Department of Biological Control System, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
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ABSTRACT |
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Mori, Shigemi, Toshihiro Matsui, Bunya Kuze, Mitsuru Asanome, Katsumi Nakajima, and Kiyoji Matsuyama. Stimulation of a Restricted Region in the Midline Cerebellar White Matter Evokes Coordinated Quadrupedal Locomotion in the Decerebrate Cat. J. Neurophysiol. 82: 290-300, 1999. In the reflexively standing acute decerebrate cat, we have previously shown that pulse train microstimulation of the hook bundle of Russel in the midline of the cerebellar white matter, through which crossed fastigiofugal fibers decussate, augments the postural tone of neck, trunk, fore-, and hindlimb extensor muscles. In the present study we examined the possible role of such stimulation in evoking locomotion as the animal is supported by a rubber hammock with its feet contacting the moving surface of a treadmill. We were able to provoke well-coordinated, bilaterally symmetrical, fore- and hindlimb movements, whose cycle time and pattern were controlled by appropriate changes in stimulus intensity and treadmill speed. We carefully and systematically mapped this cerebellar locomotor region (CLR) through repeated dorsoventral penetrations with a glass-coated tungsten microelectrode in a single animal and between animals. We found that the optimal locus for evoking locomotion was centered on the midline, at Horsley-Clarke coordinates H0 and P7.0, and extended over a rostrocaudal and dorsolateral range of ~0.5 mm. The lowest effective stimulus intensity at the optimal site was in the range of 5-8 µA. Along penetration tracks to left or right of the midline, effective stimulus intensity increased and evoked locomotor patterns were no longer symmetrical, but rather shifted toward the contralateral limbs. In the same animals, controlled locomotion was evoked by stimulating the mesencephalic locomotor region (MLR). With concomitant stimulation of the optimal sites in the CLR and the MLR, each at subthreshold strength, locomotor movements identical to those seen with suprathreshold stimulation of each site alone were evoked. With concomitant stimulation at suprathreshold strength for each site, locomotion became vigorous, with a shortened cycle time. After making ablative lesions at either the CLR or MLR (unilateral or bilateral), controlled locomotion was still evoked at the prior stimulus strength by stimulating the remaining site. Together, these results demonstrate that selective stimulation of the hook bundle of Russel in the midsagittal plane of the cerebellar white matter evokes "controlled" locomotion identical to that evoked by stimulating the MLR. We have shown that the fastigial nucleus is one of the supraspinal locomotion inducing sites and that it can independently and simultaneously trigger brain stem and spinal locomotor subprograms formerly believed to be the domain of various brain stem regions including the MLR and the subthalamic locomotor region.
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INTRODUCTION |
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What is the role of the cerebellum and the cerebellar nuclei such
as the fastigial nucleus in the control of posture and locomotion? Orlovsky (1970b) first demonstrated in decerebellated
cats, whose nervous system was simplified by transection of the
neuraxis at the thalamic or rostral mesencephalic level, that
stimulation of the subthalamic locomotor region (SLR) in the lateral
hypothalamic area and the mesencephalic locomotor region (MLR) in the
posterior midbrain was still capable of evoking locomotor movements on
the surface of a moving treadmill. In these animals, however,
coordination of the fore- and hindlimbs was greatly disturbed, partly
because of exaggerated straightening of the forelimbs due to very
strong development of extensor rigidity. In the mesencephalic cat, the disturbances in coordination were more pronounced than those in the
thalamic cat. These findings led to the general conclusion that the
cerebellum is not directly involved in the initiation of locomotor
movements but is concerned mainly with coordination of limb movements
during ongoing locomotion (Armstrong 1978
;
Grillner 1981
; Orlovsky and Shik 1976
;
Shik and Orlovsky 1976
).
In humans and other mammals, it has been well established that midline
cerebellar injuries and atrophies have, as their main consequence,
instabilities of posture (trunkal ataxia) and locomotor movements
(Dow and Moruzzi 1958). The cerebellar vermal cortex integrates proprioceptive, exteroceptive, visual, and vestibular afferent information that originates from a wide variety of sources (Armstrong 1978
; Armstrong et al. 1997
;
Arshavsky et al. 1986
). It is therefore likely that the
vermis and the fastigial nucleus (to which efferent fibers from the
vermis project) are concerned with maintaining posture and with
elaborating associated body and neck movements, as suggested by
Chambers and Sprague (1955a
,b
). Yu and Eidelberg
(1983)
demonstrated in cats that fastigial lesions, bilaterally, produced ataxic locomotor movements. Because
fastigioreticular, fastigiovestibular, and fastigiospinal pathways
originate from the fastigial nucleus in addition to fastigiothalamic
pathways (Asanuma et al. 1983a
,b
; Homma et al.
1995
; Walberg et al. 1962a
,b
; Wilson et
al. 1978
), Armstrong (1986)
proposed that the
fastigial nucleus influences posture and locomotion by coordinating the activities of axial muscle groups via the reticulo- and vestibulospinal tracts. It has been well established that both the vestibulo- and
reticulospinal fibers descend through the ventral and ventrolateral quadrants of the spinal cord (Holstege and Kuypers 1982
;
Kuypers 1981
; Kuze et al. 1999
;
Matsuyama et al. 1988
, 1997
).
