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Brainstem command systems for locomotion in the lamprey: localization of descending pathways in the spinal cord
Brain Research, 457 (1988) 338-349 Elsevier
338 BRE 13857
Brainstem command systems for locomotion in the lamprey" localization of descending pathways in the spinal cord Andrew D. McClellan Department of Physiology and Biophysics, University of lowa, Iowa City, 1A 52242 (U.S.A.) (Accepted 8 March 1988)
Key words: Locomotion; Command system; Descending pathway; Brainstem locomotor region; Lamprey
The lamprey brainstem contains a 'command system' which descends into the spinal cord to activate motor networks and initiate locomotion. In the present study, partial lesions were made in the rostral spinal cord in order to spare various tracts and determine which tracts carry the descending command signal to the spinal cord. Sparing the medial areas of the rostral spinal cord usually blocked both sensory-evoked and spontaneous locomotion, while sparing the lateral regions of the rostral spinal cord did not abolish voluntary locomotor activity. Either the ventrolateral or dorsolateral spinal tracts could support the initiation of locomotion. Brainstem structures rostral to the mesencephalon were not necessary for the initiation of locomotor behavior. The data indicate that the lateral spinal tracts contain a significant part of the descending command pathway for locomotion. In contrast, the medial spinal tracts were neither necessary nor usually sufficient to support locomotor behavior, suggesting that the larger reticulospinal Muller cells, which project in these tracts, do not contribute significantly to the initiation of locomotion. INTRODUCTION Spinal m o t o r networks for locomotion are thought to be activated by descending c o m m a n d or initiation centers in the brainstem (reviewed in refs. 1, 12, 17, 37). In a number of vertebrates, ' l o c o m o t o r regions' have been d e m o n s t r a t e d in the brainstem with electrical and/or chemical microstimulation and are organized as parallel strips on each side of the midline from the mesencephalon to the lower brainstem (reviewed in ref. 17). F o r example, microstimulation within the 'mesencephalic l o c o m o t o r region' ( M L R ) and in medial reticular nuclei can elicit well coordinated locomotion in the spinal cord 5'8'12'20'37"40. Finally, spinal lesion studies in a n u m b e r of vertebrates indicate that the lateral or ventrolateral tracts are necessary for the initiation of locomotion 3' 17,3S,41,42,46. The lamprey, a lower v e r t e b r a t e , has recently become a useful p r e p a r a t i o n for l o c o m o t o r system studies 9'31. The l a m p r e y nervous system is comparatively simpler than those of higher vertebrates and can produce l o c o m o t o r activity under in vitro conditions (reviewed in refs. 9, 18, 31). F o r example, in an in vitro
brainstem/spinal cord p r e p a r a t i o n , l o c o m o t o r activity can be elicited by sensory stimulation of the head and by electrical or chemical microstimulation of 'locomotor c o m m a n d regions' in the b r a i n s t e m 16,1s.2°. In order to u n d e r s t a n d how these brainstem command systems function, it is necessary to d e t e r m i n e the locations of neurons in the system so that they can be characterized with intracellular recordings. In the present study, partial lesions of the rostral spinal cord were used to spare various descending tracts in o r d e r to determine which descending tracts might be used by brainstem c o m m a n d neurons for locomotion. After partial spinal lesions, l o c o m o t o r capabilities were tested in whole-animals with natural tactile stimuli. These experiments indicate that the lateral spinal tracts can support the initiation of locomotion but the medial spinal tracts are n e i t h e r necessary nor usually sufficient to support l o c o m o t o r behavior. Preliminary accounts of this study have a p p e a r e d 17'19. MATERIALS AND METHODS The purpose of this study was to d e t e r m i n e which
Correspondence: A.D. McClellan, Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
339 spinal tracts are necessary for the initiation of locomotion in the lamprey. The basic paradigm used in this project involved several steps: (a) partial lesions were made in the very rostral spinal cord in order to spare various tracts; (b) the behavioral capabilities of the spinal-lesioned animals were observed; and (c) the extent of the partial spinal lesions was histologically determined. The effects of partial spinal lesions were tested in whole-animals because it is much easier to elicit locomotor activity than, for example, in in vitro preparations. Locomotor capabilities were tested with tactile stimuli rather than with microstimulation of brainstem locomotor regions, which might not project into all of the descending spinal tracts normally used for the initiation of locomotion. Spinal lesions. The effects of partial lesions were tested in several types of lampreys: adult silver lampreys (I. unicuspis; n = 59), feeding and spawning phase sea lampreys (P. marinus; n = 110), and larval sea lamprey (n = 50). Animals were anesthetized with methanesulphanate (MS-222, 0.1 mM) and placed on ice. A small dorsal incision was made between the first and second gill slit and a section of the rostral spinal cord was exposed. Localized spinal lesions were made at segments 2 - 4 (distance from anterior rim of sucker: 2.5-3.0 cm in adults, 1.25 cm in lavrae), which is about 0.5-1.5 cm caudal to the obex, depending on the size of the animal. Rostral spinal lesions were used so that there would be a relatively short length of spinal cord above the lesion, thus making it less likely for propriospinal pathways (i.e. spinal coordinating systems) or sensory feedback to couple locomotor activity across the lesion to the lower body (cf. refs. 3, 44). Complete spinal transections at this same level always eliminated the initiation of locomotor behavior. Partial spinal lesions that encompassed the entire dorsoventral thickness of the spinal cord (e.g. medial, lateral and hemisection lesions) were made with iris scissors. These manipulations would destroy both the axonal tracts as well as possible cell bodies in the lesioned part of the spinal cord. Lesions which encompassed only the dorsal or ventral parts of the thin spinal cord were made by teasing away the outermost layers of the spinal cord. In the case of ventral lesions, the exposed spinal cord was freed for 5-10 mm and twisted to expose the ventral surface. After making the lesions, the incision was sutured and the animals were indi-
vidually marked and returned to their tanks for at least 24 h. All animals were kept at 5 °C.
Behavioral observations and EMG recordings. Lampreys were placed in a tank, either 40 x 70 cm or 22 x 40 cm depending on the size of the animal, which contained 2 - 6 cm of water at 5-10 °C. The locomotor responses of all animals were recorded with a standard video system (Panasonic PV-1535 recorder and PK-452 camera) at 30 frames/s. In some animals, the video record was time-linked to simultaneous recordings of muscle activity from various points along the body. Muscle recordings were made with pairs of copper wire (50-100 p m diameter, depending on animal size), insulated except at the tip, which were inserted into body musculature with a syringe needle. Behavioral responses were analyzed by tracing sequential frames of the video record displayed on a monitor screen. In normal animals, brief tactile stimulation applied to the anterior head area or to the tail elicits a flexure response, which usually moves the stimulated body region away from the stimulus, followed by escape locomotion 16'2°. Voluntary locomotion can also occur spontaneously. In spinal-lesioned animals, special attention was given to several features of locomotor performance: spontaneous swimming, locomotor responses elicited by tactile stimulation of the head or the tail, turning, exploratory behavior (i.e. long-duration swimming along the perimeter of the tank), equilibrium, and backward swimming.
