Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Jenkinson, Edward W. and Mitchell Glickstein. Whiskers, Barrels, and Cortical Efferent Pathways in Gap Crossing by Rats. J. Neurophysiol. 84: 1781-1789, 2000. Rats can readily be trained to jump a gap of around 16 cm in the dark and a considerably larger gap in the light for a food reward. In the light, they use vision to estimate the distance to be jumped. In the dark, they use their vibrissae at the farthest distances. Bilateral whisker shaving or barrel field lesions reduce the gap crossed in the dark by about 2 cm. Information from the barrel fields reaches motor areas via cortico-cortical, basal ganglia, or cerebellar pathways. The cells of origin of the ponto-cerebellar pathway are segregated in layer Vb of the barrel field. Efferent axons of Vb cells occupy a central position within the basis pedunculi and terminate on cells in the pontine nuclei. Pontine cells, in turn, project to the cerebellar cortex as mossy fibers. We trained normal rats to cross a gap in the light and in a dark alley that was illuminated with an infra-red source. When the performance was stable, we made unilateral lesions in the central region of the basis pedunculi, which interrupted connections from the barrel field to the pons while leaving cortico-cortical and basal ganglia pathways intact. Whisking was not affected on either side by the lesion, and the rats with unilateral peduncle lesions crossed gaps of the same distance as they did pre-operatively. Shaving the whiskers on the side of the face that retains its input to the pontine nuclei reduced the maximal gap jumped in the dark by the same amount as bilateral whisker shaving. Performance in the light was not affected. Regrowth of the shaved whiskers was associated with the recovery of the maximum distance crossed in the dark. In control cases, shaving the whiskers on the other side of the face did not reduce the distance jumped in the dark or in the light. These results suggest that the cerebellum must receive whisker information from the barrel fields for whisker-guided jumps.
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INTRODUCTION |
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To use sensory input to guide
movements, sensory areas of the brain must be connected to the areas
that control movement. There are many possible routes by which these
areas are linked, but which pathway or pathways subserve any given task
remains unclear. Normal rats use whiskers to guide many of their
movements. Blind rats or rats in darkness rely almost solely on their
whiskers to guide their movements (Vincent 1912;
Watson 1907
). In the brain, each whisker is represented
by a column of cortex in register with a barrel-shaped cluster of cells
in layer IV of the contralateral cortex, the whisker's "barrel"
(Welker and Woolsey 1974
; Woolsey and Van der
Loos 1970
). The cortical representations of all the vibrissae
form the whisker barrel field or postero-medial-barrel subfield (PMBSF)
that makes up a large portion of sensory cortex of the rat
(Welker 1971
).
Ablation of a cortical barrel prevents a blind rat from using the input
from the corresponding single whisker to guide its movements across a
gap in a runway (Hutson and Masterton 1986). Although
Hutson and Masterton's rats were unable to use the input from the
whisker to control movements, the animals could still perceive passive
movement of the whisker. Hutson and Masterton suggested two
possibilities that might account for the observed deficit. Destruction
of the barrel may impair a "higher-order integration" that is
necessary for a complex task like sensory-guided movement but not a
simpler task such as detecting passive movements of the whiskers.
Alternatively they suggested that the motor system that controls gap
crossing might require sensory input from the cortex. According to this
interpretation, the barrel field lesion would have caused a
"sensory-motor disconnection", i.e., the motor system that
underpinned the movement no longer had access to tactile whisker
information from the cortex needed to perform the gap-crossing task.
One plausible route whereby cortical whisker representation is relayed
to motor structures is by way of the cerebellum. The cerebellum
receives massive input from sensory as well as motor cortical areas by
way of a single synapse in the pontine nuclei. In rats all of the
cerebral cortex, including sensory cortex projects to the pons
(Legg et al. 1989). Cells in lamina Vb of the PMBSF respond at very short latency to movement of their principle whisker (Armstrong-James et al. 1987
), and it is these same
lamina Vb cells that relay whisker information to the pontine nuclei
(Mercier et al. 1990
; Wise and Jones
1977
). Thus the cerebellum receives the type of information
that would subserve a system that utilizes sensory information to
control on-going, rapid, accurate movements.
