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NEUROSCIENCE |
1 Universités Bordeaux 2 and 1, CNRS, Bordeaux, France
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Abstract |
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(Received 26 June 2006;
accepted after revision 18 December 2006;
first published online 21 December 2006)
Corresponding author J.-R. Cazalets: CNRS Unité Mixte de Recherche 5227, Université Bordeaux 2, Zone nord Bat 2, 146, rue Léo Saignat, 33076 Bordeaux Cedex, France. Email: jean-rene.cazalets{at}u-bordeaux2.fr
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Introduction |
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The neuronal networks that generate the basic motor patterns underlying limb movements (central pattern generator; CPGs), have been located within the lumbar spinal cord for the hindlimbs (for review see Rossignol, 1996; Cazalets, 2000) and the cervical spinal cord for the forelimbs (Ballion et al. 2001), and details on intra- and interhindlimb coordination (Rossignol, 1996; Cazalets, 2000; Butt et al. 2002; Butt & Kiehn, 2003), as well as on fore- and hindlimb coordination (Juvin et al. 2005), have been reported. An unresolved question is whether these identified neuronal networks contribute to the coordinated behaviour of the entire body during actual locomotion. We have recently found that the spinal cord of newborn rat contains propriospinal pathways involved in a metachronal propagation of motor activity along the spinal axis (Cazalets, 2005). However, in this earlier sequential study the motor activation was observed during extreme pharmacological manipulation including a generalized blockade of fast inhibitory synaptic transmission. In the present study, we have analysed the interactions between the various parts of the cord during centrally generated spinal cord activity under more physiological conditions. To assess the global functioning of spinal circuitry and to understand how the thoracic, lumbar and sacral segments interact, we have recorded from up to 16 ventral roots simultaneously along the thoraco-lumbo-sacral spinal cord axis during sequences of fictive locomotor activity. Furthermore, we have combined in vitro observations with analysis in intact animals to study the simultaneous functioning of the trunk and hindlimbs during actual locomotion. We found that motor bursts propagate rostrally and caudally from the lumbar region to the most distant cord segments.
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Methods |
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Two-dimensional kinematic measurements
Four-day-old rat pups were used for kinematic analysis (n = 5). Each animal was tagged with nine dots (1 mm diameter) using a felt tip marker (Fig. 1B) at the following locations: tail (tip, middle and base), spine (sacrum, pelvis, midway between scapuli and pelvis and scapuli), head and right hindfoot. Rats were induced to walk on a rubber strip over 10 trials (approximately eight steps each) and animals were filmed with a CCD video camera (Sony, 25 frames s–1). Sustained locomotion was reliably initiated using the protocol of olfactory stimulation as developed by Jamon & Clarac (1998). Briefly, a tube containing nest material was presented to the pup. As the animal moved towards the scent of the material, the experimenter slowly withdrew the tube so that the animal followed it. Only forward walking was elicited with this method. In a given sequence, the first and last steps were excluded from the subsequent analysis. Video sequences were analysed with ImageJ software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/, 1997–2005) and a manual tracking plug-in (implemented by F.P. Cordelières, Institut Curie, Orsay, France). Skin slippage was not compensated for because it appeared minimal in the back region. The x and y coordinates of individual dots were determined manually on each frame under visual inspection. Angles were computed using Microsoft Excel and stick diagrams and angles were plotted using Igor-Pro software (Wavemetrics, OR, USA).
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Pups (n = 13) from P2 to P4 were anaesthetized by hypothermia until reflexes were lost and placed on a cold pad in order that they remained anaesthetised during tracer injection. They showed no reflex reactions such as withdrawal in response to the muscle injections. The retrograde tracer, cholera toxin B subunit Alexa Fluor 488 conjugate (CTB at 1% in distilled water, Molecular Probes, OR, USA) was injected using a Hamilton syringe into the right back muscles (longissimus or multifidus) and into the left gastrocnemius. The rats were then returned to their mother for 2 days, after which they were killed and their spinal cords were dissected out and fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (3 h at 4°C). After dehydration, the spinal cord was cleared in methyl-salicylate for 1 h, and then examined using a fluorescence microscope.
In vitro isolated spinal cord
Locomotor-like activity was elicited by bath-application of a mixture of either serotonin (5-HT, 2 x 10–5 M) or dopamine (DA, 2 x 10–4 M) plus N-methyl-D,L-aspartate (NMA, 1.5–2 x 10–5 M) (Sqalli-Houssaini et al. 1993; Barriere et al. 2004).
