| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, California
Submitted 27 June 2007; accepted in final form 19 August 2007
 |
ABSTRACT |
|---|
|
|
|
INTRODUCTION |
|---|
|
; Kiehn 2006; Roberts et al. 1998; Stein et al. 1997). During locomotor behavior, CPGs may be required to produce synchronized (in-phase) motor output between the right and left sides of the body, such as that seen in flying (locust: Wendler 1977; Wilson 1961; goose: Sholomenko et al. 1991), galloping (cat: English 1979; Miller et al. 1975), hopping (frog: Peters et al. 1996; rat-kangaroo: Webster and Dawson 2003), and fast swimming in frogs (Ostry et al. 1991; Peters et al. 1996); or to produce alternating (out-of-phase) motor output between the right and left sides, as in swimming (newt: Delvolve et al. 1997; lamprey: Cohen and Wallen 1980; Poon 1980; Xenopus embryo: Kahn et al. 1982; turtle: Field and Stein 1997; fish: Masino and Fetcho 2005) and walking (cat: Miller et al. 1975; turtle: Walker 1971; goose: Sholomenko et al. 1991; chick: Jacobson and Hollyday 1982). During locomotion, out-of-phase side-to-side coordination is carried out at least in part by spinal commissural pathways because disruption of these pathways by photoinactivation (lamprey: Buchanan and McPherson 1995), midsagittal spinal lesions (Xenopus embryo: Kahn and Roberts 1982; lamprey: Cangiano and Grillner 2003; neonatal rat: Kjaerulff and Kiehn 1996), or application of the glycine receptor antagonist strychnine (lamprey: Cohen and Harris-Warrick 1984; Hagevik and McClellan 1994; McPherson et al. 1994; mudpuppy: Jovanovic et al. 1999; neonatal rat: Juvin et al. 2005) disrupts right–left alternation. However, other studies indicate that descending propriospinal pathways can play an important role in maintaining proper right–left alternation of locomotor output under some conditions (cat: Kato 1988; Xenopus embryo: Soffe and Roberts 1982; lamprey: Jackson et al. 2005; neonatal rat: Cowley and Schmidt 1997; Kremer and Lev-Tov 1997).
Although most previous work assessed the role of commissural pathways in side-to-side alternation during electrically or chemically evoked motor patterns in reduced preparations, our study investigates coordinating mechanisms in the turtle spinal cord during voluntary locomotion in otherwise intact animals. Voluntary forward swimming in freshwater turtles typically consists of 1:1 out-of-phase movement of contralateral limbs (right–left forelimbs: RF–LF; and right–left hindlimbs: RH–LH) and of ipsilateral limbs (RF–RH and LF–LH), and 1:1 nearly in-phase movement of diagonal limbs (RF–LH and LF–RH) (Davenport et al. 1984; Field and Stein 1997; Gillis and Blob 2001; Stein 1978; Zug 1971), similar to the interlimb coordination seen during trot in quadruped mammals (Hildebrand 1989). During the normal hindlimb forward swim motor pattern, hip extensor electromyographic discharge alternates with ipsilateral hip flexor activity and contralateral hip extensor activity (Earhart and Stein 2000; Stein 2005). To assess the necessity of commissural axons in the hindlimb enlargement (spinal segments D8–S2) or the hindlimb enlargement plus the first preenlargement segment (D7–S2) for maintaining right–left hindlimb alternation during voluntary forward swimming, we measured interlimb phase before and after bisection of these segments. Preliminary results have been published in abstract form (Samara and Currie 2005).
|
|
METHODS |
|---|
|
Red-eared turtles, Trachemys scripta elegans (n = 9), with plastron lengths of 12–16 cm, were placed in crushed ice for at least 2 h before surgery to induce hypothermic anesthesia (Lennard and Stein 1977). Turtles remained partially submerged in ice for all surgical procedures. The hindlimb-region spinal cord was exposed from segments D6–S2 by dorsal laminectomy and transected at the posterior end of S2. In six turtles, bilateral dorsal rhyzotomies were performed to deafferent segments D8–S2 (n = 1) or D7–S2 (n = 5) at the start of the experiment, before prebisection behavioral testing. Three turtles were not deafferented before prebisection testing because this procedure tended to decrease swimming activity. One of the turtles in this group was deafferented after the D7–S2 cord bisection. All cord exposures were covered with dental utility wax (Heraeus Kulzer, South Bend, IN), glued to the surrounding shell with Permabond adhesive.
