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Spinal cord projections from the medial cerebellar nucleus in tree shrew (Tupaia glis)
Brain Research, 171 (1979) 383-400 © Elsevier/North-Holland Biomedical Press
383
Research Reports
SPINAL CORD PROJECTIONS FROM THE MEDIAL CEREBELLAR N U C L E U S 1N T R E E S H R E W (TUPAIA GLIS)
CAROLYN B. WARE* and ELLIOTT J. MUFSON** Department of Anatomy and Program in Biological Psychology, Downstate Medical Center, Brooklyn, N. Y. (U.S.A.)
(Accepted November 16th, 1978)
SUMMARY Electrolytic lesions were placed in the medial cerebellar nucleus in tree shrews (Tupaia glis) or in fibers issuing from this nucleus. Brains and spinal cords were processed according to Fink-Heimer procedure following survival times of 2-7 days. In control animals lesions were placed in the cerebellar cortex and, in one case, in the olfactory bulb. Degenerating fibers were seen entering the cervical spinal cord and continuing to thoracic and lumbar levels. The projection is relatively profuse in the cervical cord, becoming sparse as the fibers proceed to more caudal levels. Fibers run in the lateral funiculus, predominantly contralateral to the lesion. Some fibers are observed to travel directly through the intermediate gray matter of the spinal cord. Preterminal degeneration is seen primarily in the intermediate gray of the spinal cord. Results are discussed in relation to typical locomotor behavior oT tree shrew. INTRODUCTION The existence of direct fiber connections from the deep cerebellar nuclei to the spinal cord has been both asserted and denied. Thomas (in dogs and cats) 40 and Luna (in dogs) z7 reported degenerating fibers extending as far as lumbar levels of the spinal cord in material stained by the Marchi method. In contrast, Probst 34, also on the basis of Marchi prepared material from dogs and cats, reported fibers from the deep cerebellar nuclei extending caudally only as far as the first cervical segment. Subsequent studies have indicated descending degeneration limited to the brain stem in guinea pig 2 * Present Address : Department of Psychology, Office of Academic Affairs, State University of New York at Binghamton, Binghamton, N.Y. 13901, U.S.A. ** Present Address: Department of Psychology, Clark University, Worcester, Mass. 01610, U.S.A.
384 and monkey 6,s,35 while other reports have demonstrated fibers of cerebellar origin extending into the thoracic spinal cord region in monkey 12,3.) or at least into the upper cervical segments of the cord in monkey 13, opossum 17, rabbit18, ")6,44 and cat 7, t9,,~6,36. The advent of modern reduced silver methods has yielded similarly variable results. Achenbach and Goodman 1 reported projections from the medial cerebellar nucleus in the rat extending through cervical, thoracic, and lumbar spinal segments as demonstrated by the Nauta method. Other investigations, on the other hand, found degeneration limited to the brain stem in cat 1° 46 or dissipating in the upper cervical segments of the spinal cord in pangolin 5, cat 11,41, hedgehog 1~ and opossum es. In a recent study using autoradiography projections were confined to the cervical spinal cord 4. In these reports the issue of cerebellar projections to the spinal cord often has been relatively unemphasized. The finding of degenerated axons in the spinal cord has been overshadowed by data relating to other more profuse ascending and descending fiber connections of cerebellar nuclear origin. Comparisons among observations reported in various studies are difficult. In addition to differences in histological preparation, comparisons are inconclusive because discrepancies exist between species examined, in method and locus of lesion, and in size of lesion. In the course of our ongoing studies of the projections of the deep cerebellar nuclei in the tree shrew (an animal whose typical locomotor behavior is remarkably rapid and accurately aimed) degenerating fibers have been found (in Fink-Heimed 6 prepared material) as far caudal as lumbar spinal cord segments. This projection is relatively profuse in the cervical cord, becoming quite sparse as the fibers extend into the more caudal lumbar levels. This report presents evidence for the existence in the tree shrew of direct fiber connections from the medial cerebellar nucleus throughout cervical, thoracic, and lumbar spinal cord segments. The extent of the projeCtion seen, which apparently exceeds that in other mammalian species, may be related to the ballistic quality of the locomotor activity of the tree shrew. METHOD Fifteen tree shrews (Tupaia glis) were used in this study. In each of 12 animals a lesion was placed either in the medial cerebeltar nucleus or among fibers exiting from this nucleus. In two animals the lesion invaded cerebellar cortex and white matter but did not penetrate the nucleus. In one animal a lesion was made in the olfactory bulb in order to obtain an operated control in which no damage had been done either to any cerebellar structure or to any other possible source of a spinal cord projection. Surgery was carried out under aseptic conditions. Twenty minutes prior to induction of anesthesia by intraperitoneal injection of sodium pentobarbital, animals were premedicated with 0. l ml of sparine (intramuscular) and 0.1 ml of atropine (subcutaneous). Animals were immobilized in a stereotaxic apparatus and a 'Off steel insect pin electrode coated with formvar except for 0.5 mm at the tip was directed toward the medial cerebetlar nucleus by means of a micromanipulator. Precise lesion placement, however, proved to be more accurate when landmarks on the skull and dural surface of the individual animal were used as a guide rather than stereotaxic coordinates. The