Pups (n = 33) from P1 to P3 were anaesthetized by hypothermia until reflexes were lost, then decapitated and eviscerated. As described by Squalli et al. (1991), the spinal cord was isolated and pinned down ventral side up in a recording chamber. All dissection and recording procedures were performed under continuous perfusion with Krebs saline containing (mM): NaCl 130, KCl 3, CaCl2 2.5, MgSO4 1.3, NaH2PO4 0.58, NaHCO3 25 and glucose 10; bubbled with 95% O2–5% CO2, adjusted to pH 7.4 with HCl and maintained at room temperature (24–26°C). Spinal cords were sectioned at the T1 level at the beginning of the experiment.
Up to 16 ventral roots were recorded simultaneously (Fig. 4), using stainless steel pin electrodes insulated from the bath with Vaseline (the location of the extracellular electrodes is indicated with dots in the figures). During an episode of fictive locomotion, a steady state was reached within 10 min (Sqalli-Houssaini et al. 1993), after which measurements were made. Signals, amplified 5000 x using custom-made amplifiers, were acquired at 2 kHz on 16 channels using a Digidata 1322A interface driven by Axograph software (Axon Instruments, CA, USA). They were then processed and analysed using Axograph analysis plug-ins. Cord sections were made in the recording chamber with sharp MC-52 scissors (Moria, Paris).
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Statistical analyses were performed on raw data by descriptive statistics of circular distribution (Zar, 1984). In each experiment, the phase (
) was calculated and the mean phase 
was determined with the formula:
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| (1) |
Phase values were plotted in a circular representation (0–360 deg in the trigonometric direction), with the mean phase being indicated by the direction of the vector, and its length (range from 0 to 1) indicating the strength of the mean. The latter was calculated by the formula:
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| (2) |
The Rayleigh test was used to determine the coupling strength. Circular statistical analyses (circular linear correlations) were performed using R software (Team, 2005) or Oriana (KCS, UK). The significance threshold was taken to be P < 0.05 unless otherwise specified. Other analyses (linear regression, correlation and one-way ANOVA) were performed using Prism software (Graphpad software, CA, USA). All data values in the text are means ± S.E.M.
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Results |
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Body movements were studied in newborn rats in order to provide behavioural data that could be correlated to the activity recorded under in vitro conditions. Figure 1A presents images obtained at three different time intervals during one step of olfactory stimulus-induced locomotion. Visual examination of marker dots on the pup clearly shows that the back flexes alternately during stepping. The locomotor parameters (period, stance and swing duration) that we observed with our kinematic analysis were comparable to those previously reported by Jamon & Clarac (1998). The mean cycle period for all steps collected in five different animals was 0.8 ± 0.03 s. The schematic diagram in Fig. 1Ba shows the model that was used for analysing the region-specific movements illustrated in Fig. 1Bb where the various colour lines show the trajectories for various points on the back and tail following manual tracking. Figure 1C shows stick diagrams established for a single step that was broken down into swing (Fig. 1Ca) and stance (Fig. 1Cb) phases with respect to the reference right hindlimb. During both phases, there was a clear bending in the trunk axis as the tail orientation concomitantly changed. Figure 2 presents the angular variations at various trunk levels measured from a pup as shown schematically in Fig. 2A. For each plot in Fig. 2B and C, the coordinate axis corresponds to the average changes in angular position during a duplicated cycle (duty cycle normalized from 0 to 1 in the x axis). During each step, the angle at all levels changed cyclically so that the trunk curvature alternated symmetrically between the right (above 180 deg) and left sides (below 180 deg).
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Anatomical identification of motoneurons
Trunk and tail movements are mainly due to the involvement of the epaxial muscular system, including the multifidus, longissimus and sacroccygeus muscles which run laterally along the body axis. Before further investigating the properties and generation of trunk motor activity, we sought to identify the origin and central distribution of motoneurons supplying axial musculature in the newborn rat because these motoneurons have only been partially localized in adult rats (Brink et al. 1979). This was achieved by injecting the retrograde marker cholera toxin B subunit (see Methods) at various levels into the right axial muscles as indicated in Fig. 3A, as well as into the contralateral (left) gastrocnemius. Figure 3B shows three examples of labelled motoneuron pools, with three different injection sites (tail, Fig. 3Ba; lower back muscles, Fig. 3Bb; mid-back muscles, Fig. 3Bc). Tail muscle injections always resulted in bilateral labelling due to small muscle size. Figure 3Bb shows an example of the labelling on the left side resulting from an injection into the left gastrocnemius (G) and the labelling on the right side corresponding to a right back muscle injection (B) at level 4 indicated in Fig. 3A. Under our experimental conditions, a back muscle injection was found to label motor columns spanning two segments. Moreover, at the lumbar level, there was a substantial overlap between motoneuron pools innervating the back muscles (B) and the motoneuron pools supplying the hindlimb (G, Fig. 3Bb). The summary diagram of Fig. 3C illustrates the motoneuron location (dots), identified following injections at the various sites indicated in Fig. 3A. We have observed a relationship between the rostro-caudal location of the injected muscle site and the rostro-caudal location of the corresponding motor columns, with motoneurons innervating the tail being located in the most distal sacral and coccygeal cord regions. These data therefore showed that motoneurons innervating the axial musculature are distributed along the entire spinal cord, from thoracic to coccygeal segments and including the lumbar segments where they colocalize with motoneurons that innervate the hindlimbs.
