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Interlimb coordination during stepping in the cat: The role of the dorsal spinocerebellar tract
EXPERIMENTAL
NEUROLOGY
87,96-108
(1985)
Inter-limb Coordination during Stepping in the Cat: The Role of the Dorsal Spinocerebellar Tract ARTHUR W.ENGLISH' Department of Anatomy and Ye&es Regional Primate Research Center, Emory University, Atlanta, Georgia 30322 Received July 19. 1984 The role of the dorsal spinocerebellar tract (DSCT) in the neural control of normal interlimb coordination during overground stepping in adult cats was investigated using select spinal cord lesions. Previously, it had been shown that lesions of the caudal thoracic dorsal columns (DCs) which might involve the DSCT or its afferent fibers resulted in a marked change in the patterns of forelimb-hind limb coupling during locomotion. In the present study, more rostral DC lesions, which probably included the DSCT or its afferent fibers considerably less, resulted in nearly identical changes in the patterns of interlimb coordination during stepping. Lesions of the dorsolateral funniculus (DLF) at similar spinal levels resulted in no significant changes in interlimb coordination. These lesions did destroy the DSCT, since retrograde transport of horseradish peroxidase (HRP) from the anterior cerebellar vermis to the nucleus dorsahs was blocked caudal to the lesion. These results are consistent with the notion that the DSCT plays little if any role in the precise timing of step cycles of the different limbs. 0 1985 Academic PI~SS. hc.
INTRODUCTION
Recent studies from this laboratory (2, 3) attempted to identify neural elements that might be involved in the control of interlimb coordination during locomotion in cats. Lesions to the caudal thoracic parts of the dorsal columns (DCs) resulted in profound alterations in patterns of interlimb coordination during overground stepping. The particular effect of these lesions was not a disruption or elimination of normal interlimb coupling Abbreviations: DSCT-dorsal spinocerebellar tract, DC-dorsal column, DLF-dorsolateral funiculus, HRP-horseradish peroxidase, TMB-tetramethylbenzidine. I The aid of Dr. R. L. McBride with HRP injections and William Farnsworth with the histology is greatly appreciated. This work was completed with support of grants NS 15452 and NS 1753 I from the U.S. Public Health Service. 96 0014-4886185 $3.00 Copyright Q 1985 by Academic Press, Inc. All rigbls of reproduction in any form rewed.
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but a shift from one pattern of coordination to another. Lesions to the cranial cervical (C,) DCs had no effect on interlimb coordination. Based on these results, it was proposed that neurons in the dorsal columns at caudal thoracic but not cranial cervical spinal levels were involved in the control of normal interlimb coordination. Three neuronal systems were identified for further studies: (i) secondary dorsal column neurons [e.g., (9)] which arise from lumbar levels and ascend partly in the dorsal columns, partly in the dorsolateral funniculi; (ii) long ascending propriospinal neurons which arise from lumbar levels and ascend to cervical levels, at least in part in the dorsal columns (7); and (iii) the dorsal spinocerebellar tract (DSCT), which begins as thoracolumbar neurons, but courses mainly in the dorsolateral funniculus (DLF) (4). The DSCT is of particular interest since it is known to provide the anterior cerebellum with rather precise and detailed information as to activity in hind limb proprioceptors during controlled locomotion in decerebrate cats [see (10) for reviews]. Although the DSCT has not been proposed as a pathway directly involved with interlimb control, the disruption of its input by dorsal column lesions might be expected to influence cerebellar outflow during stepping. This in turn might give rise to a change, rather than a disruption, in patterns of interlimb coordination. I now report the results of additional lesion experiments aimed at elucidating the potential role of the DSCT in normal interlimb control. It is concluded that the available evidence lies in favor of the DSCT playing little, if any, role in normal interlimb control. MATERIAL AND METHODS Stepping Experiments. Experiments were conducted on six adult cats which had been acclimated to the laboratory environment. Each stepping trial consisted of the recording of multiple locomotor sequences as cats stepped overground along a 1 X 8-m wooden walkway. The animals stepped unrestrained at comfortable speeds and were encouraged to traverse the walkway for a food reward. No aversive stimuli were applied. Each stepping trial resulted in sampling locomotion at a variety of stepping speeds. To monitor the timing of step cycles of the different limbs during these trials, the EMG was recorded from a single extensor muscle in each limb [see (1) for details of technique and rationale]. An example of typical results is shown in Fig. IA. Each extensor muscle sampled was active during the last portion of the step cycle of its respective limb, so that the temporal spacing of the different EMG bursts provided an index of the timing of step cycles of the different limbs. From the EMG, the timing of stepping patterns of the limbs was determined as measures of step duration (e.g., Fig. 1A: SDLF) and EMG burst duration (e.g., Fig. 1A: BDLF). The coupling of step cycles
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FIG. 1. A-typical data and measurements taken. Phasic burst of EMG activity in the left lateral head of m. triceps brachii (LTLa) and in the left m. vastus lateralis (LVL) during overground walking are shown. The time between successive terminations of EMG bursts is the step cycle period, or step duration (SDF and SD,.,). The duration of each burst (BDF and BDu) was used to calculate the duty factor for each step, i.e., the ratio of BD to SD. Interlimb coupling relationships were determined from the latency (LR() between burst terminations of the two muscles, expressed as a function of SD, or as phase. B-interlimb coupling relationships in intact cats. The polar graph at the top plots duty factor along the radii and phase about the circumference. Duty factor is the ratio of B4, to SDu for the left hind limb step cycle, as measured from left VL EMG activity. Phase is determined from the ratio of the latency between step cycles of the limbs, as determined from left TL, and left VL EMG activity, to the left hind limb cycle period (!Q,). Small duty factors lie at the center of the circles, larger duty factors toward the outside. Phase values of 0” (360”) are found at the three o’clock position. Phase values increase at more counterclockwise positions, i.e., 180” at 9 o’clock. Each point on this and other plots corresponds to values determined from a single step cycle. The two graphs below the polar graph are bivariate plots of forelimb-hind limb step cycle latency (LM as defined above) (middle graph) and forelimb SD (bottom graph) against hind limb SD. In each case the data were fitted with a straight line using the method of least squares. In the center graph the slope of the line, which is an overall measure of phase, was not significantly different from 0.5 (180”). The slope of the line in the bottom graph was not different from unity.
