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INTRODUCTION |
All spinal motor systems require longitudinal coordination. This is obvious in animals like fish, where the neural systems that drive rhythmic contraction of the trunk muscles during locomotion are distributed along the cord. In the lamprey, the capacity to generate swimminglike rhythms has been demonstrated in isolated sections of spinal cord from along its whole length (Grillner et al. 1982
). This potential for widespread independent rhythmicity must be coordinated during real behavior. In the lamprey, our understanding of longitudinal coordination is based largely on modeling the pattern-generating network as a chain of coupled oscillators (Cohen et al. 1982
; Kopell and Ermentrout 1986
, 1988
). Different forms of longitudinal coordination appropriate to different motor patterns may be produced by altering the nature of the coupling between oscillators (Grillner 1981
, 1985
). In other vertebrates, the longitudinal distribution of motor systems is less uniform. Within the lumbar cord, more rostral regions may show stronger rhythmicity than more caudal regions, or may supply their sole rhythmicity (Deliagina et al. 1983
; Gelfand et al. 1988
; Kjaerulff and Kiehn 1996
; Mortin and Stein 1989
). Longitudinal coordination is still required but may be linked to basic rhythmicity. Although the locations of some pathways involved in coordination are known (Kjaerulff and Kiehn 1996
), the mechanisms remain unclear. The relationship between mechanisms for coordination and those for rhythm generation is even less clear. Several recent studies have shown that glycinergic inhibition plays a key role in coordinating a "normal" pattern of locomotor output (Cowley and Schmidt 1995
; Ho 1997
; Noga et al. 1993
). The same studies suggest that glycinergic inhibition is not essential for a basic rhythmicity; but it is unlikely that coordination can be achieved without influencing the rhythm in some way.
Both of the current theoretical models for longitudinal coordination in the lamprey, the excitatory gradient model (Matsushima and Grillner 1990
, 1992
) and asymmetric coupling model (Sigvardt 1993
), incorporate both ascending and descending longitudinal connections. This is also true of similar models in invertebrates (crayfish: Mulloney et al. 1993
; leech: Friesen and Pearce 1993
). Descending excitatory coupling appears to be a widespread requirement. However, in the lamprey asymmetric coupling model, the "dominant" coupling is ascending. As in other preparations, the anatomic and physiological basis for coupling is poorly understood (see, e.g., Sigvardt and Williams 1996
).
We have now used a simple vertebrate system, the young Xenopus tadpole, to examine the role of ascending connections, and specifically the effects of ascending glycinergic inhibition. The longitudinal organization of the locomotor system in these animals is analogous to that of limbed vertebrates in that the distribution of rhythmicity is not uniform. Unlike the lamprey, isolated sections of the cord do not all show equal rhythm-generating capability: more caudal regions show progressively less ability to sustain a rhythm (Roberts and Alford 1986
). Descending excitatory and inhibitory connections have been described in Xenopus during swimming (Dale 1985
; Dale and Roberts 1985
; Perrins and Roberts 1995a
,b
; Soffe and Roberts 1982
). However, little is known about ascending connections (Roberts and Alford 1986
). The single class of glycinergic premotor interneurons, the commissural interneurons, has crossed-ascending as well as crossed-descending axons (Roberts 1989
; Roberts and Clarke 1982
). This means that these neurons could contribute to longitudinal coordination as well as simple reciprocal interactions between the two sides. However, nothing is known about the role of ascending inhibition in either rhythm generation or coordination.
Because the tadpoles show two different forms of rhythmic behavior, we have been able to compare the effects of ascending inhibition during distinct motor patterns. Both motor patterns can be evoked by sensory stimulation without the necessity for pharmacological excitants (Soffe 1991
, 1993
). Rhythmic swimming has short cycle periods (40-100 ms) and motor bursts (<10 ms), and activity occurs with a rostrocaudal sequence as with the swimming of fish and lamprey (Green and Soffe 1996
; Kahn and Roberts 1982a
). Rhythmic struggling has longer cycle periods (usually >100 ms) and bursts (>20 ms), and a reversed, caudorostral sequence (Kahn and Roberts 1982b
). Unlike swimming, these three parameters scale together during struggling (Green and Soffe 1996
). It therefore seemed likely that ascending inhibition might affect the basic rhythm and longitudinal coordination differently during the two patterns. Because caudal activity leads on each cycle during struggling, ascending connections might play a particularly important role. The Xenopus tadpole spinal cord is neuroanatomically relatively simple (see Roberts 1989
). We have therefore been able to use specific surgical and pharmacological lesions to show that ascending connections can strongly influence struggling, but not swimming. The effects of our manipulations can be explained in terms of disruption of crossed-ascending inhibition mediated by axons of the glycinergic commissural interneurons. The contrast in effect between swimming and struggling may result from the opposite longitudinal delays in their motor and premotor discharge, and their quite different burst durations.