The results of following stimulation and ablation studies supported the
proposition that the reticulospinal pathway arising from the
pontomedullary reticular formation (PMRF) is one of the major pathways
mediating locomotor driving signals to the spinal cord, where the
central pattern generator (CPG) for locomotion is located
(Grillner 1975, 1981
; Jordan
1991
). In the decerebrate cat, Shik et al.
(1968)
stimulated the pyramidal tract at a site rostral to a
bilateral transection and evoked locomotion in the same fashion as
evoked by the MLR stimulation. Such stimulation was considered to
activate collaterals of the pyramidal tract fibers, which presumably
terminated in the PMRF. Studies in chronic adult cats (Afelt
1974
; Eidelberg 1981
) suggested that pathways in
the ventral and ventrolateral quadrants are essential for the recovery
of voluntary quadrupedal locomotion. Recent study by Brustein
and Rossignol (1998)
showed that the cats with a massive ventral and ventrolateral spinal lesion at low thoracic levels (T11 or T13) suffered from
pronounced locomotor and postural deficits in the early period after
the lesion, although all the cats eventually recovered quadrupedal
voluntary locomotion possibly due to compensatory mechanisms.
In acute precollicular-postmammillary decerebrate cats maintaining a
reflexively standing posture, we have recently reported that
stimulation of a very restricted region of the cerebellar white matter
along its midline resulted in augmentation of neck, lumbar back, fore-,
and hindlimb extensor activities, bilaterally (Asanome et al.
1998; Mori et al. 1998a
). The effective stimulus region corresponded to the midline region of the hook bundle of Russel
(Rasmussen 1933
), through which fastigioreticular,
fastigiovestibular, and fastigiospinal fibers decussate (Brodal
1981
; Homma et al. 1995
; Matsushita and
Iwahori 1971
). We report here that stimulation of the same
midline cerebellar region is capable as well of evoking locomotion in
the mesencephalic cat on the surface of a moving treadmill. From the
comparison of the characteristics of cerebellar and MLR-evoked
locomotion in a single animal, we have suggested that locomotor driving
signals arising from the fastigial nucleus and the MLR are relayed to
the spinal cord, in part, by common reticulospinal pathways
(Mori et al. 1998b
). Results of the present study now
support the proposition that the cerebellum is involved in the
triggering of locomotor-related subprograms that reside within the
brain stem and the spinal cord, and also shed considerable light on the
functional role of the fastigial nucleus in the control of posture and
locomotion in the cat. Preliminary results have been published
elsewhere (Mori et al. 1998a
,b
).
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METHODS |
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Experiments were performed on 25 adult cats. Under
halothane-nitrous oxide gas anesthesia, both carotid arteries were
ligated. After surgical decerebration at the
precollicular-postmammillary level, the head of the animal was fixed to
a stereotaxic instrument together with dorsal spinal processes of the
first three thoracic vertebrae. All wound margins and pressure points
were infiltrated with local anesthetic. The body of the animal was
supported by a rubber hammock, and the feet were placed on the surface
of a treadmill (Mori 1987, 1989
;
Mori et al. 1978
). The dorsal part of the occipital bone
and the tentorium were removed to allow access to the midline regions
of the cerebellum and the MLR. Electromyograms (EMGs) were recorded by
implanting bipolar electrodes made of thin (100 µm) copper wires into
selected extensor and flexor muscles of the left and right fore-
(biceps brachii, BB; triceps brachii, TB) and hindlimbs (quadriceps
femoris, QF; gastrocnemius-soleus, GS; tibialis anterior, TA). Recorded
EMGs were displayed on a storage oscilloscope in various combinations
and also stored on a digital tape recorder for later analysis. These
EMGs were rectified and integrated when necessary.
Details of the systematic search of the cerebellar white matter for
sites affecting locomotor behaviors have been published elsewhere
(Asanome et al. 1998). Briefly, tungsten-in-glass
microelectrodes with a tip diameter of 15-25 µm (impedance; 0.6-1.0
M
) were used for stimulation of the cerebellar white matter and the
MLR. By means of independent micromanipulators, one electrode was
introduced into the cerebellar white matter along its midline, and
another into the left or the right MLR according to Horsley-Clarke
coordinates. In five animals, a third electrode was also inserted into
the alternate right or left MLR. The first (cerebellar) electrode was
advanced dorsoventrally in the cerebellar midline at steps of 0.1-0.25
mm through the depths from H + 2 to H
2. Separate penetrations,
at intervals of 0.1-0.25 mm, covered the A-P range from P6.5 to P7.5.
Quasi-midline penetrations were made as well, from L2.0 to R2.0, and
covered the same depth and rostrocaudal range as the midline (LR0)
search. The second and the third electrodes were also inserted
dorsoventrally into the MLR at 0.1- to 0.25-mm steps (P1.5 to 2.5, LR3.5 to 4.5, H + 1 to
1). The stimuli consisted of rectangular
pulses of 0.2 ms duration in pulse trains of a frequency of 50 pulses/s. Stimulus intensity ranged from 5 µA to a maximum of 50 µA. In five experiments, the treadmill speed was changed in a
stepwise manner over the range from 0.4 to 1.5 m/s while observing
the effects of changes in the stimulus intensity delivered to the
cerebellar locomotor region (CLR) and the MLR. The treadmill
movement was routinely started before the CLR and MLR stimulation. In
three experiments, stimulus frequency was increased from 20 to 300 Hz,
and the relationships between stimulus frequency and stimulus threshold
for evoking locomotion were studied.