of the different limbs was determined by measures of the latency (e.g., Fig. 1A: Lrn) between bursts in different limbs. For each experiment, the EMG activity from four extensor muscles was recorded on tape and later input into a laboratory computer system, which sampled the EMG at 1 kHz. The four EMG signals were then displayed on a graphics device and a moveable cursor was used to measure the times of onset and termination of EMG activity from the different muscles. From these values the duration of the EMG burst and of the step cycles as well as interlimb latencies were determined, as indicated in Fig. 1A. From those values, two measures of inter-limb coordination were determined: duty factor and phase. Duty factor is the ratio of burst duration to step duration and has been shown to be
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roughly inversely related to stepping speed (1). Phase values were determined by dividing interlimb latency by step duration. Phase was expressed as degrees to emphasize the continuous nature of interlimb coordination patterns. Step cycles of two limbs which are exactly in-phase are also 360” out of phase [see (1) for a more detailed discussion]. To determine patterns of interlimb coordination, phase values from a number of stepping sequences were determined. Patterns are described as those phase values which occurred most frequently. Animals were tested in stepping trials before and after placement of select spinal cord lesions or sham operations. Interlimb stepping patterns were compared pre- and postoperatively using nonparametric statistical tests (Mann-Whitney U test) as described elsewhere (2). Surgical Procedures. Each cat was its own control. After at least one successful recording session, animals were anesthetized i.m. with a 1: 1 mixture of ketamine HCl and Xylacine. This produces a deep surgical anesthesia but is much shorter acting than pentobarbital. Animals were often alert and recovered from anesthesia in 4 to 6 h after spinal surgery. Use of this short-acting anesthetic combination greatly enhanced “functional recovery” from spinal cord lesions, relative to animals anesthetized with pentobarbital. Animals which received the longer-acting anesthetic agent often did not become alert for as long as 24 h postoperatively and seemed subjectively debilitated for as long as a week. When the shorter-acting anesthetic agent was used, recording could commerce on the 2nd or 3rd postoperative day in all cases. Despite the use of such short anesthesia no cats displayed any overt behaviors suggestive of their being in great pain. On the first postoperative day, they could be handled much as preoperatively. Similar observations have been made using short-acting barbiturates, such as thiopental (E. Eidelberg, personal communication). Under surgical anesthesia the spinal cord was exposed through a limited laminectomy and incision of the dura, and selective spinal cord lesions were made using irridectomy scissors under visual control. After placement of the lesions (or in sham-operated controls), the dura, superficial and deep musculature, and skin were closed with separate sutures. The animal then received a single dose of long-acting penicillin. Recording commenced on the 3rd postoperative day, when the animals appeared to step along the walkway without excessive encouragement and display no stepping deficits which could be obviously related to the skin incisions. Two different spinal cord lesions were used. The dorsal columns were transected at the level of the T6 vertebra. This lesion was designed to destroy the dorsal column fibers without destruction or de-afferentation of the neurons of the DSCT, which at the T6 level is almost entirely situated in the DLF. The second lesion used was a destruction of the dorsolateral portion of the cord, either
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at T6 or T12. This lesion attempted to interrupt neurons travelling in the outer edge (ca.1 mm) of the DLF. Fibers of the DSCT were probably destroyed by this lesion. In two cats, sham operations were carried out, one at T6, one at T12. The spinal cord was exposed as described above, and then all wounds were closed. In both cases, no effects on interlimb stepping patterns were observed. The animals were able to step with the same patterns of coordination as noted preoperatively on the 3rd postoperative day. In all cases, the extent of spinal cord lesions was verified histologically as has been reported (2), but in order to examine the efficacy of the lesions to the DSCT (lesion 2), the viability of this pathway was examined using the retrograde transport of horseradish peroxidase (HRP). After at least two successful postoperative recording trials, the animals were anesthetized with pentobarbital (40 mg/kg, i.p.) and a solution of 30% HRP (Sigma type VI) in normal saline was injected bilaterally into the anterior vermis of the cerebellum. The amounts of HRP injected varied between 1 and 6 ~1 but were always large to attempt to insure spinocerebellar labeling. Afier 72 h, the animals were reanesthetized and perfused with saline followed by a mixed aldehyde solution. Serial sections of the lumbar spinal cord and the regions just rostra1 and caudal to the lesion site were cut at 40 pm and reacted for demonstration of HRP using tetramethylbenzidine (TMB) as a chromogen. All sections were saved and were counterstained with pyrinineO-methyl green. Labeled cells were easily recognized by the presence of a blue-black TMB reaction product and their locations were charted onto tracings of cross sections of the spinal cord. RESULTS Dorsal Column Lesions at T6. The effects of dorsal column lesions at T6 on interlimb stepping patterns are shown in Figs. 1B and 2. In Fig. 1B are the relevant interlimb coupling data recorded preoperatively. The same types of data are shown in Fig. 2 for the same animal after a lesion to the DCs, at T6. At the top of each panel is a polar plot of duty factor (radius) vs. phase (circumference). Large duty factors lie toward the outside of the circles, smaller duty factors toward the center. Phase values of 0” (360’) lie at three o’clock, and ascend in a counterclockwise direction (i.e., 180” lies at 9 o’clock, etc.). Each polar plot considers the duty factor for the left hind limb extensor muscle and the phase relationship between step cycles of the left forelimb and left hind limb. Duty factor was calculated as the ratio of EMG burst duration:step duration for the left hind limb. Phase was calculated as the ratio of interlimb latency to the duration of the left hind