Movement and EMG recording
We videotaped turtle swimming from below by a horizontal digital video camera (Canon Optura 20 mini-DV), aimed at a 45° mirror beneath the clear Plexiglas bottom of the swim tank [Fig. 1A; tank measurements: 40 x 35 x 16 cm (length x width x height)]. Brightly colored markers (3-mm plastic beads) were sutured to the skin at the ventral–medial side of the wrists (h and i in Fig. 1B) and the ventral–anterior side of the knees (fand g) to track movement of the limbs relative to fixed reference points that were painted on the ventral plastron with white Liquid Paper correction fluid (a–e). Reference points a and c in Fig. 1B marked the rostral and caudal ends of the ventral midline; b marked the rostrocaudal level of the anterior border of the lateral shell bridge and an approximate pivot point of the pectoral girdle; d and e marked the positions of the right and left hip joints, respectively (note that images were inverted in the mirror). Video recordings were obtained with a 30-Hz frame rate and 1/250-s shutter speed, and were reviewed and annotated with a Sony GV-1000 Video Walkman. Selected swim episodes were then viewed frame by frame on a laboratory PC so that marker locations on the limbs and plastron could be manually digitized with Motus 8 software (Peak Performance Technologies, Englewood, CO). The same software was used to calculate limb movement amplitudes and interlimb phase values. We defined cycle onsets and cycle offsets in hip and forelimb movement recordings as the minimum (peak flexed) and maximum (peak extended) limb angles, respectively (Field and Stein 1997; Stein 1978). Hip angle was defined as the angle between the thigh line (d–f on the right or e–g on the left in Fig. 1B) and a line parallel to the ventral midline (a–c) with its origin at the hip joint (dashed line in Fig. 1B). The hip joint (acetabulum) is relatively fixed in the horizontal plane, even during the large pelvic girdle rotations that accompany vigorous locomotion (Walker 1971; PSG Stein, RF Samara, and SN Currie, unpublished observations). Forelimb angle was defined as 
abh on the right and abi on the left, and was measured relative to a stationary point on the ventral midline (b; see earlier text); this point corresponded to the rostrocaudal level of right and left pivot points on the pectoral girdle, where the scapular prongs articulate with the dorsal carapace (Walker 1971). We used this arbitrarily defined "forelimb angle" rather than "shoulder angle" because the shoulder joint (glenoid cavity) is not fixed during turtle locomotion, but moves through an arc between anterolateral and lateral positions as the pectoral girdle rotates (Walker 1971). Without X-ray cinematography, we were thus unable to estimate the position of the shoulder joint in video images. Head angle was defined as abj, where an angle of 0° indicated that the head was pointed straight forward and positive or negative angles indicated that the head was turned to the right or left, respectively.
|
; Walker 1973), using previously described techniques (Earhart and Stein 2000). EMGs were amplified, filtered (30–1,000 Hz), digitized (PCM Model 3000A, Vetter), and stored on VHS videotape with a voice channel and synchronization marker (see following text). Swim episodes with EMGs were used only if they exhibited distinct bursts of muscle activity followed by periods of quiescence for a minimum of four consecutive cycles. Episodes chosen for figures were formatted with Datapac 2K2 (Run Technologies, Laguna Hills, CA) and CorelDraw (Corel, Ottawa, Canada) software. A remote video synchronization unit (Peak Performance Technologies) was used to synchronize EMG recordings with kinematic video data. When manually activated, the synchronization unit produced a voltage deflection on an unused channel of the EMG record and a simultaneous sound pulse on the microphone input of the digital video camera. The kinematic sequence containing the onset of the sound pulse was marked and synchronized with the corresponding EMG recording in CorelDraw.
Bisections
We longitudinally bisected the dorsally exposed spinal cord from segments D7–S2 or D8–S2 (Fig. 2). Bisections were performed by first perforating the pia mater with fine iridectomy scissors at the anterior side of the exposure, then cutting posteriorly down the midline along the visible midsagittal sulcus. A #11 scalpel was then passed down this initial scissor cut until the right and left halves of the cord were completely separated over the specified region.
|
At the end of each experiment, turtles were reanesthetized in crushed ice and the D6–S2 cord was removed and placed in 4% paraformaldehyde for approximately 1 wk. After cord removal, turtles were killed by freezing. Cords were sectioned in the transverse plane (80 µm), mounted, and allowed to dry overnight. The next day, sections were stained with 0.1% cresyl echt violet (CellPoint Scientific, Rockville, MD) and coverslipped. Several sections were mounted from each of the seven exposed spinal segments, to verify the longitudinal extent, lateral position, and completeness of the D7–S2 or D8–S2 bisection (Fig. 2). Selected sections were photographed with a Nikon Microphot FX-A microscope using bright-field illumination.
Data analysis
The same 25 forward swim cycles were analyzed for both phase and amplitude parameters. Selected digitized episodes, in the form of time-varying angle measurements, were exported from Motus 8 as ASCII files and imported into Datapac 2K2 for further analyses. Because turtles restrained by band clamp rarely swim for extended periods, particularly after spinal surgery, we accepted swim episodes consisting of four or more cycles. The first cycle and last cycle were never quantitatively analyzed because their amplitudes were rarely representative of the entire swim episode.
Dual-referent interlimb phase values were calculated with Datapac 2K2 because dual-referent analysis is appropriate for cyclical behaviors that exhibit variable duty cycles (Berkowitz and Stein 1994; Field and Stein 1997). For this purpose, one limb was selected as "referent" (RF or RH) and the other as "target." The onsets of referent-limb extension were defined by phase values of 0.0 and 1.0. The offsets of referent extension (onsets of referent flexion) were defined by a phase value of 0.5 (for a more detailed description of phase calculations see Field and Stein 1997). To rule out the possibility of phase drift, we analyzed phase as a function of time over the course of many swim episodes (data not shown). Interlimb phase values were always similar between the early and late swim cycles for any given episode; there was no evidence of drift. Circular statistics (Batschelet 1981; Mardia and Jupp 2000; Zar 1999) and plots were calculated for phase data using Oriana 2.0 software (Kovach Computing Services, Anglesey, Wales, UK). The Rayleigh test used vector length to determine whether phase distributions were significantly clustered around the mean (nonuniform). In addition to obtaining these statistics for individual turtles, prebisection and postbisection phase values were each pooled across experiments for turtles that received the same surgical manipulations (D7–S2 or D8–S2 cord bisection, afferented or deafferented). Mean phase and circular SD were calculated for these pooled data (Table 1). The Watson–Williams test was used to determine whether postbisection mean phase values were significantly different from prebisection means (Batschelet 1981; Zar 1999).