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Next we recorded motor activity in the isolated spinal cord preparation during chemically evoked sequences of locomotor-like activity. Figure 4 illustrates simultaneous recordings from 16 ipsilateral ventral roots (indicated by the dots in Fig. 4A) during bath-application of a mixture of 5-HT and NMA (see Methods). One characteristic of the multiple-site motor pattern recorded under these conditions was that the signal-to-noise ratio (compare background activity in Fig. 4Ba to pharmacologically induced motor activity in Fig. 4Bb) was always greater in the low thoracic and lumbar ventral roots, suggesting that more units are active at these levels. In addition, there was typically a one-to-one relationship between bursts recorded in all the recorded ventral roots. Occasionally (4 out of 29 experiments) we observed two bursts of activity in either low lumbar (L4–L6) or sacral ventral roots (Cazalets et al. 1992; Cazalets & Bertrand, 2000a). When viewed on an expanded time scale, it is apparent that the motor bursts were not synchronous, as shown in Fig. 5, where the onset of bursts at various cord levels is compared to burst onset in L2 (Fig. 5A) and S4 (Fig. 5B). This shows that there was a progressive delay in onset of burst generation which was observed both in sacral and lumbo-thoracic segments. In both cases, the propagation of motor activity was in a caudal-to-rostral direction. In these series of experiments, we always recorded all lumbar and sacral ventral roots and the positions of the electrodes on the thoracic ventral roots were varied from one experiment to another in order to monitor the entire length of the thoracic cord region to establish the phase diagram as seen in Fig. 7.
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) being expressed in degrees (0–360 deg).
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In addition to the relative values provided by the polar plots and phase diagram of Fig. 7, we plotted the mean total propagation time (Fig. 8A, black bars, left-hand y axis) through the lumbo-thoracic spinal cord (L2–T2), the sacral spinal cord (S1–S4) and the associated propagation time between individual segments (Fig. 8A, grey bars, right-hand y axis). The intersegmental delay was much shorter in the thoracic segments (67 ms segment–1 or 0.015 mm ms–1 because each segment measured 1 mm) than in the sacral segments (185 ms segment–1 or 0.005 mm ms–1). Moreover plots of latency versus motor burst period (filled circles in Fig. 8B) for representative segments from the three different zones, revealed that there was a systematic and significant increase in latency with the burst period (correlation analysis between the motor period and the latency, P < 0.01). Slope comparisons indicated that the variation in latency in the thoracic versus sacral segments was significantly different (P < 0.01), with slope being less in the thoracic region (T10 in the present study) than in the lumbar (L5) or sacral (S2) areas. By contrast, the plots of phase value versus period (open squares, Fig. 8B) show that there was no correlation between burst phase and the motor period. On this basis, therefore, it appears that spinal motor networks adjust the intersegmental latency to the ongoing motor period in order to maintain a constant phase relationship of activity along the spinal axis.