Histological processing of lesioned spinal cords. Animals were anesthetized (see above), and a section of the spinal cord/notochord containing the partial spinal lesion was removed. The tissue was fixed in 10% Formalin, and histologically processed for sectioning at 5-20 p m in paraffin. The histological sections containing the spinal lesions were traced with a camera lucida. RESULTS
Behavioral observations: adult Controls. In normal adult lampreys (n = 25) with intact spinal cords (Fig. l-A1), locomotion could be initiated spontaneously or by tactile stimulation of the anterior area of the head 16, both of which presumably activate a brainstem command system that descends into the spinal cord to excite locomotor net-
340 works 16'17. F o r example, stimulation of the left side of the head elicited an S-shaped flexure response, in which the head turned to the right away from the stimulus, followed by escape locomotion during which body undulations travelled towards the tail (Fig. l - A 2 ) . The flexure c o m p o n e n t was largely produced by contraction of rostral b o d y musculature on the same side as the direction of turning (initial burst in trace 1, Fig. l - A 3 ) . Subsequent l o c o m o t o r activity was characterized by two features45'47: a l e f t - r i g h t alternation of muscle activity at the same segmental level ( 1 - 2 and 3 - 4 , Fig. l - A 3 ) and a rostrocaudal phase-lag in muscle activity on the same side ( 1 - 4 and 2 - 3 ) . Flexure responses and l o c o m o t i o n could also be elicited by tactile stimulation of the tail, which activates an ascending c o m m a n d p a t h w a y 2°. Continued activation of this ascending pathway can give
rise to slow, m o n o t o n o u s l o c o m o t o r patterns even in spinal animals. H o w e v e r , in n o r m a l animals brief tail stimulation can trigger long-duration, goal-directed episodes of locomotion, including turning, acceleration, deceleration, and exploratory behavior, that would p r e s u m a b l y require the participation of locom o t o r centers in the brainstem 17. Hemisections of the rostral spinal cord. Some of the adult lampreys in which the right or left regions of the spinal cord were spared displayed relatively n o r m a l l o c o m o t o r behavior (n = 7). In other animals (n = 11), however, there was a directional bias of the flexure reflexes and/or the l o c o m o t o r responses to the same side as the spared half of the spinal cord. F o r example, in an animal with the left areas of the rostral spinal cord spared (Fig. l-B1), tactile stimulation of either side of the head elicited turning re-
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Fig. 1. In Figs. 1-4, various tracts in the very rostral spinal cords were spared in lampreys (A1,B1). Sequential locomotor movements were recorded (A2,B2; proceeding left to right) and time linked to simultaneous recordings of activity in body musculature (A3,B3; 14, positions marked in movement panels). The dots in A2 (B2) serve as a fixed reference line as well as frame markers that correspond in time to the dots below the record in A3 (B3). A: normal adult silver lamprey (length, 30 cm; distance from head: 1,2 - 7.0 cm, 3,4 15.0 cm). S-shaped flexure responses, directed away from the stimulus, followed by escape locomotor activity elicited by brief tactile stimulation applied to the left anterior part of the head (A2, arrow). Note the left-right alternation (1-2 and 3-4) and ipsilateral phase-lag (1-4 and 2-3) during locomotor activity. B: adult silver lamprey (length, 31 cm) with a rostral lesion sparing the left half of the spinal cord (distance from head: lesion - 2.75 cm, 1,2 - 7.0 cm, 3,4 - 15.5 cm). Brief tactile stimulation of the left side of the head (B2, arrow) elicited a C-shaped flexure reflex, which was always directed towards the left, followed by locomotor activity (B2,B3). Interframe interval (IFI) = 67 ms.
341 sponses to the left (Fig. 1-B2) and flexure m o t o r activity on the left side (Fig. 1-B3; initial burst in trace 2). The subsequent locomotor responses and m o t o r activity could be symmetrical (Fig. 1-B2, 1-B3), or the direction of swimming could be biased toward the intact side of the spinal cord so that animals swam in a circle. In some cases, circling during swimming was accompanied by spinning movements and impaired equilibrium. Sparing the medial spinal tracts. In the lamprey, the ventromedial spinal cord contains the descending axons of the larger reticulospinal neurons (i.e. Muller cells 29) (see Fig. 7B). Since these neurons may be involved in the descending control of locomotor responses 29'31, the behavioral capabilities of lampreys with spared medial regions of the rostral spinal cord were examined (Fig. 2-A1,SE). The majority of animals (77%, 26 of 34 animals) with this type of lesion were unable to initiate flexure reflexes or locomotion either spontaneously or in response to tactile stimulation of the head (Fig. 2-A2, arrow; Fig. 2-A3, bar).
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In many ways these animals resembled spinalized lampreys 2°, in which tail-fin stimulation could elicit slow undulations but rostral stimulation was ineffective. In the remaining 23% of the animals with spared medial areas of the rostral spinal cord, initiation of locomotion was possible to varying degrees (Fig. 5D). However, in some of these latter animals the spared medial areas of the spinal cord were somewhat large and included parts of the intermediate spinal cord. Thus, in the majority of cases, the medial spinal tracts were insufficient to support the initiation of locomotion, suggesting that the lateral spinal tracts are more important in the descending control of locomotor behavior. Sparing the lateral spinal tracts. Of the 32 lampreys that were tested in which the lateral regions of the rostral spinal cord were spared (Fig. 2-B1,5A), all were capable of initiating relatively normal locomotion spontaneously and in response to tactile stimulation of the head or tail fin. For example, tactile stimulation of the left side of the head (Fig. 2-B2, arrow)
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Fig. 2. A: effects of sparing the medial regions of the rostral spinal cord (A1) in an adult silver lamprey (length, 28 cm; distance from head: lesion - 2.5 cm, 1,2 - 6.75 cm, 3,4 - 13.25 cm), Continuous tactile stimulation of the right part of the head (A2, arrow; A3, bar) was ineffective in eliciting locomotor responses. Notice the very weak bending movements of the tail. B: partial rostral lesions that spared the lateral areas of the rostral spinal cord (B 1) in an adult silver lamprey (length, 27 cm; distance from head: lesion - 3.0 cm, 1,2 7.0 cm, 3,4 - 14.0 cm). Brief tactile stimulation of the left part of the head (B2, arrow) elicited relatively normal flexure activity, directed away from the stimulus, followed by escape locomotor activity (B2,B3). Left-right alternation of segmental motor activity (1-2 and 3-4) and an ipsilateral phase lag (1-4 and 2-3) were present during locomotor patterns. IFI = 667 ms (A), 100 ms (B). -
342 elicited an S-shaped flexure response, which was directed away from the stimulus (Fig. 2-B2) and was produced primarily by rostral body muscle activity on the right side (initial burst in trace 1, Fig. 2-B3). The subsequent escape locomotor movements (Fig. 2B2) were accompanied by a left-right alternation (1-2 and 3-4, Fig. 2-B3) and a rostrocaudal phaselag (1-4 and 2-3). These results suggest that the lateral spinal tracts are important for the initiation of locomotion, and that the medial tracts are not necessary for normal locomotion. Coupled with the results from sparing the medial spinal tracts, the data imply that the reticulospinal Muller cells, which have medial descending axons (Fig. 7B), do not contribute significantly to the initiation of locomotor responses. Sparing the dorsolateral or ventrolateral spinal tracts. When the ventrolateral areas of the rostral spinal cord were spared (Fig. 3-A1), 86% of the lampreys (25 of 29) still were capable of initiating locomotion, either spontaneously or in response to tactile stimulation of the head. For example, tactile stimulation of the anterior part of the head elicited a flexure
reflex, which included a weak withdrawal response, followed by escape locomotion (Fig. 3-A2). The motor activity produced during these types of locomotor responses (Fig. 3-A3) was indistinguishable from those in normal, unlesioned animals. Similarly, in lampreys in which the dorsolateral regions of the rostral spinal cord were spared (Fig. 3-B1), locomotor responses and motor activity (Fig. 3-B2, 3-B3) appeared quite normal in 64% of the animals (11 of 17). Since the teasing method used to make this latter type of lesion may have traumatized the thin, ribbonlike lamprey spinal cord, it is not surprising that some of the animals were unable to locomote. Taken together, the data indicate that either the dorsolateral or ventrolateral spinal tracts can support the initiation of relatively normal locomotor behavior (Fig. 5B,C).
Other partial spinal lesions. Partial spinal lesions were also made that either spared the intermediate regions of the rostral spinal cord (n = 6), including the dorsal and ventral horns, or spared all areas of the rostral spinal cord except the intermediate re-
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Fig. 3. A: adult silver lamprey (length, 27 cm) with the ventrolateral tracts spared in the rostral spinal cord ( A I ) (distance from head: lesion - 3.0 cm, 1,2 - 6.0 cm, 3,4 - 14.0 cm). Brief tactile stimulation of the medial part of the anterior head (A2, arrow) elicited a backward flexure m o v e m e n t followed by escape locomotion (A2,A3). B: effects of sparing the dorsolateral spinal tracts (B 1) in an adult silver lamprey (length, 32 cm; distance from head: lesion - 2.5 cm, 1,2 - 7.0 cm, 3,4 - 15.5 cm). Brief tactile stimulation of the right part of the head (B2, arrow) elicited a flexure response and, after a delay, escape locomotor activity (B2,B3). IFI = 100 ms (A), 167 ms (B).