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METHODS |
Stage 37/38 (Nieuwkoop and Faber 1956
) Xenopus laevis tadpoles were obtained by induced pairing. For electrophysiological recording, tadpoles were anesthetized in MS222 (tricaine, Sigma, 0.5 mg/ml) and their dorsal fins slit to allow access of the neuromuscular blocker
-bungarotoxin. After recovery from anesthesia, the tadpoles were immobilized in 1 ml of
-bungarotoxin (Sigma, 77 µg/ml) until they no longer swam to normally effective stimuli (typically ~10-20 min). The tadpoles were then pinned through the notochord to a rotatable silicone elastomer (Sylgard) platform in a 1.5-ml bath, using etched tungsten pins, and continuously perfused with saline of composition (in mM) 115 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 2.4 NaHCO3, and 10 N-[2-hydroxyethyl]piperazene-N'-[2-ethanesulfonic acid] (HEPES), at pH 7.4. Extracellular recordings were made by means of glass suction pipettes (30-40 µm diam) applied to the intermyotome clefts (cf. Kahn and Roberts 1982a
). Tadpoles were stimulated electrically using a glass suction pipette (60 µm diam) applied to the rostral trunk skin. Pulse widths of ~0.5 ms were used, up to a maximum current of ~50 µA. Single pulses were used to evoke swimming; 30-40 pulses at a frequency of 33 Hz were applied to evoke struggling (cf. Soffe 1991
).
Lesions and pharmacological manipulations
Lesions of the spinal cord were performed using fine etched tungsten pins. In experiments involving lesions to the spinal cord, recordings of two or three control episodes of fictive locomotion were made from the ventral roots of each intact tadpole before performing the lesion. The ventral root recording electrodes and the stimulating electrode were then removed, taking care not to damage the tissue. Following the lesion, the tadpole was allowed to recover from the operation for
20 min before both recording and stimulating electrodes were replaced, and further recordings of fictive locomotion were made. Where possible, both recording and stimulating electrodes were replaced in their original positions or, failing this, at adjacent segments. In experiments involving pharmacological block of caudal neural activity, the spinal cord was first exposed by removing the dorsal margins of the myotomes, to improve access to pharmacological agents. Either MS222 (1 mg/ml) or tetrodotoxin (TTX, 0.5 µM) was perfused over the caudal spinal cord using a perfusion jet (tip diameter ~100 µm) until ventral root activity in the caudal spinal cord was abolished (typically 30-60 s). The level of each application was taken as the rostrocaudal position along the body of the tadpole at which the tip of the perfusion jet was placed. To minimize diffusion of the drugs in the rostral direction, the barrel of the perfusion jet was aimed in a caudal direction along the tadpole, and the direction of flow of the perfusing saline in the bath was also rostrocaudal, carrying the perfused drugs caudally over the spinal cord. The flow from the perfusion jet was visualized using the dye Fast Green (cf. Tunstall and Roberts 1991
). Global applications of strychnine were performed by changing the saline perfusing the recording bath to one of identical composition, but containing 10-100 nM strychnine.
Measurements and statistics
Longitudinal positions of recording sites, lesions, and pharmacological applications were measured relative to the snout. These measurements were converted into equivalent postotic segmental levels for graphic purposes (see Fig. 1). Segmental positions quoted in the text are all derived from absolute measurements. Extracellular recordings were stored and analyzed using the Digitimer Digistore system. Measurements were taken of cycle period, burst duration, and delay in burst activity. Swimming cycles were measured over an interval of 1.5 s, starting 0.5 s after the onset of swimming. Struggling cycles were measured throughout the period of repetitive stimulation used to evoke activity (see METHODS). Cycle period measurements were made between the midpoints of consecutive bursts of activity (cf. Wallen and Williams 1984
; see Green and Soffe 1996
for justification of using burst midpoint). The start and finish of each burst was usually easy to define. In rare cases, a more subjective estimate was made. This could have resulted in a small underestimate of burst duration and a similar inaccuracy in defining the midpoint. However, we did not consider this a significant source of error. Defining the number of bursts in a sequence was aided by comparing recordings from rostral and caudal electrodes. Except where stated otherwise: burst durations are quoted as absolute values; and longitudinal delays were measured as the time difference between the midpoints of rostral and caudal bursts of activity and expressed in milliseconds per millimeter. Mean values for cycle period, burst duration, and delay were calculated for control and treatment experiments, respectively, by pooling the individual measurements from two or three episodes of fictive behavior in each condition. Tests of significance were performed by analysis of variance (ANOVA), unless stated otherwise.

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| FIG. 1.
Effect of spinal transection on the rostral swimming motor pattern. A: stage 37/38 Xenopus laevis tadpole. Measurements were made as distance from the snout and converted to equivalent postotic segment. Longitudinal level is expressed only as segment in all future figures. The approximate site used for skin stimulation is indicated (*). B: fictive swimming, recorded from a rostral ventral root, was unchanged in cycle period and burst duration following spinal cord transection at the 4th postotic segment (see diagram: dorsal view of CNS and segmental myotomes indicating position of transection and ventral root recording, vr). Records start 1.5 s after the start of a swimming episode. C: changes in mean cycle period values ( cycle period) following spinal transection are variable and show no dependence on the rostrocaudal level of the transection (r = 0.13, n = 13, P 0.05). The significance of changes in each individual tadpole is indicated (P < 0.05, ; P > 0.05, ). Cycle periods were taken as the mean of 90 cycles (30 cycles each from 3 different episodes) from each tadpole before and after transection. Data are also included for tadpoles pharmacologically transected with MS222 (P < 0.05, ; P > 0.05, ;n = 4).