|
70% of the actual ratio for a given turtle, meaning that the thigh stayed within 70% of the horizontal plane throughout the swim sequence. Pre- and postbisection data were normalized to the mean prebisection amplitude for each limb then pooled together across experiments based on which surgical procedures were performed. Mean amplitude and SD were obtained for these pooled, normalized data. The Mann–Whitney U test was used to determine whether pre- and postbisection amplitudes were significantly different (Table 2).
|
|
|
RESULTS |
|---|
|
PHASE ANALYSIS.
Forward swim episodes were analyzed only if they exhibited 1) out-of-phase 1:1 forelimb movements on the right and left sides with interlimb phase values between 0.4 and 0.6; 2) a forelimb cycle period no greater than 1.5 s; and 3) a head angle no greater than ±10° off-center to the right or left, which indicated that the turtle was attempting to swim forward and not turn. Using these criteria in swimming prebisection turtles, contralateral hindlimbs (LH–RH) always displayed strong out-of-phase coordination with mean phase values close to 0.5 (range = 0.47–0.53; Table 1). Ipsilateral forelimb–hindlimb pairs (RH–RF) were also out of phase, with mean phase values near 0.4 (range = 0.40–0.43), whereas diagonal limbs (LH–RF) were nearly in phase for every turtle, with mean phase values close to 1.0 (range = 0.89–0.95). We used circular statistics to evaluate the degree of clustering or directionality of phase measurements. The Rayleigh statistic tests the hypothesis that a circular distribution is nonuniform (i.e., significantly clustered around a mean value). For every turtle in the present study, the distribution of phase values was clustered for all four sets of measurements (LH–RH, LF–RF, RH–RF, and LH–RF; P < 0.0001). In determining the degree of clustering for a set of data points, the Rayleigh test uses the length of the mean vector (r), which ranges from 0 to 1. A vector length of 0 signifies no coupling between limbs, whereas a length of 1 signifies perfect coupling (Batschelet 1981). For each prebisection turtle, limbs were strongly coupled, with vector lengths ranging from 0.84 to 0.98 (LH–RH), 0.91–0.98 (LF–RF and LH–RF), and 0.85–0.99 (RH–RF) (Figs. 3 and 4). Bursts of EMG activity were closely correlated with motion data, with HE burst onsets occurring near the minimum hip angle (peak hip flexion) for each cycle and HF burst onsets occurring near the maximum hip angle (peak hip extension) for each cycle.
|
|
For individual prebisection turtles, mean forelimb movement amplitudes ranged between 71.0 ± 5.0 and 93.8 ± 7.6°, peak to peak. Mean hip excursions ranged from 43.5 ± 22.4 to 102.4 ± 16.6°. For purposes of comparison with postbisection data, pooled forelimb and hip amplitude data were both normalized, resulting in prebisection means of 100.0 for all four measures (right forelimb, left forelimb, right hip, left hip) (Table 2).
Swimming after D8–S2 and D7–S2 spinal cord bisections
PHASE ANALYSIS.
We tested the necessity of commissural axons in the hindlimb enlargement for out-of-phase coordination between the right and left hindlimbs during swimming by splitting the cord from segments D8–S2 (n = 5), or D7–S2 (n = 6, including three turtles that previously had the D8–S2 bisection). Table 1 shows that mean interlimb phase values after D8–S2 and D7–S2 cord bisections (including afferented and deafferented turtles) changed only little compared with prebisection controls: contralateral hindlimbs (LH–RH) still displayed strong out-of-phase coordination with mean phase values close to 0.5 (range = 0.42–0.51; Table 1). Ipsilateral forelimb–hindlimb pairs (RH–RF) still exhibited mean phase values near 0.4 (range = 0.33–0.48), and diagonal limbs (LH–RF) were still nearly in phase, with mean phase values close to 1.0 (range = 0.90–0.92). Neither D8–S2 nor D7–S2 bisections weakened the out-of-phase coordination between contralateral hindlimbs (hips), nor did these lesions affect the strength of phase coupling between other limbs, including contralateral forelimbs, or forelimb–hindlimb pairs (ipsilateral and contralateral) (Table 1; Figs. 3 and 4). Although a subset of mean phase values showed small but significant differences from prebisection controls (Watson–Williams test, P < 0.05) (Table 1), interlimb coordination remained very strong after bisection because all four phase distributions (LH–RH, LF–RF, RH–RF, and LH–RF) were significantly clustered (nonuniform) for every turtle (Rayleigh test, P < 0.0001). Strong interlimb coupling after cord bisection was also indicated by vector lengths close to 1.0 (LH–RH: 0.81–0.97; RH–RF: 0.86–0.97; LH–RF: 0.69–0.95). As stated previously (see METHODS), one turtle was deafferented after D7–S2 bisection. In this case, LH–RH coordination postbisection, predeafferentation, and postdeafferentation remained out of phase (predeafferentation: 0.48 ± 0.06; postdeafferentation: 0.43 ± 0.07) and strongly coupled (Rayleigh test, P < 0.0001). Although we wanted to confine our studies to forward swimming, turtles commonly displayed head angles greater than 10° accompanied by turning behavior, although turning was rarely vigorous enough to observe bilateral hindlimb movements postbisection. When bilateral movements were present, even in deafferented animals, we observed 1:1, qualitatively out-of-phase right–left hindlimb coordination, similar to previous observations in intact turtles (Field and Stein 1997).