The output from the cerebellum is via the cerebellar nuclei. Ascending
fibers from the cerebellar nuclei project to motor areas of cortex via
the ventral thalamus and directly to the red nucleus and the superior
colliculus. The descending cerebellar projections terminate in the
pontine reticular formation, nucleus reticularis tegmentis pontis,
inferior olive, the midbrain reticular formation, and the spinal cord
(for a review of the efferent connections of the rat cerebellar nuclei,
see Voogd 1995). Thus the cerebellum has direct and
powerful access to the cells of origin of all the major descending
motor tracts. The qualities of the sensory information and the
directness of the pathways linking sensory to motor areas of the brain
are consistent with the hypothesis that this pathway by way of the
cerebellum plays a central role in the sensory guidance of movement.
Basis for experimental technique
In typical lesion studies the function of an area of the brain is
tested by destroying a pathway or structure and observing the resulting
deficits. Although this approach has been used for nearly 200 years to
test the function of the cerebellum, it has some potential drawbacks. A
particular region of the body is represented by many small-scattered
patches across the cerebellar cortex (Shambes et al.
1978). The divergent "fractured somatotopy" would make it
difficult to remove selectively all the areas of cerebellar cortex that
receive sensory input from a given sensory area or structure such as
the vibrissae.
The pontine nuclei receive sensory input from the cerebral cortex and
provide the largest source of sensory input to the cerebellum by way of
mossy fibers. In principle it might be possible to abolish vibrissal
sensory inputs arriving from the cortex by making lesions within the
pons. However, like the mossy fiber input to the cerebellum, axons from
the PMBSF terminate in several isolated patches throughout the pontine
nuclei (Mihailoff et al. 1985). Thus a selective lesion of all the cortical vibrissal terminals in the pons would be difficult.
Interrupting the efferent fibers from the cortex to the pons offers a
straightforward way to block selectively cortical whisker input to the
cerebellum. The basis pedunculi of rats contains all of the fibers
originating in the cerebral cortex and projecting to the pontine
nuclei. The position of the fibers within the peduncle reflect the area
of cortex from which they arise (Glickstein et al.
1992). Fibers arising from the cells in the frontal cortex run
in the ventro-medial portion of the peduncle. Fibers from cells in the
occipital and temporal cortex run in the dorso-lateral region of the
peduncle. Fibers from cells in parietal somatosensory areas are in the
middle of the peduncle.
One approach to analyze the functions of the cerebro-ponto-cerebellar circuit in vibrissae guided movements would be to cut the fibers descending from the PMBSF within the peduncle, thus preventing vibrissal information from reaching the pontine nuclei, and therefore the cerebellum. The lesion would test the hypothesis that Hutson and Masterton's "sensory-motor disconnection" specifically arises from a disconnection of the cerebellum from the barrel field.
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METHODS |
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Details of behavioral technique
TRAINING PROCEDURE. Eight Hooded Lister rats were trained to jump across a gap in an elevated runway in complete darkness and in the light. The animals were initially trained to jump in the light; when they were proficient at crossing large gaps using visual cues, training the animals to cross in the dark began. The dark condition was designed to test the rats' ability to cross the gap using only tactile information in the absence of visual cues. Testing the animals in the light allowed us to monitor the animals ability to use visual information to cross the gap and served as an important control to rule out the possible effects of the lesions on motor function.