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FIG. 2. Interlimb coupling relationships are shown for the same cat as in Fig. lB, 3 days after a bilateral lesion to the dorsal columns at T6. Data for the same cat 3 weeks postoperatively were the same. The format is the same as Fig. 1B. The slope of the line in the bottom graph
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limb step cycle. A similar set of data exist for each case for the right limbs. Each point in these graphs is from a single step cycle. All three plots in each panel are from a single case but show data which are representative of all similar cases. Below each polar plot is a bivariate plot of left hind limb step duration (abscissa) vs. the latency between the left forelimb and left hind limb step cycles (ordinate). The data were fitted with a straight line using the least squares method and a correlation coefficient calculated. The slope of this line is an average measure of forelimb-hind limb phase. The bottom graph in each panel is a plot of left hind limb step duration vs. left forelimb step duration. The data were fitted with a straight line and a correlation coefficient calculated, as in the middle graph of each panel. By comparing the panels in Figs. 1B and 2 the effect of T6 dorsal column lesions is discerned. In the polar plots significantly more data points lie to the right of the vertical after the lesion than before. This change from a predominance of coupling of homolateral forelimb and hind limb step cycles which is nearly 180” out-of-phase to a coupling which is more often closer to in-phase was statistically significant and was virtually identical to the effect of more caudal dorsal column lesions reported elsewhere. The effect is also noted in comparing the middle panels of Figs. 1B and 2. In control cases, a significant correlation between latency and step duration existed and the slope of the best fit linear relationship was not significantly different from 0.5 (equivalent to 180” phase). After the lesion, the correlation between latency and step duration was also significant, but the slope was significantly different from 0.5, but not from zero, indicating the shift in interlimb coupling patterns. Comparison of the bottom panels of the two figures demonstrates that both pre- and postoperatively, the duration of forelimb and hind limb cycles was highly correlated. The slope of the line fitted to these data points was not significantly different from unity. Similar analysis of the phase relationships between step cycles of the two forelimbs and between step cycles of the two hind limbs (not shown) indicated that no significant differences were found between pre- and postoperative trials. Thus the change in forelimb-hind limb coupling that occurred after T6 dorsal column lesions was not brought about by an uncoupling of step cycles of the different limbs. Lesions to the Dorsal Spinocerebellar Tract. The effect of lesions to the edge of the DLF that involve the DSCT is shown in Fig. 3. No control data are shown, as they were virtually the same as shown in Fig. 1B and reported elsewhere. Figure 3 is arranged in the same format as Figs. 1B and 2. By was not significantly different from unity. The slope of the line in the center graph was not signficantly different from zero. Note that even though duty factors were somewhat smaller than controls, step duration was not.
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FIG. 3. Interlimb coupling relationships are shown for a cat 3 days after receiving a small bilateral lesion to the dorsolateral funniculus. The format is the same as in Fig. 2. The slope of the line in the bottom graph was not significantly different from unity. The slope of the line
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comparing Fig. 3 with Fig. lB, it is seen that lesions to the DLF produced little or no effect on interlimb stepping patterns. Most data points in the polar plot of Fig. 3 lay in the region of 180” and at comparable duty factors to Fig. 1B. The bivariate plot of latency vs. step duration could be fitted with a straight line of slope not significantly different from 0.5. The duration of forelimb and hind limb step cycles was highly correlated and the slope of the straight line fit to the data points was not significantly different from unity. In similar analyses, the coupling of the two forelimb step cycles and that of the two hind limb step cycles (not shown) were unaffected by the lesion. Figure 4 summarizes the histologic confirmation of the lesion in this case. Immediately rostral to the lesion, neurons containing the HRP/TMB reaction product were found in the nucleus dorsalis, which is the main locus of cells of origin of the DSCT, just ventral and lateral to the central canal. Labeled neurons were also found in the dorsal horn (laminae I through VI), but not organized into a nuclear mass. Caudal to the lesion no labeled cell bodies were found in the position of the nucleus dorsalis (CG) but labeled cells in the ventral horn (laminae VIII and IX) and intermediate grey (lamina VII) were found. These latter cells were similar in shape and locus to neurons which have been described as belonging to the ventral spinocerebellar tract (4). The number of labeled neurons in regions other than the nucleus dorsalis was much as noted after similar injections in intact cats and as reported (4). Very few labeled cells in the region of the nucleus dorsalis were noted. DISCUSSION In a first attempt to identify neuronal elements contributing to normal interlimb coordination during locomotion, destruction of the dorsal columns at caudal thoracic levels was shown to exert a pronounced effect on interlimb coupling patterns (2). Similar lesions at the C2 spinal level had no effect. It was concluded that the destruction of some neural element that is present in the DCs at TlO but not at C2 was responsible for the change in interlimb coupling. Because the type of change involved mainly a change in the phasing of the ipsilateral forelimb and hind limb step cycles rather than a loss or disruption of coordination, the role normally played
in the middle graph was not significantly different from 0.5. Note especially that these relationships were not significantly different from controls, even though the abundance of step cycles with longer durations indicates that the animal was walking more slowly.
