal cord [START_REF] Ramanathan | Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis[END_REF]. Thus NO could now have a completely different regulatory role such as occurs in the developing brains of the chick and rodent, where NO is positively engaged in the late-phase refinement of dendritic topographies [START_REF] Inglis | The role of nitric oxide and NMDA receptors in the development of motor neuron dendrites[END_REF] and the final adult tuning of synaptic connectivity [START_REF] Wu | The role of nitric oxide in development of topographic precision in the retinotectal projection of chick[END_REF].

In addition to a likely developmental role, NO has been found to act as a potent modulator of locomotory rhythmicity in post-embryonic Xenopus tadpoles where it has been shown to decrease the duration and frequency of swimming episodes through both direct and indirect influences at spinal and brainstem levels, respectively (McLean andSillar, 2000, 2002). Our preliminary data suggest that NO continues to modulate locomotor activity through metamorphosis, albeit in a strikingly different fashion to the hatchling embryo. In contrast to the pre-metamorphic animal where NO has a net inhibitory effect on swimming, exogenously-applied NO to pro-metamorphic spinal cord/brainstem preparations activates both axial and limb motor rhythms, while removal of endogenous NO has the opposite effect (Combes et al., unpublished observations).

Presumably this switch in NO's modulatory action reflects developmental alterations in brainstem nitrergic systems and/or in the responsiveness to NO of the downstream spinal networks themselves.

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 (Figure 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 its normal environment (Fig. 5A). Thus at critical stages of metamorphosis, the two propulsive mechanisms together with their underlying neural machinery must operate within the same animal, prior to the adult system progressively superseding the larval system as the latter disappears. This replacement of one already functional system by another in Xenopus contrasts with metamorphosis in urodele amphibians, such as the salamander Pleurodeles waltlii, which also experiences the emergence of limbs in primary axial-swimming larvae and gains the capacity for secondary quadrupedal locomotion. However Pleurodeles and Xenopus differ in two major ways: firstly, the salamander does not lose its tail during metamorphosis and thereby conserves the ability for both axial-and limb-based locomotion in adulthood. Moreover, while the neural machinery for both locomotor strategies co-exist within the adult salamander's spinal cord, in contrast to the metamorphosing Xenopus, the two behavioural modes are rarely co-expressed, with undulatory swimming being used solely in an aquatic environment while quadrupedal walking is employed during terrestrial locomotion [START_REF] Delvolve | Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt, Pleurodeles waltl[END_REF]; see Figure 5B). Furthermore, computer simulations have suggested that both behaviours may actually derive from different functional configurations of the same basic spinal circuitry [START_REF] Bem | From swimming to walking: a single basic network for two different behaviors[END_REF], a likelihood that is supported by a second fundamental difference between Xenopus and Pleurodeles: during walking in the latter, the flexion/extension movements of limbs at the same (scapular or pelvic) girdle operate in left/right alternation (rather than in synchrony as in Xenopus adults), in a same manner as the axial musculature during undulatory swimming.

Fundamental differences also exist between the developmental transformations of Xenopus and insect metamorphosis. For example, holometabolous insects such as the moth Manduca sexta, and the fruitfly Drosophila melanogaster, undergo a complete transformation from a crawling larva to a walking/ flying adult (Figure 5C). In contrast to amphibians, this metamorphic transition is mediated by a resting pupal stage in which all locomotory activity ceases. A further important difference is that in Manduca and Drosophila, although the assembly of adult-specific neural circuitry relies to a certain extent on neurogenesis during larval or pupal stages, most adult motoneurons and remaining interneurons derive from a functional and anatomical re-specification of preexisting larval circuitry [START_REF] Consoulas | Behavioral transformations during metamorphosis: remodeling of neural and motor systems[END_REF]. This again contrasts with anuran metamorphosis during which primary axial motoneurons and associated spinal circuitry disappear with tail resorption, while entirely new populations of secondary motoneurons and sensory neurons are born to service the emerging limbs [START_REF] Van Mier | The development of the motor system in the clawed toad Xenopus laevis[END_REF].

Finally, in hemimetabolous insects, such as the locust and cricket (Figure 5D) in which metamorphosis is incomplete and there is no resting pupal phase, most adultspecific motor networks are assembled during embryogenesis, but await appropriate levels of maturation before becoming functional. In the flightless nymphal locust, for example, the central flight motor circuitry is already present and potentially operational, but it is either actively inhibited until the wings become fully developed or awaits permissive modulatory signals that appear in adulthood [START_REF] Stevenson | Demonstration of functional connectivity of the flight motor system in all stages of the locust[END_REF].

Although evidently different from the metamorphic assembly of limb motor circuitry in Xenopus, an equivalent precocious development has been reported in the bullfrog Rana catesbeiana where functional neural networks responsible for adult aerial respiration are already present at gill-breathing premetamorphic stages, but only become active after metamorphosis when a developmental removal of GABAergic inhibition occurs [START_REF] Straus | Developmental disinhibition: turning off inhibition turns on breathing in vertebrates[END_REF].

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 [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF], 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 accompanying metamorphosis that hitherto have not been experimentally tractable. Since the rise in plasma levels of thyroid hormones is necessary and sufficient to orchestrate the entire metamorphic process, THs are well positioned at the pinnacle of a signalling cascade which, in the spinal cord, triggers neuronal differentiation, synaptogenesis, apoptosis and many other events that allow the new limb circuitry to develop within the framework of an existing axial system. Whilst the signals in this hierarchy are not yet known, one possibility is that THs trigger the expression of NOS in the spinal cord in a regionallyand temporally-specific pattern and that NO then engages an ensemble of subordinate developmental and modulatory pathways that in turn regulate the emergence of the limb circuit and the disappearance of the axial system [START_REF] Ramanathan | Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis[END_REF]. At earlier embryonic stages of tadpole development, around the time of hatching, NO functions as a metamodulator to shift the balance of influence of two aminergic systems with opposing actions on swimming (serotonin and noradrenaline) towards the noradrenergic form of output (McLean and Sillar, 2004). It is therefore of considerable interest that the two amines also exert opposing modulatory actions on the expression of tail and limb rhythms during metamorphosis, albeit different influences to those occurring earlier in pre-metamorphic development. Might NO also differentially regulate the interactions between these two spinal locomotory systems via the metamodulation of descending aminergic (and other) pathways?

Another avenue for exploration resides at the molecular developmental level. Much is now known about gene expression and the transcription factors that regulate the differentiation of spinal circuitry and the ontogeny of limb movements in mammals.