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The above data show that coordinated motor patterns with a specific temporal organization are expressed in the isolated spinal cord. This raises two questions: from where does the propagated motor activity originate, and what determines the phase shifts? To address these questions, we performed cord section experiments in order to assess whether the different zones (T, L and S) identified in Fig. 8B have the capacity for rhythmogenesis. In the control recordings from an intact isolated spinal cord as in Fig. 9A, motor bursts were coordinated 1: 1 in all the ventral roots recorded with a burst period of 2.8 ± 0.05 s (Fig. 9C). Right and left alternation occurred in thoracic segments in all experiments where thoracic segments were recorded bilaterally (n = 5). We further performed cord transection experiments (n = 8 preparations), at levels indicated by the vertical dashed lines in Fig. 9B. Rhythmic motor patterns were still expressed in all three separated pieces of spinal cord, albeit with substantial differences in activity (Fig. 9C). As previously reported (Cazalets & Bertrand, 2000a), a sustained rhythmic activity was observed in the isolated lumbar area with a burst period (3.2 ± 0.1 s, n = 30 cycles) similar to that recorded in the intact cord, whereas a slightly slower rhythmic bursting pattern was still observed in the isolated sacral region (3.9 ± 0.2 s, n = 30 cycles). An alternation between the L2 (flexor units) and the L5 (extensor units) persisted. Similarly a left–right alternating motor root pattern also occurred in the isolated thoracic region although at an even slower cycle period (4.85 ± 0.1 s, n = 30 cycles). Such slow and sometimes irregular rhythmic motor activity was observed in the isolated thoracic T2–T12 spinal cord in six out of eight experiments, with bath-application of NMA plus either 5-HT or DA. Although slower and even more irregular, rhythmic activity was recorded in shorter pieces (T5–T12 segments) of the thoracic spinal cord. As bursting properties can be expressed in the various spinal areas including the thoracic cord, it is likely that all segments actively participate in motor wave propagation.
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Discussion |
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The exploration of the ability of spinal motor networks to generate rhythmic motor output through section experiments (Figs 9 and 10), revealed that three zones (T, L and S; Figs 7B and 9B) possess the intrinsic capacity for rhythmogenesis. While this was already described for the sacral (Cazalets & Bertrand, 2000a; Lev-Tov & Delvolve, 2000) and lumbar cord areas (Cazalets et al. 1995; Cazalets & Bertrand, 2000a), we demonstrate here that the isolated thoracic spinal cord can also generate coordinated activity with rhythmic left–right alternation. Previous studies (Cowley & Schmidt, 1997; Kremer & Lev-Tov, 1997; Ballion et al. 2001) presented contradictory data. Kremer & Lev-Tov (1997) noted that rhythmic activity (both alternating or non-alternating) or random bursting activity could be recorded in some thoracic segments, but this activity disappeared following a cord section at the low thoracic level. A similar finding was reported by Ballion et al. (2001). On the other hand, Cowley & Schmidt (1997) noted that thoracic segments when isolated could generate rhythmic activity, but their study did not give any indication of right–left phase relationships. The discrepancy between these different studies may be attributable to different experimental conditions and to the fact that a complete systematic study on the rhythm-generating properties of the thoracic spinal cord was not performed.
The rhythmic activity generated by the three different cord areas is qualitatively different, with the thoracic (Fig. 9) and sacral (Fig. 10; Cazalets & Bertrand, 2000a) regions generating slower rhythms than the lumbar area. The model of Fig. 11 correponds closely to that recently proposed by Magnuson et al. 2005), who studied the functional consequences of a spinal cord contusion at the lumbar level in adult rats. According to their model, rhythmic hindlimb locomotor activity elicited physiologically by stimulation of descending commands (see green symbols in Fig. 11) or by drug application is dominated by circuitry in the L1 and L2 segments of the intact spinal cord. In addition, the finding that further reduced preparations (e.g. isolated L3–L6) can exhibit drug-induced bursting (Kjaerulff & Kiehn, 1996; Cowley & Schmidt, 1997; Kremer & Lev-Tov, 1997; Magnuson & Trinder, 1997; Bonnot et al. 1998) demonstrates that neuronal circuitry capable of rhythmogenesis (‘rhythmic elements’) is distributed throughout the lumbar enlargement. This in turn led to the proposition (Magnuson et al. 2005) that the CPG located in the rostral part of the lumbar enlargement provides rhythmic locomotor output to neighbouring rhythmic elements that in turn transfers and modulates the output to segmental motor neurons in the adult animal. The model that we propose in Fig. 11 expands on this view to incorporate the entire spinal cord. In an intact spinal cord, the lumbar area probably imposes its own timing on the thoracic spinal cord generators, as it does for the sacral segmental rhythm (Cazalets & Bertrand, 2000a). The pre-eminent role of lumbar locomotor networks was also postulated in a recent study on the coordination between the motor circuitry controlling the forelimbs and hindlimbs (Juvin et al. 2005). In various studies (Kjaerulff & Kiehn, 1996; Cowley & Schmidt, 1997; Kremer & Lev-Tov, 1997; Bonnot et al. 1998), bursting has been found to occur in isolated pieces of the spinal cord under pharmacological activation. It cannot be excluded that some of these observations may in fact be attributable to bursting occurring in axial motoneurons and that the rhythmogenic ability of the various spinal compartments controlling axial musculature may have contributed to different interpretations concerning the organization of hindlimb locomotor networks (Cazalets, 2000; Cazalets & Bertrand, 2000b).