385 medial nucleus was located relatively consistently 0.5-1.0 m m lateral to the midline and 1-2 m m posterior to the transverse sinus. Lesions were placed in the left medial cerebellar nucleus. A Grass lesion maker model M 1 DC 5 was used in order to deliver 0.5 mA for 3-5 sec. Combiotic was administered routinely postoperatively. Following survival times of 2-7 days, the animals were anesthetized with sodium pentobarbitol and perfused through the heart with 0.9 % saline followed by 10 % fortool saline. Animals were then decapitated and the vertebral columns removed. The calvarium was removed to expose the brain, vertebral laminae and dorsal processes were removed, and spinal cords and brains in their remaining skeletal compartments were placed in 10% formalin for at least 3 days. Brains and spinal cords were then removed and placed in 10 % formalin for a minimum of 7 additional days after which the spinal cords were divided according to level (cervical, thoracic, or lumbar). Brains and spinal cord lengths were placed in 30% sucrose until the tissue reached equilibrium. All tissues were embedded in a matrix of egg yolk and gelatin. Spinal cord pieces were embedded so that alternate segments were cut in transverse and horizontal planes for each level (cervical, thoracic, or lumbar) of the cord. Brain and spinal cord blocks were cut on a freezing microtome at 25 nm. Three brains were sectioned in the horizontal and one in the parasagittal plane; the remaining brains were cut in the coronal plane. A series of every fourth section was prepared by the Fink-Heimer II procedure 16. Adjacent sections were stained with cresyl violet for cytoarchitectural analysis. The extent of the lesions and loci of degenerating particles were plotted with the aid of a microprojector. Only those particles which were irregular and varicose and which were not in apparent continuity with any reticular fibers associated with vessels when traced in sequential planes of focus were accepted as degenerating fibers. In sections from the spinal cords of all animals including the case with the olfactory bulb lesion (TS 144), cells were seen which appeared spotted with small spherical argyrophilic particles not associated with apparent fiber degeneration. This form of silver deposition seemed to be unrelated to lesion placement and was considered to be most likely an example of the artifactual silver deposits described by Heimer '~1 on cells of the olfactory bulb and interlaminar thalamic nuclei. RESULTS The medial cerebellar nucleus in the tree shrew is located on either side of the midline in the white matter at the base of the cerebellum (Figs. I A, B and 2-4). A substantial bundle of fibers completely separates the ceils of the medial nucleus from the interpositus and lateral nucleus. The line of demarcation between interpositus and lateral nuclei is somewhat ambiguous since fibers do not separate the cell groups completely 2°. Clarification of the exact boundaries between the interpositus and lateral cerebellar nuclei no doubt will depend on studies of their fiber connections and embxyological origin which are beyond the scope of this report. At the pontomedullary junction where the middle and inferior cerebellar peduncles join the cerebellum to the brain stem, cells of the lateral and superior vesti-
386 bular nuclei invade the cerebellar white matter (Figs. 1B and 3). It is critical to the interpretation of degeneration resulting from lesions of the cerebellar nuclei to avoid damage to the neuronal somata of the vestibular nuclei.
Locus of lesion Lesions were placed in the left medial cerebellar nucleus or in the fiber bundles rostral and lateral to the nucleus. The pattern and type of degeneration seen in the spinal cord following these lesions will be illustrated in cases with survival times of 4 (TS 138, TS 141, TS 170) and 7 (TS 115) days. Degenerating axons were present throughout cervical, thoracic, and lumbar spinal levels in those cases which survived 4 or more days. In cases in which the survival time was limited to two days (TS135, TS 136), small argyrophilic particles were present in the cord, but these were not associated with fragments identifiable as degenerated axons caudal to the first cervical seg, ment. In none of these cases was the entire medial nucleus destroyed, and in no case was any damage to the cells of the vestibular nuclei evident under light microscopic analysis. Cells of the vestibular nuclei were normal in Nissl-stained sections with no differences observed ipsilateral or contralateral to the lesion. In Fink-Heimer prepared sections, the degenerating axons exiting from the cerebellum through the vestibular nuclei were not observed to be augmented in number, nor was there any apparent dis-
Fig. 1. Photomicrographs of cresyl violet-stained sections of lesions in medial cerebellar nucleus. Tupaiaglis. A: TS 170; horizontal section through the cerebellum, midbrain, and diencephalon. Note that the lesion is marked by dense gliosis in the region of the left medial cerebellar nucleus and that the tissue damage is confined to the nucleus. B: TS 13B; parasagittal section through the pontomedullary junction. Gliosis at the lesion site extends into the fiber bundles between the medial and interpositus nuclei. Note that tissue damage is confined to white matter within the cerebellum and does not approach cells of the vestibular nucleus.
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TS 170 Fig. 2. Drawing of horizontal sections through the lesion in the medial cerebellar nucleus in TS 170. The lesion is shown in black, indicating tissue disruption. Striations indicate gliosis and cell loss resulting from the lesion. Note that the gliosis does not extend through the most ventral portion of the nucleus.
ruption o f tissue as was obvious in the i m m e d i a t e vicinity o f the lesion within the cerebellum.
Trajectoo' of efferent fibers F o l l o w i n g a lesion in the medial cerebellar nucleus, degenerating fibers stream both ipsilaterally and c o n t r a l a t e r a l l y (Fig. 5). Degenerating axons pass f r o m the lesion dorsal, ventral, and rostral to the c o n t r a l a t e r a l medial cerebellar nucleus. Some o f
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TS 115 Fig. 3. Drawing ol the lesion in TS 115 in coronal sections through the cerebelluni and brain stem, The lesion is confined to the caudal half of the medial cerebellar nucleus.
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Fig. 4. Drawing of the lesion in TS 141 in coronal sections through the cerebellum and brain stem. The lesion is confined to the rostral portion of the nucleus.