During the experiments, the animal's rectal temperature and mean blood pressure were monitored and kept at 36-38°C and >90 mmHg, respectively. End-tidal CO2 was measured and maintained between 4 and 6%. Warmed physiological saline solution was frequently poured onto the exposed cerebellar surface. After identifying the stimulus sites within the midline cerebellar white matter, and the left and right MLR for evoking locomotion with the lowest stimulus intensity, small electrolytic lesions were made in a sequence by passing a DC cathodal current of 10 µA for 10 s. From the marked lesions, the optimal stimulus sites for evoking locomotion were identified. In a different group of animals (n = 5), large electrolytic lesions were made at the optimal stimulus sites of the cerebellar white matter or the MLR. For this, a DC cathodal current of 30-50 µA was passed through the stimulating electrode for 30-40 s. These ablative lesions were made for the purpose of studying the effects of stimulating the intact CLR or unilateral/bilateral MLR while the counterpart was inactivated by destructive lesion.
At the end of each experiment, the cats were deeply anesthetized with
pentobarbital sodium (40 mg/kg) and perfused transcardially for 20 min
with 0.01 M phosphate-buffered saline followed by 4% Formalin
solution. After the perfusion, both the cerebellum and brain stem were
removed. After fixation in 10% Formalin solution for a few days, the
tissues were immersed for at least 5 days at 4°C in 0.1 M phosphate
buffer containing 20% sucrose. Serial transverse 50-µm sections of
the cerebellum and the brain stem were made on a cryotome. Serial
parasagittal 50-µm sections were also made in a different group of
animals. The sections were then mounted on gelatin-coated glass slides,
lightly counterstained with 1% cresyl violet, dehydrated, and cover
slipped for observation. The locations of the effective stimulus
regions within the midline cerebellar white matter for evoking
locomotion and those in the MLR were determined with reference to the
stereotaxic atlas of Berman (1968). The dorsoventral,
mediolateral, and rostrocadual extent of ablative lesions, which were
made within the cerebellar white matter and the MLR, was also estimated
from the serial transverse sections of the cerebellum and the brain stem.
In five animals, a small volume of cholera-toxin b subunit conjugated
peroxidase (CTb-HRP), which was dissolved in 0.01 M sodium phosphate
buffer (0.4%, 50 nl, List Biological Laboratories), was focally
injected into the lesioned site of the CLR by using a double-barreled
pipette composed of a stimulating microelectrode and an injection glass
pipette (Asanome et al. 1998; Ohta et al. 1988
). The stimulating microelectrode was used for evoking
locomotion as well as ablating the fibers of passage coursing through
the lesioned site. For this purpose, a DC current of 10 µA was passed through this electrode for ~30 s. The attached pipette was used for
microinjection of CTb-HRP solution into the lesioned stimulus sites.
After survival periods of 12-48 h under continuous anesthesia with
intravenous administration of physiological saline solution and warming
of animals with a heating pad, the animals were deeply anesthetized and
perfused transcardially with physiological saline solution followed by
4% Formalin solution.
In cats with CTb-HRP microinjection, the transverse 50-µm sections of
the cerebellum and the brain stem were reacted for HRP-histochemistry using the tetramethyl benzidine (TMB) procedure (Mesulam
1982). All CTb-HRP-stained sections were examined under a
light microscope equipped with a bright-field condenser, and the
trajectory of fibers of passage was studied from serial sections as far
caudally as possible. Photomicrographs were taken with a microscope
(MICROPHOTO-FXA, NIKON, Tokyo, Japan) connected to a camera, and then
scanned. To get an optimal reproduction of the staining, we modified
the contrast and luminosity of the raw scan with Adobe Photoshop 4.0 on
a Macintosh computer. The illustration was then printed with a digital
color printer (Pictrography 3000, Fujifilm, Tokyo, Japan).
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RESULTS |
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We inserted stimulating microelectrodes along dorsoventral tracks in the cerebellar midline or quasi-midline and were able to evoke locomotion on the surface of the moving treadmill with coordinated fore- and hindlimb movements in all animals. At optimal sites along the appropriately placed tracks, we could elicit such movement with stimulus intensity as low as 5 µA. At such sites, the maximum effective stimulus intensity decreased sharply with increase of frequency over the range of 20-50 Hz, and then remained almost constant over the range of 50-300 Hz. We limited test stimulus intensity to a maximum of 50 µA at all sites.
The first experiments consisted of a systematic mapping of the sites within the cerebellar white matter from which such locomotor behavior could be elicited. We also studied the trajectory of fibers passing through these locomotor regions by using the CTb-HRP tracing technique. We then investigated the possibility of controlling the pattern of the CLR-evoked locomotion with systematic changes of stimulus intensity and treadmill speed. And finally, we sought to clarify the relationship of cerebellar-induced locomotor mechanisms to those of other systems relaying through the PMRF such as the MLR.
Rostrocaudal, dorsoventral, and mediolateral extent of the effective cerebellar regions for evoking locomotion
We first searched out the rostrocaudal and dorsoventral extent of the optimal region for evoking locomotion in the cerebellar midline. Figure 1 shows responses to stimulation along three electrode tracks in one animal. The loci of optimal stimulus sites along tracks a, b, and c (at P6.85, P7.1, and P7.35, respectively) are plotted on the midsagittal (left) and frontal (right) planes of the cerebellum in Fig. 1A. We found all effective sites to be rostral to or just adjacent to the rostral pole of the fastigial nucleus (FN). Along the most rostral and caudal tracks, locomotor behavior occurred only with stimuli at points a and c (with intensities of 30 and 20 µA, respectively). The vertical bar through point b represents the range over which stimuli were effective. The points a and c, along with the bar through b, trace out an envelope of cerebellar white matter from which stimulus-evoked locomotion occurred in this animal.
