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Université de Bordeaux; Centre National de la Recherche Scientifique, Laboratoire Mouvement Adaptation Cognition (UMR 5227), Bordeaux; France
Submitted 9 January 2008; accepted in final form 12 July 2008
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ABSTRACT |
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INTRODUCTION |
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). Over the last 30 years, many studies on a variety of animal species have analyzed the functional organization of spinal and supraspinal structures involved in the orchestration of rhythmic locomotor movements (for reviews, see Cazalets and Bertrand 2000; Grillner 2006; Kiehn 2006). In quadrupedal vertebrates, the locomotor programs for anterior and posterior limb movements are elaborated by so-called "central pattern generators" (CPGs) (for reviews, see Grillner 1981; Kiehn 2006) that are located in the cervical (Ballion et al. 2001; Juvin et al. 2005) and lumbar spinal enlargements, respectively (Cazalets et al. 1995; Langlet et al. 2005). 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 (Falgairolle and Cazalets 2007; Gramsbergen et al. 1999), cat (Koehler et al. 1984; Zomlefer et al. 1984), and humans (Thorstensson et al. 1982). However, although earlier experiments on decerebrate paralyzed cat suggested a propriospinal origin of coordinated limb-trunk muscle contractions (Koehler et al. 1984), the neural basis of such coupling and the extent to which it is governed by rhythmogenic spinal circuitry remains largely unknown (Falgairolle and Cazalets 2007). Similarly, little is known of the changing functional relationship that must occur between locomotory and postural control systems during postnatal 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 Shi 2000). 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 limb-based propulsion in the adult. In premetamorphic tadpoles, body displacement is driven by waves of bilaterally alternating muscle contractions that are directed rostrocaudally along the body axis, whereas in postmetamorphic 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 (Combes et al. 2004; Rauscent et al. 2007). 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 premetamorphic larvae, to assume a dynamic postural function after their transformation into nonlocomotory back muscles in postmetamorphic adults?
To begin to address these questions, we have analyzed the bilateral coordination of axial/back muscle activity in freely behaving, premetamorphic Xenopus tadpoles and, together with the locomotor activity of hindlimb muscles, in postmetamorphic 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 rostrocaudal 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 brain stem-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 postmetamorphic Xenopus. Given the developmental switch in function of the more rostral axial muscles in larvae to nonpropulsive 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 (Beyeler et al. 2007).
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METHODS |
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) and 50 postmetamorphic juvenile adults (stage 66, <1 mo after metamorphosis) of the South African clawed toad, X. 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 EMG 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). Axial muscles were recorded at the level of the fifth 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 (Vallois 1922), 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). 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) 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, 50 mg/l, Sigma-Aldrich) and positioned dorsal side up in a Petri dish filled with frog saline (which contained, in mM: 112 NaCl, 2 KCl, 20 NaHCO3, 2.8 CaCl2, 1 MgCl2, and 17 glucose). 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. To separate the thoracic spinal cord from other regions of the nervous system, three types of lesion were made separately or in combination: a transection at the level of the last cervical cord segment, a longitudinal (sagittal) lesion that extended through only the three thoracic segments, and 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 h 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 prometamorphic 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 postmetamorphic 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 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 3 days to allow retrograde dye migration across the neuromuscular junction and along motoneuron axons to the CNS, animals were killed 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) equipped with a CCD camera (ORCA-AG Hamamatsu). Images were acquired using Simple PCI software (Compix).