In five turtles, EMGs were recorded from selected hindlimb muscles to verify that the often small-amplitude postbisection swimming movements of the hips were attributable to active muscle contractions rather than being caused by passive mechanical coupling to other moving limbs. During pre- and postbisection swimming, EMG and motion analysis data were qualitatively correlated, with HE onsets occurring near the minimum hip angle for each cycle and HF onsets occurring near the maximum hip angle for each cycle (Fig. 4). Hip extensor EMG bursts on the right and left sides continued to exhibit strict alternation after D7–S2 cord bisection (Figs. 3 and 4).
We also tested the possibility that phasic sensory inputs, resulting from weak passive movement, were responsible for maintaining postbisection coordination of the right and left hindlimbs by triggering reflex muscle contractions within the moving limbs. Spinal segments D8–S2 (n = 1) or D7–S2 (n = 5; see METHODS) were deafferented by bilateral dorsal rhizotomy. Deafferentation did not eliminate or reduce the strongly coupled out-of-phase coordination between right and left hindlimbs (LH–RH), nor did it affect the coupling between other limb pairs (Table 1; Fig. 4). This indicated that postbisection interlimb coordination was maintained by CNS synaptic connections alone.
AMPLITUDE ANALYSIS.
Although splitting the cord from D8–S2 or D7–S2 had surprisingly little effect on right–left hindlimb coordination, these lesions did significantly reduce the amplitude of hip movements and hip extensor EMG bursts on both sides (Mann–Whitney U test, P < 0.05) (Table 2, Fig. 3). D7–S2 bisections produced much greater deficits than D8–S2 bisections in hip movement amplitudes. Furthermore, if a turtle had previously received a D8–S2 bisection, extending the bisection anteriorly one more segment, through D7, always reduced the mean hip amplitude further (n = 3). These effects of cord bisection were more pronounced in deafferented turtles (D7–S2 dorsal roots cut before obtaining prebisection control data) than in afferented turtles (D7–S2 dorsal roots intact). Hindlimb movement deficits were not the result of a general postsurgical sluggishness (which should affect hindlimbs and forelimbs similarly) because hindlimb amplitudes decreased significantly more than right or left forelimb amplitudes (Mann–Whitney U test, P < 0.001) in all but one animal (afferented D8–S2 bisection, in which RF vs. LH, LF vs. RH, and LF vs. LH were all not significantly different; P > 0.05). Because we assessed only carapace-restrained animals, it is unclear whether the deficits we observed in hindlimb movement amplitude would have prevented effective swimming in freely behaving (unrestrained) turtles.
|
|
DISCUSSION |
|---|
|
Commissural pathways in turtle hindlimb enlargement can mediate right–left coordination in some circumstances
There is ample evidence that commissural pathways in the turtle spinal cord can contribute to right–left hindlimb coordination under certain conditions. Many studies with low-spinal immobilized turtles have observed coordinated right–left alternation in hindlimb motor output during unilaterally and bilaterally evoked fictive scratching (Currie and Gonsalves 1997, 1999; Currie and Lee 1997; Currie and Stein 1989; Stein et al. 1995, 1998). Crossed inhibitory and excitatory effects were also observed in low-spinal immobilized turtles during fictive flexion reflex (Currie and Lee 1996) and during interactions between fictive flexion reflex and contralateral scratch motor patterns (Currie and Stein 1989). Because these turtles were low-spinal, with no possible contribution from the disconnected forelimb enlargement, commissural axons within the hindlimb enlargement and/or preenlargement segments must have mediated the crossed motor effects and coordination. Additional experiments showed out-of-phase coupling of right and left side rhythmic motor discharge elicited by bath-applied 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) in isolated preparations of the D7–D8 turtle spinal cord (Currie 1999) and scratchlike fictive motor patterns evoked by cutaneous nerve stimulation in isolated D8 preparations (Currie and Lee 1996), thus demonstrating functional crossed coordinating mechanisms in these segments. Intracellular recording, combined with Neurobiotin injections, has been used successfully to identify subsets of spinal interneurons in the turtle hindlimb enlargement that possess crossed axons within the enlargement and are active during one or more forms of fictive scratch and flexion reflex (Berkowitz 2005; Berkowitz et al. 2006). Such cells may contribute to right–left coupling.