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FIG. 4. Histological confirmation of the DLF lesion. These data are from the same case used to demonstrate interlimb coupling relationships in Fig. 3. Upper left-a drawing of a cross section of spinal cord immediately (1 mm) rostra1 to a lesion made in the DLF at T6, showing the extent of the lesion as dark areas in the dorsolateral white matter. Shown also are the loci of several neurons which were retrogradely labeled after injection of HRP into the anterior cerebellar vermis. These appear as dots in the region of the nucleus dorsahs, the primary cells of origin of the DSCT (4). The remainder of the figure is a summary of cell counts made caudal to the lesion in this case. Top center-a plot of the total number of labeled cells found at the different spinal levels indicated. All sections were counted so that this plot reflects the absolute number of labeled neurons. Top right-a diagram of the grcy matter divided into different regions corresponding to different collections of cytoarchitectonic laminae. Cell counts were made in each region, as indicated, and are summarized below. The regions analyzed are thus the dorsal horn (laminae I-VI), two parts of the intermediate zone (medial and lateral lamina VII), the ventral horn (laminae VIII and IX, analyzed separately) and the region of the nucleus dorsalis termed the central gmy (CG). Note the abundance of labeled cells in all regions except CG.
by the destroyed neural element was considered to be fairly subtle. Three candidates for this neural element were proposed. Secondary dorsal column neurons which are found in the DCs for part of their course and in the
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DLF rostra1 to TlO, and course from the lumbar cord to the DC nuclei (9) might be involved. They might exert their effect via brain stem connections with descending neurons, such as the vestibulospinal system. Secondary DC neurons passing in the DCs throughout their course are probably not involved, because of the ineffectiveness of upper cervical DC lesions. Neurons in the DSCT might be involved, either through destruction directly in the DCs or by destruction of afferent fibers to the nucleus do&is in the DCs. The neurons of the DSCT might exert their effect via a transcerebellar route. Finally long ascending propriospinal neurons, which are lumbar interneurons that terminate at cervical levels, might be involved, if at least some of them course in the DCs, as has been suggested (7). These neurons would be expected to exert their effect on interlimb coordination via an intraspinal pathway (5, 6). Our results provide evidence that the former two suggested pathways contribute little to normal interlimb control and are thus probably not involved in the response to DC lesions described above. Lesions to the DCs at T6 would be expected to have little effect on either secondary DC neurons which course in the DLF, or on neurons of the DSCT. Most of the latter neurons might be expected to lie in the DLF at that level, so that any DSCT destruction or deafferentation would seem minimal. Because the results of T6 DC lesions are virtually the same as more caudal lesions, one would have to favor the third proposed pathway, long ascending propriospinal neurons in the DCs, as they would be the only one of the three proposed neural elements in the DCs at T6. This conclusion, based on a process of elimination, is corroborated by the lesions to the DLF. The extent of damage to secondary DC neurons in the DLF was not determined directly, so that the destruction of secondary DC neurons, coursing entirely in .the DLF, by the DLF lesions cannot be assured. Thus a role in interlimb coupling of these neurons cannot be ruled out, especially considering the extensive projections of DC nuclear cells which receive their inputs (9). However, secondary DC neurons are probably not involved in the changes in patterns of interlimb coordination resulting from more caudal DC lesions. The extent of destruction of the DSCT by lesions to the edge of the DLF was determined. In cats with these lesions the neurons of origin of the DSCT could be retrogradely labeled by HRP cranial to, but not caudal to the lesion. Neurons of origin of the ventral spinocerebellar tract were not appreciably affected. Such lesions had no discernable effect on interlimb coupling. Thus, the results of the present study support the notion that the DSCT probably exerts little or no effect on normal interlimb coupling. A role played by the DSCT which is more compatible with available anatomic
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and physiologic evidence is that proposed by Wetzel and Stuart (lo), that the DSCT provides the cerebellum with a precise copy of segmental sensory inflow during stepping. Destruction of the DSCT might deprive the cerebellum of this “alference copy” of ongoing limb movements, but the loss of this mossy fiber cerebellar input alone is apparently insufficient to alter cerebellar output enough to change interlimb coupling. The role of the DSCT in locomotor control might be more subtle, such as a detailed balancing of muscular synergies, as has been suggested by Orlovsky and Shik (8), but the DSCT probably is not involved in the precise timing of locomotor events. REFERENCES 1. ENGLISH, A. W. 1979. Interlimb coordination during stepping in the cat: an electromyograpbic analysis. J. Neurophysiol. 42: 229-243. 2. ENGLISH, A. W. 1980. Interlimb coordination during stepping in the cat: effects of dorsal column section. J. Neurophysiol. 44: 270-279. 3. ENGLISH, A. W., J. TIGGES, AND P. R. LENNARD. 1984. The anatomical organization of long ascending propriospinal neurons in the cat spinal cord. Submitted. 4. MATSUSHITA, M., Y. HOSOYA, AND M. IKEDA. 1979. Anatomical organization of the spinocerebellar system in the cat, as studied by retrograde transport of horseradish peroxidase. J. Comp. Neural. 184: 8 l-106. 5. MILLER, S., J. VAN DER BURG, AND F. G. A. VAN DER MECHB. 1975. Locomotion in the cat: basic programmes of movement. Brain Res. 91: 239-254. 6. MILLER, S., AND F. G. A. VAN DER MECHB. 1976. Coordinated stepping of all four limbs in the high spinal cat. Brain Res. 109: 395-398. 7. MOLENAAR, I., AND H. G. J. M. KUYPERS. 1978. Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels: a HRP study in cat and rhesus monkey. Brain Res. 152: 429-450. 8. ORLOVSKY, G. N., AND M. L. SHIK. 1976. Control of locomotion: a neurophysiological analysis of the cat locomotor system. Int. Rev. Physiol. Neurophysiol. II 10: 282-3 17. 9. RUSTIONI, A., AND I. MOLENAAR. 1975. Dorsal column nuclei atlerents in the lateral funniculus of the cat: distribution pattern and absence of sprouting after chronic deafferentation. Exp. Brain Res. 23: l-l 2. 10. WETZEL, M. C., AND D. G. STUART. 1976. Ensemble characteristics of cat locomotion and its neural control. Prog. Neurobiol. 7: l-98.