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The patterns of locomotor responses were stimulus-site-specific as shown in the three sets of EMGs in Fig. 1B. With stimulation at site a with the lowest effective stimulus intensity of 30 µA, the animal extended the forelimbs with an increased activity of the TB muscles, bilaterally, and exhibited hindlimb walking with alternating bursting discharges of TA and GS muscles. Mean interburst interval of these muscles was 0.77 s. During stimulation at site c with the lowest effective stimulus intensity of 20 µA, the animal flexed the hindlimbs, with an increased activity of the TA muscles, bilaterally, and now exhibited forelimb walking with alternating bursting discharges of BB and TB muscles. Mean interburst interval of these muscles was 0.53 s. By stimulating regions located further rostrally or caudally to the stimulus sites a and c, no locomotor movements were evoked even with stimulus intensity as high as 50 µA.
By stimulating site b, which corresponded to the dorsoventral depth of H + 0.3, we evoked full locomotion with a cycle time of 0.73 s at the lowest stimulus intensity of 15 µA. Along this track, we could also evoke locomotion by stimulating the sites at H + 0.1 and H + 0.5, but now with the minimum stimulus intensity of 20 µA. By stimulating the regions located further dorsally or ventrally to that marked by the vertical bar on the midsagittal plane in Fig. 1A, left, we could no longer evoke postural changes and/or locomotor movements with the stimulus intensity as high as 50 µA. By stimulating optimal sites at the depth of H + 0.2 to H + 0.4 along the P7.0 and P7.2 midline tracks, we evoked locomotion with stimulus intensities of 20-25 µA. Although the stimulation delivered to the best loci provoked fore- and hindlimb coordinated locomotion, stimulation of sites located rostrally or caudally from the best loci occasionally provoked locomotion with slightly different frequencies of fore- and hindlimb movements. In this animal, we did no further systematic stimulation study because electrolytic lesions were made at each of the stimulus sites a, b, and c.
The two photomicrographs in Fig. 2 were taken from the midsagittal and frontal sections of the cerebellum and the brain stem from two animals. The photomicrograph in Fig. 2A shows three stimulus tracks and a single lesion site from the animal shown in Fig. 1A, left. The leftmost (most rostral) track was made as a reference track. Along the next track as indicated by a thin vertical arrow, the microlesion of a site a is marked by the thick arrow. The rightmost track is the one on which the stimulus site b is located. The microlesion at site b and the stimulus track along the stimulus site c were not clearly seen on this midsagittal section. The photomicrograph in Fig. 2B illustrates the microlesion (thick arrow) that was made at the most effective stimulus site (P7.0, LR0, H0) in the animal shown in Fig. 3A. The position of the cerebellum was slightly distorted in relation to that of the brain stem. The effective stimulus site for evoking full locomotion was located within the fiber bundle dorsal to the most ventral part of the gray matter in the midline of the cerebellum.
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In the animal in Fig. 3A, the dorsoventral extent of
effective stimulus sites along the midline of the cerebellum was
studied. The stimulus track was located at the P7.0 level. Stimulus
intensity was fixed to 15 µA. By stimulating three sites along the
midline at H + 0.25 (a), H0 (b), and H-0.25
(c), full locomotion was provoked with initial rhythmic
movements of the hindlimbs. The latency, which was measured from the
onset of cerebellar stimulation to the beginning of the first bursting
discharge of GS muscles, was as short as 1.0 s for the locomotion
evoked by stimulating the site at H0 (b). The cycle time of
locomotion was ~0.70 s. By stimulating the site at H0, full
locomotion was still evoked even when the stimulus intensity was
decreased to 8 µA, but the cycle time was prolonged to 0.90 s.
Stimulation at H + 0.25 (a) or H-0.25 (c), with
the same intensity of 8 µA, failed to evoke locomotion. Stimulation of sites at H + 0.1 and H 0.1 with minimum intensity of 10-12 µA evoked locomotion. With the stimuli delivered to more dorsally or
ventrally located midline regions above H + 0.25 or below H
0.25, we could not evoke locomotor movement even with the highest stimulus intensity of 50 µA.
In a different animal, we stimulated sites on different sides of the midline (Fig. 3B). We inserted electrodes directed at the rostral pole of the fastigial nucleus at L2.0 and R2.0, as well as LR0 at the rostrocaudal level between P7.0 and P7.3. The stimulus intensity applied along each track was fixed at 20 µA. Three sets of EMGs in Fig. 3B were recorded from the left and the right forelimb muscles. In this animal, stimulation of the midline region at the depth of H + 0.25 at P7.0 level provoked full locomotion with the lowest stimulus intensity of 8 µA, and with cycle time of 0.46 s (b). During the midline-evoked locomotion, the BB and TB muscles exhibited regular and symmetrical alternating bursting discharges.