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Fig. 4. Locomotor responses in larval sea lamprey with partial lesions in the rostral spinal cord. A: effects of sparing the medial areas of the spinal cord (A1) in a larval lamprey (length, 16 cm; distance from head: lesion - 1.2 cm, 1,2 - 3.0 cm, 3,4 - 7.5 cm). Continuous tactile stimulation of the medial part of the head (A2, arrow; A3, bar) was ineffective in eliciting flexure or locomotor responses. B: sparing the lateral regions of the spinal cord (B 1) in a larval lamprey (length, 15.5 cm; distance from head: lesion - 1.1 cm, 1.2 - 3.25 cm, 3.4 - 7.5 cm). Light tactile stimulation of the left part of the head (B2, arrow) elicited a delayed flexure response that quickly gave rise to escape locomotor activity (B2,B3). Note the left-right alternation of segmental motor activity ( 1 - 2 and 3 - 4 ) and the ipsilateral phase-lag ( 1 - 4 and 2 - 3 ) during the initial locomotor patterns. IFI = 667 ms (A), 167 ms (B).
gions (n = 3). With both types of partial spinal lesions, the initiation of flexure reflexes and locomotion was not blocked (not shown).
Behavioral observations: larval lampreys The results from larval lampreys, both normal animals and those with partial spinal lesions, were similar to those obtained from adults (Fig. 5). Specifically, sparing the medial regions of the rostral spinal cord (n = 18) blocked spontaneous as well as sensory-evoked locomotor responses in 89% of the animals (Fig. 4A). In contrast, sparing the lateral areas of the rostral spinal cord (n = 22) allowed the initiation of locomotor movements and motor activity in all animals (Fig. 4B). Thus, in larval animals the descending command pathways for the initiation of locomotion appear to be located in similar lateral parts of the spinal cord as those in adult lampreys (Fig. 5).
Brainstem lesions Two types of lesions were made in the brainstem in order to determine the rostral extent of the locomotor command systems. A complete transection between the diencephalon and mesencephalon (Fig. 6A, n = 4) did not impair the initiation of locomotion, either spontaneously or in response to tactile stimulation of the head (Fig. 6B). These lesioned animals were virtually indistinguishable from normal animals, and could explore their surroundings, accelerate or decelerate, turn and make directional adjustments, and withdraw or swim backwards. Complete transections were also attempted between the mesencephalon and rhombencephalon (not shown), but many of the animals did not survive this lesion, perhaps because their respiratory centers were compromised 43. However, in one animal that was respiring after this lesion, spontaneous and sensory evoked lo-
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Fig. 5. Summary of the effects on locomotion of sparing various regions of the rostral spinal cord. A-C; sparing the lateral, ventrolateral, or dorsolateral areas of the spinal cord did not abolish the initiation of locomotion, either spontaneously or in response to tactile stimulation of the head. D,E: in 77% of the animals in which the medial areas of the spinal cord were spared (E), locomotion could not be initiated by any means, while in 23% of the animals with this type of lesion (I3) the initiation of locomotion was still possible to varying degrees.
comotion were abolished. Taken together, these results suggest that structures above the mesencephalon are not essential for the initiation of locomotion, and that the mesencephalon may be an important motor integration center in the lamprey. DISCUSSION The basic protocol in the present study involved assessing the behavioral capabilities of lampreys in which various tracts in the rostral spinal cord were spared. There are a couple of possible problems with these procedures. For example, because of trauma or anoxia, not all of the spared parts of the spinal cord that were identified histologically might be functional and conduct action potentials. This is probably not a significant factor, since the results are rather consistent but a mechanism based on trauma would be
quite variable. Anoxia is probably not a significant problem since the lamprey spinal cord is not invaded by blood vessels but receives its oxygen and nutrients via diffusion from the cerebrospinal fluid 31. It is likely that reticulospinal neurons initiate locomotion in the lamprey (see below), but several other mechanisms might be involved. First, it is possible that mechanosensory feedback (cf. ref. 44) might couple activity to the spinal cord below the lesion and produce locomotion. This seems unlikely since the length of spinal cord above the rostral lesions that could produce locomotor activity was relatively short (i.e. 2 - 4 segments), and animals with complete spinal transections at this level could not initiate locomotion. Second, descending propriospinal pathways in the lateral parts of the spinal cord 3,32 might evoke activity below a partial spinal lesion and produce locomotion. One argument against this possibility is
345 10, 12, 17, 21, 37, 41, 42, 46), yet these episodes can result spontaneously (i.e. without trigeminal activation) or from brief tactile stimulation of the head which would activate trigeminal afferents only transiently. Also, in in vitro brain/spinal cord preparations 16 in which the brain is bathed in low-calcium Ringer's (spinal cord in normal Ringer's), tactile stimulation of the head should still activate sodiumdependent action potentials in trigeminal afferents ~4 (and their descending axons) but does not initiate locomotor activity in the spinal cord (unpublished). Thus, trigeminal afferents probably mediate motor responses by activating neurons in the brainstem command system for locomotion. The above arguments suggest that the locomotor networks in spinallesioned animals in this study were activated by reticulospinal neurons that were part of the descending command system.
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Fig. 6. A: diagram of the brainstem from an adult silver lamprey (length, 29 cm; distance from head: 1,2 - 7.0 cm, 3,4 - 14.0 em) in which a complete transection was performed at the rostral border of the mesencephalon. B,C: simultaneous movements (B) and muscle activity (C) (see Materials and Methods, and Fig. 1). Brief tactile stimulation applied to the right side of the head (B2, arrow) elicited an S-shaped flexure reflex followed by escape locomotor activity. Note the left-right alternation of segmental activity (1-2 and 3-4) and the ipsilateral rostrocaudal phase-lag (1-4 and 2-3) during locomotion. IFI = 100 ms.
that pharmacological activation of locomotor patterns in a rostral section of an in vitro spinal cord produces very little or no ventral root burst activity in a more caudal section of the spinal cord bathed in normal Ringer's (ref. 3 and unpublished). And again, the length of spinal cord above the rostral lesions was relatively short. Third, tactile stimulation of the head activates trigeminal sensory neurons 15'34 which have relatively short descending axonal branches in the dorsolateral spinal cord 23 that might directly activate locomotor patterns in the spinal cord (cf. ref. 10). This also seems unlikely because long-duration episodes of locomotion would appear to require continuous activation by descending pathways (cf. refs. 6,
The results from partial lesions in the rostral spinal cord indicate that the lateral spinal tracts are always sufficient for initiating locomotion (Fig. 2B), and that in most cases these tracts are also necessary for locomotor behavior (Figs. 2A, 5E). This strongly suggests that locomotor command centers in the lamprey brainstem send a significant descending projection in the lateral tracts in order to activate spinal locomotor networks. Nonetheless, the descending command signal for locomotion may be transmitted in multiple pathways rather than in a single spinal tract. Both the dorsolateral or ventrolateral tracts can support the initiation of locomotion, suggesting that there may be two separate descending pathways with perhaps overlapping roles. In addition, broad areas of the lateral spinal cord, from the very lateral edge to intermediate areas, can support locomotor behavior, although this could be a physical constraint imposed by the thin, ribbon-like profile of the lamprey spinal cord. Finally, occasional shifts in the positions of descending axons have been reported 33, and this may explain in part why a relatively small fraction of the animals with spared medial spinal tracts could still initiate locomotion (Fig. 5D). In lampreys with a rostral spinal hemisection, the initiation of flexure and locomotor responses was still possible but in many cases the responses were biased
346 in the direction of the spared half of the spinal cord (Fig. 1B). This suggests that the descending command pathways in each lateral sector of the spinal cord activate predominantly but perhaps not exclu-
sively the spinal motor networks on the same side. Interestingly, most if not all the reticulospinal neurons have uncrossed descending axons 22'29'31.
Role of the large reticulospinal Muller cells There is presently some uncertainty whether the large reticulospinal Muller cells are involved in the initiation of locomotor behavior 27-29'31. For example, Muller cells are active during spontaneous episodes of locomotion 13 which is one of the properties that might be expected of neurons involved in initiating behavior. In addition, several lines of evidence indicate that these cells have inputs to the spinal locomotor networks 2"27"28. However, in other vertebrates, brainstem systems that are not involved in initiating locomotion (e.g. vestibulospinal) also have these properties 24"35. In the lamprey, the Muller cells send descending axons in the ventromedial spinal cord near the central canal 29 (see Fig. 7B), and the present results indicate that the medial spinal tracts are neither necessary (Fig. 2B) nor usually sufficient (Fig. 2A) for supporting locomotor responses. This would suggest that Muller cells do not contribute significantly to the activation of spinal locomotor networks 17"18. At present the results do not clarify the role of the Muller cells. This relatively fast-conducting descending system might contribute weakly to locomotor initiation, modulate on-going locomotion that is activated by other descending systems 19, or perhaps activate other responses (e.g. flexure reflexes). Further experiments are needed to resolve this issue.
