Laboratoire de Neurobiologie et Physiologie Comparées, Université Bordeaux I and Centre National de la Recherche Scientifique, 33120 Arcachon, France
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ABSTRACT |
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Delvolvé, Isabelle, Tiaza Bem, and Jean-Marie Cabelguen. Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt, Pleurodeles waltl. J. Neurophysiol. 78: 638-650, 1997. We have investigated the patterns of activation of epaxial musculature during both swimming and overground stepping in an adult newt (Pleurodeles waltl) with the use of electromyographic (EMG) recordings from different sites of the myomeric muscle dorsalis trunci along the body axis. The locomotor patterns of some limb muscles have also been investigated. During swimming, the epaxial myomeres are rhythmically active, with a strict alternation between opposite myomeres located at the same longitudinal site. The pattern of intersegmental coordination consists of three successively initiated waves of EMG activity passing posteriorly along the anterior trunk, the midtrunk, and the posterior trunk, respectively. Swimming is also characterized by a tonic activation of forelimb (dorsalis scapulae and extensor ulnae) and hindlimb (puboischiotibialis and puboischiofemoralis internus) muscles and a rhythmic activation of muscles (latissimus dorsi and caudofemoralis) acting both on limb and body axis. The latter matched the activation pattern of epaxial myomeres at the similar vertebral level. During overground stepping, the midtrunk myomeres express single synchronous bursts whereas the myomeres of the anterior trunk and those of the posterior trunk display a double bursting pattern in the form of two waves of EMG activity propagating in opposite directions. During overground stepping, the limb muscles and muscles acting on both limb and body axis were found to be rhythmically active and usually displayed a double bursting pattern. The main conclusion of this investigation is that the patterns of intersegmental coordination during both swimming and overground stepping in the adult newt are related to the presence of limbs and that they can be considered as hybrid lampreylike patterns. Thus it is hypothesized that, in newt, a chain of coupled segmental oscillatory networks, similar to that which constitutes the central pattern generator (CPG) for swimming in the lamprey, can account for both trunk motor patterns if it is influenced by limb CPGs in a way depending on the locomotor mode. During swimming, the segmental networks located close to the girdles receive extra tonic excitation coming from the limb CPGs, whereas during stepping, the axial CPGs are entrained to some extent by the limb oscillators.
Axial movements during aquatic or terrestrial locomotion in lower vertebrates consist primarily of rhythmic lateral bending of the trunk and tail. The neural networks generating the rhythmic contractions of axial muscles that underlie lateral bending during swimming have been extensively investigated in the lamprey (reviewed in Grillner et al. 1995 Experiments were performed on 18 fully metamorphosed amphibian urodeles (P. waltl) with snout vent lengths (SVLs) ranging from 78 to 102 mm. All the animals were obtained from Centre de Biologie du Développement (CNRS UMR 9925, France) and kept in an aquaria at the room temperature. No locomotor training was performed before experiments.
EMG electrode implantation
EMG electrode implantation was performed while the animal was under general anesthesia induced by immersion in a 0.1% aqueous solution of tricaine methanesulfonate. Under a dissecting microscope, pairs of fine 70-µm insulated stainless steel wires with bared ends (exposure 0.5 mm) were inserted 3-4 mm relative to the dorsal midline through small skin incisions into myomeres(2-4 electrode pairs in each animal) of the muscle dorsalis trunci. The electrode tips were separated by ~1.0 mm and the insulated portions of the wires proximal to the tips were glued to the skin overlying the dorsalis trunci muscle with cyanoacrylate adhesive. The electrode location was verified by inducing muscle contraction with electrical stimulation via the same wires and by dissection at the end of the recording session. The rostrocaudal position of the recording electrodes was expressed as fraction of the SVL. In each experiment the activity in the myomere located at 0.60 SVL level on the right side of the animal ("0.60 SVL myomere") was recorded as the reference point in the locomotor cycle (see below).