Young animals of about 6-wk were handled on a regular basis. The most tame of these animals continued to be handled and were introduced to the testing apparatus until they were the appropriate weight (around 400 g) for training to began. During training, the rats were kept at 80% of their normal maximum body weight to ensure that they were hungry. The rats were first trained to jump in the light by having them run from the start box to the end of the runway to receive a food reward before any gap was introduced. The gap was then initially set to 2 cm, if the animal crossed successfully on three consecutive trials the gap was increased by a further 2 cm. If the animal refused to cross on two consecutive trials, the gap was decreased by 4 cm or closed completely if the gap was already 4 cm or less. Using this modified staircase method the animals were first trained to jump large gaps in the light using vision to guide their movements. When performance in the light was stable, the rats were trained to cross the gap in the dark. It was important that the animals were trained in the light first. If initially trained to cross the gap in the dark, animals tended to become over reliant on their whiskers and were difficult to train to jump across larger gaps using visual information. During trials in the dark, the apparatus was lit with infra-red light (880 ± 25 nm; mean ± SD) and the animal observed using infra-red "night-vision" equipment. The regime for training the rats in the dark was the same as that used to train them to jump in the light. At small distances, the rats would simply step across the gap. At intermediate distances, 10 and 14 cm for an average rat, it would lean across the gap and reach for the other side with its paw or make contact with its snout before crossing. At larger distances, an animal would stop and lean across the gap, stretching their necks and extending their vibrissae to make contact with the far side of the gap with its whiskers. If the whiskers contacted the far side, it would cross; if not, it would turn back. The combination of intense infra-red illumination, and the magnification of the infra-red viewer allowed us to observe the whiskers in the dark. When the animal leaned across the gap, it would protract its whisker to their maximum extent and hold them relatively still in this position to make contact with the far side of the gap. The whiskers were always obviously seen making contact with the far side of the gap before the animal crossed. All the animals used in the study were trained to cross the maximum distance in the dark that they could reach across using their whiskers. For six animals, this distance was 16 cm. In one case the distance was 15 cm, and in another single case the distance was 17 cm. The differences in the maximum distances crossed in these two cases were simply due to the fact that one of these animals was slightly smaller and one slightly larger than the rest of the animals in the study.TESTING PROCEDURE. After a rat had received a lesion in the right cerebral peduncle, it was allowed to recover from the effects of surgery for an appropriate amount of time while being fed ad lib. Following this period, the animal was retrained as it had been before surgery to jump in the dark using its whiskers to guide its movements, while using vision to guide much larger jumps in the light. The rats were trained to presurgical levels of performance and were considered stable when the animal crossed the same distance on three successive days. At this point, the animal would have all of its whiskers on one side of its face cut to their bases.
Ascending sensory efferents that carry whiskers information to the cortex cross en route to the thalamus, so the whiskers on the left side of the face are represented in the right PMBSF. From the barrel fields, descending fibers travel to the pontine nucleus in the ipsilateral basis pedunculi. Therefore a lesion of fibers in the appropriate part of the right cerebral peduncle will block sensory information from the left whiskers reaching the cerebellum via the PMBSF. If then the right whiskers are cut, the remaining set of whiskers are lacking in the pathway that projects from the cortex to the pons. If the left whiskers are cut, the remaining right whiskers retain their connection from the left barrel fields to the pontine nucleus. The animals' behavior after the whisker shave would reflect the usefulness of the vibrissae for making a jump versus no-jump decision. After whiskers had been shaved, the animal was tested until its behavior was stable over a period of 3 days. Two normal animals had whiskers cut either unilaterally on one side of the snout or bilaterally with all the mystacial whiskers cut to their bases. The animals were tested after these procedures to ascertain the effect of unilateral and bilateral whisker cutting on otherwise intact animals. As a precaution that the surgery performed on the rats had not caused their vibrissae to become insensate, a simple behavioral test was performed on two of the rats. Small food pellets were held in a pair of forceps. In the dark, the forceps were brought into contact with parts of the body surface and the whiskers. The test is normally used to test orientation-localization behavior, and it is usually scored for accuracy and speed of response (see Glassman 1994Details of surgical and histological procedures
Surgery was performed on six rats to cut the middle section of
the right cerebral peduncle that contains the descending fibers that
originate in the PMBSF (Glickstein et al. 1992). The
operations were done under halothane anesthesia (1.5-2.5%
fluothane/oxygen mix at 2 l/min), the lesion was created using a
radio-frequency lesion maker (Radionics, model RFG-4A). When the probe
was at the appropriate depth within the brain, its tip was raised to a
temperatures between 85 and 95°C for a duration between 4.5 and 10 min.
At the conclusion of behavioral testing, to confirm that the barrel
fields had been disconnected, the pontine nuclei of three rats were
filled with wheat-germ agglutinin conjugated to horse-radish peroxidase
(WGA-HRP). After histological processing, the corresponding whisker
barrel fields on the two sides of the brain were examined for the
presence of retrogradely labeled cells that would indicate an intact
connection with the pontine nuclei. In two other cases, the extent of
the disconnection was studied using anterograde tracers. One rat
received bilateral PMBSF injections of WGA-HRP, and one received a
similar injection with biotinylated-dextran amine. In both cases, the
pontine nuclei were studied after histological processing for the
presence of labeled terminal fibers, which would indicate the presence
or absence of intact connections between the pontine nuclei and their
corresponding barrel fields. At the conclusion of the experiment, in
all cases, serial sections were cut and stained for fibers and cells to
verify the position of the lesion (Hutchins and Weber
1983).