An interesting feature of the phase-lag diagram of Fig. 7B is the temporal nature of motor burst propagation in the sacral area. In the intact spinal cord, sacral activity propagates caudo-rostrally, in a phase-opposite sequence to the ipsilateral L2 segments. Our cord section experiments also revealed important insights into the underlying synaptic organization (see Fig. 11). First, they indicate that rhythm generation does not require cross-cord connections (horizontal blue lines in Fig. 11) as also demonstrated in the Xenopus embryo (Soffe, 1989) and recently in the lamprey (Cangiano & Grillner, 2003). The vertical blue lines in Fig. 11 indicate that the reciprocal organization of ipsilateral flexor (L2) and extensor (L5) activity bursting (Cazalets et al. 1992) also does not rely on cross-cord connections but is completely organized ipsilaterally (Kudo & Yamada, 1987). By contrast, the ipsilateral L2/sacral antiphase pattern relies on contralaterally projecting connections (vertical brown lines in Fig. 11) because in a complete hemicord preparation (Fig. 10B), sacral bursting activity switched to an in-phase motor pattern with L2 bursting. It is surprising that when the spinal cord was split only from the sacral to L5 segments, the antiphase pattern persisted, although the intersegmental latency was dramatically reduced to produce almost single phase sacral motor activity (Fig. 10C). This suggests that different cross-cord connections are involved both in setting the timing of intersegmental phase shifts as well as in the reciprocal bilateral organization. Commissural interneurons that are active during locomotion have been shown to play a major role in setting the contralateral relationships in various species (Buchanan, 1996; Soffe et al. 2001; Butt & Kiehn, 2003; Jankowska et al. 2005; Zhong et al. 2006). In neonatal rat, however, the involvement of different classes of these commissural interneurons (both segmental and intersegmental) with their firing patterns occurring at all phases of the locomotor cycle has led to the suggestion that they may also be involved in other aspects of locomotor rhythm generation (Butt & Kiehn, 2003; Zhong et al. 2006). The results of our study suggest that one of these roles is in setting the phase delay between segments along the spinal cord as has also been suggested in Xenopus embryo cord (Green & Soffe, 1998). This should be further investigated by determining whether the anatomical distribution of commissural interneurons along the spinal cord is commensurate with such a specific role.
Temporal organization of motor activity in the spinal cord
Our phase analysis revealed several discrete zones along the spinal axis in which motor activity propagates sequentially. This demonstration was possible because we recorded from neighbouring ventral roots, which allowed the establishment of the phase relationships between adjacent segments. In lower vertebrates, such as lamprey and frog tadpole, axial body movements involved in locomotion result from a rostro-caudal metachronal wave of motoneuron discharge that gives rise to muscle contractions with appropriate phase delay in adjacent body segments (Cohen, 1987; Matsushima & Grillner, 1992; Roberts et al. 1998; McClellan & Hagevik, 1999; Grillner & Wallen, 2002). Similarly, our in vitro data strongly suggest that the metachronal changes observed in trunk bending (Fig. 2) rely on intrinsic spinal cord properties. In a previous study performed under particular pharmacological conditions (strychnine and bicuculline), it was concluded that the spinal cord contains axial propriospinal pathways that may be involved in intersegmental coordination (Cazalets, 2005). Although this system may account for the phase lag observed here, the reported speed of longitudinal propagation in the presence of strychnine and bicuculline was much higher (Cazalets, 2005). In the present study without blockade of inhibitory synaptic transmission, the mean propagation of locomotor bursts along the thoracic cord was 800 ms (Fig. 8), indicating that propagation between each segment may also involve local interactions that effectively slow propagation. If long spinal fibres that distribute to each segmental level were solely involved (red lines in Fig. 11), presumably propagation would be faster because it would only take the time for spike conduction along the fibres, as well as synaptic delay (i.e. about 50 ms). As suggested in previous work (Cazalets, 2005), and as also shown in the lamprey (McClellan & Hagevik, 1999; Miller & Sigvardt, 2000), it is therefore likely that both long intersegmental neuronal tracts and local circuit interactions between adjacent segmental oscillators are involved in coupling. Furthermore, the fact that both thoracic and sacral areas can express intrinsic bursting properties (Fig. 9), albeit weaker than the lumbar region, may also contribute to slowing motor propagation. To date the exact nature of the propriospinal systems involved in the coordinating process is unknown. In the lamprey, the excitatory interneurons that traverse from two to six segments have been suggested to account for intersegmental coordination (Dale, 1986). Juvin et al. (2005) have suggested that interactions between lumbar and cervical cord regions may be mediated by asymmetric propriospinal pathways arising in the lumbar area and relaying through the thoracic level caudo-rostrally. In the newborn rat, a class of ipsilateral excitatory interneuron (Kiehn & Butt, 2003) could also be partly responsible for the segment–segment interactions. In addition, we have previously shown that long propriospinal pathways probably exist that participate in motor burst propagation (Cazalets, 2005).