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RESULTS |
Blocking caudal activity during struggling and swimming motor patterns
Several classes of neuron within the spinal cord of the Xenopus tadpole possess ascending axons (Roberts and Clarke 1982
) and may therefore influence the generation of locomotor patterns more rostrally. We first attempted to remove all ascending influences from the caudal spinal cord and examined the characteristics of swimming and struggling motor patterns generated rostrally and evoked by electrical stimulation of the rostral trunk skin (Fig. 1A,*). After recording rostral ventral root discharge in intact animals, we removed ascending influences originating caudal to a particular level either surgically, by transecting the spinal cord at that level, or pharmacologically, by blocking caudal neural activity with the anesthetic MS222. The combination of these two approaches allowed the advantages of each to be exploited. Surgical transection allowed the rostrocaudal position of the block to be precisely determined. Pharmacological block with MS222 had the advantages of being reversible and not necessitating the removal and repositioning of recording and stimulating electrodes between control and experimental recording. However, the precise level of the pharmacological block was harder to determine. A ventral root electrode placed caudal to the level of the MS222 application ensured that neural activity was completely blocked. Both surgical and pharmacological blocks were performed at different rostrocaudal levels along the spinal cord between 1.5 and 3.0 mm from the snout (postotic segments 3-11; Fig. 1A).
Effect of pharmacological and surgical spinal cord transection on swimming
Surgical transection of the spinal cord had no significant effect on the cycle period or burst duration of the rostral swimming motor pattern (Fig. 1B). In individual tadpoles there was sometimes a significant change in cycle period after the transection (for 7 of 13 tadpoles, P < 0.05), but the direction of the change (increase or decrease) was variable and showed no relationship with the rostrocaudal level of the transection (Fig. 1C). In the group as a whole there was no significant difference in the mean cycle period before and after transection (n = 13, P > 0.05). Blocking activity caudal to the fourth segment with MS222 (n = 4) similarly had no detectable effect on the swimming motor pattern (Fig. 1C).
Variation in cycle period measured between different control swimming episodes in the same tadpole was significantly greater than the variation in cycle period measured in each episode (P < 0.05). This variation between episodes may mask more subtle changes in the motor pattern resulting from removal of caudal activity and may be responsible for the significant changes in mean cycle period that were sometimes observed after spinal transection. One possible source of variation in spinal activity is descending input from the brain, originating for example in the pineal photoreceptor system (Foster and Roberts 1982
). Removing the effects of such descending influences will reduce variability resulting from this source. More subtle effects on the swimming pattern were therefore investigated using MS222 to remove caudal activity in tadpoles transected rostrally at the level of the otic capsule (n = 5). The level of MS222 application was kept constant at 1.7 mm from the snout (postotic segment 4) in all cases. More rostral applications were prevented by the need to place a rostral ventral root electrode to record motor activity. Control cycle periods obtained after rostral transection at the otic capsule still showed a significant variation between episodes in the same treatment group (P < 0.05). A nested ANOVA of cycle period and burst duration values (with episode number nested within treatment group), still showed no significant difference between control and caudally transected tadpoles (P > 0.05, n = 5). It remains possible that removal of caudal activity has an effect on the rostral swimming motor pattern, but if so it is small compared with the normal variation in the motor pattern.
Effect of pharmacological and surgical spinal cord transection on struggling
In contrast with swimming, the effects of blocking caudal spinal cord activity on the rostral struggling motor pattern were very clear. In most spinally transected tadpoles (n = 14) and tadpoles in which caudal neural activity had been blocked pharmacologically with MS222 (n = 19), there was a marked increase in both cycle period and motor burst duration following block (Fig. 2A). The increase in both cycle period and burst duration was significantly correlated with the rostrocaudal level of the block (Fig. 2, B and C). More rostral transections and MS222 applications produced greater increases in both cycle period and burst duration, although there was a large variation in the magnitude of this effect between different tadpoles transected at the same level. In general, removing activity caudal to a level 2.5 mm from the snout (postotic segment 8) produced no significant effect on the motor pattern. Removing activity more rostral to this produced a variety of effects in individual tadpoles ranging from extreme cases in which there was no significant effect on the cycle period (in 6 of 33 tadpoles, P > 0.05) or burst duration (in 8 of 33 tadpoles, P > 0.05), to cases where there was a breakdown in rhythm generation altogether (3 of 33 tadpoles). Because of this variation between tadpoles, the dependence of effects on the level of the transection was investigated further by using MS222 to block caudal activity at different spinal levels in the same tadpoles. In all tadpoles tested (n = 4), the percentage increase in both burst duration and cycle period was progressively greater with progressively more rostral MS222 applications (Fig. 3A). Once again, however, there was considerable variation between the magnitude of the effect and the level of the treatment between different tadpoles. The effect of MS222 on the burst duration and cycle period was quickly reversible on washing (Fig. 3B). In four additional experiments, TTX was applied caudally to block activity in intact tadpoles. Its effects were like those of MS222 (and are included in Fig. 2C) but were not readily reversible.

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| FIG. 2.