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Fig. 7. A: diagram of lamprey brain (rostral up), showing the location of reticular nuclei26 in the mesencephalon (MT), isthmus (ARRN) and rhombencephalon (MRRN, PRRN). B: schematic drawing of a cross-section of the lamprey spinal cord, showing many of the large descending axons of the Muller cells29in the ventromedial tracts of the spinal cord. The very large Mauthner cell axons are shown in the intermediate areas of the lateral spinal cord 29. MT, mesencephalic tegmentum; ARRN, anterior rhombencephalic reticular n.; MRRN, medial rhombencephalic reticular n.; PRRN, posterior rhombencephalic reticular n. (A) redrawn from ref. 26; (B) redrawn from ref. 29.
In the lamprey brainstem, 4 nuclei (MT, mesencephalic tegmentum; A R R N , anterior rhombencephalic reticular n. ; M R R N , medial rhombencephalic reticular n.; P R R N , posterior rhombencephalic reticular n.; Fig. 7A) contain reticulospinal neurons 22"26. Although the Muller cells, which are located in the rostral 3 nuclei, have long descending axons in the medial spinal tracts 33 (Fig. 7B), these neurons probably do not contribute significantly to the initiation of locomotion (see above). Some of the smaller reticuiospinal neurons in the M R R N and P R R N are known to descend in the ventrolateral and dorsolateral spinal tracts, respectively 3°-32, which
347 are important descending pathways for the initiation of locomotion (Fig. 5). Preliminary results indicate that retrograde transport of horseradish peroxidase (HRP) through ventrolateral and dorsolateral spinal tracts labels neurons largely in the MRRN and PRRN, respectively19. Chemical stimulation in these areas with excitatory amino acids, or their agonists, can elicit locomotor patterns in in vitro preparations ~s. Thus, at least some of the medium to smaller neurons in the MRRN and PRRN with lateral descending axons are very likely to be involved in activating spinal motor networks for locomotion. Other techniques will be necessary to determine which of the neurons in these nuclei participate in the initiation of locomotion.
Organization of the brainstem command system Transections made at various levels in the brainstem suggest that the rostral extent of the locomotor command system is in the mesencephalon (Fig. 6). The above discussion suggests that the descending or output neurons in the command system may reside in the MRRN and/or PRRN. These results imply that the brainstem command system has several levels, which might include an input area (e.g. mesencephalon) for integration of information and a descending relay area (e.g. MRRN and/or PRRN) for activating spinal motor networks 17 (also see ref. 12). Comparison to other vertebrates In several classes of vertebrates, the lateral spinal tracts have been shown to carry a descending signal for activating locomotion (refs. 5, 38, 41, 48; reviewed in refs. 12, 17). For example, in the cat the ventrolateral spinal tracts must be intact for the initiation of locomotion, either spontaneously 5'48 or by stimulation in the mesencephalic locomotor regions 3s (MLR). Similar results stressing the importance of the ventrolateral spinal tracts for locomotion have been obtained in the monkey 6, stingray 46, and birds 41. In general, the dorsolateral spinal tracts alone could not support the initiation of locomotor behavior, although recent evidence in the cat suggests that descending pathways in either the ventrolateral or dorsolateral spinal tracts can produce locomotion 49. Furthermore, in spinal animals, stimulation in the lateral spinal funiculi can elicit locomotion in several classes of vertebrates (cf. refs. 10, 42, 46;
reviewed in ref. 17), although in the stingray, stimulation in the dorsolateral or ventrolateral tracts was effective46. In contrast, the results described here for the lamprey indicate that the ventrolateral and dorsolateral spinal tracts can both support the initiation of locomotion. However, it should be stressed that the lamprey spinal cord is relatively flat compared to that in higher vertebrates, and so it is difficult to make strict comparisons of spinal tracts. In the cat, structures rostral to the mesencephalon are important for the normal initiation of behaviors that incorporate locomotion 1"37. In contrast, lampreys with a transection at the rostral border of the mesencephalon appear to locomote and behave normally. In lower vertebrates the mesencephalon is thought to be a major sensorimotor integration area 22'25, while in higher vertebrates more rostral structures such as the basal ganglia, thalamus, subthalamus, hypothalamus, and perhaps the cortex seem to be important for integrating locomotor responses 1"37.However, in a wide range of vertebrates, including the lamprey, electrical and/or chemical stimulation in roughly similar locomotor regions extending from the mesencephalon to the caudal brainstem can elicit locomotion (refs. 6-8, 11, 21, 40; reviewed in refs. 12, 17, 37). In particular, the gigantocellular reticular formation in mammals and birds appears to be an important brainstem relay for the descending activation of spinal motor networks 11'12' 36,39 and stimulation in this area can elicit locomotion 7's,4°. These descending brainstem relay areas in higher vertebrates might be roughly analogous to some of the descending neurons in the MRRN and/or PRRN in the lamprey.
CONCLUSIONS The present results in the lamprey show that the lateral spinal tracts are important for the normal initiation of locomotion. Relatively small reticulospinal neurons in the MRRN and PRRN with lateral descending axons may be involved in activating spinal motor networks for locomotion. The medial spinal tracts, which contain the descending axons from Muller cells, were neither necessary nor generally sufficient to support the normal initiation of locomotion, suggesting that these cells do not contribute signifi-
348 cantly to t h e initiation of l o c o m o t i o n l T h e l o c o m o t o r
ACKNOWLEDGEMENTS
c o m m a n d system is l o c a t e d b e t w e e n the rostral bord e r of t h e m e s e n c e p h a l o n and the c a u d a l b r a i n s t e m
I w o u l d like to t h a n k D. L a w r e n c e and B. N e i l s e n
and m a y h a v e several levels for i n t e g r a t i n g i n f o r m a -
for technical s u p p o r t , and C h a r l o t t e W o h l e n b e r g and
tion and relaying activity to the spinal m o t o r net-
H e l e n D u e r for h e l p with the histology. S u p p o r t e d by
works. W o r k is n o w in p r o g r e s s to d e t e r m i n e h o w this
N I H G r a n t NS23216, S C R F G r a n t 501-5, and I A S
c o m m a n d system functions in activating and m o d -
G r a n t 85-3 a w a r d e d to A . D . M .
ulating l o c o m o t o r p a t t e r n s to a d a p t t h e m to the on-
grateful for initial s u p p o r t and r e s o u r c e s p r o v i d e d by P. G e t t i n g f r o m N I H G r a n t NS17328.
going n e e d s of the animal.