NEUROLOGY
87,96-108
(1985)
Inter-limb Coordination during Stepping in the Cat: The Role of the Dorsal Spinocerebellar Tract ARTHUR W.ENGLISH' Department of Anatomy and Ye&es Regional Primate Research Center, Emory University, Atlanta, Georgia 30322 Received July 19. 1984 The role of the dorsal spinocerebellar tract (DSCT) in the neural control of normal interlimb coordination during overground stepping in adult cats was investigated using select spinal cord lesions. Previously, it had been shown that lesions of the caudal thoracic dorsal columns (DCs) which might involve the DSCT or its afferent fibers resulted in a marked change in the patterns of forelimb-hind limb coupling during locomotion. In the present study, more rostral DC lesions, which probably included the DSCT or its afferent fibers considerably less, resulted in nearly identical changes in the patterns of interlimb coordination during stepping. Lesions of the dorsolateral funniculus (DLF) at similar spinal levels resulted in no significant changes in interlimb coordination. These lesions did destroy the DSCT, since retrograde transport of horseradish peroxidase (HRP) from the anterior cerebellar vermis to the nucleus dorsahs was blocked caudal to the lesion. These results are consistent with the notion that the DSCT plays little if any role in the precise timing of step cycles of the different limbs. 0 1985 Academic PI~SS. hc.
INTRODUCTION
Recent studies from this laboratory (2, 3) attempted to identify neural elements that might be involved in the control of interlimb coordination during locomotion in cats. Lesions to the caudal thoracic parts of the dorsal columns (DCs) resulted in profound alterations in patterns of interlimb coordination during overground stepping. The particular effect of these lesions was not a disruption or elimination of normal interlimb coupling Abbreviations: DSCT-dorsal spinocerebellar tract, DC-dorsal column, DLF-dorsolateral funiculus, HRP-horseradish peroxidase, TMB-tetramethylbenzidine. I The aid of Dr. R. L. McBride with HRP injections and William Farnsworth with the histology is greatly appreciated. This work was completed with support of grants NS 15452 and NS 1753 I from the U.S. Public Health Service. 96 0014-4886185 $3.00 Copyright Q 1985 by Academic Press, Inc. All rigbls of reproduction in any form rewed.
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but a shift from one pattern of coordination to another. Lesions to the cranial cervical (C,) DCs had no effect on interlimb coordination. Based on these results, it was proposed that neurons in the dorsal columns at caudal thoracic but not cranial cervical spinal levels were involved in the control of normal interlimb coordination. Three neuronal systems were identified for further studies: (i) secondary dorsal column neurons [e.g., (9)] which arise from lumbar levels and ascend partly in the dorsal columns, partly in the dorsolateral funniculi; (ii) long ascending propriospinal neurons which arise from lumbar levels and ascend to cervical levels, at least in part in the dorsal columns (7); and (iii) the dorsal spinocerebellar tract (DSCT), which begins as thoracolumbar neurons, but courses mainly in the dorsolateral funniculus (DLF) (4). The DSCT is of particular interest since it is known to provide the anterior cerebellum with rather precise and detailed information as to activity in hind limb proprioceptors during controlled locomotion in decerebrate cats [see (10) for reviews]. Although the DSCT has not been proposed as a pathway directly involved with interlimb control, the disruption of its input by dorsal column lesions might be expected to influence cerebellar outflow during stepping. This in turn might give rise to a change, rather than a disruption, in patterns of interlimb coordination. I now report the results of additional lesion experiments aimed at elucidating the potential role of the DSCT in normal interlimb control. It is concluded that the available evidence lies in favor of the DSCT playing little, if any, role in normal interlimb control. MATERIAL AND METHODS Stepping Experiments. Experiments were conducted on six adult cats which had been acclimated to the laboratory environment. Each stepping trial consisted of the recording of multiple locomotor sequences as cats stepped overground along a 1 X 8-m wooden walkway. The animals stepped unrestrained at comfortable speeds and were encouraged to traverse the walkway for a food reward. No aversive stimuli were applied. Each stepping trial resulted in sampling locomotion at a variety of stepping speeds. To monitor the timing of step cycles of the different limbs during these trials, the EMG was recorded from a single extensor muscle in each limb [see (1) for details of technique and rationale]. An example of typical results is shown in Fig. IA. Each extensor muscle sampled was active during the last portion of the step cycle of its respective limb, so that the temporal spacing of the different EMG bursts provided an index of the timing of step cycles of the different limbs. From the EMG, the timing of stepping patterns of the limbs was determined as measures of step duration (e.g., Fig. 1A: SDLF) and EMG burst duration (e.g., Fig. 1A: BDLF). The coupling of step cycles
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FIG. 1. A-typical data and measurements taken. Phasic burst of EMG activity in the left lateral head of m. triceps brachii (LTLa) and in the left m. vastus lateralis (LVL) during overground walking are shown. The time between successive terminations of EMG bursts is the step cycle period, or step duration (SDF and SD,.,). The duration of each burst (BDF and BDu) was used to calculate the duty factor for each step, i.e., the ratio of BD to SD. Interlimb coupling relationships were determined from the latency (LR() between burst terminations of the two muscles, expressed as a function of SD, or as phase. B-interlimb coupling relationships in intact cats. The polar graph at the top plots duty factor along the radii and phase about the circumference. Duty factor is the ratio of B4, to SDu for the left hind limb step cycle, as measured from left VL EMG activity. Phase is determined from the ratio of the latency between step cycles of the limbs, as determined from left TL, and left VL EMG activity, to the left hind limb cycle period (!Q,). Small duty factors lie at the center of the circles, larger duty factors toward the outside. Phase values of 0” (360”) are found at the three o’clock position. Phase values increase at more counterclockwise positions, i.e., 180” at 9 o’clock. Each point on this and other plots corresponds to values determined from a single step cycle. The two graphs below the polar graph are bivariate plots of forelimb-hind limb step cycle latency (LM as defined above) (middle graph) and forelimb SD (bottom graph) against hind limb SD. In each case the data were fitted with a straight line using the method of least squares. In the center graph the slope of the line, which is an overall measure of phase, was not significantly different from 0.5 (180”). The slope of the line in the bottom graph was not different from unity.