Of course, extensive evidence from other vertebrate preparations shows that commissural pathways play important roles in maintaining right–left phase coupling, although the data come largely from isolated spinal cord or semiintact preparations in which locomotor activity was artificially activated. Midsagittal sectioning of isolated neonatal rat spinal cords from segments L3–L6 or Th12–L2 greatly reduced the right–left coupling of 5-HT/NMDA-induced lumbar ventral root bursts, even when these lesions were limited to the ventral commissure (Kjaerulff and Kiehn 1996). In lampreys, various lesion studies have suggested essential roles for commissural pathways in right–left alternation by using midline bisections of isolated-cord or semiintact-immobilized preparations (see Fig. 5 in Cangiano and Grillner 2003; see Fig. 6 in Jackson et al. 2005), midline bisections of moving preparations (see Fig. 4 in Jackson et al. 2005), photoablation of commissural interneurons (Buchanan and McPherson 1995), or strychnine blockade of glycinergic synapses, which converted right–left motor output from alternating to synchronized (Cohen and Harris-Warrick 1984; Hagevik and McClellan 1994).
Longitudinal propriospinal pathways mediate forelimb–hindlimb coordination
Past in vivo experiments suggested that turtle forelimb–hindlimb coordination was maintained at least in part by propriospinal pathways. The fact that high-spinal turtles produce coordinated forelimb–hindlimb motor output in response to unpatterned electrical stimulation of the cervical dorsolateral funiculus (DLF) (Stein 1978) showed that forelimb–hindlimb coordination did not depend on supraspinal input. Studies involving electrical brain stem stimulation in decerebrate-immobilized turtles (Currie 2003) indicated that sensory input was also not required for proper forelimb–hindlimb coordination during fictive swimming. Alternating activity in the forelimb enlargement may entrain alternating hindlimb activity by descending propriospinal axons. This is supported by recent mammalian studies. For example, Akay et al. (2006) showed in decerebrate cats walking on a laterally split treadmill (separate treadmills for the forelimbs and hindlimbs) that lowering forelimb treadmill speed reduced the step frequencies of both forelimbs and hindlimbs, whereas lowering hindlimb treadmill speed affected only the hindlimb step frequency. This suggested that interlimb coordination was predominantly maintained by a descending inhibitory pathway. At present, we cannot say whether the propriospinal pathways that coordinate forelimb and hindlimb movements in turtles are crossed (coupling diagonal limbs), uncrossed (coupling ipsilateral limbs), or both; however, work by Orsal et al. (1990) indicated that forelimb and hindlimb networks interact primarily by crossed coupling between diagonal limbs during treadmill stepping in thalamic cats.
Longitudinal propriospinal pathways can mediate right–left coordination
Although most studies examining the role of commissural pathways in producing rhythmic, coordinated output used pharmacological or electrical stimulation of locomotor-like activity in surgically reduced preparations, our experiments assessed the role of commissural pathways in the voluntary locomotion of largely intact animals. In reduced preparations, it is possible that some descending pathways, which may be sufficient to maintain right–left alternation, are eliminated. As far as we can determine, the only other in vivo study indicating that longitudinal propriospinal tracts can contribute to right–left hindlimb coordination was conducted in cats, where midsagittal bisections of the lumbar cord did not disrupt right–left hindlimb alternation during voluntary walking in otherwise intact animals (Kato 1988).
Various in vitro preparations also provide evidence that spinal commissural pathways are not necessary for right–left alternation. The thoracolumbar cord of neonatal rat pups produced alternating bilateral ventral root output during 5-HT or 5-HT/NMDA-evoked fictive locomotion, even after midsagittal cord bisections from the thoracolumbar junction through the lumbar enlargement. This implied that descending supralumbar input was sufficient to maintain side-to-side alternation of the lumbar ventral roots (Cowley and Schmidt 1997; Kremer and Lev-Tov 1997). Similar experiments in larval lampreys, using both whole animal and in vitro brain–spinal cord preparations, showed that descending drive from brain locomotor centers and intact, rhythmically active regions of the rostral spinal cord, could drive rhythmic locomotor output from both sides of the midsagittally split caudal spinal cord (right and left caudal hemicords) (Jackson et al. 2005). In turtles, Kusuma and ten Donkelaar (1980) histologically traced the cell bodies and axon terminals of long propriospinal axons that interconnected the limb enlargements, suggesting that these fibers contributed to interlimb coordination. A subset of long descending propriospinal axons that traveled in the ventral and lateral funiculi at midbody originated from cell bodies in the ventral gray matter of the forelimb enlargement and terminated in the ventral and intermediate gray of the hindlimb enlargement. Evidence from our laboratory (Samara and Currie 2004; unpublished observations) showed that unilateral lesions of this ventrolateral white matter in the interenlargement region of the turtle spinal cord disrupted both ipsilateral forelimb–hindlimb coordination and hindlimb–hindlimb coordination during voluntary swim behavior. The effect on hindlimb–hindlimb coordination may have been due to the disrupted balance of descending propriospinal drive to the right and left hindlimb circuitry.