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I am particularly
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349 Korn (Eds.), Neurobiology of the Mauthner Cell, Raven, New York, 1978, pp, 245-269. 30 Rovainen, C.M., Eiectrophysiology of vestibulospinal and vestibuloreticulospinal systems in lamprey, J. Neurophysiol., 42 (1979) 745-766. 31 Rovainen, C.M., The neurobiology of lampreys, Physiol. Rev., 59 (1979) 1007-1077. 32 Rovainen, C.M., Effects of groups of propriospinal interneurons on fictive swimming in the isolated spinal cord of the lamprey, J. Neurophysiol., 54 (1985) 299-317. 33 Rovainen, C.M., Johnson, P.A., Roach, E.A. and Mankowsky, J.A., Projections of individual axons in lamprey spinal cord determined by tracing through serial sections, J. Comp. Neurol., 149 (1973) 193-201. 34 Rovainen, C.M. and Yan, Q., Sensory responses of dorsal cells in the lamprey brain, J. Comp. Physiol., 156 (1985) 181-183. 35 Russell, D.F. and Zajac, F.E., Effects of stimulating Deiters' nucleus and medial longitudinal fasciculus on the timing of the fictive locomotor rhythm induced in cats by DOPA, Brain Research, 177 (1979) 588-592. 36 Shefchyk, S.J., Jell, R.M. and Jordan, L.M., Reversible cooling of the brainstem reveals areas required for mesencephalic locomotor region evoked treadmill locomotion, Exp. Brain Res., 56 (1984) 257-262. 37 Shik, M.L. and Orlovsky, G.N., Neurophysiology of locomotor automatism, Physiol. Rev., 56 (1976) 465-501. 38 Steeves, J.D. and Jordan, L.M., Localization of a descending pathway in the spinal cord which is necessary for controlled treadmill locomotion, Neurosci. Lett., 20 (1980) 283-288. 39 Steeves, J.D. and Jordan, L.M., Autoradiographic demonstration of the projections from the mesencephalic locomotor region, Brain Research, 307 (1984) 263-276. 40 Steeves, J.D., Sholomenko, G.N. and Webster, D.M.S., Stimulation of the pontomedullary reticular formation ini-
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tiates locomotion in decerebrate birds, Brain Research, 401 (1987) 205-212. Steeves, J.D. and Weinstein, G.N., Brainstem areas and descending pathways for the initiation of flying and walking in birds, Soc. Neurosci. Abstr., 10 (1984) 30. Stein, P.S.G., Swimming movements elicited by electrical stimulation of the turtle spinal cord: the high spinal preparation, J. Comp. Physiol., 124 (1978) 203-210. Thompson, K.J., Organization of inputs to motoneurons during fictive respiration in the isolated lamprey brain, J. Comp. Physiol., 157 (1985) 291-302. Wallen, P., Spinal mechanisms controlling locomotion in dogfish and lamprey, Acta Physiol. Scand., Suppl. 503 (1982) 1-45. Wallen, P. and Williams, T., Fictive locomotion in the lamprey spinal cord in vitro compared with swimming in the intact and spinal animal, J. Physiol. (Lond.), 347 (1983) 225-239. Williams, B.J., Livingston, C.A. and Leonard, R.B., Spinal cord pathways involved in initiation of swimming in the stingray, Dasyatis sabina: spinal cord stimulation and lesions, J. Neurophysiol., 51 (1984)578-591. Williams, T., Mechanical and neural patterns underlying swimming by lateral undulations: review of studies on fish, amphibia and lamprey. In S. Grillner, P. Stein, D. Stuart, H. Forssberg and R. Herman, Neurobiology of Vertebrate Locomotion, Wenner-Gren Symposium Series, Vol. 45, MacMillan, London, 1986, pp. 141-155. Windle, W.F., Smart, J.O. and Beers, J.J., Residual function after subtotal spinal cord transection in adult cats, Neurology, 8 (1958) 518-521. Yamaguchi, T., Descending pathways eliciting forelimb stepping in the lateral funiculus: experimental studies with stimulation and lesion of the cervical cord in decerebrate cats, Brain Research, 379 (1986) 125-136.
338 BRE 13857
Brainstem command systems for locomotion in the lamprey" localization of descending pathways in the spinal cord Andrew D. McClellan Department of Physiology and Biophysics, University of lowa, Iowa City, 1A 52242 (U.S.A.) (Accepted 8 March 1988)
Key words: Locomotion; Command system; Descending pathway; Brainstem locomotor region; Lamprey
The lamprey brainstem contains a 'command system' which descends into the spinal cord to activate motor networks and initiate locomotion. In the present study, partial lesions were made in the rostral spinal cord in order to spare various tracts and determine which tracts carry the descending command signal to the spinal cord. Sparing the medial areas of the rostral spinal cord usually blocked both sensory-evoked and spontaneous locomotion, while sparing the lateral regions of the rostral spinal cord did not abolish voluntary locomotor activity. Either the ventrolateral or dorsolateral spinal tracts could support the initiation of locomotion. Brainstem structures rostral to the mesencephalon were not necessary for the initiation of locomotor behavior. The data indicate that the lateral spinal tracts contain a significant part of the descending command pathway for locomotion. In contrast, the medial spinal tracts were neither necessary nor usually sufficient to support locomotor behavior, suggesting that the larger reticulospinal Muller cells, which project in these tracts, do not contribute significantly to the initiation of locomotion. INTRODUCTION Spinal m o t o r networks for locomotion are thought to be activated by descending c o m m a n d or initiation centers in the brainstem (reviewed in refs. 1, 12, 17, 37). In a number of vertebrates, ' l o c o m o t o r regions' have been d e m o n s t r a t e d in the brainstem with electrical and/or chemical microstimulation and are organized as parallel strips on each side of the midline from the mesencephalon to the lower brainstem (reviewed in ref. 17). F o r example, microstimulation within the 'mesencephalic l o c o m o t o r region' ( M L R ) and in medial reticular nuclei can elicit well coordinated locomotion in the spinal cord 5'8'12'20'37"40. Finally, spinal lesion studies in a n u m b e r of vertebrates indicate that the lateral or ventrolateral tracts are necessary for the initiation of locomotion 3' 17,3S,41,42,46. The lamprey, a lower v e r t e b r a t e , has recently become a useful p r e p a r a t i o n for l o c o m o t o r system studies 9'31. The l a m p r e y nervous system is comparatively simpler than those of higher vertebrates and can produce l o c o m o t o r activity under in vitro conditions (reviewed in refs. 9, 18, 31). F o r example, in an in vitro
brainstem/spinal cord p r e p a r a t i o n , l o c o m o t o r activity can be elicited by sensory stimulation of the head and by electrical or chemical microstimulation of 'locomotor c o m m a n d regions' in the b r a i n s t e m 16,1s.2°. In order to u n d e r s t a n d how these brainstem command systems function, it is necessary to d e t e r m i n e the locations of neurons in the system so that they can be characterized with intracellular recordings. In the present study, partial lesions of the rostral spinal cord were used to spare various descending tracts in o r d e r to determine which descending tracts might be used by brainstem c o m m a n d neurons for locomotion. After partial spinal lesions, l o c o m o t o r capabilities were tested in whole-animals with natural tactile stimuli. These experiments indicate that the lateral spinal tracts can support the initiation of locomotion but the medial spinal tracts are n e i t h e r necessary nor usually sufficient to support l o c o m o t o r behavior. Preliminary accounts of this study have a p p e a r e d 17'19. MATERIALS AND METHODS The purpose of this study was to d e t e r m i n e which
Correspondence: A.D. McClellan, Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
339 spinal tracts are necessary for the initiation of locomotion in the lamprey. The basic paradigm used in this project involved several steps: (a) partial lesions were made in the very rostral spinal cord in order to spare various tracts; (b) the behavioral capabilities of the spinal-lesioned animals were observed; and (c) the extent of the partial spinal lesions was histologically determined. The effects of partial spinal lesions were tested in whole-animals because it is much easier to elicit locomotor activity than, for example, in in vitro preparations. Locomotor capabilities were tested with tactile stimuli rather than with microstimulation of brainstem locomotor regions, which might not project into all of the descending spinal tracts normally used for the initiation of locomotion. Spinal lesions. The effects of partial lesions were tested in several types of lampreys: adult silver lampreys (I. unicuspis; n = 59), feeding and spawning phase sea lampreys (P. marinus; n = 110), and larval sea lamprey (n = 50). Animals were anesthetized with methanesulphanate (MS-222, 0.1 mM) and placed on ice. A small dorsal incision was made between the first and second gill slit and a section of the rostral spinal cord was exposed. Localized spinal lesions were made at segments 2 - 4 (distance from anterior rim of sucker: 2.5-3.0 cm in adults, 1.25 cm in lavrae), which is about 0.5-1.5 cm caudal to the obex, depending on the size of the animal. Rostral spinal lesions were used so that there would be a relatively short length of spinal cord above the lesion, thus making it less likely for propriospinal pathways (i.e. spinal coordinating systems) or sensory feedback to couple locomotor activity across the lesion to the lower body (cf. refs. 3, 44). Complete spinal transections at this same level always eliminated the initiation of locomotor behavior. Partial spinal lesions that encompassed the entire dorsoventral thickness of the spinal cord (e.g. medial, lateral and hemisection lesions) were made with iris scissors. These manipulations would destroy both the axonal tracts as well as possible cell bodies in the lesioned part of the spinal cord. Lesions which encompassed only the dorsal or ventral parts of the thin spinal cord were made by teasing away the outermost layers of the spinal cord. In the case of ventral lesions, the exposed spinal cord was freed for 5-10 mm and twisted to expose the ventral surface. After making the lesions, the incision was sutured and the animals were indi-
vidually marked and returned to their tanks for at least 24 h. All animals were kept at 5 °C.