of the different limbs was determined by measures of the latency (e.g., Fig. 1A: Lrn) between bursts in different limbs. For each experiment, the EMG activity from four extensor muscles was recorded on tape and later input into a laboratory computer system, which sampled the EMG at 1 kHz. The four EMG signals were then displayed on a graphics device and a moveable cursor was used to measure the times of onset and termination of EMG activity from the different muscles. From these values the duration of the EMG burst and of the step cycles as well as interlimb latencies were determined, as indicated in Fig. 1A. From those values, two measures of inter-limb coordination were determined: duty factor and phase. Duty factor is the ratio of burst duration to step duration and has been shown to be
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roughly inversely related to stepping speed (1). Phase values were determined by dividing interlimb latency by step duration. Phase was expressed as degrees to emphasize the continuous nature of interlimb coordination patterns. Step cycles of two limbs which are exactly in-phase are also 360” out of phase [see (1) for a more detailed discussion]. To determine patterns of interlimb coordination, phase values from a number of stepping sequences were determined. Patterns are described as those phase values which occurred most frequently. Animals were tested in stepping trials before and after placement of select spinal cord lesions or sham operations. Interlimb stepping patterns were compared pre- and postoperatively using nonparametric statistical tests (Mann-Whitney U test) as described elsewhere (2). Surgical Procedures. Each cat was its own control. After at least one successful recording session, animals were anesthetized i.m. with a 1: 1 mixture of ketamine HCl and Xylacine. This produces a deep surgical anesthesia but is much shorter acting than pentobarbital. Animals were often alert and recovered from anesthesia in 4 to 6 h after spinal surgery. Use of this short-acting anesthetic combination greatly enhanced “functional recovery” from spinal cord lesions, relative to animals anesthetized with pentobarbital. Animals which received the longer-acting anesthetic agent often did not become alert for as long as 24 h postoperatively and seemed subjectively debilitated for as long as a week. When the shorter-acting anesthetic agent was used, recording could commerce on the 2nd or 3rd postoperative day in all cases. Despite the use of such short anesthesia no cats displayed any overt behaviors suggestive of their being in great pain. On the first postoperative day, they could be handled much as preoperatively. Similar observations have been made using short-acting barbiturates, such as thiopental (E. Eidelberg, personal communication). Under surgical anesthesia the spinal cord was exposed through a limited laminectomy and incision of the dura, and selective spinal cord lesions were made using irridectomy scissors under visual control. After placement of the lesions (or in sham-operated controls), the dura, superficial and deep musculature, and skin were closed with separate sutures. The animal then received a single dose of long-acting penicillin. Recording commenced on the 3rd postoperative day, when the animals appeared to step along the walkway without excessive encouragement and display no stepping deficits which could be obviously related to the skin incisions. Two different spinal cord lesions were used. The dorsal columns were transected at the level of the T6 vertebra. This lesion was designed to destroy the dorsal column fibers without destruction or de-afferentation of the neurons of the DSCT, which at the T6 level is almost entirely situated in the DLF. The second lesion used was a destruction of the dorsolateral portion of the cord, either
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at T6 or T12. This lesion attempted to interrupt neurons travelling in the outer edge (ca.1 mm) of the DLF. Fibers of the DSCT were probably destroyed by this lesion. In two cats, sham operations were carried out, one at T6, one at T12. The spinal cord was exposed as described above, and then all wounds were closed. In both cases, no effects on interlimb stepping patterns were observed. The animals were able to step with the same patterns of coordination as noted preoperatively on the 3rd postoperative day. In all cases, the extent of spinal cord lesions was verified histologically as has been reported (2), but in order to examine the efficacy of the lesions to the DSCT (lesion 2), the viability of this pathway was examined using the retrograde transport of horseradish peroxidase (HRP). After at least two successful postoperative recording trials, the animals were anesthetized with pentobarbital (40 mg/kg, i.p.) and a solution of 30% HRP (Sigma type VI) in normal saline was injected bilaterally into the anterior vermis of the cerebellum. The amounts of HRP injected varied between 1 and 6 ~1 but were always large to attempt to insure spinocerebellar labeling. Afier 72 h, the animals were reanesthetized and perfused with saline followed by a mixed aldehyde solution. Serial sections of the lumbar spinal cord and the regions just rostra1 and caudal to the lesion site were cut at 40 pm and reacted for demonstration of HRP using tetramethylbenzidine (TMB) as a chromogen. All sections were saved and were counterstained with pyrinineO-methyl green. Labeled cells were easily recognized by the presence of a blue-black TMB reaction product and their locations were charted onto tracings of cross sections of the spinal cord. RESULTS Dorsal Column Lesions at T6. The effects of dorsal column lesions at T6 on interlimb stepping patterns are shown in Figs. 1B and 2. In Fig. 1B are the relevant interlimb coupling data recorded preoperatively. The same types of data are shown in Fig. 2 for the same animal after a lesion to the DCs, at T6. At the top of each panel is a polar plot of duty factor (radius) vs. phase (circumference). Large duty factors lie toward the outside of the circles, smaller duty factors toward the center. Phase values of 0” (360’) lie at three o’clock, and ascend in a counterclockwise direction (i.e., 180” lies at 9 o’clock, etc.). Each polar plot considers the duty factor for the left hind limb extensor muscle and the phase relationship between step cycles of the left forelimb and left hind limb. Duty factor was calculated as the ratio of EMG burst duration:step duration for the left hind limb. Phase was calculated as the ratio of interlimb latency to the duration of the left hind