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RESULTS |
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Figure 1 shows the behavioral performance of case 1 tested as normal and then tested again unoperated but with only its left whiskers remaining: tested again with a lesion of the right cerebral peduncle and a full complement of whiskers, tested again after the peduncle lesion and only the left whiskers remaining, and finally after the peduncle lesion and retaining only the right whiskers remaining. Figure 2 shows the reconstruction of the lesion in the right cerebral peduncle in this case.
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Initially case 1 consistently crossed a gap of 15 cm in the dark using only its whiskers to gauge the gap. At smaller gaps of 11 and 13 cm, the animal would use a combination of paw, nose, and whiskers to judge the distance to the far edge. At gaps greater than 15 cm in the dark, the rat could not reach the far side even with its whiskers. If the rat could not make contact with the far edge, it would not jump (Fig. 1A).
To test whether the animal could use a single set of whiskers to guide the jump across the gap, all the whiskers on the right side of the snout were cut to their bases. With only the left whiskers remaining, the rat continued to cross a gap of 15 cm in the dark. The rat was observed to always touch the far side of the gap with the remaining left whiskers before jumping across. At gaps of 11 and 13 cm, the animal would touch the far side with either its nose or paw before crossing (Fig. 1B).
The right whiskers were allowed to regrow, after which the animal received a lesion in its right cerebral peduncle. The animal displayed no visible motor effects following surgery, and retraining began 5 days after surgery. After the peduncle lesion with both sets of whiskers intact, the animal could still cross a gap of 15 cm in the dark using its whiskers to guide its movement (Fig. 1C).
At this point, all the whiskers on the right side of the face were again cut to their bases. Even though the remaining left mystacial whiskers could be seen making contact with the far side of the gap, the rat refused to cross a gap of 15 cm. The rat would still cross gaps of 11 and 13 cm in the dark, distances that it could reach with either its nose or paw. As the whiskers on the right side of the snout regrew, the distance the animal would cross increased. When the whiskers on the right reached their original length the rat would once again cross 15 cm in the dark. The result suggest that the rat was capable and willing to use relevant information collected with the nose or paw despite its unwillingness or inability to use similar information collected with its left vibrissae (Fig. 1D).
When the performance at 15 cm in the dark using a full complement of whiskers was consistent, the whiskers on the left side of the face were cut to their bases. Cutting the left whiskers had no effect on the threshold for gap crossing. The rat continued to cross a gap of 15 cm in the dark using the remaining regrown right mystacial whiskers to judge the far edge of the gap (Fig. 1E). The results indicate that the inability to use vibrissal information to guide a movement across a gap in this case was specific to the left whiskers whose barrel field had been disconnected from the pontine nuclei.
We used a varied sequence of whisker shaving combined with peduncle lesions in different animals. Regardless of the order in which the whisker cuts and peduncle lesions were carried out, the same results were seen following the same combination of peduncle lesion and whisker cut. Figure 3 shows the results of case 6 in which there was a different sequence of ablations from that of case 1.
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Case 6 was trained and tested as normal before undergoing surgery to cut the right cerebral peduncle, tested after the unilateral peduncle lesion with a full complement of whiskers, tested again after all the whiskers on the left side of the face were cut to their bases. Finally the animal's whiskers were then allowed to regrow, then all the whiskers on the right side of the muzzle were cut to their bases and the animal tested. Figure 4 shows reconstruction of the lesion in the right cerebral peduncle in this case.
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With all its whiskers intact the rat crossed a gap of 16 cm in the dark using only its vibrissae to detect the far edge of the gap. At the smaller gaps of 12 and 14 cm, the rat also crossed consistently, often using its paw or nose as well as its whiskers to find the far side of the gap before crossing (Fig. 3A). The fact that this animal would cross a slightly larger gap than the previous animal is simply due to its larger size, allowing it to reach further either with its nose, paw, or vibrissae.