Similar molecular mechanisms also appear to be engaged during the development of the zebrafish swimming system, suggesting a common embryonic template for spinal motor system assembly. However, the construction of the limb circuitry in Xenopus takes place at comparatively much later stages in development and, rather than the limb and axial spinal networks emerging in parallel, the limb network forms once the axial system is already mature. Is the same palette of molecular signals expressed at these later stages, and how does this secondary developmental programme for the construction of new neuronal types and connections coordinate and interact with the molecular substrate of already existing tail circuitry? Resolving such issues should help provide insights into the developmental assembly and operation of related neural networks for behaviour in general. [START_REF] Nieuwkoop | Normal Tables for Xenopus laevis[END_REF] until the onset of metamorphosis at stage 54. TH levels increase during prometamorphosis (stages 54-58) to peak at the climax (stages 59-64) when the major changes in body format occur. THs then decline until the completion of metamorphosis at stage 66. The entire metamorphic process lasts ~4 weeks. AU: arbitrary units. (Adapted from [START_REF] Leloup | La triiodothyronine: hormone de la métamorphose des amphibiens[END_REF].

Figure 1 .

 1 Figure 1. Timetable of development in Xenopus laevis and the correlation of changes in

Figure 2 .

 2 Figure 2. Fictive swimming in isolated spinal cord/brain stem preparations from pre-

Figure 3 .

 3 Figure 3. Fictive swimming in isolated spinal cord/brain stem preparations from a

Figure 4 .

 4 Figure 4. Summary of changes in co-ordination within and between limb and tail motor

Figure 5 .

 5 Figure 5.

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).


Anna Beyeler

Charles Métais

Denis Combes

John Simmers

Didier Le

Didier Le Ray
email: didier.leray@u-bordeaux2.fr

Metamorphosis-induced changes in the coupling of spinal thoraco-lumbar motor outputs during swimming in Xenopus laevis

Keywords: Amphibian, motor networks, propriospinal pathways, development, locomotion, posture

Anuran metamorphosis includes a complete remodeling of the animal's biomechanical apparatus, requiring a corresponding functional reorganization of underlying central neural circuitry. This involves changes that must occur in the coordination between the motor outputs of different spinal segments in order to harmonize locomotor and postural functions as the limbs grow and the tail regresses. In pre-metamorphic Xenopus laevis tadpoles, axial motor output drives rostro-caudally propagating segmental myotomal contractions that generate propulsive body undulations. During metamorphosis, the anterior axial musculature of the tadpole progressively evolves into dorsal muscles in the post-metamorphic froglet, in which some of these back muscles lose their implicit locomotor function to serve exclusively in postural control in the adult. To understand how locomotor and postural systems interact during locomotion in juvenile Xenopus, we have investigated the coordination between postural back and hindlimb muscle activity during free forward swimming. Axial/dorsal muscles, which contract in bilateral alternation during undulatory swimming in pre-metamorphic tadpoles, change their left-right coordination to become activated in phase with bilaterally-synchronous hindlimb extensions in locomoting juveniles. Based on in vitro electrophysiological experiments as well as specific spinal lesions in vivo, a spinal cord region was delimited in which propriospinal interactions are directly responsible for the coordination between leg and back muscle contractions. Our findings therefore indicate that dynamic postural adjustments during adult Xenopus locomotion are mediated by local intraspinal pathways through which the lumbar generator for hindlimb propulsive kicking provides caudo-rostral commands to thoracic spinal circuitry controlling the dorsal trunk musculature.

INTRODUCTION

Many behavioral tasks in invertebrates and vertebrates alike are driven by primary motor programs that arise from neuronal network assemblages within the central nervous system. Vertebrate locomotion is one of the best understood of such functions, the basic motor program for which being generated by local spinal networks that are controlled by higher centers in the brainstem and are continually shaped by sensory information from the periphery [START_REF] Rossignol | Dynamic sensorimotor interactions in locomotion[END_REF]. Over the last thirty years, many studies on a variety of animal species have analyzed the functional organization of spinal and supra-spinal structures involved in the orchestration of rhythmic locomotor movements (for reviews, see [START_REF] Cazalets | Ubiquity of motor networks in the spinal cord of vertebrates[END_REF][START_REF] Grillner | Biological pattern generation: the cellular and computational logic of networks in motion[END_REF][START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF].

In quadrupedal vertebrates, the locomotor programs for anterior and posterior limb movements are elaborated by so-called "central pattern generators" (CPGs; for reviews, see [START_REF] Grillner | Control of locomotion in bipeds, tetrapods and fish[END_REF][START_REF] Kiehn | Locomotor circuits in the mammalian spinal cord[END_REF]) that are located in the cervical [START_REF] Ballion | Forelimb locomotor generators and quadrupedal locomotion in the neonatal rat[END_REF]Juvin et al. 2005) and lumbar spinal enlargements, respectively [START_REF] Cazalets | Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat[END_REF][START_REF] Langlet | Mid-lumbar segments are needed for the expression of locomotion in chronic spinal cats[END_REF]. For meaningful locomotory behavior, however, musculature in other body regions such as the trunk and neck must be coordinated with limb movements to ensure postural stability during body displacements. Rhythmic activation of axial trunk muscles in time with appendicular locomotor activity has been reported in rat (Gramsbergen et al. 1999, Falgairolle and[START_REF] Falgairolle | Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat[END_REF], cat [START_REF] Koehler | Phasic modulation of trunk muscle efferents during fictive spinal locomotion in cats[END_REF][START_REF] Zomlefer | Electromyographic study of lumbar back muscles during locomotion in acute high decerebrate and low spinal cats[END_REF] and humans (Thorstensson et al. 1982). However, although earlier experiments on decerebrate paralyzed cat suggested a propriospinal origin of coordinated limb-trunk muscle contractions [START_REF] Koehler | Phasic modulation of trunk muscle efferents during fictive spinal locomotion in cats[END_REF], the neural basis of such coupling and the extent to which it is governed by rhythmogenic spinal circuitry remains largely unknown [START_REF] Falgairolle | Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat[END_REF]. Similarly, little is known of the changing functional relationship that must occur between locomotory and postural control systems during post-natal development.

Metamorphosis from tadpole to frog in anuran amphibians like Xenopus laevis constitutes one of the most striking developmental transformations in biology, involving fundamental alterations in virtually all of the animal's physiological systems and body structure (for a review, see [START_REF] Shi | Amphibian metamorphosis: from morphology to molecular biology[END_REF]. One of the most dramatic changes occurs in the biomechanical apparatus, whereby the tail is resorbed and new limbs are formed as the organism changes its mode of locomotion from tail-based undulations in larvae to limbbased propulsion in the adult. In pre-metamorphic tadpoles, body displacement is driven by waves of bilaterally-alternating muscle contractions that are directed rostro-caudally along the body axis, whereas in post-metamorphic juveniles, bilaterally-synchronous hindlimb kicking is principally used to propel the animal forward. Previous studies strongly suggested that this metamorphosis-induced transition in locomotor strategy results principally from the gradual emergence of a lumbar CPG specifically dedicated to the control of the newly developed hindlimbs [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF][START_REF] Rauscent | Development and neuromodulation of spinal locomotor networks in the metamorphosing frog[END_REF]). However, the functional destiny of original axial motor circuitry that persists in the adult spinal cord remains to be determined. Although the caudal spinal cord segments that control axial movements in tadpoles disappear with tail resorption after metamorphic climax, the segments above the lumbar enlargement are preserved in adulthood and must also adapt to the new body format. To what extent do developmental changes in the organization of segmental networks relate to the animal's needs for postural control during swimming? More specifically, what coordinating processes enable certain axial muscles, which are directly engaged in body propulsion in the pre-metamorphic larvae, to assume a dynamic postural function after their transformation into non-locomotory back muscles in post-metamorphic adults?