389
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LUMBAR
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Fig. 5. A : TS 170; drawing of degeneration shown in a horizontal section through pons, medulla and the first cervical spinal segment. Due to the cephalic flexure, the spinal cord is cut obliquely so that ventral horn, intermediate gray, and dorsal horn all appear in the section. Nerve roots are shown in solid black. Degenerating fibers are indicated by broken lines while preterminal degeneration is shown by dots. An enlargement of the area outlined in the spinal cord is reproduced as a photomicrograph in Fig. 8A. B : drawing of degeneration in TS 170 in representative transverse sections through cervical, thoracic, and lumbar spinal cord segments. these crossing fibers traverse the nucleus while others form bundles o f fibers rostral to the nucleus. One bundle o f decussating fibers follows a course directed between the interpositus and medial nuclei to enter the brain stem medial to the restiform body. Degenerating fibers pass t h r o u g h and between the lateral and inferior vestibular nuclei. C o n t r a l a t e r a l to the lesion, a g r o u p o f degenerating axons continues dorsal to or traverses the interpositus nucleus to form a curved bundle o f fibers d o r s a l to the b r a c h i u m conjunctivum, the uncinate fasciculus. F r o m this bundle, a few fibers descend the b r a i n stem c o n t r a l a t e r a l to the lesion. As seen in Fig. 5, the descending fibers on b o t h sides travel t h r o u g h the pons a n d m e d u l l a in a r a t h e r diffuse fashion. A significant n u m b e r a p p r o a c h the midline a n d some cross the raph6; others pass within the fiber bundles extending l o n g i t u d i n a l l y on b o t h sides o f the medial plane, including the regions o f the tectospinal t r a c t a n d the medial longitudinal fasciculus. The most a b u n d a n t g r o u p o f fibers extends t h r o u g h the
390 medial reticular formation. At the level of the decussation of the pyramids, cerebetlar efferent fibers either enter the gray matter ot the spinal cord directly or pass into the lateral funiculus (Figs. 7, 8B and 10A. C). Instead of forming a compact bundle, these efferent fibers course through the white matter as loosely scattered axons. A few fibers emerging from the medial reticular formation enter the anterior funiculus. In addition to fibers from the nucleus, some corticofugal fibers issuing from the electrode track exit from the cerebellum. These fibers end in vesti bular nuclei and reticular formation ipsilateral to the cerebellar cortical lesion. In horizontal sections through the spinal cord, the degenerating axons are seen to follow the dorsal half of lateral funiculus (Fig. 6). At all levels the fibers of passage seen in white matter are less numerous than those seen branching into the gray matter. (Figs. 7 and 9A, B). Degenerating axons seen in lumbar (Fig. 8A, B) and thoracic (Fig. 9) levels were sparse in comparison to those observed in cervical segments (Fig. 7C). Within the spinal cord preterminal degeneration appears m the intermediate gray and ventral horn and is seen in laminae V, VI, VII. and VIH : at upper cervical levels the density of preterminat degeneration in the intermediate gray rivals that seen in the reticular formation in the medulla (Figs. 5 and 7A, C). Fragments of preterminal degenerating fibers are seen in close relation to neuronal somata in the intermediate gray and ventral horn (Figs. 10. 11 and 12): argyrophilic particles appear both in the neuropil and over neuronal somata. ROSTRAL
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Fig. 6. Drawing of degeneration in TS 141 in representative horizontal sections through cervical, thoracic, and lumbar spinal segments. The enlargement of the area outlined in the lateral funiculus of the cervical section is reproduced as a photomicrograph in Fig. 10A.
391
Fig. 7. P h o t o m i c r o g r a p h s o f degeneration ( F i n k - H e i m e r ) in TS 170. A: low power picture o f fibers e n t e r i n g the lateral funiculus a n d intermediate gray. Outlined areas are reproduced at a higher m a g n i fication in 7B a n d 7C. x 25. B: degenerating fibers are seen entering the lateral funiculus a n d intermediate gray. Fibers coursing nearer to or within the intermediate gray are o f relatively smaller diameter t h a n those travelling in the lateral funiculus, x 100. C: degenerating fibers within the intermediate gray of the cervical spinal cord. × 100.
392
Fig. 8. Photomicrographs of degeneration (Fink-Heimer) in a transverse section through the lumbar spinal cord contralateral to the lesion in TS 115. A: montage of low power photomicrographs showing a hemisection of the spinal cord of TS ! 15 contralateral to the lesion for orientation. The dorsal horn is free of fragments of degenerated fibers such as those which appear in the ventral h o r n . . 25. Area outlined is reproduced at a higher magnification in Fig. 9B. B: degenerating fibers a n d preterminal degeneration in the lumbar spinal cord, TS 115. ~ 250.
393
Fig. 9. Photomicrographs of degenerating fibers (Fink Heimer) in the lateral funiculus contralateral to the lesion as seen in horizontal sections through cervical and thoracic spinal cord segments. A: degenerating fibers in the cervical cord, TS 170. Illustration is a montage of two photomicrographs taken at different planes of focus. ~ 125 B : degenerating fibers in the thoracic spinal cord, TX 138. x 125. C: degenerating fibers in the thoracic spinal cord, TS 170. x 250.
394 Following unilateral lesions of the medial cerebellar nucleus, degeneration necessarily appears bilaterally, since the fibers decussating within the cerebellum are so intimately related to all surfaces of the nucleus that injury of one medial cerebellar nucleus cannot be produced independently of damage to fibers originating from the other. In TS 170 the lesion, while large, is closely confined to the limits of the nucleus itself (Fig. 2). Degeneration seen following this lesion is predominantly contralateral (Figs. 5 and 6). In contrast, in TS 141 the damage to the nucleus itself is relatively minor; fibers crossing rostral to the nucleus are, however, significantly involved (Fig. 4). The pattern of degeneration in the spinal cord following this lesion is bilaterally symmetrical (Fig. 6). Since the ipsilateral degenerating axons seem to b e related to interruption of fibers crossing from the contralateral nucleus, the projection apparently is predominantly crossed.
Appearance of control mater&l In the spinal cord TS 144 in which a lesion was placed in the olfactory bulb sporadic argyrophilic fragmented fibers are seen in widely separated sections. These occur with no particular bias toward one or the other side and are equally rare in cervical, tholacic and lumbar segments. Except for these randomly occurring fragments and occasional cells with particulate artifact as described previously, the spinal cord sections in control material are clear of argyrophilic particulate aggregation.
Fig. 10. Photomicrograph of degenerated fibers and preterminal degeneration (Fink-Heimer) associated with neuronal soma and processesin transverse section through the cervical spinal cord, TS 170. Degenerating fibers and panicles of preterminal degeneration associated with two neuronal somata and their processes, x 500.
395
Fig. 11. Horizontal section through the thoracic spinal cord in TS 138. A degenerating fiber turns around the neuronal soma (Fink-Heimer). A montage of four photomicrographs taken at different planes of focus, x 500.
Fig. 12. Degenerating fibers and preterminal degeneration (Fink Heimer) seen in the lumbar spinal cord. TS 138. Degeneration in the lumbar spinal cord. × 125.