Protocol
After electrodes had been implanted, animals were placed in a tank (80 × 13 cm) filled with 10 cm of tap water and allowed to recover from anesthesia for 1-2 h. After recovery, the newts were easily induced to swim the length of the tank or to walk on a wet, stainless steel surface by touching or gently squeezing the base of the tail. A wet walking surface was used because the locomotor movements of the newt are smooth and continuous under these conditions (Roos 1964
Data collection and analysis
The voltage signals from electrodes were differentially amplified 10,000 times (band pass 0.3-10 kHz with a 50-Hz notch filter), displayed on an oscilloscope, and stored on a eight-channel data recorder (DAT Biologic). Thereafter the data were played back on a multichannel electrostatic printer (Gould Recorder ES-1000). The EMGs from those sections of data obtained during episodes of steady locomotion were sampled (1 kHz per channel) with the use of an A-D converter (Cambridge Electronic Design 1401). The digital EMGs were full-wave rectified and smoothed by a software filter (<100 Hz). An interactive software was then used to mark manually with a cursor (resolution < 1 ms) the onset and cessation of bursts of EMG activity. The criterion used for delineation of a single EMG burst was a signal-to-noise ratio of
General observations
As shown in Fig. 1, the EMG pattern during swimming (A) is characterized by a rhythmic activation of myomeres and a tonic activation of the limb muscles, whereas during stepping (B), myomeres and limb muscles all displayed a rhythmic activity. These differences in patterns of activation of limb muscles reflect the differences in limb movement during the two locomotor modes: during swimming the limbs are held back against the body wall, whereas they move rhythmically during stepping (cf. also Davis et al. 1990
EMG patterns in epaxial muscles during swimming
During swimming episodes, the EMG activity on the two sides of the body at the same level was strictly alternating in all animals (compare i mid trk and co mid trk in Fig. 4). Therefore in our experiments we recorded EMG activity mainly on one side of the body (usually the right side) because it reflected a similar pattern, albeit in antiphase, to the contralateral side. The mean duration of the EMG bursts calculated for several classes of cycle duration (400-550 ms) at different trunk sites ranged from 103 ± 17 to 203 ± 21 ms (Table 1), which corresponded to 21.7 ± 4.0% and 39.0 ± 4.2% of cycle duration, respectively.
EMG patterns in epaxial muscles during stepping
The expression of EMG activity by epaxial muscles during stepping, in contrast to swimming, varies according to the longitudinal location along the body axis (Fig. 7).
Relative timing of epaxial and limb muscle activities during swimming and stepping
The limb muscles examined in our study displayed stepping patterns that were quite similar to those reported in studies on other salamanders (Ashley-Ross 1995
Epaxial muscle activation
Our results demonstrate that the rhythmic activation of epaxial myomeres during swimming in adult P. waltl results from three temporally distinct EMG waves passing posteriorly and in succession along the length of the body. Some nonuniformity in the timing of EMG activity in epaxial musculature during swimming in the salamander has been already reported (Frolich and Biewener 1992 Limb muscle activation
During stepping, the main EMG activity of dorsalis scapulae and puboischiofemoralis internus occurs during the swing phase and that of extensor ulnae and puboischiotibialis occurs during the stance phase of the limb (Ashley-Ross 1995 Neural mechanisms of intersegmental coordination
Our results do not allow direct inferences regarding the structure of neural networks underlying the patterns of muscle activation during swimming and stepping in the newt. However, it has previously been shown in a variety of vertebrates that the major features of the EMG pattern during steady locomotion in intact animals are similar to those of the electroneurographic patterns recorded during fictive locomotion in reduced or in vitro preparations (cat: Perret 1983
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
) and Xenopus tadpole (Roberts et al. 1986
). By contrast, the neural circuits producing lateral bending during terrestrial locomotion in lower tetrapods have not been studied directly. Consequently, any attempt to compare and contrast the central neural circuits controlling the axial motor system during swimming and stepping has not been performed.
). However, an essential prerequisite for an accurate identification and characterization of in vitro axial locomotor patterns is a detailed knowledge of the patterns of activation of axial muscles during locomotor movements in the intact animal.
). These waves of EMG activity have been related to the lateral undulations of the body observed in kinematic studies (Ashley-Ross 1994
; Daan and Belterman 1968
; Frolich and Biewener 1992
; Roos 1964
). In the second study, the contribution of hypaxial musculature to lateral bending in Dicampton ensatus has been investigated (Carrier 1993
). However, in both studies, EMG recordings were made only from a few longitudinal sites and, although some general features of the motor patterns during the two locomotor modes were described, we still lack a precise description of the timing of axial muscle activity along the entire body axis.
). Moreover, in an attempt to relate neural circuits controlling axial movements and those controlling limb movements, we have also examined the EMG pattern of some limb muscles during the two modes of locomotion.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Székely et al. 1969
). The muscles were identified according to Francis (1934)
.
). The swimming episodes were sometimes preceded by a period of low-speed paddling by the limbs (see Fig. 1A). This mode of aquatic locomotion has not been analyzed in the present study. Immediately after each experiment, animals were killed by overanesthesia.
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FIG. 1.