Effect of spinal transection on the rostral struggling motor pattern. A: ventral root recordings of the struggling motor pattern show an increase in cycle period and burst duration following spinal transection. B: increases in cycle period (top) and burst duration (bottom) were correlated with transection level (r = 0.62 and r = 0.66, respectively, n = 14, P < 0.05). C: following pharmacological transection with MS222 ( and , n = 19) or tetrodotoxin (TTX; and , n = 4), cycle period (top) and burst duration (bottom) were correlated with application level (r = 0.51 and r = 0.53, respectively, n = 20, both P < 0.05). In B and C, changes in most individual tadpoles were significant ( and , P < 0.05), although some were not ( and , P > 0.05). In 3 additional cases ( ), MS222 application produced continuous bursting (CB, not included in regression analysis). Changes in burst duration and cycle period were strongly correlated with each other following both surgical (D) and pharmacological (E) transection (r = 0.89, n = 14 and r = 0.97, n = 20, both P < 0.05).
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| FIG. 3.
Effect of pharmacological transection on rostral struggling in individual tadpoles. A: progressively more rostral applications of MS222 in the same tadpoles (n = 4) resulted in progressively greater increases in the cycle period (top) and burst duration (bottom). B: these effects were fully reversible on washing (illustrated for the maximum effects shown in A). CB indicates continuous bursting ( ).
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The increases in cycle period and burst duration following caudal transection or pharmacological block showed a strong positive correlation (P < 0.05). With both treatments, the increase in burst duration was greater than the increase in cycle period: 1.7 times greater for spinally transected tadpoles (Fig. 2D) and 1.3 times greater for pharmacologically blocked tadpoles (Fig. 2E). Overall, the results of these experiments support the idea that the caudal spinal cord exerts an important influence on the generation of the struggling motor pattern rostrally. This influence is presumably mediated by ascending axons of more caudal neurons belonging to the premotor circuitry.
Effect of midsagittal section on the rostral struggling and swimming motor patterns
Neuroanatomic studies of the spinal cord motor system have characterized spinal premotor interneuron classes that have uncrossed-ascending axons, and one class that has crossed-ascending axons (Roberts and Clarke 1982
). The latter are the glycinergic inhibitory commissural interneurons (Dale et al. 1990
). A second neuron class with crossed axons, the dorsolateral commissural interneurons, is not active during swimming and struggling (Soffe 1993
) (see DISCUSSION). We used midsagittal section of the caudal spinal cord to cut selectively the crossed, and therefore crossed-ascending axons, while leaving uncrossed pathways in the spinal cord intact (Fig. 4A). This allowed us to test whether the selective effects of removing caudal activity on the struggling motor pattern but not swimming could be accounted for by the loss of this crossed-ascending inhibition. Midsagittal section of the spinal cord becomes increasingly difficult as it narrows caudally (<70-80 µm diam). Tadpoles were therefore first transected at a level of 3.5 mm from the snout (postotic segment 13). As shown above, transection at this level has no significant effect on either struggling or swimming motor patterns. After making control recordings, midsagittal sections were performed; the rostral extent of the section was 1.7 mm from the snout (postotic segment 4) in all cases. If the effects of caudal block described above resulted from the loss of crossed-ascending connections, midsagittal division should have an effect equivalent to a spinal transection at 1.7 mm from the snout. Ventral root recordings were made rostral to the rostral extent of the division (and also more caudally, see below), between 1.5 and 1.7 mm from the snout (postotic segments 3-4), before and after the operation.

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| FIG. 4.
Effect of midsagittal section on rostral struggling. A: ventral root discharge before and after midline section caudal to the 4th postotic segment (see diagram). B: both cycle period and burst duration were significantly increased (n = 10, P < 0.05). C: changes in burst duration and cycle period were strongly correlated with each other following midline section (r = 0.76, n = 10, P < 0.05).
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As with the surgical or pharmacological transection experiments, midsagittal section had no overall significant effect on either motor burst duration or cycle period during swimming (n = 10, P > 0.1). However, during struggling (Fig. 4A), there was again a significant increase in most cases in both burst duration (for 7 of 10 tadpoles, P < 0.05; Fig. 4B) and cycle period (for 7 of 10 tadpoles, P < 0.05; Fig. 4B). The increases were consistent with those seen after spinal transection at the same level, and were again significantly correlated (P < 0.05; Fig. 4C). These results show that loss of crossed-ascending inhibitory connections can account for the increases in the duration and cycle period of motor bursts observed during struggling when the influence of the caudal spinal cord has been removed. It remains possible that other ascending connections could also influence the generation of the struggling motor pattern rostrally (see DISCUSSION).
If activity were not maintained caudally following midsagittal division, the effect of the operation could have been explained simply by the loss of all ascending connections originating in more caudal regions. The operation would then have been directly equivalent to cord transection. To confirm that rhythmic activity was maintained caudal to the rostral extent of a sagittal division, caudal ventral root recordings were made from the same tadpoles. In all cases, rhythmic discharge was maintained caudally during both swimming and struggling.