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FIG. 2. Interlimb coupling relationships are shown for the same cat as in Fig. lB, 3 days after a bilateral lesion to the dorsal columns at T6. Data for the same cat 3 weeks postoperatively were the same. The format is the same as Fig. 1B. The slope of the line in the bottom graph
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limb step cycle. A similar set of data exist for each case for the right limbs. Each point in these graphs is from a single step cycle. All three plots in each panel are from a single case but show data which are representative of all similar cases. Below each polar plot is a bivariate plot of left hind limb step duration (abscissa) vs. the latency between the left forelimb and left hind limb step cycles (ordinate). The data were fitted with a straight line using the least squares method and a correlation coefficient calculated. The slope of this line is an average measure of forelimb-hind limb phase. The bottom graph in each panel is a plot of left hind limb step duration vs. left forelimb step duration. The data were fitted with a straight line and a correlation coefficient calculated, as in the middle graph of each panel. By comparing the panels in Figs. 1B and 2 the effect of T6 dorsal column lesions is discerned. In the polar plots significantly more data points lie to the right of the vertical after the lesion than before. This change from a predominance of coupling of homolateral forelimb and hind limb step cycles which is nearly 180” out-of-phase to a coupling which is more often closer to in-phase was statistically significant and was virtually identical to the effect of more caudal dorsal column lesions reported elsewhere. The effect is also noted in comparing the middle panels of Figs. 1B and 2. In control cases, a significant correlation between latency and step duration existed and the slope of the best fit linear relationship was not significantly different from 0.5 (equivalent to 180” phase). After the lesion, the correlation between latency and step duration was also significant, but the slope was significantly different from 0.5, but not from zero, indicating the shift in interlimb coupling patterns. Comparison of the bottom panels of the two figures demonstrates that both pre- and postoperatively, the duration of forelimb and hind limb cycles was highly correlated. The slope of the line fitted to these data points was not significantly different from unity. Similar analysis of the phase relationships between step cycles of the two forelimbs and between step cycles of the two hind limbs (not shown) indicated that no significant differences were found between pre- and postoperative trials. Thus the change in forelimb-hind limb coupling that occurred after T6 dorsal column lesions was not brought about by an uncoupling of step cycles of the different limbs. Lesions to the Dorsal Spinocerebellar Tract. The effect of lesions to the edge of the DLF that involve the DSCT is shown in Fig. 3. No control data are shown, as they were virtually the same as shown in Fig. 1B and reported elsewhere. Figure 3 is arranged in the same format as Figs. 1B and 2. By was not significantly different from unity. The slope of the line in the center graph was not signficantly different from zero. Note that even though duty factors were somewhat smaller than controls, step duration was not.
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comparing Fig. 3 with Fig. lB, it is seen that lesions to the DLF produced little or no effect on interlimb stepping patterns. Most data points in the polar plot of Fig. 3 lay in the region of 180” and at comparable duty factors to Fig. 1B. The bivariate plot of latency vs. step duration could be fitted with a straight line of slope not significantly different from 0.5. The duration of forelimb and hind limb step cycles was highly correlated and the slope of the straight line fit to the data points was not significantly different from unity. In similar analyses, the coupling of the two forelimb step cycles and that of the two hind limb step cycles (not shown) were unaffected by the lesion. Figure 4 summarizes the histologic confirmation of the lesion in this case. Immediately rostral to the lesion, neurons containing the HRP/TMB reaction product were found in the nucleus dorsalis, which is the main locus of cells of origin of the DSCT, just ventral and lateral to the central canal. Labeled neurons were also found in the dorsal horn (laminae I through VI), but not organized into a nuclear mass. Caudal to the lesion no labeled cell bodies were found in the position of the nucleus dorsalis (CG) but labeled cells in the ventral horn (laminae VIII and IX) and intermediate grey (lamina VII) were found. These latter cells were similar in shape and locus to neurons which have been described as belonging to the ventral spinocerebellar tract (4). The number of labeled neurons in regions other than the nucleus dorsalis was much as noted after similar injections in intact cats and as reported (4). Very few labeled cells in the region of the nucleus dorsalis were noted. DISCUSSION In a first attempt to identify neuronal elements contributing to normal interlimb coordination during locomotion, destruction of the dorsal columns at caudal thoracic levels was shown to exert a pronounced effect on interlimb coupling patterns (2). Similar lesions at the C2 spinal level had no effect. It was concluded that the destruction of some neural element that is present in the DCs at TlO but not at C2 was responsible for the change in interlimb coupling. Because the type of change involved mainly a change in the phasing of the ipsilateral forelimb and hind limb step cycles rather than a loss or disruption of coordination, the role normally played
in the middle graph was not significantly different from 0.5. Note especially that these relationships were not significantly different from controls, even though the abundance of step cycles with longer durations indicates that the animal was walking more slowly.