Patterns of electromyographic (EMG) activity during locomotion in the newt Pleurodeles waltl. A: characteristic body shape of newt (left) and EMG recordings (right) during swimming. B: characteristic body shape of newt (left) and EMG recordings (right) during overground stepping. Two locomotor episodes were obtained from same individual a few min apart. Vertical line indicated by arrow in A: time at which swimming episode begins. Traces in A and B, from top to bottom, were recorded from right dorsalis scapulae, right extensor ulnae, left latissimus dorsi, midtrunk, and tail myomeres on right side of animal (see drawings at left and text for electrode locations). Note that for each channel, voltage amplification was identical in A and B. i, ipsilateral; co, contralateral; trk, trunk; post, posterior; SVL, snout vent length.
2:1 and a time separation from other bursts >20 ms.
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FIG. 4.
EMG patterns in epaxial muscles during swimming. From top to bottom: anterior trunk (i ant trk), midtrunk (i mid trk), and posterior trunk (i post trk) myomeres on right side and midtrunk myomere on left side (co mid trk). See text for electrode locations. For each channel, EMG activity was full-wave rectified, filtered, and averaged over n = 20-23 consecutive swimming cycles. Averaging was triggered by onset of activity of right 0.60 SVL myomere (i mid trk). Vertical lines: time interval ("delay") between activation of anterior trunk myomere and that of reference myomere on same side. Data are from same individual.
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TABLE 1.
Characteristics of EMG activity of epaxial musculature during swimming
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TABLE 2.
Dependence of the delay and phase lag on the swimming cycle duration
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
).
). Another salient observation in our experiments was that the latissimus dorsi (Fig. 1) and the caudofemoralis muscles, which act both on the limb and the body axis, fired rhythmically during the two locomotor modes.
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FIG. 2.
Comparison of cycle duration during swimming and stepping. Each histogram plots cycle duration (abscissa, binwidth 25 ms) vs. occurrence (ordinate). Cycle duration was calculated as time interval between 2 successive EMG bursts in 0.60 SVL myomere on right side. Number of cycles (N) and mean ± SD are indicated to right of each histogram. Data are from same individual.
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FIG. 3.
Interlimb coordination during overground stepping. A and B: histograms plot phase difference between 2 limbs (shaded areas in left insets) on abscissa and occurrence on ordinate. In A (diagonally opposed limbs), phase difference, expressed as % of cycle duration, was calculated by dividing time interval between onsets of main EMG bursts (see text) of right dorsalis scapulae and left puboischiofemoralis internus by duration of step cycle. Bin width: 2% of cycle duration. In B (bilaterally homologous limbs), time interval between onsets of main bursts of right and left puboischiofemoralis internus was divided by duration of step cycle. Bin width: 2% of cycle duration. Number of cycles (N) and mean ± SD are indicated to right of each plot. Data are from same individual in A and B. RF, right forelimb; LH, left hindlimb; RH, right hindlimb.
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FIG. 5.
EMG patterns in epaxial muscles during swimming. A: right anterior and midtrunk myomeres. B: right mid- and posterior trunk myomeres. See text for electrode locations. Conventions as in Fig. 4. Data are from 2 different swimming episodes (n = 9-20 cycles) for same individual.
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FIG. 6.
Intersegmental coordination during swimming. A and B: dependence of rostrocaudal delay (ordinate) on position of myomere along body axis (abscissa). Each symbol represents mean rostrocaudal delay obtained for observed range of swimming cycle duration (indicated in inset). Negative values of delay: myomeres activated before reference myomere (i.e., 0.60 SVL recording site). Positive values: myomeres activated after reference myomere. C: dependence of rostrocaudal phase lag (ordinate) on position of myomere along body axis (abscissa). Rostrocaudal phase lag was calculated as rostrocaudal delay divided by corresponding cycle duration. Each symbol represents mean value of rostrocaudal phase lag in range of swimming cycle durations indicated in inset. Data were from single individual in A and pooled from 5 individuals in B and C. In A-C, vertical bars indicate SDs of delay and of phase lag, respectively.
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FIG. 7.
EMG patterns in epaxial muscles during overground stepping. A: right anterior trunk myomeres. B: right midtrunk myomeres. C: right posterior trunk myomeres. See text for electrode locations. Same type of representation as in Fig. 4. Data are from same individual in A and B(n = 12 cycles) and from 2 individuals in C (n = 11-21 cycles).
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FIG. 8.