Effect of spinal hemisection on the rostral struggling and swimming motor patterns
Because of the technical difficulty in performing midsagittal sections on the small tadpole spinal cord, we used a different preparation to determine whether the effects of removing crossed-ascending inhibitory connections vary with rostrocaudal level, like those following spinal transection. Spinal hemisection (Soffe and Roberts 1982
), involving a complete transverse section through one side of the spinal cord (Fig. 5A), removes descending excitation to more caudal neurons on the cut side. As a result, they do not fire during swimming. On the intact side of the cord, ipsilateral pathways remain intact, and caudal neurons continue to fire. Like midsagittal section, this lesion removes crossed-ascending influences, in this case arising caudal to the hemisection because 1) crossed-ascending axons on the cut side are lesioned at the hemisection and 2) crossed-ascending axons on the intact side are from neurons with somata on the cut side caudal to the transection, which do not fire during swimming (Soffe and Roberts 1982
). We confirmed that they also do not fire during struggling by making ventral root recordings from four tadpoles caudal to the hemisection. These showed no activity during swimming or struggling. The hemisection operation differs from midsagittal section primarily in that uncrossed-ascending connections are also lost on one side (the cut side). Hemisections were performed at a series of different rostrocaudal levels between 1.5 and 2.5 mm from the snout (postotic segments 3-8). Ventral root recordings were made rostral to the level of the lesion, and between 0.7 and 1.8 mm (4-10 segments) caudal to the level of the lesion (see next section) on the intact side of the cord, both before and after the operation.

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| FIG. 5.
Effect of spinal hemisection on swimming and struggling. A: swimming discharge recorded from rostral (Rvr) and caudal (Cvr) ventral roots before and after spinal hemisection (see diagram). B: changes in swimming cycle period following hemisection were variable and not correlated with longitudinal level (r = 0.25, n = 22, P > 0.1). The significance of changes in each individual tadpole is indicated (P < 0.05, ; P > 0.05, ). C: struggling discharge recorded from rostral and caudal ventral roots before and after spinal hemisection. D: increases in cycle period (top) and burst duration (bottom) were correlated with hemisection level (r = 0.47 and r = 0.50, respectively, n = 28, P < 0.05). The significance for individual tadpoles is represented as in B. E: changes in burst duration and cycle period were strongly correlated with each other following hemisection (r = 0.94, n = 25, P < 0.05).
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During swimming, there was no significant effect of the spinal hemisection on the cycle period or duration of rostral motor bursts (Fig. 5A); although as with transection there were individual significant, but small and inconsistent, differences (both increases and decreases) in cycle period (n = 18, P < 0.05). Changes overall showed no relationship with the rostrocaudal level of the hemisection (Fig. 5B; n = 22, P > 0.1). During struggling there was an increase in the cycle period and duration of rostral motor bursts (Fig. 5C). The effect was significantly correlated with the level of the operation: more rostral hemisections generally produced greater increases in both cycle period (Fig. 5D; r =
0.47, n = 28, P < 0.05) and burst duration (Fig. 5D; r =
0.50, n = 28, P < 0.05). The increase in these two parameters showed a strong positive correlation: the increase in burst duration was 1.5 times greater than the increase in cycle period (r = 0.91, n = 27, P < 0.05; Fig. 5E). These influences on the rostral motor pattern are consistent with those seen after either caudal transection or midsagittal section (described above).
Effect of spinal hemisection on caudal motor bursts during struggling
During struggling in the intact tadpole, ventral root bursts recorded caudally (2.7-3.6 mm from the snout: segments 9-14) were in most cases significantly longer in duration than those measured more rostrally (1.5-2.5 mm from the snout: segments 3-8; 18 of 28 individual tadpoles, P < 0.05; overall, P < 0.05). The mean proportion of the cycle period occupied by rostral bursts was 0.40 ± 0.01 (n = 28) and caudal bursts was 0.51 ± 0.02 (n = 28). After hemisection, rhythmic activity was recorded 0.7-1.8 mm (4-10 segments) caudal to the lesion on the intact side in 26 of 28 tadpoles. In the other two tadpoles, ventral root recordings caudal to the lesion showed continuous discharge with no apparent rhythmicity. In all cases, ventral root activity rostral to the lesion was rhythmic. Motor bursts caudal to the lesion increased in duration after hemisection, like those rostrally. The mean absolute magnitude of this increase was 1.4 times greater caudally than rostrally (r = 0.93, n = 25, P < 0.05). However, because the caudal bursts were longer anyway, the percentage increases rostrally and caudally were similar (only 1.1 times greater caudally r = 0.94, n = 25, P < 0.05).
Effect of spinal hemisection on longitudinal delay during swimming and struggling
Previous work has shown that the cycle period, duration, and longitudinal delay of motor bursts all scale together during struggling and during transitions from struggling to swimming (although not during swimming itself), suggesting that neural mechanisms controlling these three characteristics are linked (Green and Soffe 1996
). Because our earlier manipulations affected both cycle period and burst duration during struggling, we wished to know whether longitudinal delay was also altered. Specifically, because the caudorostral longitudinal delay increases during struggling as burst duration and cycle period increase (Green and Soffe 1996
), we predicted that the increase in cycle period and burst duration produced by spinal hemisection might similarly be associated with an increased caudorostral longitudinal delay. Using the same tadpoles as in the previous section, we measured longitudinal delay between the midpoints of motor bursts recorded rostrally and just caudal to the lesion on the intact side of the cord.