To begin to address these questions, we have analyzed the bilateral coordination of axial/back muscle activity in freely-behaving, pre-metamorphic Xenopus tadpoles and, together with the locomotor activity of hindlimb muscles, in post-metamorphic young adults. In the latter, moreover, simultaneous electromyographic (EMG) recordings were made from back and limb muscles on both sides of the body during free straight-ahead swimming, then a series of spinal cord lesions was performed to better understand the neural origins of the temporal relationships between activity in these muscle sets in vivo. We show that the rostro-caudal recruitment sequence and left-right alternation of thoracic axial muscles in the tadpole is replaced during metamorphosis by a different adult coordination pattern in which back and leg muscles contract synchronously during swimming, in a bilaterally in-phase pattern. Based on in vivo lesions and in vitro experiments on isolated brainstem-spinal cord preparations, it appears that the lumbar CPG for hindlimb locomotion is also directly responsible for driving thoracic motor output to dorsal muscles in post-metamorphic Xenopus. Given the developmental switch in function of the more rostral axial muscles in larvae to non-propulsive trunk musculature in the adult frog, the Xenopus model should help to provide new insights into the dynamic interactions between locomotory and postural control systems in general. Part of this work has been presented previously in abstract form [START_REF] Beyeler | Propriospinal coordination of posturo-locomotor function in post-metamorphic Xenopus laevis[END_REF].

MATERIALS AND METHODS

Experiments were performed on four pre-and three pro-metamorphic tadpoles (stages 54 to 57 and to 60-61, respectively; [START_REF] Nieuwkoop | Normal table of Xenopus laevis (DAUDIN). A systematical and chronological survey of the development from the fertilized egg till the end[END_REF] and 50 postmetamorphic juvenile adults (stage 66, <1 month after metamorphosis) of the South African clawed toad, Xenopus laevis, bred from an in-house laboratory colony.

Tadpoles were raised until near stage 50 at 20°C and then kept at room temperature throughout metamorphosis. EMG recordings were performed on animals at room temperature in separate aquaria, while in vitro experiments were performed at 16-18°C.

All procedures were in keeping with the animal care guidelines of the Bordeaux University and the CNRS.

In vivo electromyographic recordings EMG activity of rostral axial musculature in larvae and back and leg muscles in juveniles was recorded using pairs of 50 µm insulated wire electrodes connected through a grounded cable to a differential AC amplifier (Model 1700, AM-System Inc., WA). Axial muscles were recorded at the level of the 5 th segment of the tadpole spinal cord, corresponding to the segment that commands the future back muscles in postmetamorphic juveniles. In the latter, EMG recordings were made on both sides of the trunk from the third myomere of the dorsalis trunci, which is located dorsally along the vertebral column at mid-distance between the fore-and hindlimbs [START_REF] Vallois | Les transformations de la musculature de l'épisome chez les Vertébrés[END_REF], and from the ankle extensor plantaris longus (often called gastrocnemius in frogs; Peters 2005) of both hindlimbs. The implantation of EMG recording wires was performed under light anesthesia after a small incision had been made in the overlying skin, then the electrodes were fixed to the muscle surface with a spot of Vetbond 3M adhesive (World Precision Instruments, UK). The skin was then replaced and attached with glue.

EMG signals were directed to a computer through a CED Micro 1401 interface (Cambridge Electronic Design Ltd, UK) for storage and later analysis using Spike 2 (CED) software.

Surgery for spinal lesions in juveniles

Juvenile frogs were individually anesthetized with freshly dissolved tricaine methanesulfonate (MS 222, 50mg/L, Sigma-Aldrich) and positioned dorsal side up in a Petri dish filled with frog saline (in mM: NaCl 112; KCl 2; NaHCO 3 20; CaCl 2 2,8; MgCl 2 1; Glucose 17). After an incision was made in the dorsal skin, muscles were carefully removed and the underlying vertebrae were opened dorsally along the thoracic and/or lumbar cord regions. In order to separate the thoracic spinal cord from other regions of the nervous system, three types of lesion were made separately or in combination:

(i) a transection at the level of the last cervical cord segment;

(ii) a longitudinal (sagittal) lesion that extended through only the three thoracic segments;

(iii) a hemi-or whole-cord transection between the last thoracic and first lumbar segments;

After lesioning, the spinal cord was cleaned and covered with a small piece of gauze compress soaked in saline. The skin was then replaced and attached with Vetbond glue.

Animals were allowed 24 hr to recover from anesthesia and surgery before EMG recordings commenced.

The extent of spinal lesions was verified after experimental recordings, and only data from animals with appropriate lesions were analyzed in this study. Control experiments on operated juvenile Xenopus but with the spinal cord remaining intact (n=4) showed no significant changes in either their patterns of free swimming or the coordination between dorsalis trunci and plantaris longus muscle activity (data not shown). EMG recordings in pre-and pro-metamorphic tadpoles were restricted to rostral axial muscles at a level that corresponded to the adult thoracic cord region.

Retrograde staining of dorsalis motoneurons

The somata of motoneurons innervating the dorsalis trunci muscles were located in the spinal cord by means of retrograde axonal staining from target muscles in vivo. Four post-metamorphic animals were anesthetized in MS 222 and placed on ice in a Petri dish. A short incision was made in the skin along the dorsal midline in order to gain access to the back muscles. Small crystals of two fluorescent dyes (alexa fluor 488 and 546 coupled to dextran 10,000) were inserted separately with a fine pin into the left and right third myomeres of dorsalis trunci. The skin was then resealed with glue, and animals were placed in separate aquaria for recovery. After three days to allow retrograde dye migration across the neuromuscular junction and along motoneuron axons to the CNS, animals were sacrificed and their spinal cords dissected out and fixed overnight at 4°C in 4% paraformaldehyde in phosphate buffer 0.1 M, pH=7.4. After dehydration, the cords were cleared in glycerol and examined with a fluorescent microscope (Leica-DMRB, France) equipped with a CCD camera (ORCA-AG Hamamatsu, France). Images were acquired using Simple PCI software (Compix Inc., PA, USA).