Significance of reduced hindlimb (hip) swim amplitudes after bisection of the D7 segment
In addition to indicating that descending propriospinal drive alone can maintain right–left hindlimb coordination, our results may provide insight into how descending pathways that excite spinal locomotor CPGs ("locomotor command pathways") are organized in turtles to activate contralateral hindlimb movements. As shown in Table 2, D7–S2 bisections decreased hindlimb swim amplitudes much more than D8–S2 bisections. One possible explanation is that bisection of the D7 segment cuts a major fraction of the descending locomotor command where it crosses the midline anterior to the D8–S2 hindlimb enlargement. This is consistent with previous evidence showing that electrical stimulation of the right D3 DLF in low-spinal turtles with movement continued to evoke swimlike movements in the left hindlimb after surgical removal of the right side of the segments D8–S2 (Samara and Currie 2006). Another spinal lesion study in low-spinal immobilized turtles suggested that these command pathways cross the midline posterior to the D5 spinal segment (Currie 2000). Thus it is likely that the crossed locomotor command tracts that have been demonstrated in several turtle preparations (decerebrate with movement: Kazzenikov et al. 1980; decerebrate immobilized: Currie 2003; high-spinal with movement: Stein 1978; low-spinal with movement: Lennard and Stein 1977; low-spinal immobilized: Juranek and Currie 2000; voluntarily swimming intact: Samara and Currie 2004) activate contralateral hindlimb locomotion by a pathway that crosses the midline largely within segments D7 and/or D6, just anterior to the hindlimb enlargement. Further experiments are required to identify the cells of origin and characterize the timing and spatial distribution of descending axonal signals that turn on and modulate turtle locomotion.
Can descending propriospinal drive originating in the forelimb enlargement directly activate hindlimb motor output?
The low-amplitude hindlimb movements and hip EMG bursts that continued after we split the lumbosacral cord from D7–S2 may have been activated 1) by surviving command fibers (uncrossed or crossing anterior to D7) that were left intact by the spinal bisection, 2) by the same descending propriospinal signals from the forelimb enlargement that also coordinated forelimb and hindlimb movements, or 3) a combination of both. What is the evidence that interenlargement propriospinal activity by itself can directly drive motor output? In isolated and partitioned neonatal rat spinal cords, descending 5-HT or 5-HT/NMDA-induced drive originating in the cervical enlargement was demonstrated to be sufficient to induce fictive locomotor output from the hindlimb enlargement (Ballion et al. 2001). In our future experiments, similar studies with in vitro turtle spinal cords that include both limb enlargements could determine the extent to which localized, chemically activated locomotor activity in the forelimb enlargement can directly drive hindlimb motor discharge.
|
|
GRANTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. N. Currie, Department of Neuroscience, University of California, Riverside, CA 92521 (E-mail: currie{at}mail.ucr.edu)
|
|
REFERENCES |
|---|
|
Ballion B, Morin D, Viala D. Forelimb locomotor generators and quadrupedal locomotion in the neonatal rat. Eur J Neurosci 14: 1727–1738, 2001.[CrossRef][Web of Science][Medline]
Batschelet E. Circular Statistics in Biology. London: Academic Press, 1981.
Berkowitz A. Physiology and morphology indicate that individual spinal interneurons contribute to diverse limb movements. J Neurophysiol 94: 4455–4470, 2005.
Berkowitz A, Stein PSG. Activity of descending propriospinal axons in the turtle hindlimb enlargement during two forms of fictive scratching: phase analyses. J Neurosci 14: 5105–5119, 1994.[Abstract]
Berkowitz A, Yosten GLC, Ballard MR. Somato-dendritic morphology predicts physiology for neurons that contribute to several kinds of limb movements. J Neurophysiol 95: 2821–2831, 2006.
Buchanan JT, McPherson DR. The neuronal network for locomotion in the lamprey spinal cord: evidence for the involvement of commissural interneurons. J Physiol (Paris) 89: 221–233, 1995.[CrossRef][Web of Science][Medline]
Butt SJ, Lebret JM, Kiehn O. Organization of left–right coordination in the mammalian locomotor network. Brain Res Rev 40: 107–117, 2002.[CrossRef][Medline]
Cangiano L, Grillner S. Fast and slow locomotor burst generation in the hemispinal cord of the lamprey. J Neurophysiol 89: 2931–2942, 2003.
Cohen AH, Harris-Warrick RM. Strychnine eliminates alternating motor output during fictive locomotion in the lamprey. Brain Res 293: 164–167, 1984.[CrossRef][Web of Science][Medline]
Cowley KC, Schmidt BJ. Regional distribution of the locomotor pattern-generating network in the neonatal rat spinal cord. J Neurophysiol 77: 247–259, 1997.
Currie SN. Fictive hindlimb motor patterns evoked by AMPA and NMDA in turtle spinal cord-hindlimb nerve preparations. J Physiol (Paris) 93: 199–211, 1999.[CrossRef][Web of Science][Medline]
Currie SN. Descending pathways that activate fictive swim motor patterns in the turtle spinal cord. Soc Neurosci Abstr 26: 746.1, 2000.
Currie SN. Fictive locomotion evoked by electrical stimulation of the brainstem in decerebrate immobilized turtles. Soc Neurosci Abstr 29: 276.9, 2003.
Currie SN, Gonsalves GG. Right–left interactions between rostral scratch networks generate rhythmicity in the preenlargement spinal cord of the turtle. J Neurophysiol 78: 3479–3483, 1997.
Currie SN, Gonsalves GG. Reciprocal interactions in the turtle hindlimb enlargement contribute to scratch rhythmogenesis. J Neurophysiol 81: 2977–2987, 1999.
Currie SN, Lee S. Glycinergic inhibition in the turtle spinal cord regulates the intensity and pattern of fictive flexion reflex motor output. Neurosci Lett 205: 75–78, 1996.[CrossRef][Web of Science][Medline]
Currie SN, Lee S. Glycinergic inhibition contributes to the generation of rostral scratch motor patterns in the turtle spinal cord. J Neurosci 17: 3322–3333, 1997.