Postmortem study of histological sections showed that the most effective stimulus sites targeting LR0 and R2 were located along the midline and 2 mm lateral from the midline of the cerebellum, but the site targeting R2.0 was located at R2.3. The stimulus sites along the lateral tracks were located adjacent to the most rostral and dorsal part of the left and the right fastigial nuclei, respectively. With stimulation of the optimal site located at L2.0, H-0.2, the animal walked with marked deviation of the right fore- and hindlimbs to the right-hand side. Bursting discharges of the left BB and TB muscles were remarkably diminished while those of the right BB and TB muscles were augmented (a). The left fore- and hindlimbs also deviated to the right-hand side as if the animal was trying to turn or to walk diagonally on the treadmill. With stimulation of the optimal site located at R2.3, H-0.2, locomotor movements with a pattern opposite to those evoked by stimulating L2.0 region were evoked. Now the animal walked with marked deviation of the left and the right fore- and hindlimbs to the left-hand side. The bursts of activity in the left and the right forelimb muscles were augmented and diminished, respectively (c). The cycle time of the locomotion evoked by stimulating the two sites at L2.0 and R2.3 was ~0.57 and 0.58 s, respectively. By decreasing the stimulus intensity from 20 to 15 µA at these lateral sites, the degree of body tilting was slightly diminished, but asymmetry of the locomotor movements persisted. The cycle time of the locomotion was also prolonged by 0.1-0.2 s.
In this animal, the dorsoventral extent of the effective regions along the midline track was ~0.5 mm as in the first animal illustrated in Fig. 1. In contrast, the dorsoventral extent of effective regions along the laterally located tracks increased to ~0.6-0.8 mm. Along the lateral tracks, optimal stimulus sites were located slightly ventral to those in the midline. Locomotion evoked by stimulating the sites located along the lateral tracks always evoked asymmetric movements of the fore- and the hindlimbs, and the closer the lateral stimulus sites were to the midline, the smaller was the asymmetry of the evoked locomotion. These results suggest that locomotor movements can be evoked by stimulating a barrel-shaped neural region, which lies transversely within the cerebellar white matter between the rostral poles of the left and right fastigial nuclei.
Representative location of a CTb-HRP-injected microlesion and trajectory of CTb-HRP-labeled fibers
By focally injecting CTb-HRP in the lesioned effective site for evoking locomotion, it was possible to trace the anterogradely labeled fibers that passed through the lesioned sites. Across animals with different survival periods up to 48 h, we obtained essentially the same results. The photomicrograph in Fig. 4A illustrates the lesioned site on the frontal plane at P7.1. The shape of the microlesion was oval with a diameter of ~0.3 mm, and it was surrounded by a laterally extended diffusion area of CTb-HRP. The two photomontages in Fig. 4, B and C, were made from the photomicrographs, which were taken from the sections slightly rostral to the CTb-HRP injection site. CTb-HRP-labeled fibers appeared as densely stained, rod-shaped structures of various calibers. The length of rods was shorter at the quasi-midline region and longer at the lateral region. A small portion of these fibers passed through the rostral portion of the left and the right fastigial nuclei. These findings indicate that the fibers run rostrolaterally from the CTb-HRP injection site and then transversely at the lateral region.
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The photomontages in Fig. 4, B and C, show a
bundle of labeled fibers running transversely to the left and to the
right. The labeled fibers curved along the dorsal part of the left and
the right brachium conjunctivum (BC), respectively. These fibers then descended ventrally along the lateral border of superior and lateral vestibular nuclei. Some of them continued to run to the PMRF and the
vestibular complex. The trajectory of the CTb-HRP-labeled fibers was
essentially the same as that identified by focal microinjection of an
anterograde neural tracer, Phaseolus vulgaris
leucoaggulutinin, into the fastigial nucleus (Homma et al.
1995). These results together with demonstration of the
selective location of the retrogradely labeled cells in the fastigial
nuclei (Asanome et al. 1998
; Mori et al.
1998a
) demonstrated that the cerebellar-evoked locomotion was
due to activation of the hook bundle of Russel, through which crossed
fastigiofugal fibers decussate.
Comparison of CLR- and MLR-evoked locomotion, and summation effects of the CLR and MLR stimulations
To elucidate the characteristics of CLR-evoked locomotion, effects of CLR and MLR stimulation were first compared. Stimulation of the CLR (P7.0, LR0, H + 0.2) for 5 s with the stimulus intensity of 10 µA and with the treadmill speed of 0.6 m/s evoked coordinated locomotion with latency of ~1.5 s after the stimulus onset as shown in the EMG records in Fig. 5A. Cycle time was ~0.70 s. After termination of the stimulation, locomotion continued for ~8-10 steps with a gradual prolongation of cycle time and weakening of the bursting discharges of the left and the right TA and GS muscles. Stimulation of the MLR in the same animal (P2.0, L4.0, H0) with the same parameters of stimulus intensity and duration evoked locomotion with latency of ~1.5 s and with cycle time of ~0.68 s (Fig. 5B). The CLR- and MLR-evoked locomotion were qualitatively indistinguishable.
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We also found that concomitant stimulation of CLR and MLR, at subthreshold intensity for each locus, evoked locomotion like that evoked by separate suprathreshold stimulation (not illustrated). Integrated EMGs made from the records obtained during separate and simultaneous CLR and MLR stimulation are illustrated in Fig. 6. The stimulus site in the CLR was at P6.9, LR0, H + 0.4, and that in the MLR was at P2.0, L4.2, H0. The treadmill speed was kept constant at 0.8 m/s. MLR stimulation alone at stimulus intensity of 15 µA evoked locomotion with cycle time of ~0.60 s (Fig. 6A). CLR stimulation alone at stimulus intensity of 10 µA also evoked locomotion with cycle time of 0.60 s (Fig. 6B). When these stimulations were combined, locomotion became vigorous. Individual bursting discharges of both the TB and GS muscles became stronger in both amplitude and duration compared with those observed during the locomotion evoked by either the MLR or the CLR alone.