Isolated brainstem-spinal cord preparations

Dissection and recording procedures were similar to those previously described by [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]. The skin and cranial cartilage of juvenile frogs were opened under deep anesthesia with tricaine methanesulfonate and the forebrain removed above the mesencephalon. The spinal cord and remaining brainstem were dissected out together with the thoracic ventral roots and the nerve branches that innervate the hindlimb tibialis anterioris and the plantaris longus, which were previously identified as being ankle flexor and extensor muscles, respectively (d'Avella et al. 2003;d'Avella & Bizzi, 2005). Isolated preparations were then transferred into a recording chamber and kept under oxygenated frog saline at 16-18°C. Suction electrodes were used to record motor activity from left and right thoracic ventral roots, while Vaseline-insulated wire electrodes were used to record bilateral extensor and flexor activity "en passant" in distal limb motor nerve branches. Ventral root and nerve signals were amplified, displayed and stored in the same way as for EMG recordings.

All spinal motor output patterns, including those related to swimming, occurred spontaneously without additional chemical or electrical stimulation. Unfortunately, in vitro preparations subjected to cervico-thoracic or thoraco-lumbar transections were found incapable of producing bouts of coordinated fictive locomotion, either spontaneously or under classical pharmacological stimulation (with bath-applied NMDA, bicuculline, serotonin, noradrenalin, either separately or together). However, as with the intact spinal cord, isolated preparations with a sagittally-lesioned thoracic cord continued to generate episodes of spontaneous swimming motor output. Only data from these latter experiments were analyzed and are illustrated here.

Data analysis

All analyses of electrical recordings were performed using homemade scripts running under Spike 2 (Spike 2 language, CED). Raw signals were first integrated, and only activity sequences obtained during episodes of rectilinear forward swimming were analyzed further. The onsets of motor bursts occurring during such episodes were automatically detected in the integrated traces, and pooled data according to animal groups (control, longitudinal spinal cord lesion, etc.) were then transferred to Oriana software (Kovach Computing Services, UK) for circular phase analysis of the temporal relationship between activity in selected pairs of muscles or nerves. The results of this analysis gave the mean vector µ and its length r, and two tests were used to examine the circular distributions: (i) the Rayleigh test (Z) which tested the concentration of phase values around the mean vector, with a random distribution indicating a lack of coordination between the two compared burst activities and (ii), the V-test (u) which tested for a preferential direction (angle) of a given phase distribution (indicated in the text as u(direction°)). In order to simplify graphical representations, phase values were plotted as grand means of the means of relative burst onsets throughout individual locomotor episodes. Nevertheless, the similarity between distributions (for example between control and lesioned animal groups) was verified for whole populations of events using the Mardia-Watson-Wheeler test (W). The angular dispersion around the mean vector µ was also calculated in order to assess the effect of a given lesion on the power of coupling between different muscle or nerve discharges, with the size of the angular dispersion value being inversely proportional to the strength of coupling. A two-tailed non-parametric t-test was used to compare phase dispersions in groups of control and lesioned animals.

2D kinematics in juvenile animals

Intact and lesioned juvenile frogs were video-taped with a digital camera

RESULTS

Developmental changes in bilateral axial muscle coordination during swimming in metamorphosing Xenopus

In pre-metamorphic Xenopus laevis (stages 54-57), forward locomotion is achieved by rhythmic body undulations produced by bilaterally-alternating spinal ventral root bursts that drive myotomal muscle contractions in rostro-caudally directed waves along the tail [START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF][START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]. To observe such axial muscle activity in freely-behaving tadpoles, we recorded from the dorsal region of the myotome of the fifth spinal cord segment, which in the adult corresponds to the second thoracic segment controlling the back musculature. Consistent with earlier observations from spinal ventral root recordings in vitro [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]), EMG recordings from left and right rostral axial muscles (hereafter referred to as axial/dorsal (ad)) in intact tadpoles were activated alternately during episodes of actual locomotion (Fig. 1A). The strongly clustered distribution of left versus right muscle burst onsets (n=7 episodes; µ=170.00°; r=0.99; Fig. 1A right) was further indicative of a strong phase-coupling (Z=6.84; p<0.001) with a phase difference of near 180° (u(180°)=3.64; p<0.001) corresponding to symmetrical left-right alternating contractions necessary for rectilinear propulsion.

During metamorphosis, anurans like Xenopus develop articulated limbs that become functional while the larval tail continues to play an active locomotor role for 1 to 2 weeks prior to its degeneration [START_REF] Nieuwkoop | Normal table of Xenopus laevis (DAUDIN). A systematical and chronological survey of the development from the fertilized egg till the end[END_REF][START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF].

During this transitional period, both axial and appendicular locomotor systems can Following metamorphosis (stage 66; 17 animals) the more rostral axial muscles, which now correspond to postural back muscles (dorsalis trunci, referred to as "dt" in Fig. 1C and following figures), continue to be co-activated during swimming that is now solely limb-based following resorption of the tail. Thus, although uncoordinated bursts may also occur occasionally (not illustrated), circular analysis of left versus right dorsalis trunci bursts revealed a relatively narrow phase distribution (n=37 episodes; µ=358.59°; r=0.98; Z=35.39; p<0.001) that had a preferential direction toward phase 0° (u(0°)=8.41; p<0.001). These findings therefore show that during metamorphosis, dorsal axial muscles alter their pattern of activation during swimming from strict bilateral alternation during tail-based locomotion to predominant synchrony during limb-based propulsion. Moreover, in mixed metamorphic stages when both tail-and limb-based propulsion occurs, the ad muscles of a given animal can switch, even within the same swimming episode, between either coordination pattern according to the type of locomotion being expressed. While the neural basis of axial muscle recruitment during swimming in pre-metamorphic Xenopus tadpoles has been well described [START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF], the coordinating mechanisms engaged in driving these muscles during limb-based locomotion in the adult are unknown. The following in vivo and in vitro experiments were therefore designed to explore the neural origin of the synchronous activation of bilateral dorsal trunk muscles during forward swimming in post-metamorphic animals.

Coordination of hindlimb and dorsal muscles during rectilinear swimming in juvenile adults

The hindlimb muscle activity patterns that allowed a distinction to be made between rectilinear forward swimming and turning behavior can be seen in the kinematics analysis and associated EMG recordings of Figure 2. Episodes of linear swimming are characterized by bilaterally-symmetrical kick trajectories of the hind legs (see stick diagrams in Fig. 2A1), unlike the uncoordinated limb movements associated with nonlinear swimming (Fig. 2B1). As can be seen in the traces of the hindlimb knee and ankle joint excursions in Figure 2A2, B2 (upper traces), the coordinate movements of the two right limb joints occurred as an in-phase mirror image of the homologous left limb joint excursions during a linear swim episode (Fig. 2A2). In contrast, the excursions of these same joints became asymmetric and irregular during turning maneuvers (Fig. 2B2).