During swimming there was a significant increase in the rostrocaudal longitudinal delay after hemisection in 12 of 22 tadpoles (P < 0.05); in the remaining 10 tadpoles there was no significant change. In the group as a whole, the increase was significant (n = 22, P < 0.05). The percentage increase in delay did not correlate with the rostrocaudal level of the hemisection (Fig. 6A). During struggling, there was also an increase in the longitudinal delay after hemisection, although in this case the delay was caudorostral (Fig. 6A). Overall, the change in delay during struggling was dependant on the longitudinal level of the hemisection, although there was no significant linear correlation. Instead, hemisections caudal to a level ~2.25 mm from the snout (7th postotic myotome) had no significant effect on delay (7 of 7 tadpoles, P > 0.05); hemisections rostral to this level produced a significant increase in delay in 11 of 18 tadpoles (P < 0.05). The delay continued to show a positive correlation with both the change in burst duration (r = 0.72, n = 25, P < 0.05) and cycle period (r = 0.85, n = 25, P < 0.05; Fig. 6B), but these correlations were weaker than the correlation between cycle period and burst duration (Fig. 5).

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| FIG. 6.
Effect of spinal hemisection on the longitudinal delay during swimming and struggling. A: the overall increase in delay during swimming is not correlated with hemisection level (top: n = 22, P > 0.05). The increase in delay during struggling in contrast was significantly correlated (r = 0.42, n = 25, P < 0.05). The significance of changes in each individual tadpole is indicated (P < 0.05, ;P > 0.05, ). B: the increase in delay during struggling was correlated with the increases in both cycle period (top: r = 0.85, n = 25, P < 0.05) and burst duration (bottom: r = 0.72,n = 25, P < 0.05). C: delay during struggling between the starts of caudal and rostral burst (D1) was larger than between the ends (D2). In the intact tadpoles D1 is significantly greater than D2 (16 of 20 tadpoles, P < 0.05). Following hemisection there is a significant increase in D1 (15 of 20 tadpoles, P < 0.05), whereas D2 remains largely unchanged.
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As described in the previous section, the duration of caudal motor bursts during struggling is greater than the duration of rostral bursts. This clearly has implications for the way that longitudinal delay is measured. In addition to measurements based on burst midpoints, we also made separate measurements of delays between the starts and ends of bursts (Fig. 6C; delays D1 and D2). The longitudinal delay measured between the midpoints of bursts is equal to (D1 + D2)/2. The difference in rostral and caudal burst durations (described earlier) meant that delays D1 and D2 cannot be the same. In the intact tadpole, D1 was significantly greater than D2 (16 of 20 tadpoles, P < 0.05; Fig. 6D). After hemisection there was a significant increase in D1 (15 of 20 tadpoles P < 0.05), whereas D2 generally remained unchanged or decreased slightly. To combine measurements from different tadpoles, we normalized the delay values by expressing them as phase delays (delay·cycle period
1) per mm. The mean D1 phase delay (0.204 ± 0.012 mm
1, n = 20) was significantly greater than the mean D2 phase delay (0.096 ± 0.010 mm
1, n = 20) in the intact tadpole (P < 0.05). The mean D1 phase delay after hemisection (0.252 ± 0.014 mm
1, n = 20) was significantly greater than the mean D1 phase delay in the intact tadpole (P < 0.05, n = 20), whereas the D2 phase delay after hemisection (0.090 ± 0.016 mm
1, n = 20) was not significantly different (P > 0.05, n = 20).
In summary, the increase measured in caudorostral delay during struggling following spinal hemisection is not due to an overall shift in the timing of bursts. Instead, it results predominantly from a greater advance in motor burst onset caudally than rostrally on each cycle. Similar measurements were not made during swimming because the motor bursts are very brief and vary little in duration, making D1 and D2 difficult to determine with accuracy.
Effect of strychnine on struggling and swimming motor patterns
The manipulations described above were designed to remove either all ascending influences from more caudally situated neurons or to remove selectively those mediated by crossed-ascending axons. Because the effects of these manipulations can most easily be explained as the result of removing the influence of crossed-ascending glycinergic inhibition (see DISCUSSION), we wished to know whether the effects we described could be produced simply by a nonspecific reduction in the overall level of glycinergic inhibition. We therefore investigated the effects of global applications of relatively low concentrations of the glycinergic antagonist strychnine. Ventral root recordings were made from the rostral and caudal spinal cord during swimming and struggling, before and after bath application of 10-100 nM strychnine.
During swimming there was no significant effect of strychnine at these low concentrations on either the burst duration (n = 7, P > 0.1), cycle period (n = 7, P > 0.1), or longitudinal delay (n = 6, P > 0.1). During struggling, strychnine concentrations of 100 nM caused a total disruption of the motor pattern resulting in purely tonic activity in six of six tadpoles. Strychnine concentrations in the range 10-60 nM were sufficient to produce a significant decrease in cycle period (5 of 6 tadpoles, P < 0.05; Fig. 7). The effect on burst duration was less clear: rostral burst duration was increased in five of six tadpoles, but in only two was this significant (P < 0.05; Fig. 7). Expressed as a proportion of cycle period, however, burst duration increased significantly (P < 0.05) in all cases. Strychnine applications produced a significant decrease in caudorostral longitudinal delay thereby making motor bursts at different longitudinal levels effectively more synchronous (6 of 6 tadpoles, P < 0.05, Fig. 7). Globally weakening glycinergic inhibition during struggling therefore affects both cycle period and longitudinal delay in an opposite way to the surgical removal of crossed-ascending inhibitory connections.

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| FIG. 7.
Effect of low concentrations of strychnine on struggling. Application of 10-60 nM strychnine usually decreased cycle period (5/6, P < 0.05). Burst duration showed occasional increase (2/6, P < 0.05). Delay was significantly decreased in all cases (6/6, P < 0.05). The significance of changes in each individual tadpole is indicated (P < 0.05, ; P > 0.05, ).