Behavioral observations and EMG recordings. Lampreys were placed in a tank, either 40 x 70 cm or 22 x 40 cm depending on the size of the animal, which contained 2 - 6 cm of water at 5-10 °C. The locomotor responses of all animals were recorded with a standard video system (Panasonic PV-1535 recorder and PK-452 camera) at 30 frames/s. In some animals, the video record was time-linked to simultaneous recordings of muscle activity from various points along the body. Muscle recordings were made with pairs of copper wire (50-100 p m diameter, depending on animal size), insulated except at the tip, which were inserted into body musculature with a syringe needle. Behavioral responses were analyzed by tracing sequential frames of the video record displayed on a monitor screen. In normal animals, brief tactile stimulation applied to the anterior head area or to the tail elicits a flexure response, which usually moves the stimulated body region away from the stimulus, followed by escape locomotion 16'2°. Voluntary locomotion can also occur spontaneously. In spinal-lesioned animals, special attention was given to several features of locomotor performance: spontaneous swimming, locomotor responses elicited by tactile stimulation of the head or the tail, turning, exploratory behavior (i.e. long-duration swimming along the perimeter of the tank), equilibrium, and backward swimming.
Histological processing of lesioned spinal cords. Animals were anesthetized (see above), and a section of the spinal cord/notochord containing the partial spinal lesion was removed. The tissue was fixed in 10% Formalin, and histologically processed for sectioning at 5-20 p m in paraffin. The histological sections containing the spinal lesions were traced with a camera lucida. RESULTS
Behavioral observations: adult Controls. In normal adult lampreys (n = 25) with intact spinal cords (Fig. l-A1), locomotion could be initiated spontaneously or by tactile stimulation of the anterior area of the head 16, both of which presumably activate a brainstem command system that descends into the spinal cord to excite locomotor net-
340 works 16'17. F o r example, stimulation of the left side of the head elicited an S-shaped flexure response, in which the head turned to the right away from the stimulus, followed by escape locomotion during which body undulations travelled towards the tail (Fig. l - A 2 ) . The flexure c o m p o n e n t was largely produced by contraction of rostral b o d y musculature on the same side as the direction of turning (initial burst in trace 1, Fig. l - A 3 ) . Subsequent l o c o m o t o r activity was characterized by two features45'47: a l e f t - r i g h t alternation of muscle activity at the same segmental level ( 1 - 2 and 3 - 4 , Fig. l - A 3 ) and a rostrocaudal phase-lag in muscle activity on the same side ( 1 - 4 and 2 - 3 ) . Flexure responses and l o c o m o t i o n could also be elicited by tactile stimulation of the tail, which activates an ascending c o m m a n d p a t h w a y 2°. Continued activation of this ascending pathway can give
rise to slow, m o n o t o n o u s l o c o m o t o r patterns even in spinal animals. H o w e v e r , in n o r m a l animals brief tail stimulation can trigger long-duration, goal-directed episodes of locomotion, including turning, acceleration, deceleration, and exploratory behavior, that would p r e s u m a b l y require the participation of locom o t o r centers in the brainstem 17. Hemisections of the rostral spinal cord. Some of the adult lampreys in which the right or left regions of the spinal cord were spared displayed relatively n o r m a l l o c o m o t o r behavior (n = 7). In other animals (n = 11), however, there was a directional bias of the flexure reflexes and/or the l o c o m o t o r responses to the same side as the spared half of the spinal cord. F o r example, in an animal with the left areas of the rostral spinal cord spared (Fig. l-B1), tactile stimulation of either side of the head elicited turning re-
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Fig. 1. In Figs. 1-4, various tracts in the very rostral spinal cords were spared in lampreys (A1,B1). Sequential locomotor movements were recorded (A2,B2; proceeding left to right) and time linked to simultaneous recordings of activity in body musculature (A3,B3; 14, positions marked in movement panels). The dots in A2 (B2) serve as a fixed reference line as well as frame markers that correspond in time to the dots below the record in A3 (B3). A: normal adult silver lamprey (length, 30 cm; distance from head: 1,2 - 7.0 cm, 3,4 15.0 cm). S-shaped flexure responses, directed away from the stimulus, followed by escape locomotor activity elicited by brief tactile stimulation applied to the left anterior part of the head (A2, arrow). Note the left-right alternation (1-2 and 3-4) and ipsilateral phase-lag (1-4 and 2-3) during locomotor activity. B: adult silver lamprey (length, 31 cm) with a rostral lesion sparing the left half of the spinal cord (distance from head: lesion - 2.75 cm, 1,2 - 7.0 cm, 3,4 - 15.5 cm). Brief tactile stimulation of the left side of the head (B2, arrow) elicited a C-shaped flexure reflex, which was always directed towards the left, followed by locomotor activity (B2,B3). Interframe interval (IFI) = 67 ms.
341 sponses to the left (Fig. 1-B2) and flexure m o t o r activity on the left side (Fig. 1-B3; initial burst in trace 2). The subsequent locomotor responses and m o t o r activity could be symmetrical (Fig. 1-B2, 1-B3), or the direction of swimming could be biased toward the intact side of the spinal cord so that animals swam in a circle. In some cases, circling during swimming was accompanied by spinning movements and impaired equilibrium. Sparing the medial spinal tracts. In the lamprey, the ventromedial spinal cord contains the descending axons of the larger reticulospinal neurons (i.e. Muller cells 29) (see Fig. 7B). Since these neurons may be involved in the descending control of locomotor responses 29'31, the behavioral capabilities of lampreys with spared medial regions of the rostral spinal cord were examined (Fig. 2-A1,SE). The majority of animals (77%, 26 of 34 animals) with this type of lesion were unable to initiate flexure reflexes or locomotion either spontaneously or in response to tactile stimulation of the head (Fig. 2-A2, arrow; Fig. 2-A3, bar).
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In many ways these animals resembled spinalized lampreys 2°, in which tail-fin stimulation could elicit slow undulations but rostral stimulation was ineffective. In the remaining 23% of the animals with spared medial areas of the rostral spinal cord, initiation of locomotion was possible to varying degrees (Fig. 5D). However, in some of these latter animals the spared medial areas of the spinal cord were somewhat large and included parts of the intermediate spinal cord. Thus, in the majority of cases, the medial spinal tracts were insufficient to support the initiation of locomotion, suggesting that the lateral spinal tracts are more important in the descending control of locomotor behavior. Sparing the lateral spinal tracts. Of the 32 lampreys that were tested in which the lateral regions of the rostral spinal cord were spared (Fig. 2-B1,5A), all were capable of initiating relatively normal locomotion spontaneously and in response to tactile stimulation of the head or tail fin. For example, tactile stimulation of the left side of the head (Fig. 2-B2, arrow)
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Fig. 2. A: effects of sparing the medial regions of the rostral spinal cord (A1) in an adult silver lamprey (length, 28 cm; distance from head: lesion - 2.5 cm, 1,2 - 6.75 cm, 3,4 - 13.25 cm), Continuous tactile stimulation of the right part of the head (A2, arrow; A3, bar) was ineffective in eliciting locomotor responses. Notice the very weak bending movements of the tail. B: partial rostral lesions that spared the lateral areas of the rostral spinal cord (B 1) in an adult silver lamprey (length, 27 cm; distance from head: lesion - 3.0 cm, 1,2 7.0 cm, 3,4 - 14.0 cm). Brief tactile stimulation of the left part of the head (B2, arrow) elicited relatively normal flexure activity, directed away from the stimulus, followed by escape locomotor activity (B2,B3). Left-right alternation of segmental motor activity (1-2 and 3-4) and an ipsilateral phase lag (1-4 and 2-3) were present during locomotor patterns. IFI = 667 ms (A), 100 ms (B). -
342 elicited an S-shaped flexure response, which was directed away from the stimulus (Fig. 2-B2) and was produced primarily by rostral body muscle activity on the right side (initial burst in trace 1, Fig. 2-B3). The subsequent escape locomotor movements (Fig. 2B2) were accompanied by a left-right alternation (1-2 and 3-4, Fig. 2-B3) and a rostrocaudal phaselag (1-4 and 2-3). These results suggest that the lateral spinal tracts are important for the initiation of locomotion, and that the medial tracts are not necessary for normal locomotion. Coupled with the results from sparing the medial spinal tracts, the data imply that the reticulospinal Muller cells, which have medial descending axons (Fig. 7B), do not contribute significantly to the initiation of locomotor responses. Sparing the dorsolateral or ventrolateral spinal tracts. When the ventrolateral areas of the rostral spinal cord were spared (Fig. 3-A1), 86% of the lampreys (25 of 29) still were capable of initiating locomotion, either spontaneously or in response to tactile stimulation of the head. For example, tactile stimulation of the anterior part of the head elicited a flexure
reflex, which included a weak withdrawal response, followed by escape locomotion (Fig. 3-A2). The motor activity produced during these types of locomotor responses (Fig. 3-A3) was indistinguishable from those in normal, unlesioned animals. Similarly, in lampreys in which the dorsolateral regions of the rostral spinal cord were spared (Fig. 3-B1), locomotor responses and motor activity (Fig. 3-B2, 3-B3) appeared quite normal in 64% of the animals (11 of 17). Since the teasing method used to make this latter type of lesion may have traumatized the thin, ribbonlike lamprey spinal cord, it is not surprising that some of the animals were unable to locomote. Taken together, the data indicate that either the dorsolateral or ventrolateral spinal tracts can support the initiation of relatively normal locomotor behavior (Fig. 5B,C).