396 DISCUSSION
The weight of the evidence supports the existence of projections trom the medial cerebeltar nucleus to at least cervical levels or the spinal cord in cataa,l'a,~.:~a,36, 41, monkey 4,~a, hedgehog 1~, opossum17, 28 and pangolinn; but assertions of fiber connections from cerebellar nuclei to thoracic and lumbar spinal segments have been few and widely separatedl,ZT,30,3~, 4o. Projections have been reported in the lumbar cord based on both Marchi z7,4° and Nauta I techniques, yet in other studies such connections were not seen. Recently, Earle and MatzkO 5, using Fink-Heimer and Nauta methods in hedgehog, specifically denied the existence of cerebellar projections to lower spinal cord levels. Before addressing the significance of spinal cord projections from the cerebellum in the tree shrew, the possibility that some of these degenerating fibers originate from areas other than the medial cerebellar nucleus must be excluded. Sporadic cell death has been reported in the lateral cerebeltar nucleus of the cat 14 and the lateral vestibular nucleus of the rat 39. Indeed, an occasional fragmented argyrophilic fiber can be seen in spinal cord sections of control material (TS 144). These did not, however, occur with the frequency, regularity of distribution, laterality or consistency of the degenerated fibers which were evident following medial cerebellar nucleus lesions. The likelihood of inadvertent damage to the vestibular nuclei in the course of placing lesions in the cerebellar nuclei has long been recognized7, 40,4~.44 In the present material, lesions made electrolytically are discrete and relatively small. The extent of tissue damage, as seen both in cresyl violet-stained and Fink-Heimer prepared sections, does not involve cells of the vestibular nuclei which appear to be normal upon light microscopic examination. No observable differences are seen between the vestibular nuclei ipsilateral and contralateral to the lesion. Although direct damage to the vestibular nuclei can be excluded, the possibility of retrograde or transneuronal reaction to the cerebellar lesion must be considered 9. Retrograde cell death in the vestibular nuclei conceivably could result from destruction of the medial cerebellar nucleus, a major target for vestibular projections. Such retrograde degeneration, undetectable at the short survival times optimal for FinkHeimer preparations, might produce changes in axon terminals. Alternatively, loss of the profuse connections supplied to the vestibular nuclei by the medial cerebellar nucleus could precipitate transneuronal changes in the vestibular nuclei which also might be reflected in degeneration of their axon terminals. The observed distribution of degeneration seen in this tree shrew material mitigates against the vestibular nuclei as the source of the argyrophilic fibers. Well over half the nucleus is destroyed in TS 170: degenerated axons seen entering the vestibular nuclei are dense bilaterally. Since the afferent fibers entering the vestibular nuclei are. apparently, equally dense on both sides, any resulting transneuronal changes should be proportionately distributed bilaterally. The degenerauon seen m TS 170 was. however, clearly heavier contralateral to the lesion. In TS 141 and TS 115. damage to the medial cerebellar nucleus is less extensive. It seems unlikely that this reduction in either vestibular axon terminals in the medial cerebetlar nucleus or loss of cerebellar affe-
397 rents to the vestibular nucleus could be significant enough to produce alterations in the vestibular cells sufficient to account for the observed anterograde changes in the spinal cord. Furthermore, in horizontal sections of the spinal cord the degenerating fibers appear in the lateral funiculus in a relatively dorsal position, whereas the fibers of the lateral vestibulospinal tract would be expected to course in the anterolateral fiber bundle if they follow the trajectory reported for other mammals 7,31,33. In this material the number of degenerating fibers does not diminish appreciably as they pass from the brain stem into the cervical spinal cord (Fig. 5). This finding is in contrast to several reports in which others, who were unable to follow cerebellar efferent fibers beyond cervical levels, stress the paucity of the degeneration observed as the projection enters the cordl~,lS, 36. Many of the fibers we observe entering the spinal cord seemed to be of relatively small diameter. Heimer 21 has suggested that axons ot very small caliber may fail to impregnate with silver to sufficient degree to be seen by the light microscope: only their preterminal enlargements are revealed. This phenomenon could account for at least some of the inconsistencies reported in various studies. The appearance of the fibers degenerating in the tree shrew spinal cord is similar to that of the degenerating fibers in the medulla and pons: to accept them as efferents of the medial cerebellar nucleus seems to be the most parsimonious interpretation of these results. This being so, the significance of this projection should be considered in relation to that of other mammals. The cerebellar nuclei project to the reticular formation, the vestibular nuclei, and the red nucleus (all of which have direct fiber connections to the spinal cord) in a large number of species studied 25, including the tree shrew 47. Recent reports have confirmed projections from cerebellar nuclei to the superior colliculus including those layers from which the tectospinal projections originate 3'~s. Medial, interpositus, and lateral nuclei of the cerebellum also project to the ventral anterior and ventrolateral nuclei of the thalamus from which efferent fibers are distributed to the motor cortex25, 27. Thus, the mammalian nervous system appears to be organized in such a way that the cerebellum maintains extensive and direct control over the nuclei which are involved in the initiation of motor behavior. It is entirely consistent with this pattern that cerebellar efferent fibers reach the spinal cord. A common pattern of organization, however, may also accomodate variation among species. The motor behavior of tree shrew may be more strongly influenced by the cerebellum than by the corticospinal system. Kornhuber 24 has proposed that the cerebellum is uniquely responsible for aiming ballistic movement, i.e. movement which occurs too rapidly to be directed continuously by sensory feedback. The direction and target localization of such a movement must be determined prior to initiation of the motor behavior. In fact, the typical locomotor behavior of tree shrews fits this description most precisely. Tupaia glis, the species used in this study, occupies a semi-arboreal econiche. Rapid, accurately directed short leaps are characteristic of the locomotor activity of these animals ')3. 