Currie SN, Stein PSG. Interruptions of fictive scratch motor rhythms by activation of cutaneous flexion reflex afferents in the turtle. J Neurosci 9: 488–496, 1989.[Abstract]
Davenport J, Munks SA, Oxford PJ. A comparison of swimming in marine and freshwater turtles. Proc R Soc Lond B Biol Sci 220: 447–475, 1984.
Delvolve I, Bem T, Cabelguen JM. Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt Pleurodeles waltl. J Neurophysiol 78: 638–650, 1997.
Earhart GM, Stein PSG. Step, swim, and scratch motor patterns in the turtle. J Neurophysiol 84: 2181–2190, 2000.
English AW. Interlimb coordination during stepping in the cat: an electromyographic analysis. J Neurophysiol 42: 229–243, 1979.
Field EC, Stein PSG. Spinal cord coordination of hindlimb movements in the turtle: interlimb temporal relationships during bilateral scratching and swimming. J Neurophysiol 78: 1404–1413, 1997.
Gillis GB, Blob RW. How muscles accommodate movement in different physical environments: aquatic vs. terrestrial locomotion in vertebrates. Comp Biochem Physiol A Physiol 131: 61–75, 2001.[CrossRef][Medline]
Grillner S. Control of locomotion in bipeds, tetrapods, and fish. In: Handbook of Physiology. The Nervous System. Motor Control. Bethesda, MD: Am. Physiol. Soc., 1981, sect. 1, vol. II, pt. 2, p. 1179–1236.
Hagevik A, McClellan AD. Coupling of spinal locomotor networks in larval lamprey revealed by receptor blockers for inhibitory amino acids: neurophysiology and computer modeling. J Neurophysiol 72: 1810–1829, 1994.
Hildebrand M. The quadrupedal gaits of vertebrates. Bioscience 39: 766–775, 1989.[CrossRef][Web of Science]
Jackson AW, Horinek DF, Boyd MR, McClellan AD. Disruption of left–right reciprocal coupling in the spinal cord of larval lamprey abolishes brain-initiated locomotor activity. J Neurophysiol 94: 2031–2044, 2005.
Jacobson RD, Hollyday M. Electrically evoked walking and fictive locomotion in the chick. J Neurophysiol 48: 257–270, 1982.
Jovanovic K, Petrov T, Stein RB. Effects of inhibitory neurotransmitters on the mudpuppy (Necturus maculatus) locomotor pattern in vitro. Exp Brain Res 129: 172–184, 1999.[CrossRef][Web of Science][Medline]
Juranek J, Currie SN. Electrically evoked fictive swimming in the low-spinal immobilized turtle. J Neurophysiol 83: 146–155, 2000.
Juvin L, Simmers J, Morin D. Propriospinal circuitry underlying interlimb coordination in mammalian quadrupedal locomotion. J Neurosci 25: 6025–6035, 2005.
Kahn JA, Roberts A. Experiments on the central pattern generator for swimming in amphibian embryos. Philos Trans R Soc Lond B Biol Sci 296: 229–243, 1982.[CrossRef][Web of Science][Medline]
Kahn JA, Roberts A, Kashin SM. The neuromuscular basis of swimming movements in embryos of the amphibian Xenopus laevis. J Exp Biol 99: 175–184, 1982.
Kato M. Longitudinal myelotomy of lumbar spinal cord has little effect on coordinated locomotor activities of bilateral hindlimbs of the chronic cats. Neurosci Lett 93: 259–263, 1988.[CrossRef][Web of Science][Medline]
Kazzenikov OV, Selionov VA, Shik ML, Iakovleva GV. Rhombencephalic "locomotor area" of turtles (in Russian). Neirofiziologiia 12: 382–390, 1980.[Medline]
Kiehn O. Locomotor circuits in the mammalian spinal cord. Annu Rev Neurosci 29: 279–306, 2006.[CrossRef][Web of Science][Medline]
Kjaerulff O, Kiehn O. Distribution of networks generating and coordinating locomotor activity in the neonatal rat spinal cord in vitro: a lesion study. J Neurosci 16: 5777–5794, 1996.
Kremer E, Lev-Tov A. 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, 1997.
Kusuma A, ten Donkelaar HJ. Propriospinal fibers interconnecting the spinal enlargements in some quadrupedal reptiles. J Comp Neurol 193: 871–891, 1980.[CrossRef][Web of Science][Medline]
Lennard PR, Stein PS. Swimming movements elicited by electrical stimulation of turtle spinal cord. I. Low-spinal and intact preparations. J Neurophysiol 40: 768–778, 1977.
Mardia KV, Jupp PE. Directional Statistics (2nd ed.). New York: Wiley, 2000.
Masino MA, Fetcho JR. Fictive swimming motor patterns in wild type and mutant larval zebrafish. J Neurophysiol 93: 3177–3188, 2005.