Other partial spinal lesions. Partial spinal lesions were also made that either spared the intermediate regions of the rostral spinal cord (n = 6), including the dorsal and ventral horns, or spared all areas of the rostral spinal cord except the intermediate re-
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Fig. 3. A: adult silver lamprey (length, 27 cm) with the ventrolateral tracts spared in the rostral spinal cord ( A I ) (distance from head: lesion - 3.0 cm, 1,2 - 6.0 cm, 3,4 - 14.0 cm). Brief tactile stimulation of the medial part of the anterior head (A2, arrow) elicited a backward flexure m o v e m e n t followed by escape locomotion (A2,A3). B: effects of sparing the dorsolateral spinal tracts (B 1) in an adult silver lamprey (length, 32 cm; distance from head: lesion - 2.5 cm, 1,2 - 7.0 cm, 3,4 - 15.5 cm). Brief tactile stimulation of the right part of the head (B2, arrow) elicited a flexure response and, after a delay, escape locomotor activity (B2,B3). IFI = 100 ms (A), 167 ms (B).
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Fig. 4. Locomotor responses in larval sea lamprey with partial lesions in the rostral spinal cord. A: effects of sparing the medial areas of the spinal cord (A1) in a larval lamprey (length, 16 cm; distance from head: lesion - 1.2 cm, 1,2 - 3.0 cm, 3,4 - 7.5 cm). Continuous tactile stimulation of the medial part of the head (A2, arrow; A3, bar) was ineffective in eliciting flexure or locomotor responses. B: sparing the lateral regions of the spinal cord (B 1) in a larval lamprey (length, 15.5 cm; distance from head: lesion - 1.1 cm, 1.2 - 3.25 cm, 3.4 - 7.5 cm). Light tactile stimulation of the left part of the head (B2, arrow) elicited a delayed flexure response that quickly gave rise to escape locomotor activity (B2,B3). Note the left-right alternation of segmental motor activity ( 1 - 2 and 3 - 4 ) and the ipsilateral phase-lag ( 1 - 4 and 2 - 3 ) during the initial locomotor patterns. IFI = 667 ms (A), 167 ms (B).
gions (n = 3). With both types of partial spinal lesions, the initiation of flexure reflexes and locomotion was not blocked (not shown).
Behavioral observations: larval lampreys The results from larval lampreys, both normal animals and those with partial spinal lesions, were similar to those obtained from adults (Fig. 5). Specifically, sparing the medial regions of the rostral spinal cord (n = 18) blocked spontaneous as well as sensory-evoked locomotor responses in 89% of the animals (Fig. 4A). In contrast, sparing the lateral areas of the rostral spinal cord (n = 22) allowed the initiation of locomotor movements and motor activity in all animals (Fig. 4B). Thus, in larval animals the descending command pathways for the initiation of locomotion appear to be located in similar lateral parts of the spinal cord as those in adult lampreys (Fig. 5).
Brainstem lesions Two types of lesions were made in the brainstem in order to determine the rostral extent of the locomotor command systems. A complete transection between the diencephalon and mesencephalon (Fig. 6A, n = 4) did not impair the initiation of locomotion, either spontaneously or in response to tactile stimulation of the head (Fig. 6B). These lesioned animals were virtually indistinguishable from normal animals, and could explore their surroundings, accelerate or decelerate, turn and make directional adjustments, and withdraw or swim backwards. Complete transections were also attempted between the mesencephalon and rhombencephalon (not shown), but many of the animals did not survive this lesion, perhaps because their respiratory centers were compromised 43. However, in one animal that was respiring after this lesion, spontaneous and sensory evoked lo-
344 Locomotion
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Fig. 5. Summary of the effects on locomotion of sparing various regions of the rostral spinal cord. A-C; sparing the lateral, ventrolateral, or dorsolateral areas of the spinal cord did not abolish the initiation of locomotion, either spontaneously or in response to tactile stimulation of the head. D,E: in 77% of the animals in which the medial areas of the spinal cord were spared (E), locomotion could not be initiated by any means, while in 23% of the animals with this type of lesion (I3) the initiation of locomotion was still possible to varying degrees.
comotion were abolished. Taken together, these results suggest that structures above the mesencephalon are not essential for the initiation of locomotion, and that the mesencephalon may be an important motor integration center in the lamprey. DISCUSSION The basic protocol in the present study involved assessing the behavioral capabilities of lampreys in which various tracts in the rostral spinal cord were spared. There are a couple of possible problems with these procedures. For example, because of trauma or anoxia, not all of the spared parts of the spinal cord that were identified histologically might be functional and conduct action potentials. This is probably not a significant factor, since the results are rather consistent but a mechanism based on trauma would be
quite variable. Anoxia is probably not a significant problem since the lamprey spinal cord is not invaded by blood vessels but receives its oxygen and nutrients via diffusion from the cerebrospinal fluid 31. It is likely that reticulospinal neurons initiate locomotion in the lamprey (see below), but several other mechanisms might be involved. First, it is possible that mechanosensory feedback (cf. ref. 44) might couple activity to the spinal cord below the lesion and produce locomotion. This seems unlikely since the length of spinal cord above the rostral lesions that could produce locomotor activity was relatively short (i.e. 2 - 4 segments), and animals with complete spinal transections at this level could not initiate locomotion. Second, descending propriospinal pathways in the lateral parts of the spinal cord 3,32 might evoke activity below a partial spinal lesion and produce locomotion. One argument against this possibility is
345 10, 12, 17, 21, 37, 41, 42, 46), yet these episodes can result spontaneously (i.e. without trigeminal activation) or from brief tactile stimulation of the head which would activate trigeminal afferents only transiently. Also, in in vitro brain/spinal cord preparations 16 in which the brain is bathed in low-calcium Ringer's (spinal cord in normal Ringer's), tactile stimulation of the head should still activate sodiumdependent action potentials in trigeminal afferents ~4 (and their descending axons) but does not initiate locomotor activity in the spinal cord (unpublished). Thus, trigeminal afferents probably mediate motor responses by activating neurons in the brainstem command system for locomotion. The above arguments suggest that the locomotor networks in spinallesioned animals in this study were activated by reticulospinal neurons that were part of the descending command system.
13
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that pharmacological activation of locomotor patterns in a rostral section of an in vitro spinal cord produces very little or no ventral root burst activity in a more caudal section of the spinal cord bathed in normal Ringer's (ref. 3 and unpublished). And again, the length of spinal cord above the rostral lesions was relatively short. Third, tactile stimulation of the head activates trigeminal sensory neurons 15'34 which have relatively short descending axonal branches in the dorsolateral spinal cord 23 that might directly activate locomotor patterns in the spinal cord (cf. ref. 10). This also seems unlikely because long-duration episodes of locomotion would appear to require continuous activation by descending pathways (cf. refs. 6,
The results from partial lesions in the rostral spinal cord indicate that the lateral spinal tracts are always sufficient for initiating locomotion (Fig. 2B), and that in most cases these tracts are also necessary for locomotor behavior (Figs. 2A, 5E). This strongly suggests that locomotor command centers in the lamprey brainstem send a significant descending projection in the lateral tracts in order to activate spinal locomotor networks. Nonetheless, the descending command signal for locomotion may be transmitted in multiple pathways rather than in a single spinal tract. Both the dorsolateral or ventrolateral tracts can support the initiation of locomotion, suggesting that there may be two separate descending pathways with perhaps overlapping roles. In addition, broad areas of the lateral spinal cord, from the very lateral edge to intermediate areas, can support locomotor behavior, although this could be a physical constraint imposed by the thin, ribbon-like profile of the lamprey spinal cord. Finally, occasional shifts in the positions of descending axons have been reported 33, and this may explain in part why a relatively small fraction of the animals with spared medial spinal tracts could still initiate locomotion (Fig. 5D). In lampreys with a rostral spinal hemisection, the initiation of flexure and locomotor responses was still possible but in many cases the responses were biased
346 in the direction of the spared half of the spinal cord (Fig. 1B). This suggests that the descending command pathways in each lateral sector of the spinal cord activate predominantly but perhaps not exclu-
sively the spinal motor networks on the same side. Interestingly, most if not all the reticulospinal neurons have uncrossed descending axons 22'29'31.