Functional implications of metachronal propagation
Body displacement in elongated animals such as tadpoles (Roberts et al. 1998; Soffe et al. 2001; Tunstall et al. 2002), lamprey (Cohen, 1987; McClellan & Hagevik, 1999; Miller & Sigvardt, 2000; Grillner & Wallen, 2002) and snakes (Gasc et al. 1989) are driven by trunk muscle contractions that occur sequentially along the body length. Rhythmic activation of back muscles during locomotion has also been reported to occur in various quadrupeds such as the cat (Carlson et al. 1979; Zomlefer et al. 1984), adult rat (Geisler & Gramsbergen, 1998), newt (Delvolve et al. 1997) and human (Thorstensson et al. 1982). By using retrograde staining, we determined here that the motoneuron pools that innervate the trunk and tail muscles are distributed along the spinal cord, in a manner that matches the distribution of muscles along the body. These results add to those of a previous study in adult rats (Brink et al. 1979), in which the central locality of motoneurons innervating muscles that specifically intervene in lordosis were determined. Moreover our in vitro electrophysiological data appear to support the physiological involvement of trunk muscle activity in locomotion. Although the thoracic ventral roots innervate other body regions, particularly the respiratory (Monteau & Hilaire, 1991) and abdominal musculature (Iscoe, 1998), because the spinal cord was transected at the T1 level in our experiment, respiratory motoneurons were not involved in the recorded rhythmic patterns (Monteau & Hilaire, 1991). Furthermore, an interesting finding is that back muscle motoneurons are colocalized with the hindlimb muscle motoneurons at the lumbar spinal cord level (Fig. 3Bb; see also Nicolopoulos). Thus, the possibility arises that segmental lumbar output may concomitantly reflect different sources of rhythmogenesis: the lumbar hindlimb generators themselves and the networks responsible for back muscle activation. Alternatively, the pre-eminent activation of hindlimb motoneurons by the lumbar generators may mask the activation of axial motoneurons in this cord region.
In the present study, we have found evidence suggesting that trunk curvature observed during locomotion is due to a sequential propagation of motor output-related activity along the spinal cord in newborn rat. Our kinematic data provide evidence for a caudo-rostral progression in the maximum angular deviation that occurs with a specific phase relationship (Fig. 2D). Our in vitro data match observations from the intact animal because a comparable caudo-rostral propagation of motor bursts was observed (Fig. 7B). Another interesting observation is that the intersegmental latency in the sacral area is longer than in the thoracic region. This may reflect the fact that the central nervous system differentially controls spinal curvature, which is greater in the lower back (Fig. 1A; compare angle variations in Fig. 2Ba and 2Bb and c, Fig. 2D) and the tail. Such an organization would therefore seem to support the dynamic control of posture by the performance of fluent movements during locomotion. Moreover our data suggest that the networks responsible for metachronal propagation of motor patterns during locomotion may correspond to those observed in invertebrates or lower vertebrates, and thus are highly conserved.
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References |
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Barriere G, Mellen N & Cazalets JR (2004). Neuromodulation of the locomotor network by dopamine in the isolated spinal cord of newborn rat. Eur J Neurosci 19, 1325–1335.[CrossRef][Medline]
Bonnot A, Morin D & Viala D (1998). Organization of rhythmic motor patterns in the lumbosacral spinal cord of neonate mouse. Ann N Y Acad Sci 860, 432–435.
Brink EE, Morrell JI & Pfaff DW (1979). Localization of lumbar epaxial motoneurons in the rat. Brain Res 170, 23–41.[CrossRef][Medline]
Buchanan JT (1996). Lamprey spinal interneurons and their roles in swimming activity. Brain Behav Evol 48, 287–296.[Medline]
Butt SJ & Kiehn O (2003). Functional identification of interneurons responsible for left-right coordination of hindlimbs in mammals. Neuron 38, 953–963.[CrossRef][Medline]
Butt SJ, Lebret JM & Kiehn O (2002). Organization of left-right coordination in the mammalian locomotor network. Brain Res Brain Res Rev 40, 107–117.[CrossRef][Medline]
Cangiano L & Grillner S (2003). Fast and slow locomotor burst generation in the hemispinal cord of the lamprey. J Neurophysiol 89, 2931–2942.