29,38,43. Forelimb function is precise, fast, and stereotyped in nature. When a meal worm is offered beyond the mesh of the cage door, a tree shrew instantly projects a forelimb through the small opening to snatch the bait, continuing the activity for
398 dozens o f repetitions without missing the w o r m ( M a r t i n , p e r s o n a l c o m m u n i c a t i o n ) . The ballistic quality o f the m o t o r b e h a v i o r o f T u p a i a g l i s has obvious a d a p t i v e value in a s e m i - a r b o r e a l habitat. I n this context, it is interesting t h a t in recent r e p o r t s o f cerebellar projections in the h e d g e h o g 16 a n d o p o s s u m 2s, based on F i n k - H e i m e r procedures, d e g e n e r a t i n g axons were n o t seen c a u d a l to the cervical spinal segments. N e i t h e r o f these n o c t u r n a l a n i m a l s are c h a r a c t e r i z e d by rapid, c o o r d i n a t e d m o t o r activity. The s h o r t survival times which are o p t i m a l for d e m o n s t r a t i o n o f d e g e n e r a t i n g axons limit the o p p o r t u n i t y for behavioral observation, while the small lesions which are desirable for a n a t o m i c a l p u r p o s e s do not p r o d u c e m a r k e d changes ill c o o r d i n a tion. All the a n i m a l s showed some i n c o o r d i n a t i o n in the first days following surgery, b u t this could be related as much to e d e m a a n d other t r a n s i t o r y p o s t - o p e r a t i v e changes as to the direct cerebellar damage. F u r t h e r studies o f cerebellar c o n t r o l o f m o t o r b e h a v i o r in the tree shrew are n o w u n d e r w a y in our l a b o r a t o r y . ACKNOWLEDGEMENTS W e t h a n k Dr. M i m i H a l p e r n , Dr. Jacqueline J a k w a y , a n d Dr. W a l t e r R i s s for their suggestions; Dr. H e n r i Begleiter a n d Dr. F r a n k Scalia for p r o v i d i n g the stereotaxic e q u i p m e n t ; Mr. lrvin W i l s o n for technical assistance; Mrs. S u d h a Joshi, Miss C a r o l J a r y n o w s k i a n d Mrs. R o s e K r a u s for m a n u s c r i p t p r e p a r a t i o n s ; Mr. J a c k lllari for p h o t o g r a p h i c assistance; a n d Mrs. Tina M a g n o , w i t h o u t w h o m the project c o u l d n o t have been accomplished.
REFERENCES 1 Achenbach, K. E. and Goodman. D. C., Cerebellar projections to pons, medulla and spinal cord in the albino rat, Brain Behav. EvoL. 1 (1968) 43-57. 2 Allen, W., Distribution of the fibers originating from the different basal cerebeltar nuclei, J. comp. Neurol., 36 (1923) 399-439. 3 Angaut, P. and Bowsher, D., Ascending projections of the medial cerebellar ffastigial) nucleus: an experimental study in the cat, Brain Research, 24 (1970) 49-68. 4 Batton, R. R., Jayaraman. A., Ruggiero, D. and Carpenter, M.. Fastigial efferent projections in the monkey: an autoradiographic study, J. comp. Neurol., 174 (1977) 281-306. 5 Bautista, N. S. and Foltz, F. M., The efferent connections of the cerebellar nuclei in the pangolin, J. comp. Neurol., 132 (1968) 213-226. 6 Bautista, N. S. and Matzke, H. A., A degeneration study of cerebellifugal fibers in the monkey, J. Hirnforsch., 8 (1965-1966) 283-299. 7 Cajal, S. Ram6n Y, Histologie du Systdme Nerveux. Consejo Superior de lnvestigateiones cientificas Instituto Ramon Cajal, Madrid, 1955, pp. 124-129. 8 Carpenter. M. B., Lesions of the fastigial nuclei in the rhesus monkey, Amer..L Anat.. 104 (1959} 1-20. 9 Carpenter. M. B., Bard, D. S. and Alling, F. A., Anatomical connections between the fastigial nuclei, the labyrinth, and the vestibular nuclei in the cat, J. comp. Neurol., 111 (1959) 1-26. 10 Carpenter, M. B., Brittin, G. M. and Pines, J., Isolated lesions of the fastigial nuclei in the cat, J. comp. Neurot., 109 (1958) 65-89. 11 Carpenter, M. B. and Nova, H., Descending division of the brachium conjunctivum in the cat: a cerebello-reticular system, J. comp. NeuroL, 114 (1960) 295-301 12 Carpenter, M. B. and Stevens, G. H., Structural and functional relationships between the deep
399 cerebellar nuclei and the brachium conjunctivum in the rhesus monkey, J. comp. Neurol., 107 (1957) 109-164. 13 Carrea, R. M. and Mettler, F. A., The anatomy of the primate brachium conjunctivum and associated structures, J. comp. Neurol., 101 (1954) 565-690. 14 Chan-Palay, V., Neuronal plasticity in the cerebellar cortex and lateral nucleus, Z. Anat. EntwicklGesch., 142 (1973) 23-35. 15 Earle, A. M. and Matzke, H. A., Efferent fibers of the deep cerebellar nuclei in hedgehogs, J. comp. Neurol., 154 (1974) 117 132. 16 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 17 Foltz, F. M. and Matzke, H., An experimental study on the origin, course, and termination of the cerebellifugal fibers in the opossum, J. comp. Neurol., 114 (1960) 107-125. 18 Gerebtzoff, M. A., Les bases anatomiques de la physiologic due cervelet, La Cellule, 49 (1941-43) 73-167. 19 Gray, L. P., Some experimental evidence on the connections of the vestibular mechanism in the cat, J. eomp. Neurol., 41 (1926) 319-364. 20 Haines, D. E., The morphology of the cerebellar nuclei of Galago and Tupaia, Amer. J. phys. Anthropol., 35 (1971) 27-42. 