Role of the large reticulospinal Muller cells There is presently some uncertainty whether the large reticulospinal Muller cells are involved in the initiation of locomotor behavior 27-29'31. For example, Muller cells are active during spontaneous episodes of locomotion 13 which is one of the properties that might be expected of neurons involved in initiating behavior. In addition, several lines of evidence indicate that these cells have inputs to the spinal locomotor networks 2"27"28. However, in other vertebrates, brainstem systems that are not involved in initiating locomotion (e.g. vestibulospinal) also have these properties 24"35. In the lamprey, the Muller cells send descending axons in the ventromedial spinal cord near the central canal 29 (see Fig. 7B), and the present results indicate that the medial spinal tracts are neither necessary (Fig. 2B) nor usually sufficient (Fig. 2A) for supporting locomotor responses. This would suggest that Muller cells do not contribute significantly to the activation of spinal locomotor networks 17"18. At present the results do not clarify the role of the Muller cells. This relatively fast-conducting descending system might contribute weakly to locomotor initiation, modulate on-going locomotion that is activated by other descending systems 19, or perhaps activate other responses (e.g. flexure reflexes). Further experiments are needed to resolve this issue.
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Fig. 7. A: diagram of lamprey brain (rostral up), showing the location of reticular nuclei26 in the mesencephalon (MT), isthmus (ARRN) and rhombencephalon (MRRN, PRRN). B: schematic drawing of a cross-section of the lamprey spinal cord, showing many of the large descending axons of the Muller cells29in the ventromedial tracts of the spinal cord. The very large Mauthner cell axons are shown in the intermediate areas of the lateral spinal cord 29. MT, mesencephalic tegmentum; ARRN, anterior rhombencephalic reticular n.; MRRN, medial rhombencephalic reticular n.; PRRN, posterior rhombencephalic reticular n. (A) redrawn from ref. 26; (B) redrawn from ref. 29.
In the lamprey brainstem, 4 nuclei (MT, mesencephalic tegmentum; A R R N , anterior rhombencephalic reticular n. ; M R R N , medial rhombencephalic reticular n.; P R R N , posterior rhombencephalic reticular n.; Fig. 7A) contain reticulospinal neurons 22"26. Although the Muller cells, which are located in the rostral 3 nuclei, have long descending axons in the medial spinal tracts 33 (Fig. 7B), these neurons probably do not contribute significantly to the initiation of locomotion (see above). Some of the smaller reticuiospinal neurons in the M R R N and P R R N are known to descend in the ventrolateral and dorsolateral spinal tracts, respectively 3°-32, which
347 are important descending pathways for the initiation of locomotion (Fig. 5). Preliminary results indicate that retrograde transport of horseradish peroxidase (HRP) through ventrolateral and dorsolateral spinal tracts labels neurons largely in the MRRN and PRRN, respectively19. Chemical stimulation in these areas with excitatory amino acids, or their agonists, can elicit locomotor patterns in in vitro preparations ~s. Thus, at least some of the medium to smaller neurons in the MRRN and PRRN with lateral descending axons are very likely to be involved in activating spinal motor networks for locomotion. Other techniques will be necessary to determine which of the neurons in these nuclei participate in the initiation of locomotion.
Organization of the brainstem command system Transections made at various levels in the brainstem suggest that the rostral extent of the locomotor command system is in the mesencephalon (Fig. 6). The above discussion suggests that the descending or output neurons in the command system may reside in the MRRN and/or PRRN. These results imply that the brainstem command system has several levels, which might include an input area (e.g. mesencephalon) for integration of information and a descending relay area (e.g. MRRN and/or PRRN) for activating spinal motor networks 17 (also see ref. 12). Comparison to other vertebrates In several classes of vertebrates, the lateral spinal tracts have been shown to carry a descending signal for activating locomotion (refs. 5, 38, 41, 48; reviewed in refs. 12, 17). For example, in the cat the ventrolateral spinal tracts must be intact for the initiation of locomotion, either spontaneously 5'48 or by stimulation in the mesencephalic locomotor regions 3s (MLR). Similar results stressing the importance of the ventrolateral spinal tracts for locomotion have been obtained in the monkey 6, stingray 46, and birds 41. In general, the dorsolateral spinal tracts alone could not support the initiation of locomotor behavior, although recent evidence in the cat suggests that descending pathways in either the ventrolateral or dorsolateral spinal tracts can produce locomotion 49. Furthermore, in spinal animals, stimulation in the lateral spinal funiculi can elicit locomotion in several classes of vertebrates (cf. refs. 10, 42, 46;
reviewed in ref. 17), although in the stingray, stimulation in the dorsolateral or ventrolateral tracts was effective46. In contrast, the results described here for the lamprey indicate that the ventrolateral and dorsolateral spinal tracts can both support the initiation of locomotion. However, it should be stressed that the lamprey spinal cord is relatively flat compared to that in higher vertebrates, and so it is difficult to make strict comparisons of spinal tracts. In the cat, structures rostral to the mesencephalon are important for the normal initiation of behaviors that incorporate locomotion 1"37. In contrast, lampreys with a transection at the rostral border of the mesencephalon appear to locomote and behave normally. In lower vertebrates the mesencephalon is thought to be a major sensorimotor integration area 22'25, while in higher vertebrates more rostral structures such as the basal ganglia, thalamus, subthalamus, hypothalamus, and perhaps the cortex seem to be important for integrating locomotor responses 1"37.However, in a wide range of vertebrates, including the lamprey, electrical and/or chemical stimulation in roughly similar locomotor regions extending from the mesencephalon to the caudal brainstem can elicit locomotion (refs. 6-8, 11, 21, 40; reviewed in refs. 12, 17, 37). In particular, the gigantocellular reticular formation in mammals and birds appears to be an important brainstem relay for the descending activation of spinal motor networks 11'12' 36,39 and stimulation in this area can elicit locomotion 7's,4°. These descending brainstem relay areas in higher vertebrates might be roughly analogous to some of the descending neurons in the MRRN and/or PRRN in the lamprey.
CONCLUSIONS The present results in the lamprey show that the lateral spinal tracts are important for the normal initiation of locomotion. Relatively small reticulospinal neurons in the MRRN and PRRN with lateral descending axons may be involved in activating spinal motor networks for locomotion. The medial spinal tracts, which contain the descending axons from Muller cells, were neither necessary nor generally sufficient to support the normal initiation of locomotion, suggesting that these cells do not contribute signifi-
348 cantly to t h e initiation of l o c o m o t i o n l T h e l o c o m o t o r
ACKNOWLEDGEMENTS
c o m m a n d system is l o c a t e d b e t w e e n the rostral bord e r of t h e m e s e n c e p h a l o n and the c a u d a l b r a i n s t e m
I w o u l d like to t h a n k D. L a w r e n c e and B. N e i l s e n
and m a y h a v e several levels for i n t e g r a t i n g i n f o r m a -
for technical s u p p o r t , and C h a r l o t t e W o h l e n b e r g and
tion and relaying activity to the spinal m o t o r net-
H e l e n D u e r for h e l p with the histology. S u p p o r t e d by
works. W o r k is n o w in p r o g r e s s to d e t e r m i n e h o w this
N I H G r a n t NS23216, S C R F G r a n t 501-5, and I A S
c o m m a n d system functions in activating and m o d -
G r a n t 85-3 a w a r d e d to A . D . M .
ulating l o c o m o t o r p a t t e r n s to a d a p t t h e m to the on-
grateful for initial s u p p o r t and r e s o u r c e s p r o v i d e d by P. G e t t i n g f r o m N I H G r a n t NS17328.
going n e e d s of the animal.
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I am particularly
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