McPherson DR, Buchanan JT, Kasicki S. Effects of strychnine of fictive swimming in the lamprey: evidence for glycinergic inhibition, discrepancies with model predictions, and novel modulatory rhythms. J Comp Physiol A Sens Neural Behav Physiol 175: 311–321, 1994.[Medline]
Miller S, Vanderburg J, van der Meche FGA. Locomotion in cat: basic programs of movement. Brain Res 91: 239–253, 1975.[CrossRef][Web of Science][Medline]
Mortin LI, Stein PSG. Spinal cord segments containing key elements of the central pattern generators for three forms of scratch reflex in the turtle. J Neurosci 9: 2285–2296, 1989.[Abstract]
Orsal D, Cabelguen JM, Perret C. Interlimb coordination during fictive locomotion in the thalamic cat. Exp Brain Res 82: 536–546, 1990.[Web of Science][Medline]
Ostry DJ, Feldman AG, Flanagan JR. Kinematics and control of frog hindlimb movements. J Neurophysiol 65: 547–562, 1991.
Peters SE, Kamel LT, Bashor DP. Hopping and swimming in the leopard frog, Rana pipiens: I. Step cycles and kinematics. J Morphol 230: 1–16, 1996.[CrossRef][Web of Science][Medline]
Roberts A, Soffe SR, Wolf ES, Yoshida M, Zhao FY. Central circuits controlling locomotion in young frog tadpoles. Ann NY Acad Sci 860: 19–34, 1998.[CrossRef][Web of Science][Medline]
Robertson GA, Mortin LI, Keifer J, Stein PSG. Three forms of the scratch reflex in the spinal turtle: central generation and motor patterns. J Neurophysiol 53: 1517–1534, 1985.
Samara RF, Currie SN. Impairment of voluntary locomotion in turtles following partial lesions of the mid-body spinal cord. Soc Neurosci Abstr 882.12, 2004.
Samara RF, Currie SN. Crossed commissural pathways in the spinal hindlimb enlargement are not necessary for right–left hindlimb alternation during turtle swimming. Soc Neurosci Abstr 630.18, 2005.
Samara RF, Currie SN. Electrical activation of hindlimb locomotor activity in the turtle spinal cord hemi-enlargement preparation. Soc Neurosci Abstr 32: 448.27, 2006.
Sholomenko GN, Funk GD, Steeves JD. Locomotor activities in the decerebrate bird without phasic afferent input. Neuroscience 40: 257–266, 1991.[CrossRef][Web of Science][Medline]
Soffe SR, Roberts A. Tonic and phasic synaptic input to spinal cord motoneurons during fictive locomotion if frog embryos. J Neurophysiol 48: 1279–1288, 1982.
Stein PSG. Swimming movements elicited by electrical stimulation of the turtle spinal cord: the high spinal preparation. J Comp Physiol A Sens Neural Behav Physiol 124: 203–210, 1978.[CrossRef]
Stein PSG. Neuronal control of turtle hindlimb motor rhythms. J Comp Physiol A Sens Neural Behav Physiol 191: 213–229, 2005.[CrossRef][Web of Science][Medline]
Stein PSG, Grillner S, Selverston AI, Stuart DG. Neurons, Networks, and Motor Behavior. Cambridge, MA: MIT Press, 1997.
Stein PSG, McCullough ML. Example of 2:1 interlimb coordination during fictive rostral scratching in a spinal turtle. J Neurophysiol 79: 1132–1134, 1998.
Stein PSG, McCullough ML, Currie SN. Spinal motor patterns in the turtle. Ann NY Acad Sci 860: 142–154, 1998a.[CrossRef][Web of Science][Medline]
Stein PSG, McCullough ML, Currie SN. Reconstruction of flexor/extensor alternation during fictive rostral scratching by two-site stimulation in the spinal turtle with a transverse spinal hemisection. J Neurosci 18: 467–479, 1998b.
Stein PSG, Victor JC, Field EC, Currie SN. Bilateral control of hindlimb scratching in the spinal turtle: contralateral spinal circuitry contributes to the normal ipsilateral motor pattern of fictive rostral scratching. J Neurosci 15: 4343–4355, 1995.[Abstract]
Walker WF. A structural and functional analysis of walking in the turtle, Chrysemys picta marginata. J Morphol 134: 195–214, 1971.[CrossRef][Web of Science][Medline]
Walker WF Jr. The locomotor apparatus of Testudines. In: Biology of the Reptilia, edited by Gans C. New York: Academic Press, 1973, vol. 4, p. 1–100.
Webster KN, Dawson TJ. Locomotion energetics and gait characteristics of a rat-kangaroo, Bettongia penicillata, have some kangaroo-like features. J Comp Physiol B Biochem Syst Environ Physiol 173: 549–557, 2003.[CrossRef][Medline]
Zar JH. Biostatistical Analysis (4th ed.). Upper Saddle River, NJ: Prentice Hall, 1999.
Zug GR. Buoyancy, locomotion, morphology of the pelvic girdle and hindlimbs, and systematics of cryptodiran turtles. Misc Publ Mus Zool Univ Mich 142: 1–98, 1971.
This article has been cited by other articles:
|
|
 |
|
  A. Borgmann, S. L. Hooper, and A. Buschges Sensory Feedback Induced by Front-Leg Stepping Entrains the Activity of Central Pattern Generators in Caudal Segments of the Stick Insect Walking System J. Neurosci., March 4, 2009; 29(9): 2972 - 2983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. F. Samara and S. N. Currie Location of Spinal Cord Pathways That Control Hindlimb Movement Amplitude and Interlimb Coordination During Voluntary Swimming in Turtles J Neurophysiol, April 1, 2008; 99(4): 1953 - 1968. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