21 Heimer, L., Selective silver impregnation of degenerating axoplasm. In W. J. H. Nauta and S. Ebbesson, (Eds.), Contemporary Research Methods in Neuroanatomy, Springer-Verlag, New York, 1970. 22 Jane, J. A., Campbell, C. B. G. and Yashon, D., Pyramidal tract : a comparison of two prosimian primates, Science, 147 (1965) 153-155. 23 Kaufman, J. H., Studies on the behavior of captive tree shrews (Tupaia glis), Folia primatol., 3 (1965) 50-74. 24 Kornhuber, H. H., Motor functions of cerebellum and basal ganglia: the cerebello-cortical saccadic (ballistic) clock, the cerebello-nuclear hold regulator, and the basal ganglia ramp (voluntary speed, smooth movement) generator, Kybernetik, 8 (1971) 157-162. 25 Larsell, O. and Jansen, J., The Comperative Anatomy and Histology of the Cerebellum: the Human Cerebellum, Cerebellar Connections, and Cerebellar Cortex, Univ. of Minnesota Press, 1972. 26 Lewandowsky, M., Beitrage zur anatomic des hirnstammes, J. P3ychol. Neurol. (Lpz.), 2 (1903) 18-29. 27 Luna, E., Contribution experimentale a la connaissance des voies de projection du cervelet, Arch. ltal. Biol., 51 (1909) 137. 28 Martin, G. F., King, J. S. and Dom, R., The projections of the deep cerebellar nuclei of the opossum, Didelphis marsupialis virginiana, J. Hirnforsch., in press. 29 Martin, R. D., Reproduction and ontogeny in tree shrews (Tupaia glis) with reference to their general behavior and taxonomic relationships, Z. Tierpsychol., 25 (1968) 409-532. 30 Mettler, F. A., Orioli, F. L., Grundfest, H. and Liss, H., The descending limb of the brachium conjunctivum, Trans. Amer. neurol. Ass., (1954) 79 81. 31 Nyberg-Hansen, R., Origin and termination of fibers from the vestibular nuclei descending in the medial longitudinal fasciculus, J. comp. Neurol., 122 (1964) 355-367. 32 Orioli, F. and Mettler, F. A., Descending limb of the brachium conjunctivum in Macaca mulatta, J. comp. Neurol., 106 (1956) 339-362. 33 Pompeiano, O. and Brodal, A., The origin of vestibulospinal fibers in the cat, Arch. ital. Biol., 95 (1957) 166-195. 34 Probst, M., Zur anatomie und physiologie des kleinhirns, Arch. Psychiat. Nervenkr., 33 (1902) 692-777. 35 Rand, R. W., An anatomical and experimental study of the cerebellar nuclei and their efferent pathways in the monkey, J. comp. Neurol., 101 (1954) 167-224. 36 Rasmussen, A. T,, Origin and course of the fasciculus uncinatus (Russell) in the cat, with observations on other fiber tracts arising from the cerebellar nuclei, J. comp. Neurol., 57 (1933) 165 184. 37 Shriver, J. E. and Noback, C. R., Cortical projections to the lower brain stem and spinal cord in the tree shrew (Tupaia glis). J. eomp. NeuroL, 130 (1967) 25-54. 38 Sorenson, M. W. and Conaway, C. H., Observations on the social behavior of tree shrews in captivity, Folia primatol., 4 (1966) 124-145. 39 Sotelo, D. and Palay, S. L., Altered axons and axon terminals in the lateral vestibular nucleus of the rat, Lab. Invest., 25 (1971) 653-671. 40 Thomas, A., Le faisceau cerebelleux descendent, C. R. Soc. Biol. (Paris), 4 (1897) 36-37.
400 41 Thomas, D. M., Kaufman, R. P., Sprague, J. M. and Chambers, W. W., Experimental studies of the vermal cerebellar projections in the brain stem of the cat (fastigiobulbar lract), J. Anal. (Lond.), 90 (1956) 371-385. 42 Tigges, J. and Shantha, T. R., A stereotaxic Brain Atlas o f the Tree Shrew ( Tupaia giis ) , Williams and Wilkins, Baltimore, 1969. 43 Vandenbergh, J. G., Feeding, activity, and social behavior of the tree shrew, Tupaiagtis, in a large outdoor enclosure, Foliaprimatol., 1 (1963)199 207. 44 Van Gehuchten, A., Le faisceau en crochet ou faisceau cerebellobulbaire, Le Nevraxe, Vll (1905J 119-159. 45 Verhaart, W. J. C., The pyramidal tract of Tupaia, compared to that of other primates, J. comp. Neurol., 126 (1966) 43-50. 46 Voogd, J., The Cerebellum o f the Cat, Davis, Philadelphia, (1961) 160 pp. 47 Ware, C. B., Efferent projections of the deep cerebellar nuclei in tree shrew (Tupaia glis), Anat. Rec., 175 (1973) 463.
383
Research Reports
SPINAL CORD PROJECTIONS FROM THE MEDIAL CEREBELLAR N U C L E U S 1N T R E E S H R E W (TUPAIA GLIS)
CAROLYN B. WARE* and ELLIOTT J. MUFSON** Department of Anatomy and Program in Biological Psychology, Downstate Medical Center, Brooklyn, N. Y. (U.S.A.)
(Accepted November 16th, 1978)
SUMMARY Electrolytic lesions were placed in the medial cerebellar nucleus in tree shrews (Tupaia glis) or in fibers issuing from this nucleus. Brains and spinal cords were processed according to Fink-Heimer procedure following survival times of 2-7 days. In control animals lesions were placed in the cerebellar cortex and, in one case, in the olfactory bulb. Degenerating fibers were seen entering the cervical spinal cord and continuing to thoracic and lumbar levels. The projection is relatively profuse in the cervical cord, becoming sparse as the fibers proceed to more caudal levels. Fibers run in the lateral funiculus, predominantly contralateral to the lesion. Some fibers are observed to travel directly through the intermediate gray matter of the spinal cord. Preterminal degeneration is seen primarily in the intermediate gray of the spinal cord. Results are discussed in relation to typical locomotor behavior oT tree shrew. INTRODUCTION The existence of direct fiber connections from the deep cerebellar nuclei to the spinal cord has been both asserted and denied. Thomas (in dogs and cats) 40 and Luna (in dogs) z7 reported degenerating fibers extending as far as lumbar levels of the spinal cord in material stained by the Marchi method. In contrast, Probst 34, also on the basis of Marchi prepared material from dogs and cats, reported fibers from the deep cerebellar nuclei extending caudally only as far as the first cervical segment. Subsequent studies have indicated descending degeneration limited to the brain stem in guinea pig 2 * Present Address : Department of Psychology, Office of Academic Affairs, State University of New York at Binghamton, Binghamton, N.Y. 13901, U.S.A. ** Present Address: Department of Psychology, Clark University, Worcester, Mass. 01610, U.S.A.