Isolated brain stem-spinal cord preparations
Dissection and recording procedures were similar to those previously described by Combes et al. (2004). The skin and cranial cartilage of juvenile frogs were opened under deep anesthesia with tricaine methanesulfonate, and the forebrain was removed above the mesencephalon. The spinal cord and remaining brain stem 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 and Bizzi 2005; d'Avella et al. 2003). 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 petroleum jelly (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 N-methyl-D-aspartate (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) 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: 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 the V-test (u), which tested for a preferential direction (angle) of a given phase distribution [indicated in the text as u(direction°)]. 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 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 nonparametric 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 (Handycam DCR-PC350E, Sony) while behaving freely in their standard aquaria or during EMG recording in a smaller experimental tank. No clear evidence was found for a perturbing effect of the fine EMG wires on the freedom of locomotor movements. The video files were transferred to a computer using Windows Movie Maker software (Microsoft) for storage and joint kinematics. The latter were performed using the free Image J software developed by W. S. Rasband (U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/, 1997–2005). Using a manual tracking plug-in supplied by F. P. Cordelières (Institut Curie), the x and y coordinates of limb joints were determined under visual inspection by mouse-clicking on individual frames. Joint angles were determined with Microsoft Excel, while stick diagrams and angle variations were plotted using CorelDraw 7 (Corel).
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RESULTS |
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In premetamorphic X. 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 rostrocaudally directed waves along the tail (Combes et al. 2004; Roberts et al. 1998). 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 (Combes et al. 2004), 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.
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; Nieuwkoop and Faber 1956). During this transitional period, both axial and appendicular locomotor systems can participate conjointly or independently in swimming behavior. As seen in the EMG recordings from a prometamorphic (stages 60–61) tadpole in Fig. 1B, 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].
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 premetamorphic Xenopus tadpoles has been well described (Roberts et al. 1998), 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 postmetamorphic 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 Fig. 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 (B1). As can be seen in the traces of the hindlimb knee and ankle joint excursions in Fig. 2, A2 and B2 (top 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, bottom traces; see also Fig. 3, B and C), whereas the activity of the same muscles became desynchronized during nonlinear 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.
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In addition to the bilateral synchrony of dorsalis trunci muscle activity during swimming (Figs. 1C and 3, B and 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 rostrocaudal propagation of alternating contractions of the equivalent muscles in the premetamorphic 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 (rc) = 0.44, P < 0.001, regression slope (a) = 0.11 and rc = 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 (rc = 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 (Tokizane et al. 1951). To this end, an in vitro brain stem/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 (Combes et al. 2004), 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). Thus homolateral antagonistic limb nerves were active in alternation (µ = 186.06°; r = 0.64; Z = 174.04; P < 0.001), while homologous bilateral motor nerves tended to express in-phase bursting (tibialis nerves: µ = 26.59°; r = 0.69; Z = 134.55; P < 0.001: plantaris nerves: µ = 343.12°; r = 0.84; Z = 190.99; P < 0.001), in a manner appropriate for producing alternating cycles of bilaterally synchronous limb extensions and flexions. In addition, burst synchrony was preserved across the isolated cord at the thoracic segmental level (Fig. 3, E and F: µ = 2.58°; r = 0.53; Z = 134.55; P < 0.001) as well as between the thoracic ventral roots and plantaris nerves originating from the lumbar cord region (µ = 2.22°; r = 0.63; Z = 97.97; P < 0.001). These results therefore clearly imply that sensory inputs generated by actual limb and body movements during locomotion are not 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 histogram pairs) 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).
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) 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 (Lalonde and Strazielle 2007; Takakusaki et al. 2004). 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 Fig. 4C, where the phase dispersion in lesioned animals (
) was compared with equivalent data obtained from intact control animals (as illustrated in Fig. 3, A–C and G). In cervico-thoracic transected animals, the activity of left and right dorsalis trunci also remained synchronized [n = 26 episodes; µ = 5.87°; r = 0.97; Z = 24.44; P < 0.001; u(0°) = 6.95; P < 0.001; Fig. 4B, left], with the phase dispersion also being significantly narrower (W = 34.21; P < 0.001) than in intact animals (Fig. 4C, left histogram pair). Similarly, back and leg muscle activity continued to display an in-phase coordination [n = 7 episodes; µ = 2.20°; r = 0.98; Z = 5.74; P < 0.001; u(0°) = 3.39; P < 0.001; Fig. 4B, right] with a burst onset phase distribution significantly different from control (W = 17.38; P < 0.001) due to a significant decrease in phase dispersion (Fig. 4C, right). As in control animals, furthermore, the intensity of dorsal and limb extension muscle bursts remained linearly correlated (rc = 0.72; P < 0.001; a = 0.82) after a cervico-thoracic transection. These data therefore indicated that the cyclic activation of dorsalis trunci muscles during locomotion was not attributable to descending commands from the brain stem. Rather the actual strengthening of thoracic cross-cord and lumbo-thoracic coupling following a rostral cord transection was more indicative of the functional versatility that brain stem influences are capable of imposing on otherwise inflexible coordination patterns arising from hardwired spinal circuitry.