Correspondingly, in simultaneous left-right EMG recordings, the ankle extensor plantaris longus muscles (referred to as "pl" in Fig. 2 and following figures) of the hindlimbs were synchronously active with regularly-recurring bursts during rectilinear swim episodes (Fig. 2A2, lower traces; see also Fig. 3B,C), whereas the activity of the same muscles became desynchronized during non-linear swimming (Fig. 2B2 bottom traces). In the remaining in vivo and in vitro experiments, therefore, we focused attention on episodes of real or fictive swimming in which synchronous bursting occurred either in the left and right plantaris muscles or in the corresponding extensor motor nerves.

In order to determine how back and hindlimb motor activity is coordinated during young adult swimming, we made simultaneous EMG recordings in vivo from the left and right plantaris longus muscles and the third myomeres of dorsalis trunci (Fig. 3A,B). An initial analysis of the phase relationship between left and right plantaris longus burst onsets showed a close coordination (n=21 episodes; µ=4.89°; r=0.98; Z=20.04; p<0.001; Fig. 3C middle polar plot) that was centered around a 0° phase value (u(0°)=6.31; p<0.001). Thus, although some phase dispersion occurred, the main tendency was for bilateral synchrony to occur between the two extensor muscles, commensurate with the generation of linear forward propulsion (see Fig. 2A, compare with 2B).

In addition to the bilateral synchrony of dorsalis trunci muscle activity during swimming (Fig. 1C and Fig. 3B,C left polar plot), a phase analysis of dorsalis EMG activity versus ipsilateral plantaris bursts (n=23 episodes; µ=3.59°; r=0.96; Z=21.37; p<0.001; Fig. 3C right polar plot) also indicated coincident discharge of these trunk and limb muscles (also see Fig. 3B), with a mean burst onset phase vector that was directed at 0° (u(0°)=6.53; p<0.001). Thus, in contrast to the rostro-caudal propagation of alternating contractions of the equivalent muscles in the pre-metamorphic tadpole, the relative timing of dorsal muscle contractions had altered with the change in mode of locomotion during metamorphosis, so that the activation of dorsalis trunci in the froglet was now phase-locked with bilaterally-synchronous hindlimb movements.

Furthermore, the strength of activation of the dorsal and hindlimb extensor muscle pairs displayed similar relationships with swimming speed (mean frequency 4.41.2 Hz; 17 animals). In both cases, area measurements of individual bursts in the integrated EMG traces revealed a linear and slightly positive correlation between burst area and the frequency of the corresponding swim cycle (regression coefficient (r c )=0.44, p<0.001, regression slope (a)=0.11 and r c =0.54, p<0.001, a=0.09 for dorsalis and plantaris muscles, respectively; n=450 cycles). Moreover, the intensities of back versus limb muscle bursts were themselves significantly and positively correlated (r c =0.24, p<0.001, a=0.37), which further pointed to the close temporal relationship between locomotor-related output produced in the thoracic and lumbar cord regions.

We next investigated whether this coupling between dorsalis trunci and plantaris longus activity was mediated by direct propriospinal interactions between the thoracic and lumbar cord regions or resulted indirectly from sensory reflexes, as has been reported to occur, for example, in the maintenance of human stance [START_REF] Tokizane | Electromyographic studies on tonic neck, lumbar and labyrinthine reflexes in normal persons[END_REF]. To this end, an in vitro brainstem/spinal cord preparation, therefore with no movement-related sensory feedback, was used to assess the effects of deafferentation on the temporal relationships between motor activity recorded in the second thoracic ventral roots that normally innervate dorsalis trunci and the distal nerve branches innervating the ankle extensor and flexor muscles (Fig. 3D). As previously reported [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]), such isolated preparations (n=7) continue to express spontaneous episodes of fictive locomotion (Fig. 3E). Although generally shorter (8.2±3.5 vs.

10.5±2.9 cycles; p<0.05) and slower (1.77±0.5 vs. 4.4±1.2 Hz; p<0.001) than swimming episodes in the intact animal, these in vitro motor patterns continued to exhibit burst phase relationships that corresponded closely to trunk and limb movements in vivo (Fig. 3F).

Effects of spinal lesions on the coordination between juvenile hindlimb and dorsal muscle activity

To further understand the phase-coupling of back and hindlimb muscle contractions during linear forward swimming, we made specific lesions to the spinal cords of juvenile frogs (see Methods) in order to define the structural substrate necessary for ensuring such a strong functional relationship.

Cervico-thoracic cord lesions

In higher vertebrates including humans, it is commonly acknowledged that the coordination between postural back muscle activity and limb locomotor movements is largely governed by descending influences from supra-spinal centers [START_REF] Takakusaki | Role of basal ganglia-brainstem systems in the control of postural muscle tone and locomotion[END_REF][START_REF] Lalonde | Brain regions and genes affecting postural control[END_REF]. To assess whether, in the lower vertebrate Xenopus, dorsalis trunci activation and its coordination with hindlimb movements might also depend on cerebrospinal commands, a series of in vivo and in vitro experiments was performed in which a complete spinal cord transection was made between the last cervical and the first thoracic segments. Unfortunately, no consistent patterns of fictive swimming could be evoked in isolated preparations after such a cervico-thoracic cord transection (n=8; not shown), and only lesions performed in vivo provided useful data (n=11; Fig. 4). Although these lesioned animals were unable to produce spontaneous locomotor-related activity, tactile stimulation of one of the hindlimbs could immediately elicit short episodes of linear swimming kick movements that were similar to those expressed in intact animals (Fig. 4A). Limb and trunk kinematics during such episodes were also comparable to those of intact animals (not shown), and again, the left and right plantaris longus muscles remained active in strict synchrony (n=7 episodes; µ=2.84°; r=0.99; Z=6.86; p<0.001; u(0°)=3.71; p<0.001; Fig. 4B, middle polar plot).

However, the coupling between these muscles was increased significantly by the rostral cord transection (W=24.68; p<0.001) as seen in the middle histogram pair of Figure 4C, right plantaris longus and dorsalis vs. plantaris muscle activity, as indicated by the substantial increase in r values for all phase relationships presented in Table 1. These latter experiments therefore supported the conclusion that propriospinal projections from the lumbar CPG circuitry to thoracic cord segments are sufficient and necessary for maintaining the strict lumbo-thoracic coupling observed during normal rectilinear swimming.

Finally, sagittal thoracic cord lesions to in vitro preparations (Fig. 5E) had effects on fictive locomotor patterns (n=4; Fig. 5F-H) that were broadly similar to those seen in vivo (compare with Fig. 5A-D). This included a persistence of overall synchrony were observed in the overall coordination between locomotor-related bursting in the plantaris longus motor nerves and thoracic ventral roots after a thoracic sagittal section in vitro (Fig. 5F, compare with recordings from intact isolated cord in Fig. 3E), although inexplicably, the strength of the coupling between both left and right limb extensor and flexor nerve bursts increased significantly (bilateral tibialis nerves, W=54.47; p<0.001: plantaris nerves, W=78.07; p<0.001) due to a decreased variability in their phase distributions (Fig. 5H; p<0.01 and p<0.001, respectively).