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To further evaluate the characteristics of CLR-evoked locomotion, we systematically changed the treadmill speed and the stimulus intensity, and studied the changes in the patterns of evoked locomotion. We then examined the patterns of MLR-evoked locomotion in a similar manner in the same animal. The results are summarized in Fig. 7. In this animal, stimulus sites of the CLR and the MLR were located at P7.0, LR0, H + 0.2, and at P2.0, L4.2, H0, respectively. EMGs in Fig. 7, A and B, were recorded during CLR-evoked locomotion in relation to the stepwise changes in the treadmill speed and the stimulus intensity, respectively. We first evoked full locomotion by stimulating the CLR with constant stimulus intensity of 10 µA. With an increase in the treadmill speed from 0.7 m/s (a) to 1.1 m/s (b) and then to 1.4 m/s (c), the duration of bursting periods of TB and GS muscles, bilaterally, decreased remarkably with shortening of cycle time from 0.72 to 0.60 s and then to 0.54 s (Fig. 7A). The animal changed its locomotor patterns from a slow walk (a) to a fast walk (b and c).
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Then, keeping the treadmill speed constant at 0.8 m/s, we changed the
stimulus intensity from 10 µA (a) to 15 µA
(b) and then to 24 µA (c in Fig.
7B). With this, evoked locomotion became vigorous with
stronger bursting discharges of TB and GS muscles and with shortening
of cycle time from 0.60 s (a) to 0.48 s
(b) and then to 0.45 s (c). At the highest
stimulus intensity of 24 µA, the left and the right TA muscles
exhibited alternating bursting discharges, and bursting periods of the
left and the right GS muscles overlapped slightly as observed during a
transitional period from a fast walk to a gallop (half-bound)
(Stuart et al. 1973). Although not illustrated, similar
EMG changes to those observed during CLR-evoked locomotion were
observed during MLR-evoked locomotion in relation to the changes in the
stimulus intensity and the treadmill speed. The plots in Fig. 7,
C and D, illustrate the changes in the cycle time
of CLR- (filled circles) and MLR-evoked locomotion (open circles) in
relation to the stepwise changes in the treadmill speed and stimulus
intensity, respectively. All these results clearly demonstrated that
the pattern of CLR-evoked locomotion can be controlled by changing the
combination of stimulus intensity and treadmill speed, findings very
similar to those seen with stimulation of MLR.
Effects of ablative lesions at either the CLR or MLR
Finally, we made ablative lesions in either the bilateral MLR or the CLR, and we studied effects of stimulating the counterpart. This study was aimed at answering the question of whether or not the CLR and the MLR are functionally independent locomotion inducing loci or mutually dependent loci for evoking locomotion. We held the location of the stimulating electrode fixed once the optimal stimulus site was identified. Observations and EMG recordings were made 5-10 min after making ablative lesions in the locomotion inducing sites. We assessed the degree of ablative lesions by stimulating the lesioned sites with the stimulus intensity >100 µA, which was 5-10 times stronger than the suprathreshold stimulus intensity for evoking locomotion in the same sites before making the lesions. With this stimulus intensity, no sign of locomotor movements was noted.
We found histologically identified lesions in the cerebellar white matter to extend ~0.5 mm, dorsoventrally, rostrocaudally, and mediolaterally, obliterating completely the effective stimulus loci for evoking locomotion. The area of the lesions made in the left and the right MLR also ablated those stimulus loci from which locomotion could usually be evoked. After making lesions, locomotion was evoked on stimulation of the remaining intact site with patterns similar to those before making the lesions. The stimulus intensity needed for evoking locomotion was similar to that before making ablative lesions in the MLR, unilaterally and bilaterally. Essentially similar results were obtained by stimulating the MLR after making ablative lesions in the CLR. These results demonstrated that the CLR and the MLR are independent locomotion inducing sites and are capable of activating brain stem-spinal locomotor subprograms commonly and separately.
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DISCUSSION |
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The results of the present study demonstrated clearly that stimulation of the hook bundle in the midline of the cerebellum is capable of evoking coordinated locomotion on the surface of a moving treadmill, suggesting that fastigial cells are capable of initiating and controlling locomotion in the mesencephalic cat.
Locomotion inducing sites and functional role of the reticulospinal cells in the initiation of locomotion
In the mesencephalic cat, Shik et al. (1966) first
demonstrated that MLR stimulation evokes "controlled" locomotion on
the surface of a moving treadmill. The MLR was considered to correspond to the caudal portion of the cuneiform nucleus. In the same
preparation, Shik et al. (1968)
also showed that
stimulation of the rostral pyramidal tract provoked locomotion provided
that the pyramidal tracts were sectioned bilaterally at the medullary
level caudal to the pyramidal stimulus sites. Such stimulation evoked
only walking or trotting, and the pyramidal tract-evoked locomotion was in general much less stable than MLR-evoked locomotion. With concomitant stimulation of the MLR and the pyramid, each at
subthreshold strength, locomotion was also evoked. In the pyramidal
tract-sectioned cat, rostral pyramidal stimulation still evoked
locomotion even after making ablative lesions in the MLR, bilaterally.