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; for a review see Roberts et al. 1998). To assess whether the switch to bilateral synchrony of dorsalis muscle activation in the adult animal might result from the establishment of cross-cord excitatory connections during metamorphosis, restricted midline cord sagittal lesion were made in froglets to separate the two sides of all three segments of the thoracic cord region. After such a sagittal lesion (n = 7; Fig. 5, A–D), animals were still able to express rectilinear swimming-like behavior (Fig. 5B), and EMG recordings as well as kinematics (not illustrated) did not reveal any major alterations in the pattern of coordination either between bilateral hindlimb movements or between the contractions of the left and right dorsalis trunci muscles. Thus the two plantaris longus muscles were still synchronously active during swimming episodes [µ = 4.28°; r = 0.80; Z = 178.57; P < 0.001; u(0°) = 18.84; P < 0.001; Fig. 5C] with a coupling strength that remained similar to intact control animals (W = 2.93; P > 0.05; and Fig. 5D, middle histogram pair). In contrast, although the left and right dorsalis trunci also retained an overall tendency for synchronous activity [µ = 14.85°; r = 0.54; Z = 72.79; P < 0.001; u(0°) = 11.66; P < 0.001; Fig. 5C, left polar plot], the burst onset phases were slightly but significantly more dispersed than in control (W = 35.52; P < 0.001; Fig. 5D, left histogram pair). However, a closer inspection of the raw data revealed that this broader distribution resulted from an increase in the occurrence of supernumerary dorsalis activity that did not correspond to hindlimb muscle contractions (e.g., L dt EMG activity in Fig. 5B). Nevertheless, these data indicated that excitatory cross-cord pathways within the thoracic segments were not primarily responsible for the bilateral synchronization of dorsal muscle activity (see following text).
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Thoraco-Lumbar Cord Lesions. 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 were only obtained from in vivo experiments (n = 11; Fig. 6, A–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. 6, A2 and left plot in B) 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.
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DISCUSSION |
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; Rauscent et al. 2007) 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 premetamorphic Xenopus tadpoles and after their transition to postural musculature in postmetamorphic 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 intersegmental 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 rostrocaudal activation of axial muscles in swimming larval Xenopus (Combes et al. 2004; Roberts et al. 1998), 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 (Combes et al. 2004) 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.
First 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 toward 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 on descending influences from the brain stem, 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 (Gramsbergen et al. 1999; Takakusaki et al. 2004). 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.
Second, 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 premetamorphic Xenopus larvae where inhibitory commissural interneurons are responsible for alternating segmental CPG activity on the two sides of the cord (Roberts et al. 1998). Evidently, these larval inhibitory interactions are not simply replaced by equivalent excitatory cross-cord pathways in thoracic segments during metamorphosis to ensure conjoint left/right dorsalis activation. Rather the persistence of dorsal muscle co-activation after thoracic cord partitioning that remained coordinated with hindlimb movements indicated that the thoracic intrasegmental 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 Combes et al. 2004). 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 postmetamorphic Xenopus, in Rana pipens, lumbar interneurons have been previously labeled that project directly into the thoracic spinal cord (Schotland and Tresch 1997). In Xenopus juveniles, furthermore, these lumbo-thoracic projections appear to be homolateral because 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 (Zomlefer et al. 1984), 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 (Cazalets 2005; Falgairolle and Cazalets 2007).