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FIG. 4. Histological confirmation of the DLF lesion. These data are from the same case used to demonstrate interlimb coupling relationships in Fig. 3. Upper left-a drawing of a cross section of spinal cord immediately (1 mm) rostra1 to a lesion made in the DLF at T6, showing the extent of the lesion as dark areas in the dorsolateral white matter. Shown also are the loci of several neurons which were retrogradely labeled after injection of HRP into the anterior cerebellar vermis. These appear as dots in the region of the nucleus dorsahs, the primary cells of origin of the DSCT (4). The remainder of the figure is a summary of cell counts made caudal to the lesion in this case. Top center-a plot of the total number of labeled cells found at the different spinal levels indicated. All sections were counted so that this plot reflects the absolute number of labeled neurons. Top right-a diagram of the grcy matter divided into different regions corresponding to different collections of cytoarchitectonic laminae. Cell counts were made in each region, as indicated, and are summarized below. The regions analyzed are thus the dorsal horn (laminae I-VI), two parts of the intermediate zone (medial and lateral lamina VII), the ventral horn (laminae VIII and IX, analyzed separately) and the region of the nucleus dorsalis termed the central gmy (CG). Note the abundance of labeled cells in all regions except CG.
by the destroyed neural element was considered to be fairly subtle. Three candidates for this neural element were proposed. Secondary dorsal column neurons which are found in the DCs for part of their course and in the
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DLF rostra1 to TlO, and course from the lumbar cord to the DC nuclei (9) might be involved. They might exert their effect via brain stem connections with descending neurons, such as the vestibulospinal system. Secondary DC neurons passing in the DCs throughout their course are probably not involved, because of the ineffectiveness of upper cervical DC lesions. Neurons in the DSCT might be involved, either through destruction directly in the DCs or by destruction of afferent fibers to the nucleus do&is in the DCs. The neurons of the DSCT might exert their effect via a transcerebellar route. Finally long ascending propriospinal neurons, which are lumbar interneurons that terminate at cervical levels, might be involved, if at least some of them course in the DCs, as has been suggested (7). These neurons would be expected to exert their effect on interlimb coordination via an intraspinal pathway (5, 6). Our results provide evidence that the former two suggested pathways contribute little to normal interlimb control and are thus probably not involved in the response to DC lesions described above. Lesions to the DCs at T6 would be expected to have little effect on either secondary DC neurons which course in the DLF, or on neurons of the DSCT. Most of the latter neurons might be expected to lie in the DLF at that level, so that any DSCT destruction or deafferentation would seem minimal. Because the results of T6 DC lesions are virtually the same as more caudal lesions, one would have to favor the third proposed pathway, long ascending propriospinal neurons in the DCs, as they would be the only one of the three proposed neural elements in the DCs at T6. This conclusion, based on a process of elimination, is corroborated by the lesions to the DLF. The extent of damage to secondary DC neurons in the DLF was not determined directly, so that the destruction of secondary DC neurons, coursing entirely in .the DLF, by the DLF lesions cannot be assured. Thus a role in interlimb coupling of these neurons cannot be ruled out, especially considering the extensive projections of DC nuclear cells which receive their inputs (9). However, secondary DC neurons are probably not involved in the changes in patterns of interlimb coordination resulting from more caudal DC lesions. The extent of destruction of the DSCT by lesions to the edge of the DLF was determined. In cats with these lesions the neurons of origin of the DSCT could be retrogradely labeled by HRP cranial to, but not caudal to the lesion. Neurons of origin of the ventral spinocerebellar tract were not appreciably affected. Such lesions had no discernable effect on interlimb coupling. Thus, the results of the present study support the notion that the DSCT probably exerts little or no effect on normal interlimb coupling. A role played by the DSCT which is more compatible with available anatomic
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and physiologic evidence is that proposed by Wetzel and Stuart (lo), that the DSCT provides the cerebellum with a precise copy of segmental sensory inflow during stepping. Destruction of the DSCT might deprive the cerebellum of this “alference copy” of ongoing limb movements, but the loss of this mossy fiber cerebellar input alone is apparently insufficient to alter cerebellar output enough to change interlimb coupling. The role of the DSCT in locomotor control might be more subtle, such as a detailed balancing of muscular synergies, as has been suggested by Orlovsky and Shik (8), but the DSCT probably is not involved in the precise timing of locomotor events. REFERENCES 1. ENGLISH, A. W. 1979. Interlimb coordination during stepping in the cat: an electromyograpbic analysis. J. Neurophysiol. 42: 229-243. 2. ENGLISH, A. W. 1980. Interlimb coordination during stepping in the cat: effects of dorsal column section. J. Neurophysiol. 44: 270-279. 3. ENGLISH, A. W., J. TIGGES, AND P. R. LENNARD. 1984. The anatomical organization of long ascending propriospinal neurons in the cat spinal cord. Submitted. 4. MATSUSHITA, M., Y. HOSOYA, AND M. IKEDA. 1979. Anatomical organization of the spinocerebellar system in the cat, as studied by retrograde transport of horseradish peroxidase. J. Comp. Neural. 184: 8 l-106. 5. MILLER, S., J. VAN DER BURG, AND F. G. A. VAN DER MECHB. 1975. Locomotion in the cat: basic programmes of movement. Brain Res. 91: 239-254. 6. MILLER, S., AND F. G. A. VAN DER MECHB. 1976. Coordinated stepping of all four limbs in the high spinal cat. Brain Res. 109: 395-398. 7. MOLENAAR, I., AND H. G. J. M. KUYPERS. 1978. Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels: a HRP study in cat and rhesus monkey. Brain Res. 152: 429-450. 8. ORLOVSKY, G. N., AND M. L. SHIK. 1976. Control of locomotion: a neurophysiological analysis of the cat locomotor system. Int. Rev. Physiol. Neurophysiol. II 10: 282-3 17. 9. RUSTIONI, A., AND I. MOLENAAR. 1975. Dorsal column nuclei atlerents in the lateral funniculus of the cat: distribution pattern and absence of sprouting after chronic deafferentation. Exp. Brain Res. 23: l-l 2. 10. WETZEL, M. C., AND D. G. STUART. 1976. Ensemble characteristics of cat locomotion and its neural control. Prog. Neurobiol. 7: l-98.