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DISCUSSION |
Role of the caudal spinal cord in pattern generation
The results of this study clearly demonstrate that relatively caudal regions of the Xenopus tadpole spinal cord can have strong effects on motor activity generated more rostrally. They also show that these effects are pattern specific: they are clear during struggling, but could not be revealed during swimming. This difference is perhaps surprising because struggling and swimming appear to be generated by common circuitry, at least to the extent that the same neuronal classes are active during both patterns and both are driven by similar components of synaptic drive (Soffe 1993
). The same types of ascending connections would therefore be expected to be operating during both struggling and swimming. We will first consider the neuronal basis for the ascending effects we have described and then consider why these effects differ between motor patterns if common ascending connections are involved.
Ascending pathways in the Xenopus tadpole spinal cord
An important feature of the Xenopus tadpole preparation is that the morphological characteristics of the eight neuronal classes found in the spinal cord are known in some detail (Roberts 1989
; Roberts and Clarke 1982
). All can possess ascending axons. There are therefore a variety of potential ascending pathways (in fact more than descending pathways) that could relay information from caudal to more rostral regions within the spinal cord, and thus mediate the ascending influences we have described.
Rohon-Beard sensory neurons, other than those stimulated directly, do not fire during swimming or struggling (Clarke et al. 1984
; Soffe 1993
). Dorsolateral commissural interneurons, and probably the far less numerous dorsolateral ascending interneurons, are inhibited during swimming and particularly during struggling, and also do not fire (Roberts and Sillar 1990
; Soffe 1993
). Some excitatory descending interneurons have short, uncrossed-ascending axons. Because these neurons are rhythmically active during both swimming and struggling (Soffe 1993
), they are likely to provide some short-range rhythmic ascending excitation during both patterns. However, the effects of blocking caudal spinal cord activity by transection were mimicked by midsagittal divisions that leave uncrossed pathways intact.
Two other sources of uncrossed-ascending axons are the ascending interneurons and the cerebrospinal fluid-contacting Kolmer-Agdhur cells. Both cell types show
-aminobutyric acid (GABA) immunoreactivity and are therefore presumed to be inhibitory (see Roberts 1989
). Both could provide pathways by which the caudal spinal cord modulates the activity of more rostral regions. However, these should again not have been influenced by midsagittal division.
The glycinergic commissural interneurons have axons that cross to the opposite side of the spinal cord and then bifurcate to produce both descending and ascending axons (Dale et al. 1986
; Roberts and Clarke 1982
; Roberts et al. 1988
; Soffe et al. 1984
). The ascending axons can project up to 1 mm (4-5 segments) rostrally (often extending into the hindbrain or further), and, although only preliminary direct physiological evidence exists (Green, unpublished observations), they are in a suitable position to make en passant synapses with other classes of premotor interneurons and motorneurons that make up the spinal locomotor pattern generator. These interneurons are well established as being rhythmically active during swimming and struggling (Dale 1985
; Soffe 1993
; Soffe et al. 1984
) and provide the midcycle inhibition seen during both patterns.
Which of the above neurons could produce the effects we have described? Specifically removing crossed-cord ascending pathways can mimic the effects on rostral struggling of removing the caudal spinal cord. Only two classes of neuron have crossed-ascending axons: the premotor commissural interneurons and the dorsolateral commissural sensory interneurons. Only the commissural interneurons are active during swimming and struggling (Soffe 1993
). We therefore conclude that the increase in burst duration and cycle period observed during struggling when the caudal spinal cord is removed results primarily from a decrease in crossed-ascending inhibition mediated by the glycinergic commissural interneurons. Our results do not preclude other ascending pathways, such as the short ascending axons of excitatory descending interneurons, playing a role in mediating information flow from the caudal to the rostral spinal cord. They do, however, suggest that crossed-ascending inhibition from commissural interneurons is the dominant ascending influence during rhythmic activity.
The effects of caudal transection on rostral motor patterns could be viewed simply as the result of reducing the number of segments able to participate in rhythm generation. Recorded rostrally, however, such a reduction would still have to operate through loss of ascending connections. Also, we have chosen to illustrate the broad relationship between the longitudinal position of a lesion and the magnitude of the motor pattern perturbation as a linear one. This may be an oversimplification, but our present results do not allow us to make a more detailed interpretation.
Effects of crossed-ascending inhibition during struggling
How might removing crossed-ascending inhibition produce an increase in the burst duration and cycle period of the rostral struggling motor pattern, and why are similar effects not seen during swimming? To answer this, we will first propose a mechanism through which loss of crossed-ascending inhibition alone could lead to the observed changes during struggling.
The neurons known to be involved in pattern generation (excitatory descending interneurons, inhibitory commissural interneurons and motorneurons) on the same side of the cord are essentially coactive at a particular longitudinal level (Roberts et al. 1986
; Soffe 1993
). Consequently, motor activity on one side at each longitudinal level will be accompanied by inhibition on the other, meditated by coactive reciprocal inhibitory commissural interneurons. Besides crossed inhibition from directly opposite, neurons in all regions of the cord (except the most caudal) could receive crossed-ascending inhibition from more caudal regions on the opposite side of the cord via the crossed-ascending axons of commissural interneurons. This arrangement is illustrated in Fig. 8.