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 nonfunctional 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 limb-based propulsion). Figure 7 summarizes the associated developmental changes in spinal network coordination on the basis of previous knowledge on larval Xenopus (Roberts et al. 1998; Tunstall and Roberts 1994) and our current findings in young adult frogs. Essentially spinal connectivity switches from a system that generates rostrocaudally propagating, bilaterally alternating motor patterns for undulatory swimming in tadpoles (Fig. 7A) to caudorostrally, 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 premetamorphic larva, becomes subordinate to new, more caudal, rhythm-generating circuitry that drives hindlimb extension/flexion from the lumbar region of the adult spinal cord.
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For effective displacement during locomotion, coordinated dynamic postural regulation is required to stabilize body orientation by controlling mainly the head and trunk musculature (Assaiante et al. 2005; Massion et al. 2004). 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 Mori et al. 2004). Although the postural trunk muscles of mammals, like in adult Xenopus, are known to contract rhythmically in time with the step cycle (Falgairolle and Cazalets 2007; Gramsbergen et al. 1999; Koehler et al. 1984), 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 (Vinay et al. 2002).
In lower vertebrates such as fish, nonanuran 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 (Deliagina et al. 2006). In postmetamorphic 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 (Massion 1994), the most likely contribution of the third myomere of dorsalis trunci during locomotion, especially during powerful swimming, is to increase body stiffness 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 (Ritter 1995, 1996).
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 (Deliagina et al. 2006; Mori 1987; Mori et al. 2004), 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 pattern-generating 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 (Macpherson and Inglis 1993). Furthermore, complex motor kinematics and EMG coordination (Bélanger et al. 1996) and body orientation and geometry (Fung and Macpherson 1999) 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 (Stuart 2005).
In conclusion, Xenopus metamorphosis is associated with a complete reorganization of the postural system from a larval architecture in which the rostrocaudal recruitment of axial musculature during swimming implies postural adjustments on a segment-by-segment basis to an adult en bloc strategy of postural control where posturo-locomotor 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Le Ray, Université de Bordeaux; Centre National de la Recherche Scientifique, Laboratoire Mouvement Adaptation Cognition (UMR 5227), 146 Rue Léo Saignat, 33076 Bordeaux; France (E-mail: didier.leray{at}u-bordeaux2.fr)
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180°). B: coordination of the equivalent muscles in a late-metamorphosing tadpole (stage 60). Same panel arrangement as in A. Note that depending on the mode of locomotion (shaded area 1 and time base expansion below: larval-like body undulations; shaded area 2 and time base expansion below: adult-like leg kicking) the dorsal muscle coordination switched from alternation to synchrony as indicated by the change in mean vector direction from 
, 
, the direction of knee excursion during hindlimb extension). Vertical scale bar: 100°). Bottom traces: simultaneous EMG recordings from ankle extensor plantaris longus of the right (R-pl) and left (L-pl) hindlimbs. Note the strict bilateral synchrony of the muscle bursts. B: nonrectilinear swimming. Same arrangement as in A. Note the irregularity and dissociation of limb trajectories (1), knee and ankle excursions (2, top), and between plantaris longus activity on the 2 sides (2, bottom).
) and in vitro (
). CNS isolation increased the dispersion of left versus right thoracic/back phase values (*P < 0.05), while the distributions of the two other phase relationships remained unaltered. A–C and D–F are from 2 different groups of animals.
, sites of EMG recordings. B: sample EMG recordings and corresponding integrated traces. C: circular analysis of indicated phase relationships (same arrangement as in 
) of either a hindlimb (hstim) or forelimb (fstim) after a thoraco-lumbar transection as indicated in the drawing above. Left: recordings show uncoordinated dorsal and hindlimb activity during hindlimb movement; right: bilateral dorsalis trunci activity in the absence of limb muscle activity. B and C: same arrangement as in 