Shik et al. (1968)
suggested that pyramidal
tract-evoked locomotion was due to an activation of reticulospinal
cells in the PMRF by way of corticoreticular fibers. Clear evidence for
an anatomic basis for this proposition was recently provided by
Matsuyama and Drew (1997)
. MLR-evoked locomotion was also
demostrated to be due to an activation of reticulospinal cells
(Garcia-Rill and Skinner 1987
; Orlovsky
1970b
,c
; Shik and Orlovsky 1976
).
The physiologically identified MLR was found, anatomically, to project
to the magnocellular (FTM) and gigantocellular (FTG) tegmental fields
of the medullary reticular formation (MRF) with ipsilateral dominancy
(Garcia-Rill et al. 1983; Steeves and Jordan 1984
). Intracelluar (Orlovsky 1970c
) and
extracellular recording studies (Garcia-Rill and Skinner
1987
) also showed that MLR stimulation results in mono- and
polysynaptic activation of reticulospinal cells in the PMRF. In
addition to the MLR, Garcia-Rill (1991)
suggested that
the pedunculopontine nucleus (PPN) is also one of the
locomotion-inducing site in the midbrain. They found that efferent
fibers from the PPN/MLR projects to the transition zone between FTM and
FTG of the MRF. These results suggested that the major pathways
mediating the stimulus effects of the MLR/PPN complex is through the
FTM, especially the reticulospinal pathways that originated from the
nucleus reticularis magnocellularis (NRMc) (Garcia-Rill et al.
1983
; Jordan 1991
).
Cerebellar and brain stem neuronal mechanisms related to initiation and control of posture and locomotion
During MLR-evoked locomotion of the decerebrate cats on the moving
treadmill, Orlovsky (1970a) first recorded the activity of reticulospinal cells in the FTG and found that these cells exhibit,
besides tonic activity, rhythmical discharges. In a similar locomotor
preparation, Orlovsky (1972)
also found rhythmically discharging fastigial cells with tonic activity. The frequency curve
for the "average" fastigial neuron showed maximum activity approximately in the swing phase of the ipsilateral hindlimb and in
phase with that of the reticulospinal cells. These observations demonstrated that fastigial and reticulospinal cells are in fact involved in the control of MLR-evoked locomotion. The finding that
fastigial cells exhibited rhythmical discharges during the period of
the pinna-evoked fictitious scratch reflex in immobilized cat
(Antziferova et al. 1980
) suggests that some of the
rhythmical fastigial cell discharges might be centrally generated, as
opposed to being due to peripheral feedback from the moving limb.
To answer the question of whether or not the fastigial nucleus is
mainly involved in the modulation of locomotion or whether it is also
capable of initiating locomotion or both, it is necessary to understand
the function of reticulospinal cells. Ito et al. (1970)
and Eccles et al. (1975)
demonstrated that stimulation in or near the fastigial nucleus resulted in orthodromic activation of
reticulospinal cells in the FTG. The fastigial nucleus seems to have
the potential capability of initiating locomotion by activating reticulospinal cells. However, highly complex effects on posture and
locomotion were evoked by stimulating the fastigial nucleus (Batini and Pompeiano 1958
; Moruzzi and Pompeiano
1956
). Such complex effects can be the result of concomitant
activation of corticofastigial and corticovestibular Purkinje cell
axons, together with cells of origin of fastigiofugal fibers and
certain cerebellar afferent fibers (Mori et al. 1998a
).
Before further discussing the fastigial contribution to the initiation
and control of locomotion, it will be necessary to understand the
specific trajectory of fastigiofugal fibers in relation to the hook
bundle and the CLR.
Trajectory of crossed fastigiofugal fibers passing through the CLR and their termination areas in the PMRF
Rasmussen (1933) first demonstrated that both
crossed and uncrossed fastigiofugal fibers originate from the fastigial
nucleus. Approximately one-half of the fastigiofugal fibers were found to decussate, pass through the rostral part of, and rostral to the
opposite fastigial nucleus to form the hook bundle of Russel before
entering the cerebellar peduncle. These fibers converged as they
approach the midline region of the cerebellar white matter (Matsushita and Iwahori 1971
). Uncrossed fibers coursed
caudally medial to the brachium conjunctivum and then ventrolaterally
to the vestibular complex. Crossed fastigiofugal fibers include
fastigioreticular, fastigiovestibular, and fastigiospinal fibers
terminating at the upper cervical cord (Fukushima et al.
1977
; Walberg et al. 1962a
,b
). Fastigiofugal
fibers, which originated from the rostral, middle, and caudal parts of
the fastigial nucleus, tended to pass through the lower, middle, and
upper portion of the hook bundle, respectively. Each of these fiber
groups terminated in the specific regions of the FTG (Homma et
al. 1995
).
In the mesencephalic cat placed on a still surface, we showed that
midline stimulation of the hook bundle, with the stimulus intensity
subthreshold for evoking locomotion, provoked general augmentation of
postural muscle tone (Asanome et al. 1998; Mori et al. 1998a
). By increasing the stimulus intensity, we found that the same animal exhibits locomotor-like movements on both the
stationary and moving treadmill surface. Before the initiation of
locomotion, the level of postural muscle tone was routinely increased.