At this point, the animal underwent surgery to interrupt cortico-pontine fibers within the right cerebral peduncle. The reconstruction of the lesion can be seen in Fig. 4. After 2 days the animal had recovered from the immediate effects of the surgery and re-training began to attain preoperative levels of performance. With a lesion in the right cerebral peduncle and both sets of whiskers intact, the animal would consistently still cross 16 cm in the dark. At this distance, it could reach the far side only with its whiskers, which it used to guide its movements. The animal also continued to cross smaller gaps using a mixture of nose, paw, and whiskers to detect the far edge. The lesion in the cerebral peduncle alone did not affect the animal's ability to guide its movements using its whiskers (Fig. 3B).
When the animal's performance was stable, all the whiskers on the left mystacial pad were cut to their bases. The animal's performance was unaffected by this procedure. The rat continued to cross a gap of 16 cm in the dark using the remaining right whiskers to guide its movements. At smaller distances, the animal continued to cross consistently using whisker, paw, and nose to judge the gap distance (Fig. 3C). The animal's whiskers were then allowed to regrow to their full length.
With both sets of whiskers, the animal continued to cross a gap of 16 cm in the dark. At this point, all the whiskers on the right side of the animal's snout were cut to their bases. With only the left mystacial whiskers remaining, the animal refused to cross a gap of 16 cm in the dark, although it could touch the far side of the gap with the remaining left whiskers. At the smaller gaps of 12 and 14 cm, the animal would cross the gap touching the far side of the gap with its nose or paw before crossing (Fig. 3D).
In this case, it was only when of a lesion in the right cerebral peduncle was combined with cutting the right whiskers that the deficit in the animal's performance was seen. To confirm these results, four more animals received a lesion in the right cerebral peduncle followed by cutting the right whiskers. In all cases, after a lesion in the right peduncle, the rat could not use its left whiskers to guide its movements across the gap.
All of the animals jumped considerably further in the light than they did in the dark. Distances jumped in the light varied between 24 and 34 cm, but in no case did a peduncle lesion, whiskers cut, or combination of a lesion and whisker cut cause a decrease in the maximum distance the animal would jump in the light. Figure 5 shows a summary of results of the effects of five different combinations of ablations on gap-crossing performance.
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In all cases the lesions were placed in an appropriate position to cut the efferent fibers descending from the barrel field in the cerebral peduncle. In all but one case, the interruption of cortico-pontine fibers from the PMBSF was verified by either the scarcity or absence of retrogradely labeled cell in the cortex after filling the pontine nuclei or by the presence or absence of terminal label in the pontine nuclei following bilateral injection of anterograde tracer in the PMBSF (see METHODS). In all cases the label, either retrograde or anterograde, was heavily biased toward the side of the brain that contained the intact peduncle. The lack of label on the side of the brain containing the sectioned peduncle indicates that the efferent fibers from the PMBSF had been cut in the peduncle. Figures 6 and 7 show the injection site and label illustrating the sparsity or absence of label on the side of the brain containing the cut peduncle.
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The two animals that were tested for the ability to make orientation responses showed no change in their behavior after surgery, nor did they display any differences between either side of their body or right or left whiskers.
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DISCUSSION |
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Nearly all behavioral studies of the vibrissal system have focused
on the vibrissae as a peripheral sensory structure. Cutting whiskers or
severing the trigeminal sensory nerve causes a deficit in sensory
discrimination tasks and the sensory guidance of movements that would
normally utilize the whiskers, thus demonstrating the importance of the
whiskers as a primary source of sensory information (Broughton
1823; Carvell and Simons 1990
; Vincent
1912
; Watson 1907
). Hutson and Masterton
(1986)
discovered that a lesion of a barrel in primary sensory
cortex prevented a rat from using the corresponding whisker to make a
sensory guided movement but did not stop the animal from detecting
passive movement of the same whisker.