Carlson H, Halbertsma J & Zomlefer M (1979). Control of the trunk during walking in the cat. Acta Physiol Scand 105, 251–253.[Medline]
Cazalets JR (2000). Organization of the spinal locomotor network in neonatal rat. In Neurobiology of Spinal Cord Injury, ed. Kalb R & Stritmatter SM, pp. 89–111. Humana Press Inc, Totowa, NJ.
Cazalets JR (2005). Metachronal propagation of motoneurone burst activation in isolated spinal cord of newborn rat. J Physiol 568, 583–597.
Cazalets JR & Bertrand S (2000a). Coupling between lumbar and sacral motor networks in the neonatal rat spinal cord. Eur J Neurosci 12, 2993–3002.[CrossRef][Medline]
Cazalets JR & Bertrand S (2000b). Ubiquity of motor networks in the spinal cord of vertebrates. Brain Res Bull 53, 627–634.[CrossRef][Medline]
Cazalets JR, Borde M & Clarac F (1995). Localization and organization of the central pattern generator for hindlimb locomotion in newborn rat. J Neurosci 15, 4943–4951.[Abstract]
Cazalets JR, Sqalli-Houssaini Y & Clarac F (1992). Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat. J Physiol 455, 187–204.
Cohen AH (1987). Intersegmental coordinating system of the lamprey central pattern generator for locomotion. J Comp Physiol A 160, 181–183.[CrossRef]
Cowley KC & Schmidt BJ (1997). Regional distribution of the locomotor pattern-generating network in the neonatal rat spinal cord. J Neurophysiol 77, 247–259.
Dale N (1986). Excitatory synaptic drive for swimming mediated by amino acid receptors in the lamprey. J Neurosci 6, 2662–2675.[Abstract]
Delvolve I, Bem T & Cabelguen JM (1997). Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt, Pleurodeles waltl. J Neurophysiol 78, 638–650.
Duysens J & Van de Crommert HW (1998). Neural control of locomotion; The central pattern generator from cats to humans. Gait Posture 7, 131–141.[CrossRef][Medline]
Gasc J-P, Cattaert D, Chasserat C & Clarac F (1989). Proplusive action of a snake pushing again a single site: its combined analysis. J Morphol 201, 315–329.[CrossRef]
Geisler HC & Gramsbergen A (1998). The EMG development of the longissimus and multifidus muscles after plugging the horizontal semicircular canals. J Vestib Res 8, 399–409.[CrossRef][Medline]
Green CS & Soffe SR (1998). Roles of ascending inhibition during two rhythmic motor patterns in Xenopus tadpoles. J Neurophysiol 79, 2316–2328.
Grillner S & Wallen P (2002). Cellular bases of a vertebrate locomotor system-steering, intersegmental and segmental co-ordination and sensory control. Brain Res Brain Res Rev 40, 92–106.[CrossRef][Medline]
Iscoe S (1998). Control of abdominal muscles. Prog Neurobiol 56, 433–506.[CrossRef][Medline]
Jamon M & Clarac F (1998). Early walking in the neonatal rat: a kinematic study. Behav Neurosci 112, 1218–1228.[CrossRef][Medline]
Jankowska E, Edgley SA, Krutki P & Hammar I (2005). Functional differentiation and organization of feline midlumbar commissural interneurones. J Physiol 565, 645–658.
Juvin L, Simmers J & Morin D (2005). Propriospinal circuitry underlying interlimb coordination in mammalian quadrupedal locomotion. J Neurosci 25, 6025–6035.
Kiehn O & Butt SJ (2003). Physiological, anatomical and genetic identification of CPG neurons in the developing mammalian spinal cord. Prog Neurobiol 70, 347–361.[CrossRef][Medline]
Kjaerulff O & Kiehn O (1996). Distribution of networks generating and coordinating locomotor activity in the neonatal rat spinal cord in vitro: a lesion study. J Neurosci 16, 5777–5794.
Koehler WJ, Schomburg ED & Steffens H (1984). Phasic modulation of trunk muscle efferents during fictive spinal locomotion in cats. J Physiol 353, 187–197.
Kremer E & Lev-Tov A (1997). Localization of the spinal network associated with generation of hindlimb locomotion in the neonatal rat and organization of its transverse coupling system. J Neurophysiol 77, 1155–1170.