By a CTb-HRP retrograde tracing method, we found that cells of origin
of fibers that projected to the CLR were located exclusively in the
fastigial nuclei, bilaterally (Asanome et al. 1998
). The
trajectory of descending fibers coursing through the CLR was found in
this study to be essentially the same as that identified by injecting
an anterograde neural tracer, Phaseolus vulgaris
leucoaggulutinin, into the fastigial nucleus (Homma et al.
1995
). These results disclose clearly that the CLR corresponds to the hook bundle in the midline of the cerebellum, and that CLR
stimulation exerts dual effects on posture and locomotion.
Effects of CLR stimulation on reticulospinal cells and on induced locomotion
The characteristic trajectory of fastigiofugal fibers indicates
that stimulation of the hook bundle results in a selective and
simultaneous activation of crossed fastigioreticular,
fastigiovestibular, and fastigiospinal fibers, descending bilaterally
to the brain stem and the spinal cord. Midline stimulation would
activate decussating fastigial fibers and in turn activate both the
left and right PMRF to a similar extent (Mori et al.
1998a). An increase in the stimulus intensity would result in
an increase in the number of excited reticulospinal cells, and in
consequence enhance locomotor driving signals descending to the spinal
cord. In contrast, stimulation of the hook bundle at a locus located
laterally to the midline would result in a predominantly unilateral
activation of fastigiofugal fibers before decussation of the fibers at
the midline, resulting in recruitment of a larger number of
reticulospinal and vestibulospinal cells located contralaterally to the
stimulus site.
We evoked widely varying patterns of locomotion, depending on the locus
of stimulation. Stimulus site-specific differences in the evoked
locomotor patterns may be related to the differences in the activated
group of fastigiofugal fibers within the hook bundle, and to
termination sites of fastigioreticular and fastigiovestibular fibers in
the PMRF (Homma et al. 1995). Based on the results
obtained by microstimulation in the intact unanesthetized cat,
Drew and Rossignol (1990a
,b
) suggested that the MRF is
topographically organized, but without usual precision of such
organization. Movements of the head were obtained from the whole extent
of the brain stem. Ipsilateral forelimb movements were preferentially
evoked by stimulation to the dorsal MRF, whereas contralateral forelimb
movements and movements of the hindlimbs were evoked from more ventral
and rostral regions. At present, however, it is not possible to
directly correlate various patterns of CLR-evoked locomotion to
activated components of the fastigioreticular and/or fastigiovestibular
fibers, and topographical organization of the PMRF.
We have recently found that reticulospinal cells in the NRGc were
monosynaptically activated by CLR stimulation (Matsui et al.
1997b; Mori et al. 1998b
). Some of these cells
were also activated, mainly polysynaptically with MLR stimulation. The
NRGc reticulospinal cells, which projected to the lumbar segments,
increased their discharge frequency remarkably during CLR-evoked
locomotion with some phasic modulation (Matsui et al.
1997b
; Mori et al. 1998b
). The conduction
velocity of these NRGc cells was faster than 55 m/s. The maximum
activity of these cells was found at diverse times of the step cycle in
the hindlimbs, as in the study of Drew et al. (1986)
in
intact cats walking on the treadmill. During the CLR-evoked tonic
discharges of these NRGc cells, we found individual spikes that were
time locked to each stimulus pulse of the CLR stimulation. This result
suggested that CLR-evoked locomotor driving signals to the spinal cord
were relayed by the NRGc reticulospinal cells. Such a proposition,
however, does not rule out the possibility that some of them are
mediated by the NRMc reticulospinal cells in parallel, because a part
of the fastigioreticular fibers terminated in the FTM (Homma et
al. 1995
).
Control of locomotion by cerebellar nuclei and the cerebellum
Major functions of the cerebellum have been considered to be
integration of multi-modal afferent information, storage of motor programs, motor learning, and triggering of the motor programs (Bloedel 1992, 1995
; Brooks and
Thach 1981
; Houk et al. 1993
; Ito
1984
). The fastigial nucleus is located ideally at a key
station of a functional spinocerebellar "closed loop" formed
between the neural elements in the cerebellum, brain stem and spinal
cord (Armstrong 1986
; Arshavsky et al.
1986
). Because execution of proper locomotion requires
simultaneous control of head, body, and limb movements in addition to
balance control, fastigiospinal, fastigioreticular, and
fastigiovestibular fibers can be considered to carry command signals
related to head-neck movements (Wilson et al. 1978
),
activation of the CPG (Grillner 1981
; Jordan
1991
), and the maintenance of balance. Our preliminary study
showed that some of the vestibulospinal cells were monosynaptically
activated by CLR stimulation (Matsui et al. 1997a
). The
present study demonstrated and suggested that cells in the fastigial
nucleus contribute to initiation and modulation of posture and
locomotion, by simultanously controlling the activity of multiple
reticulospinal and vestibulospinal cells, in addition to spinal cells
in the upper cervical segments.
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ACKNOWLEDGMENTS |
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We acknowledge the technical assistance of C. Takasu in these experiments. We thank M. Mori for photographic assistance. We also express sincere appreciation to Dr. P. Reynolds, Oregon Health Sciences University for critically reviewing the original version of this manuscript.
This study was supported by Ministry of Education, Science, Sports and Culture of Japan Grant in Aid for Scientific Research A06404087 to S. Mori.
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FOOTNOTES |
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Address for reprint requests: S. Mori, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 July 1998; accepted in final form 22 March 1999.
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REFERENCES |
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