Thoraco-lumbar cord lesions

In order to confirm that the phasic activation of the left and right dorsalis trunci derives directly from the lumbar locomotory CPG, complete spinal cord transections were made between the last thoracic and first lumbar segments. Here again, such lesions prevented locomotor-related activity in in vitro preparations (n=8; not shown), and meaningful data was only obtained from in vivo experiments (n=11; Fig. 6A-C) in which the dorsalis or plantaris muscles were activated by briefly pinching either an anterior or a posterior limb, respectively. With hindlimb stimulation (Fig. 6A1), only arrhythmic and mostly uncoordinated plantaris muscle activity was observed. In contrast, EMG-recorded dorsalis (i.e. at a level more rostral to the cord transection) expressed left and right patterns of activity (Fig. 6A2 and left plot in Fig. 6B) in response to a forelimb stimulation (which also elicited bouts of forelimb movements; not illustrated) that tended to be synchronous (µ=359.42°; r=0.73; Z=22.05; p<0.001; u(0°)=6.64; p<0.001) despite a wider phase dispersion (W=12.08; p<0.01; and Fig. 6C left) than in intact control animals. As expected, no temporal correlation was found between dorsalis activity and the occasional plantaris bursts that occurred in response to hindlimb pinching (µ=302.16°;r=0.69;but Z=1.40;p>0.05;Fig. 6B right), further confirming that dorsalis was not being driven via a possible reflex loop that was being activated by passive dorsal muscle stretching during hindlimb extensions.

Finally, in two juveniles, the thoracic and lumbar segments were separated on one side only (left) in combination with a longitudinal thoracic segment section. Such sagittally/hemi-cord lesioned animals were still able to swim spontaneously (Fig. 6D1)

or in response to limb tactile stimulation (not illustrated). Under these conditions, dorsalis activity generally occurred only on the unlesioned side (Fig. 6D1) and it remained phase-coupled to rhythmic hindlimb extensions (µ=334.12°; r=0.92; Z=6.73; p<0.001; u(0°)=3.30; p<0.001; Fig. 6E, right plot). In contrast, the occasional dorsalis activity that occurred on the side of the hemi-cord lesion expressed no clear temporal relationship with hindlimb plantaris contractions (µ=128.89°; r=0.73; Z=4.31; p<0.001; non significant V test; Fig. 6E, left plot). Consequently, the dorsalis vs. plantaris phase values for activity on the disconnected (left) cord side was significantly more dispersed than for the intact (right) cord side (p<0.001; Fig. 6F). Finally, these bilateral differences were not the result of lesion-induced damage to ipsilateral thoracic motor circuitry since strong spontaneous bilateral dorsalis activity was occasionally observed when hindlimb locomotor movements were absent (Fig. 6D2).

DISCUSSION

The fundamental modifications in locomotor behavior that occur during amphibian metamorphosis [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF][START_REF] Rauscent | Development and neuromodulation of spinal locomotor networks in the metamorphosing frog[END_REF]) require a concomitant adaptation of postural control and its coordination with locomotion. The present study explored this changing functional relationship by recording axial/thoracic motor output to locomotory muscles in pre-metamorphic Xenopus tadpoles and after their transition to postural musculature in post-metamorphic young adults. Associated changes in longitudinal coupling between thoracic and lumbar cord segments during swimming in juvenile frogs were also examined through simultaneous recordings from respective back and hindlimb muscles, from their corresponding motor nerves in isolated nervous system preparations, and after various spinal cord lesions performed either in vivo or in vitro.

Metamorphosis-induced modifications in intra-and inter-segmental coordination

Whereas in tadpoles the entire axial musculature is recruited in strict left/right alternation, the dorsal axial muscles controlled by the thoracic region of the young adult spinal cord are synchronously activated during free forward swimming. Moreover, in contrast to the progressive rostro-caudal activation of axial muscles in swimming larval

Xenopus [START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF][START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]), back and leg extensor muscles are co-activated during juvenile hindlimb propulsion. These radical changes in the coordination between left and right thoracic hemi-segments, and between thoracic and lumbar segments, therefore implied that the functional reorganization of spinal motor circuitry that occurs during Xenopus metamorphosis [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]) extends into mid cord regions that are not involved in producing actual limb movements. Several observations from our in vivo and in vitro experiments provided insights into the nature of this developmental transformation.

Firstly, all spinal lesions in froglets that isolated the thoraco-lumbar cord from sensory or supra-spinal inputs tended to narrow the phase distributions of back versus leg muscle locomotor activity towards even closer synchrony, suggesting that a preferential functional linkage between the lumbar and thoracic cord segments is established during the metamorphic process. Further clear evidence that the dorsal muscle-hindlimb extensor coupling does not rely on sensory feedback arises from the observation that the activation patterns of thoracic ventral roots and leg motor nerves in isolated in vitro preparations remained similar to those of their corresponding target muscles in vivo. The stronger synchrony observed between back and hindlimb muscle activity after disconnecting the thoraco-lumbar cord from the rostral CNS also suggested that the coupling was not reliant upon descending influences from the brainstem, a situation that differs from rodents and humans where supra-spinal inputs have been reported to be primarily responsible for coordinating back and leg muscle contractions during locomotion [START_REF] Gramsbergen | The activation of back muscles during locomotion in the developing rat[END_REF][START_REF] Takakusaki | Role of basal ganglia-brainstem systems in the control of postural muscle tone and locomotion[END_REF]).

Thus, although a remodeling of supra-spinal centers is undoubtedly required in metamorphosing Xenopus to adapt descending commands to the changing spinal circuitry, the altered thoraco-lumbar coordination occurring in young adults is mainly attributable to de novo synaptic pathways that are intrinsic to the spinal cord.