Relative timing of epaxial muscle activities during step cycle. All recordings were performed on same side of body. Step cycle (abscissa) was defined as time interval between 2 successive onsets of activity in 0.60 SVL myomere. For each longitudinal location (ordinate), bars represent bursts of EMG activity expressed as percentage of step cycle. For anterior trunk and posterior trunk sites, black bars represent in-phase bursts and white bars out-of-phase bursts. Latencies of burst onset and termination were normalized to cycle duration (range: 950-1300 ms). Thin horizontal lines to left and to right of each bar: SDs of latency of burst onset and termination, respectively.
30.6 ± 4.4% and
71.6 ± 5.7% of cycle duration per SVL for anterior and posterior trunk, respectively).
; Peters and Goslow 1983
; Székely et al. 1969
; Wheatley et al. 1992
). The dorsalis scapulae, extensor ulnae, latissimus dorsi, and puboischiofemoralis internus muscles could fire two bursts within each step cycle, one that was always present ("main burst") and the other that was not ("facultative burst"), whereas the pubioischiotibialis and caudofemoralis muscles always fired a single burst (Fig. 9, A and B).
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FIG. 9.
Relative timing of epaxial and limb muscle activities during step cycle. Conventions as in Fig. 8. All recordings were performed on same side of body. A: forelimb muscles. B: hindlimb muscles. For limb muscles, black bars represent main bursts and white bars facultative bursts. Anatomic positions of myomeres and limb muscles are indicated in drawings at left (A, dorsal view; B, ventral view).
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FIG. 10.
Relative timing of latissimus dorsi, caudofemoralis, and epaxial muscle activities during swimming cycle. All EMG recordings were performed on same side of body. Conventions as in Fig. 9. Cycle range: 400-500 ms. Bars corresponding to latissimus dorsi and caudofemoralis are illustrated according to location of their insertions along trunk (Ashley-Ross 1992 ; Francis 1934
).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The greater number of sites recorded in the present study reveals that this nonuniformity results from an actual reversal in order of activation of myomeres at two sites of the trunk (~0.55 and 0.85 SVL).
also found that the rate of EMG propagation depended on the longitudinal position, with anterior locations displaying higher speed of propagation. By contrast, in anguilliform swimmers (eel, lamprey) EMG activation appears to propagate at a constant speed (Grillner and Kashin 1976
; Wallén and Williams 1984
; Williams et al. 1989
). These differences in the propagation velocity of EMG activity along the body could be related to the presence of limbs (urodela) or pectoral fins (trout).
; Wallén and Williams 1984
). In addition, our results demonstrate that the rostrocaudal delay was linearly dependent on the cycle duration and that the phase lag was independent of cycle duration (i.e., the swimming speed) only in the tail. A scaling of the delay with the cycle duration and the frequency-independent phase lag have been reported in swimming fish (Grillner 1974
; Grillner and Kashin 1976
; Jayne and Lauder 1995
) and lamprey (Poon 1980
; Wallén and Williams 1984
) and are considered to be constant features of swimming behavior. From this it can be concluded that in the newt, only spinal neural circuits underlying swimming movements of the tail have all the properties of the anguilliform swimming network.
). Indeed, the midtrunk myomeres (i.e., 0.55-0.90 SVL) were synchronously activated in alternation with the main activity in the most rostral and caudal myomeres.
; Roos 1964
). Interestingly, the additional burst in phase with midtrunk activity was more consistent for myomeres located close to the girdles (e.g., 0.40 SVL and 1.10 SVL). The role of the double activation of those myomeres might also be to control the stiffness of the trunk close to the girdles to support the action of limbs.
; Székely et al. 1969
). Thus the midtrunk activity occurs during the stance phase of the ipsilateral forelimb indicated by ongoing extensor ulnae activity (Fig. 9A) and during the swing phase of the ipsilateral hindlimb indicated by the main burst of puboischiofemoralis internus (Fig. 9B). Therefore our results confirm the previous kinematic data showing that forelimb stance phase and hindlimb swing phase are related to lateral bending of the midtrunk on the same side (Ashley-Ross 1994
; Daan and Belterman 1968
; Roos 1964
). This coordination of the limbs with the midtrunk contributes to the progress of the animal during overground stepping by enhancing the length of the stride (Daan and Belterman 1968
; Roos 1964
).
; Peters and Goslow 1983
). The main burst of latissimus dorsi would assist in lifting up the forelimb during the flexor phase, whereas its facultative burst would contribute in drawing the forelimb backward during the propulsive phase (see Cabelguen et al. 1981
).
; lamprey: Wallén and Williams 1984
; fish: Fetcho and Svoboda 1993
; chick: Jacobson and Hollyday 1982
; tadpole: Kahn and Roberts 1982
; mudpuppy: Wheatley et al. 1992
). Thus it is very likely also that the patterns of activation of epaxial myomeres (intersegmental coordination) observed in the present study are essentially produced by the central pattern generators (CPGs) for locomotion.