384 and monkey 6,s,35 while other reports have demonstrated fibers of cerebellar origin extending into the thoracic spinal cord region in monkey 12,3.) or at least into the upper cervical segments of the cord in monkey 13, opossum 17, rabbit18, ")6,44 and cat 7, t9,,~6,36. The advent of modern reduced silver methods has yielded similarly variable results. Achenbach and Goodman 1 reported projections from the medial cerebellar nucleus in the rat extending through cervical, thoracic, and lumbar spinal segments as demonstrated by the Nauta method. Other investigations, on the other hand, found degeneration limited to the brain stem in cat 1° 46 or dissipating in the upper cervical segments of the spinal cord in pangolin 5, cat 11,41, hedgehog 1~ and opossum es. In a recent study using autoradiography projections were confined to the cervical spinal cord 4. In these reports the issue of cerebellar projections to the spinal cord often has been relatively unemphasized. The finding of degenerated axons in the spinal cord has been overshadowed by data relating to other more profuse ascending and descending fiber connections of cerebellar nuclear origin. Comparisons among observations reported in various studies are difficult. In addition to differences in histological preparation, comparisons are inconclusive because discrepancies exist between species examined, in method and locus of lesion, and in size of lesion. In the course of our ongoing studies of the projections of the deep cerebellar nuclei in the tree shrew (an animal whose typical locomotor behavior is remarkably rapid and accurately aimed) degenerating fibers have been found (in Fink-Heimed 6 prepared material) as far caudal as lumbar spinal cord segments. This projection is relatively profuse in the cervical cord, becoming quite sparse as the fibers extend into the more caudal lumbar levels. This report presents evidence for the existence in the tree shrew of direct fiber connections from the medial cerebellar nucleus throughout cervical, thoracic, and lumbar spinal cord segments. The extent of the projeCtion seen, which apparently exceeds that in other mammalian species, may be related to the ballistic quality of the locomotor activity of the tree shrew. METHOD Fifteen tree shrews (Tupaia glis) were used in this study. In each of 12 animals a lesion was placed either in the medial cerebeltar nucleus or among fibers exiting from this nucleus. In two animals the lesion invaded cerebellar cortex and white matter but did not penetrate the nucleus. In one animal a lesion was made in the olfactory bulb in order to obtain an operated control in which no damage had been done either to any cerebellar structure or to any other possible source of a spinal cord projection. Surgery was carried out under aseptic conditions. Twenty minutes prior to induction of anesthesia by intraperitoneal injection of sodium pentobarbital, animals were premedicated with 0. l ml of sparine (intramuscular) and 0.1 ml of atropine (subcutaneous). Animals were immobilized in a stereotaxic apparatus and a 'Off steel insect pin electrode coated with formvar except for 0.5 mm at the tip was directed toward the medial cerebetlar nucleus by means of a micromanipulator. Precise lesion placement, however, proved to be more accurate when landmarks on the skull and dural surface of the individual animal were used as a guide rather than stereotaxic coordinates. The
385 medial nucleus was located relatively consistently 0.5-1.0 m m lateral to the midline and 1-2 m m posterior to the transverse sinus. Lesions were placed in the left medial cerebellar nucleus. A Grass lesion maker model M 1 DC 5 was used in order to deliver 0.5 mA for 3-5 sec. Combiotic was administered routinely postoperatively. Following survival times of 2-7 days, the animals were anesthetized with sodium pentobarbitol and perfused through the heart with 0.9 % saline followed by 10 % fortool saline. Animals were then decapitated and the vertebral columns removed. The calvarium was removed to expose the brain, vertebral laminae and dorsal processes were removed, and spinal cords and brains in their remaining skeletal compartments were placed in 10% formalin for at least 3 days. Brains and spinal cords were then removed and placed in 10 % formalin for a minimum of 7 additional days after which the spinal cords were divided according to level (cervical, thoracic, or lumbar). Brains and spinal cord lengths were placed in 30% sucrose until the tissue reached equilibrium. All tissues were embedded in a matrix of egg yolk and gelatin. Spinal cord pieces were embedded so that alternate segments were cut in transverse and horizontal planes for each level (cervical, thoracic, or lumbar) of the cord. Brain and spinal cord blocks were cut on a freezing microtome at 25 nm. Three brains were sectioned in the horizontal and one in the parasagittal plane; the remaining brains were cut in the coronal plane. A series of every fourth section was prepared by the Fink-Heimer II procedure 16. Adjacent sections were stained with cresyl violet for cytoarchitectural analysis. The extent of the lesions and loci of degenerating particles were plotted with the aid of a microprojector. Only those particles which were irregular and varicose and which were not in apparent continuity with any reticular fibers associated with vessels when traced in sequential planes of focus were accepted as degenerating fibers. In sections from the spinal cords of all animals including the case with the olfactory bulb lesion (TS 144), cells were seen which appeared spotted with small spherical argyrophilic particles not associated with apparent fiber degeneration. This form of silver deposition seemed to be unrelated to lesion placement and was considered to be most likely an example of the artifactual silver deposits described by Heimer '~1 on cells of the olfactory bulb and interlaminar thalamic nuclei. RESULTS The medial cerebellar nucleus in the tree shrew is located on either side of the midline in the white matter at the base of the cerebellum (Figs. I A, B and 2-4). A substantial bundle of fibers completely separates the ceils of the medial nucleus from the interpositus and lateral nucleus. The line of demarcation between interpositus and lateral nuclei is somewhat ambiguous since fibers do not separate the cell groups completely 2°. Clarification of the exact boundaries between the interpositus and lateral cerebellar nuclei no doubt will depend on studies of their fiber connections and embxyological origin which are beyond the scope of this report. At the pontomedullary junction where the middle and inferior cerebellar peduncles join the cerebellum to the brain stem, cells of the lateral and superior vesti-
386 bular nuclei invade the cerebellar white matter (Figs. 1B and 3). It is critical to the interpretation of degeneration resulting from lesions of the cerebellar nuclei to avoid damage to the neuronal somata of the vestibular nuclei.