Secondly, that the left/right back muscle synchronization with bilateral hindlimb extensions persisted after separating the two sides of the thoracic cord suggested that the back muscle coupling does not rely principally on direct cross-cord connections at the thoracic cord level. This contrasts with the situation in pre-metamorphic Xenopus larvae where inhibitory commissural interneurons are responsible for alternating segmental CPG activity on the two sides of the cord [START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF]. Evidently, these larval inhibitory interactions are not simply replaced by equivalent excitatory cross-cord pathways in thoracic segments during metamorphosis in order to ensure conjoint left/right dorsalis activation. Rather, the persistence of dorsal muscle co-activation after thoracic cord partitioning which remained coordinated with hindlimb movements indicated that the thoracic intra-segmental coupling was being driven by the lumbar CPG itself. In direct contrast to the thoracic segments, however, the assembly of hindlimb locomotor circuitry in the lumbar cord region during metamorphosis must here include new cross-cord excitatory connections that are necessary for bilateral limb kick synchrony (see also [START_REF] Combes | Developmental segregation of spinal networks driving axial-and hindlimb-based locomotion in metamorphosing Xenopus laevis[END_REF]). Furthermore, the establishment of longitudinal projections from the lumbar to thoracic cord regions is also required to provide the necessary anatomical substrate for coupling dorsalis efferent commands to the hindlimb motor circuitry. While such ascending propriospinal pathways remain to be identified in post-metamorphic Xenopus, in Rana pipens, lumbar interneurons have been previously labeled which project directly into the thoracic spinal cord [START_REF] Schotland | Segmental and propriospinal projection systems of frog lumbar interneurons[END_REF].

In Xenopus juveniles, furthermore, these lumbo-thoracic projections appear to be homolateral, since a hemicord section in vivo that disconnected the thoracic circuitry of that side from the lumbar cord region suppressed activity of the corresponding dorsalis muscle during swimming, but did not affect hindlimb kicking-timed activation of the dorsalis on the intact contralateral side.

Finally, a complete cord transection that separated the thoracic and lumbar segments not only decoupled leg and back motor outputs during swimming, but also resulted in an overall significant decrease in coincident left-right dorsalis activity. However, this bilateral relationship never became completely random after a thoraco-lumbar separation, indicating that co-activating influences on the motor commands to these muscles may to some extent arise from cerebrospinal inputs and/or the cervical CPG circuitry responsible for rhythmic forelimb movements. Indeed, it is possible that the occasional participation of forelimb CPG-driven propulsion during hindlimb swimming may also contribute to the coordination of dorsalis muscle contractions.

Taken together, therefore, these findings indicate that descending pathways, sensory inputs and cross-cord connectivity in the thoracic spinal cord are not essential for coordinating bilateral back muscle contractions with rhythmic hindlimb extensions during rectilinear swimming in juvenile Xenopus. Rather, their coordination appears to be determined principally by ipsilateral projections that ascend from the hindlimb CPG in lumbar cord segments. Earlier work on spinal cat has also proposed that the lumbar locomotor CPG may control the activation sequence of back muscles during walking [START_REF] Zomlefer | Electromyographic study of lumbar back muscles during locomotion in acute high decerebrate and low spinal cats[END_REF], and in newborn rats, changes in trunk curvature during locomotion are time-locked with rhythmic hindlimb stepping, which here also appears to derive from lumbo-thoracic interactions [START_REF] Cazalets | Metachronal propagation of motoneurone burst activation in isolated spinal cord of newborn rat[END_REF][START_REF] Falgairolle | Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat[END_REF].

It is important to remember that the development of limb-based locomotion in

Xenopus is fundamentally different from most other vertebrates in which effective locomotory behavior depends on the progressive emergence of functional spinal circuitry from a non-functional embryonic precursor. In Xenopus, an already operational primary locomotor system (for axial-based swimming) is replaced by another during metamorphosis to satisfy completely different biomechanical requirements (for limbbased propulsion). Figure 7 summarizes the associated developmental changes in spinal network coordination on the basis of previous knowledge on larval Xenopus [START_REF] Tunstall | A longitudinal gradient of synaptic drive in the spinal cord of Xenopus embryos and its role in coordination of swimming[END_REF][START_REF] Roberts | Central circuits controlling locomotion in young frog tadpoles[END_REF]) and our current findings in young adult frogs.

Essentially, spinal connectivity switches from a system that generates rostro-caudally propagating, bilaterally-alternating motor patterns for undulatory swimming in tadpoles (Fig. 7A) to caudo-rostrally, bilaterally-synchronous activity that drives back muscle contractions in time with limb-kicking in the adult (Fig. 7B). In this way, thoracic circuitry, which contributes actively to body propulsion as an equivalent member of a chain of segmental oscillatory networks in the pre-metamorphic larva, becomes subordinate to new, more caudal, rhythm-generating circuitry that drives hindlimb extension/flexion from the lumbar region of the adult spinal cord.

Posturo-locomotor interactions

For effective displacement during locomotion, coordinated dynamic postural regulation is required to stabilize body orientation by controlling mainly the head and trunk musculature [START_REF] Massion | Why and how are posture and movement coordinated?[END_REF][START_REF] Assaiante | Development of postural control in healthy children: a functional approach[END_REF]. The interactions between locomotory and postural systems are generally considered to be based on two separate neural command networks, both of which are thought to be activated and coordinated exclusively by supra-spinal commands and sensory feedback signals (see [START_REF] Mori | Reactive and anticipatory control of posture and bipedal locomotion in a nonhuman primate[END_REF]). Although the postural trunk muscles of mammals, like in adult Xenopus, are known to contract rhythmically in time with the step cycle [START_REF] Koehler | Phasic modulation of trunk muscle efferents during fictive spinal locomotion in cats[END_REF][START_REF] Gramsbergen | The activation of back muscles during locomotion in the developing rat[END_REF][START_REF] Falgairolle | Metachronal coupling between spinal neuronal networks during locomotor activity in newborn rat[END_REF], the neural basis of this coupling has remained unclear, due undoubtedly to the close functional relationship between the locomotory and postural systems that renders them difficult to distinguish during ongoing movement. Furthermore, in most species including humans, the two systems develop simultaneously and often employ overlapping sets of muscles [START_REF] Vinay | Develoment of posture and locomotion: an interplay of endogenously generated activities and neurotrophic actions by descending pathways[END_REF].

In lower vertebrates such as fish, non-anuran amphibians and anuran larvae (including Xenopus), locomotion and posture are achieved by the same sets of axial muscles that are activated sequentially along the body length during swimming, indicating that the coordination between these two functions occurs at the individual segmental level [START_REF] Deliagina | Neural bases of postural control[END_REF]. In post-metamorphic anurans where propulsion is now limb-based, at least part of the larval dorsal axial musculature has evidently undergone a functional switch from a mainly locomotory to an essentially postural role as thoracic trunk muscles in the adult. Specifically, given its location flanking a rigidified section of the adult spinal cord, and consistent with a requirement for dynamic postural control in general [START_REF] Massion | Postural control system[END_REF], the most likely contribution of the third myomere of dorsalis trunci during locomotion, especially during powerful swimming, is to increase body stiffness in order to resist external forces, in a similar manner to epaxial musculature in lizards, for example, which serves to stabilize the trunk against ground reaction forces during locomotion [START_REF] Ritter | Epaxial muscle function during locomotion in a lizard (Varanus salvator) and the proposal of a key innovation in the vertebrate axial musculoskeletal system[END_REF][START_REF] Ritter | Axial muscle function during lizard locomotion[END_REF].