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FIG. 11.
Schematic diagrams of neural mechanisms of intersegmental coordination during swimming in lamprey (A) and during swimming (B) and stepping (C) in newt. A: schematic representation of "trailing oscillator hypothesis" (adapted from Grillner et al. 1993 ). Drawing at left: characteristic shape of lamprey during swimming. Bar diagrams: motor pattern along 1 side of body during forward and backward swimming. Bars indicate when myomeres are active. Wiring diagrams: chain of segmental oscillators (
) that underlies intersegmental pattern of coordination during forward and backward swimming. Oscillators are coupled with mutual excitation and inhibition (
) and driven by "leading oscillator" (heavy
) located at rostral end of chain during forward swimming and at caudal end of chain during backward swimming. B: proposed neural organization underlying intersegmental pattern of coordination during swimming in newt. Conventions as in A. Note 3 leading oscillators in chain (see text). The 2 boxes at right represent forelimb (top) and hindlimb (bottom) central pattern generators (CPGs) for locomotion that are tonically active during swimming. Horizontal lines: tonic excitatory inputs that come from limb CPGs and increase excitability of segmental oscillatory networks located close to pectoral and pelvic girdles. Three EMG waves are propagated posteriorly because of descending dominance of oscillators located in segments close to girdles. C: proposed neural mechanisms generating intersegmental pattern of coordination during stepping in newt. Motor pattern on one side of body during step cycle (0, T) is represented as in B. The lighter the bar, the weaker the EMG activity. Note the 2 leading oscillators during 1st half of step cycle (0, T/2) and synchronization of midtrunk oscillators during 2nd half of step cycle (T/2, T). Rhythmic inputs coming from limb CPGs increase (
) or decrease (
) excitability of other segmental oscillators.
; Matshushima and Grillner 1990
, 1992
). Segmental oscillators with a higher level of tonic excitation become the "leading segments" and drive, with a lag, segmental oscillatory networks in both the rostral and caudal directions. During forward swimming, the leading segments are located in the rostral part of the spinal cord and produce a wave of activity passing down the body axis (Fig. 11A, left), whereas during backward swimming they are located in the caudal part of the spinal cord and the wave of activity now travels in a caudorostral direction (Fig. 11A, right).
). Although the present study was not designed to address this issue, our data do suggest that, similarily to the intersegmental coordination during swimming, the intersegmental coordination during stepping in the newt is generated by neuronal networks that are basically similar to those underlying swimming in the lamprey ("lampreylike swimming CPG"). Indeed, a comparison of the pattern of activation of myomeres during stepping in the newt (Fig. 11C) with those during forward or backward swimming in lamprey (Fig. 11A) suggests that the former pattern can be considered as a hybrid lamprey pattern resulting from waves of activity initiated at different sites of the body and traveling rostrally or caudally along the body axis. In the first half of the step cycle (Fig. 11C,0-T/2), two waves are generated (one, initiated in the rostral segments, propagates down the anterior trunk, and the other, initiated in the midtail segments, propagates up from the tail), whereas in the second half of the step cycle (Fig. 11C, T/2-T) the midtrunk activity spreads up and down the anterior trunk and posterior trunk, respectively. Thus an extra excitatory input should influence a lampreylike swimming network in a phase-dependent way: the rostral and midtail segments in the first half of the step cycle, the midtrunk segments in the second half of the step cycle.
) and theoretically (Kopell and Ermentrout 1986
, 1988
; Kopell et al. 1990
). However, it is still not known whether such a network can be entrained from more than one site of the chain, and this is a possibility that should be tested in further experimental and modeling studies.
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ACKNOWLEDGEMENTS |
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We thank Drs. G. Le Masson and J. Simmers for helpful comments on the manuscript. We also thank Dr. J. Simmers. for correcting the English.
This study was supported by grants from Université Bordeaux I and Conseil Régional d'Aquitaine. I. Delvolvé received a studentship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. T. Bem was supported by a stipend from France-Poland exchange program (Centre National de la Recherche Scientifique-Polish Academy of Sciences).
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FOOTNOTES |
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Present address of T. Bem: Institute of Biocybernetics and Biomedical Engineering, 02-109 Warsaw, Trojena 4, Poland.
Address for reprint requests: J. M. Cabelguen, Lab. Neurobiologie et Physiologie Comparées, Place Peyneau, 33120 Arcachon, France.
Received 3 June 1996; accepted in final form 25 March 1997.
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