Kudo N & Yamada T (1987). N-methyl-D, L-aspartate-induced locomoter activity in a spinal cord-hindlimb muscles preparation of the newborn rat studied in vitro. Neurosci Left 75, 43–48.[CrossRef]
Lev-Tov A & Delvolve I (2000). Pattern generation in non-limb moving segments of the mammalian spinal cord. Brain Res Bull 53, 671–675.[CrossRef][Medline]
McClellan AD & Hagevik A (1999). Coordination of spinal locomotor activity in the lamprey: long-distance coupling of spinal oscillators. Exp Brain Res 126, 93–108.[CrossRef][Medline]
Magnuson DS, Lovett R, Coffee C, Gray R, Han Y, Zhang YP & Burke DA (2005). Functional consequences of lumbar spinal cord contusion injuries in the adult rat. J Neurotrauma 22, 529–543.[CrossRef][Medline]
Magnuson DS & Trinder TC (1997). Locomotor rhythm evoked by ventrolateral funiculus stimulation in the neonatal rat spinal cord in vitro. J Neurophysiol 77, 200–206.
Matsushima T & Grillner S (1992). Neural mechanisms of intersegmental coordination in lamprey: local excitability changes modify the phase coupling along the spinal cord. J Neurophysiol 67, 373–388.
Miller WL & Sigvardt KA (2000). Extent and role of multisegmental coupling in the Lamprey spinal locomotor pattern generator. J Neurophysiol 83, 465–476.
Monteau R & Hilaire G (1991). Spinal respiratory motoneurons. Prog Neurobiol 37, 83–144.[CrossRef][Medline]
Nicolopoulos-Stournaras S & Iles JF (1983). Motor neuron columns in the lumbar spinal cord of the rat. J Comp Neurol 217, 75–85.[CrossRef][Medline]
Orlovski GN, Deliagina T & Grillner S (1999). Neural control of locomotion: from mollusc to man. Oxford University Press, Oxford, UK.
Roberts A, Soffe SR, Wolf ES, Yoshida M & Zhao FY (1998). Central circuits controlling locomotion in young frog tadpoles. Ann N Y Acad Sci 860, 19–34.
Rossignol S (1996). Neural control of stereotypic limb movements. In Handbook of Physiology, section 12, Exercise: Regulation and Integration of Multiple Systems, ed. Rowell B & Sheperd JT, pp. 173–216. American Physiological Society, Bethesda, MD, U S A.
Soffe SR (1989). Roles of glycinergic inhibition and N-methyl-D-aspartate receptor mediated excitation in the locomotor rhythmicity of one half of the Xenopus embryo central nervous system. Eur J Neurosci 1, 561–571.[CrossRef][Medline]
Soffe SR, Zhao FY & Roberts A (2001). Functional projection distances of spinal interneurons mediating reciprocal inhibition during swimming in Xenopus tadpoles. Eur J Neurosci 13, 617–627.[CrossRef][Medline]
Sqalli-Houssaini Y, Cazalets JR & Clarac F (1993). Oscillatory properties of the central pattern generator for locomotion in neonatal rats. J Neurophysiol 70, 803–813.
Sqalli-Houssaini Y, Cazalets JR, Fabre JC & Clarac F (1991). A cooling/heating system for use with in vitro preparations: study of temperature effects on newborn rat rhythmic activities. J Neurosci Methods 39, 131–139.[CrossRef][Medline]
Team RDC (2005). R: A Language and Environnement for Statistical Computing. R Foundation for Statistical Computing, Vienne, Austria.
Thorstensson A, Carlson H, Zomlefer MR & Nilsson J (1982). Lumbar back muscle activity in relation to trunk movements during locomotion in man. Acta Physiol Scand 116, 13–20.[Medline]
Tunstall MJ, Roberts A & Soffe SR (2002). Modelling inter-segmental coordination of neuronal oscillators: synaptic mechanisms for uni-directional coupling during swimming in Xenopus tadpoles. J Comput Neurosci 13, 143–158.[CrossRef][Medline]
Zar JH (1984). Biostatistical Analysis, vol. 1. Prentice Hall, Englewood Cliffs.
Zhong G, Diaz-Rios M & Harris-Warrick RM (2006). Intrinsic and functional differences among commissural interneurons during fictive locomotion and serotonergic modulation in the neonatal mouse. J Neurosci 26, 6509–6517.
Zomlefer MR, Provencher J, Blanchette G & Rossignol S (1984). Electromyographic study of lumbar back muscles during locomotion in acute high decerebrate and in low spinal cats. Brain Res 290, 249–260.[CrossRef][Medline]
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). Slopes obtained with linear regression for S2 (0.8 ± 0.1), L5 (0.7 ± 0.07) and T10 (0.3 ± 0.1). Slopes were significantly different (P < 0.01). Correlation analysis of phase versus period (
).
), the hemi-spinal cord (