In lower vertebrates and mammals, both anticipatory and compensatory adjustments to postural perturbations are thought to depend on supra-spinal influences and sensory feedback from the periphery [START_REF] Mori | Integration of posture and locomotion in acute decerebrate cats and in awake, freely moving cats[END_REF][START_REF] Mori | Reactive and anticipatory control of posture and bipedal locomotion in a nonhuman primate[END_REF][START_REF] Deliagina | Neural bases of postural control[END_REF], thereby implying that spinal postural circuitry serves merely as a final output stage for higher order or reflex commands. Our findings add a further dimension to this common view by indicating that the spinal cord is itself capable of organizing a major component of postural adjustment by using intrinsic feed-forward signals from the lumbar patterngenerating networks to predict the consequences of locomotor actions. Interestingly, previous work on cats has shown that vestibular information is not required for controlling posture, since responses to postural perturbations are preserved following a bilateral labyrinthectomy [START_REF] Macpherson | Stance and balance following bilateral labyrinthectomy[END_REF]. Furthermore, complex motor kinematics and EMG coordination [START_REF] Bélanger | A comparison of treadmill locomotion in adult cats before and after spinal transection[END_REF], and body orientation and geometry [START_REF] Fung | Attributes of quiet stance in the chronic spinal cat[END_REF] persist in chronic spinal cats, indicating here also that supra-spinal commands are not essential for maintaining equilibrium during walking or quiet stance. However, to our knowledge a proactive (rather than an anticipatory or reactive) relationship between locomotory movements and postural adjustments that resides solely with propriospinal mechanisms has not previously been clearly established [START_REF] Stuart | Integration of posture and movement: Contributiuons of Sherrington, Hess, and Bernstein[END_REF].

In conclusion, Xenopus metamorphosis is associated with a complete reorganization of the postural system from a larval architecture in which the rostro-caudal recruitment of axial musculature during swimming implies postural adjustments on a segment-bysegment basis, to an adult "en bloc" strategy of postural control where posturolocomotor coordination extends over several spinal segments, from the lumbar to the thoracic cord regions. Furthermore, our findings suggest that Xenopus offers an attractive model for future studies on the developmental plasticity of posture/locomotion coupling and spinal network interactions. Especially intriguing in this context is that both larval and adult motor networks co-exist and can function separately within the spinal cord at mid-metamorphic stages of development (see Fig. 1B), thereby providing the opportunity to explore the neuronal basis of different posturo-locomotory strategies within the same organism.

  participate conjointly or independently in swimming behavior. As seen in the EMG recordings from a pro-metamorphic (stage 60-61) tadpole in Figure1B, the axial/dorsal muscles display two distinct types of bilateral coordination according to the mode of locomotion. During tail-based swimming (n=9 episodes; Fig.1B1), the left and right ad muscles express a typical larval coordination pattern in which they are activated alternately (µ=152.11°; r=0.93; Z=6.04; p<0.001; u(180°)=3.07; p<0.001), whereas following a spontaneous switch to limb-based propulsion (12 episodes; Fig.1B2), the same ad muscles are now active in synchronous bursts (µ=350.53°; r=0.98; Z=9.63; p<0.001; u(0°)=4.33; p<0.001).

  essential for maintaining either the coordination of thoracic motor output to the left and right back muscles or the longitudinal coupling between motor commands to the back and hindlimb muscles. Nonetheless, a significant increase in the phase dispersion of bilateral thoracic bursts in the isolated preparation (p<0.05; Fig. 3G, compare left pair of histogram bars) indicated that sensory information may participate in strengthening the coordination between the left and right dorsalis muscle contractions. However, other burst phase relationships (left vs. right plantaris longus and dorsalis vs. plantaris) remained similarly distributed in vitro to their corresponding muscle EMG patterns in vivo (Fig. 3G, middle and right histogram pairs).

  Fig.5C, left polar plot), the burst onset phases were slightly but significantly more

  between left and right thoracic ventral root bursts (W=5.75; p>0.05; Fig. 5G left) despite a significantly increased phase dispersion (p<0.05; Fig. 5H left), which in turn, and consistent with in vivo lesion experiments (Fig. 5D left), implied a residual contribution of thoracic cross-cord connections to bilateral coupling. More significantly, no changes

Figure 1 :

 1 Figure 1: Metamorphosis-induced modifications in bilateral axial/dorsal muscle

Figure 2 :

 2 Figure 2: Kinematics of free swimming in juvenile adult Xenopus. A: Rectilinear

Figure 3 :

 3 Figure 3: Coordination between bilateral back and hindlimb motor activity during

Figure 4 :

 4 Figure 4: Effect of a complete cervico-thoracic cord transection on back and

Figure 5 :

 5 Figure 5: Effect of a sagittal thoracic cord lesion on back and hindlimb motor

Figure 6 :

 6 Figure 6: Effect of thoraco-lumbar cord transections on back and leg muscle

Figure 7 :

 7 Figure 7: Organization of spinal lumbar and thoracic circuitry for locomotion and

TABLE LEGEND Table 1 : Effects of multiple spinal lesions in vivo.

 LEGEND1 Circular statistics of the coordination between left and right dorsalis and plantaris longus muscles, and between back and hindlimb muscles (dt/pl) following combined cervico-thoracic cord transection, sagittal thoracic section, and thoraco-lumbar rhizotomy. Results of the Rayleigh's uniformity test (Z), the V-test for the 0° direction of the mean vector (u(0°)), and the Mardia-Watson-Wheeler test (W) indicate that the three bilateral and longitudinal relationships examined were not significantly modified by these multiple lesions, although phase distribution variability decreased for l/r plantaris longus and dt/pl coupling. The control r value for each relationship is indicated in brackets.

				Beyeler et al.
				Table 1
		µ (°)	r	Z	p	u (O°)	p	W	p
	left/right	10.40	0.89	7.99 <0,001 3.93 <0,001 3.70	>0,05
	dorsalis trunci		(0,65)
	left/right	3.36	0.99	10.81 <0,001 4.64 <0,001 14.53 <0,001
	plantaris longus		(0,80)
	dt /pl	357.32 0.88	16.95 <0,001 5.82 <0,001 9.83	<0,01
			(0,59)

Acknowledgements and grants

The authors are grateful to M. Falgairolle for helping with kinematics analysis, and R. Nargeot for advice in statistical treatments. We also thank M.-J. Cabirol-Pol for technical assistance in fluorescence microscopy and L. Parra-Iglesias for animal care and maintenance. This work was funded by the CNRS (ATIP Jeunes Chercheurs).

A. Beyeler was funded by a studentship from the French "Ministère de L'Education Nationale, de l'Enseignement Supérieur et de la Recherche". The authors also thank the European Neuroscience Institute Network (ENI-Net) for valuable discussions and for covering part of the publication fees.

postural control whereby back muscle contractions are matched proactively to ongoing locomotor movements.