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| FIG. 8.
Simple prediction of the timing of crossed-ascending and crossed-descending components of inhibition received by a rostral neuron during struggling and swimming. The schematic of the spinal cord (left) shows a rhythmically active neuron (R) on the right side receiving inhibition from commissural inhibitory interneurons (i) on the left side at 7 different rostrocaudal levels. Because R is a rostral neuron, 4 levels of i are more caudal and only 2 are more rostral. Bursts of rhythmic activity of neuron R are represented by closed blocks. Bursts corresponding to the activity of commissural interneurons on the opposite side are represented by hatched blocks. The compound "inhibitory postsynaptic potentials (IPSPs)" that would result in neuron R from this pattern of connections and longitudinal timing is indicated for 1 cycle. The timing of individual components of the IPSP are shown by dashed lines from each rostrocaudal level. Notice that during struggling the onset of inhibition at R precedes the end of the corresponding burst (shaded area of IPSP) and could therefore play a role in burst termination. During swimming, the relatively short rostrocaudal delay and burst duration means that there is no equivalent overlap between IPSP and motor burst. For clarity, dashed lines indicate the timing of only 2 individual components of the IPSP (from most rostral and most caudal inhibitory interneurons) during swimming. See text for further details.
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The combination of longitudinal delay and conduction delay means that crossed-ascending inhibition from more caudal regions will arrive at a more rostral position out-of-phase with crossed inhibition from directly opposite. Because of the relatively large caudorostral delay during struggling, inhibition from progressively more caudal regions will arrive progressively earlier in each cycle than inhibition from directly opposite. Conduction delays (3-8 ms/mm) (Dale and Roberts 1985
) are relatively small in comparison with the longitudinal delays during struggling and affect the timing of activity only slightly. On each cycle, crossed-ascending inhibition from more caudal regions will start to arrive before the end of each more rostral motor burst (Fig. 8). We propose that inhibition will build up toward the end of the burst as inhibitory neurons situated less caudally become active. Since the timing of this proposed buildup of inhibition overlaps appropriately with the time at which bursts terminate during struggling (shaded, Fig. 8), we suggest that crossed-ascending inhibition normally plays a role in terminating motor bursts during struggling. Removing the elements of crossed-ascending inhibition that originate in more caudal neurons (for example by spinal transection, hemisection, or midsagittal section) will preferentially weaken the earliest part of each phase of inhibition, the part that coincides with the latter part of each rostral motor burst. Consequently, burst termination will be delayed and burst duration will be increased. Because burst duration and the duration of the associated reciprocal inhibition will increase symmetrically on the two sides, the result will also be an increase in cycle period.
Effects of crossed-ascending inhibition during swimming
We did not observe a clear effect of ascending inhibition on more rostral activity during swimming, consistent with the findings of Roberts and Alford (1986)
. This result could be explained in one of two ways: 1) the ascending pathways could be selectively suppressed during swimming or 2) differences in the operation of the circuitry or in the timing of the swimming and struggling patterns could themselves change the effectiveness. We suggest that the latter provides a sufficient explanation. Swimming shows two important differences from struggling that will affect the action of ascending inhibition on the rostral motor pattern. First, the longitudinal delay during swimming is much smaller than during struggling, and in the opposite (rostrocaudal) direction. Inhibition arriving from descending and ascending pathways is therefore much closer to being synchronous, with ascending inhibition arriving progressively later than crossed-descending inhibition. Second, swimming burst durations are very short, with each rhythmic neuron firing only once per cycle. The combination of these factors means that crossed inhibition from any level does not arrive until after rhythmic neurons have fired on each cycle; and inhibition will occupy a shorter fraction of the cycle. Crossed-ascending inhibition cannot therefore play a role in burst termination during swimming and will be less significant overall than during struggling.
Our explanations above assume that glycinergic commissural interneurons make synaptic connections from along the lengths of both their crossed-descending and crossed-ascending axons. Evidence from hemisected preparations (Soffe and Roberts 1982
) or paired intracellular recordings (Dale 1985
) suggests that the crossed-descending axons of commissural interneurons make inhibitory synapses with contralateral motoneurons along at least the first one-third to one-half (150-350 µm) of their length. Preliminary evidence also shows that during swimming rostral motoneurons can receive midcycle inhibition from more caudal neurons (up to 800 µm), presumably mediated by crossed-ascending axons (Green, unpublished observations). However, the details have yet to be established.
Control of longitudinal delay
The crossed-ascending and crossed-descending axons of the commissural interneurons provide a major anatomic pathway by which coupling between different regions of the cord could be achieved. The finding that strychnine reduces the caudorostral longitudinal delay during struggling, effectively making rostral and caudal bursts more synchronous, supports the involvement of glycinergic inhibition and therefore the crossed inhibition from commissural interneurons. The results of the lesion experiments also support a role for inhibition in determining longitudinal coordination. Manipulating the longitudinal distribution of crossed inhibition within the spinal cord by rostral hemisection can alter longitudinal delay during both struggling and swimming. However, these results do not yet allow us to propose a mechanism. Longitudinal gradients in the strength of inhibition (Tunstall and Roberts 1994
) and differences in the effectiveness of postinhibitory rebound (Tunstall and Roberts 1991
) could both influence the way that inhibition acts during each particular motor pattern.