Rats cannot use a set of whiskers in this gap-crossing task if the connection between the whisker barrel field and the pontine nuclei is cut. The deficit was not due to an impairment of the whisker movements. The rate and pattern and symmetry of whisking was unchanged after the operation. The rats would use their whiskers to judge a gap distance if they could reach across; this was true when they were crossing in the light as well as the dark. To cross the larger gaps, the animals would lean across the gap and protract their whiskers fully to reach the other side. Following the lesion, both the left and right whiskers were protracted equally, when observed either in normal light levels or using the infra-red sight. Postoperatively the animals made contact with the distant platform with the tips of both sets of whiskers as they had before the lesion. When deprived of one set of whiskers, the remaining set still made contact with the far side. We conclude that the animals were not prevented from crossing the gap because of an inability to use their whiskers properly.
Even if the animal could reach the far edge of the gap, it is possible
that the animal could not feel its left whiskers. Although as rats with
a total PMBSF lesion can still detect passive stimulation of a whisker
with sensitivity equal to that of a normal rat (Hutson and
Masterton 1986), it would seem unlikely that cutting the
efferent fibers from the PMBSF in the cerebral peduncle would affect
whisker sensitivity. However, to ensure that the animals could still
feel and react to their whiskers before and after the peduncle lesion two animals were observed during orientation testing.
Localization-orientation behavior was unchanged after the lesion nor
was there a unilateral bias in the ability to detect passive
stimulation of the body.
In all of the lesions, there was some collateral damage to the substantia nigra pars reticularis, which lies immediately dorsal to the cerebral peduncle. Damage to this structure might produce some form of general motor deficit. Also efferent cortical axons from the barrel fields project to the superior colliculus. Like cortico-pontine fibers, these fibers descend in the cerebral peduncle. They bifurcate near the position of our lesions and slightly more rostrally. Therefore some of the fibers that would normally project from the PMBSF to the superior colliculus might have been interrupted, perhaps causing a motor disturbance that could impair performance in the gap-crossing task. Several observations during our training and testing show that there was no such general degradation of motor function following any of their lesions.
After unilateral lesions were made in the cerebral peduncles, the animals were quickly retrained to the same levels of performance they had displayed prior to the peduncle section. One group of four animals was retrained soon after surgery; starting retraining on average 15 days post-operatively. These animals all reached pre-operative levels of performance, on average 36 days after surgery. If a general motor deficit was present, it is likely to have been at its most severe immediately after the surgery, thus preventing quick retraining. No motor deficit was revealed after surgery, and the animals rapidly achieved preoperative levels of performance. Another two animals were retrained following a longer lay off after surgery; one of 4 mo, one of 9 mo. Both of these animals were retrained to preoperative levels of performance following the right peduncle lesion.
Prior to, and following, the peduncle surgery, the animals were trained to jump in the light. This procedure served as an internal control against motor impairment caused by the peduncle section. Jumping large distances in the light represents a demanding motor task, one considerably more difficult than the animal had to perform to cross smaller gaps in the dark. Damage to the motor systems that control the jump would reveal itself as a decrease in the distances that the animals could jump in the light. In all cases, jumping in the light was unaffected by the surgery or whisker shaving.
Finally, when an animal with a lesion in the peduncle refused to use the corresponding whiskers to cross the gap in the dark, it could and did make use of its nose tip or its paw to gauge the distance to cross; evidence that the animal was perfectly capable of making the movements required to cross the gap following the lesion.
The animals could therefore feel their whiskers and make the required movements to cross the gap. The deficit was specifically related to the use of the left whiskers, the ability to cross the gap recovering as the right whiskers re-grew after being cut.
Hutson and Masterton (1986) showed that the ablation of
the barrel field prevented a rat from using the contralateral whiskers in a gap-crossing task. We have shown that cutting the peduncle, thus
preventing cortical vibrissal sensory information reaching the
cerebellum prevents the animal from using the corresponding whiskers
in these cases the left whiskers
to gather sensory
information to guide the animals movements. The evidence suggests that
the cortico-ponto-cerebellar pathway is part of a system that subserves fast, ongoing, sensory guidance of movement.
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ACKNOWLEDGMENTS |
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The authors thank I. Kralj-Hans for assistance throughout the study.
This research was supported by grants to M. Glickstein from the Wellcome Trust.
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FOOTNOTES |
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Address for reprint requests: E. W. Jenkinson, Dept. of Anatomy, University College London, Malet Place, London WC1E 6BT, UK (E-mail: e.jenkinson{at}ucl.ac.uk).
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 7 February 2000; accepted in final form 2 June 2000.
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REFERENCES |
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