Locus of lesion Lesions were placed in the left medial cerebellar nucleus or in the fiber bundles rostral and lateral to the nucleus. The pattern and type of degeneration seen in the spinal cord following these lesions will be illustrated in cases with survival times of 4 (TS 138, TS 141, TS 170) and 7 (TS 115) days. Degenerating axons were present throughout cervical, thoracic, and lumbar spinal levels in those cases which survived 4 or more days. In cases in which the survival time was limited to two days (TS135, TS 136), small argyrophilic particles were present in the cord, but these were not associated with fragments identifiable as degenerated axons caudal to the first cervical seg, ment. In none of these cases was the entire medial nucleus destroyed, and in no case was any damage to the cells of the vestibular nuclei evident under light microscopic analysis. Cells of the vestibular nuclei were normal in Nissl-stained sections with no differences observed ipsilateral or contralateral to the lesion. In Fink-Heimer prepared sections, the degenerating axons exiting from the cerebellum through the vestibular nuclei were not observed to be augmented in number, nor was there any apparent dis-
Fig. 1. Photomicrographs of cresyl violet-stained sections of lesions in medial cerebellar nucleus. Tupaiaglis. A: TS 170; horizontal section through the cerebellum, midbrain, and diencephalon. Note that the lesion is marked by dense gliosis in the region of the left medial cerebellar nucleus and that the tissue damage is confined to the nucleus. B: TS 13B; parasagittal section through the pontomedullary junction. Gliosis at the lesion site extends into the fiber bundles between the medial and interpositus nuclei. Note that tissue damage is confined to white matter within the cerebellum and does not approach cells of the vestibular nucleus.
387
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142
134
126
122
TS 170 Fig. 2. Drawing of horizontal sections through the lesion in the medial cerebellar nucleus in TS 170. The lesion is shown in black, indicating tissue disruption. Striations indicate gliosis and cell loss resulting from the lesion. Note that the gliosis does not extend through the most ventral portion of the nucleus.
ruption o f tissue as was obvious in the i m m e d i a t e vicinity o f the lesion within the cerebellum.
Trajectoo' of efferent fibers F o l l o w i n g a lesion in the medial cerebellar nucleus, degenerating fibers stream both ipsilaterally and c o n t r a l a t e r a l l y (Fig. 5). Degenerating axons pass f r o m the lesion dorsal, ventral, and rostral to the c o n t r a l a t e r a l medial cerebellar nucleus. Some o f
8
TS 115 Fig. 3. Drawing ol the lesion in TS 115 in coronal sections through the cerebelluni and brain stem, The lesion is confined to the caudal half of the medial cerebellar nucleus.
,I
Fig. 4. Drawing of the lesion in TS 141 in coronal sections through the cerebellum and brain stem. The lesion is confined to the rostral portion of the nucleus.
389
THORACIC
LUMBAR
A
TS 170
B
TS 170
Fig. 5. A : TS 170; drawing of degeneration shown in a horizontal section through pons, medulla and the first cervical spinal segment. Due to the cephalic flexure, the spinal cord is cut obliquely so that ventral horn, intermediate gray, and dorsal horn all appear in the section. Nerve roots are shown in solid black. Degenerating fibers are indicated by broken lines while preterminal degeneration is shown by dots. An enlargement of the area outlined in the spinal cord is reproduced as a photomicrograph in Fig. 8A. B : drawing of degeneration in TS 170 in representative transverse sections through cervical, thoracic, and lumbar spinal cord segments. these crossing fibers traverse the nucleus while others form bundles o f fibers rostral to the nucleus. One bundle o f decussating fibers follows a course directed between the interpositus and medial nuclei to enter the brain stem medial to the restiform body. Degenerating fibers pass t h r o u g h and between the lateral and inferior vestibular nuclei. C o n t r a l a t e r a l to the lesion, a g r o u p o f degenerating axons continues dorsal to or traverses the interpositus nucleus to form a curved bundle o f fibers d o r s a l to the b r a c h i u m conjunctivum, the uncinate fasciculus. F r o m this bundle, a few fibers descend the b r a i n stem c o n t r a l a t e r a l to the lesion. As seen in Fig. 5, the descending fibers on b o t h sides travel t h r o u g h the pons a n d m e d u l l a in a r a t h e r diffuse fashion. A significant n u m b e r a p p r o a c h the midline a n d some cross the raph6; others pass within the fiber bundles extending l o n g i t u d i n a l l y on b o t h sides o f the medial plane, including the regions o f the tectospinal t r a c t a n d the medial longitudinal fasciculus. The most a b u n d a n t g r o u p o f fibers extends t h r o u g h the
390 medial reticular formation. At the level of the decussation of the pyramids, cerebetlar efferent fibers either enter the gray matter ot the spinal cord directly or pass into the lateral funiculus (Figs. 7, 8B and 10A. C). Instead of forming a compact bundle, these efferent fibers course through the white matter as loosely scattered axons. A few fibers emerging from the medial reticular formation enter the anterior funiculus. In addition to fibers from the nucleus, some corticofugal fibers issuing from the electrode track exit from the cerebellum. These fibers end in vesti bular nuclei and reticular formation ipsilateral to the cerebellar cortical lesion. In horizontal sections through the spinal cord, the degenerating axons are seen to follow the dorsal half of lateral funiculus (Fig. 6). At all levels the fibers of passage seen in white matter are less numerous than those seen branching into the gray matter. (Figs. 7 and 9A, B). Degenerating axons seen in lumbar (Fig. 8A, B) and thoracic (Fig. 9) levels were sparse in comparison to those observed in cervical segments (Fig. 7C). Within the spinal cord preterminal degeneration appears m the intermediate gray and ventral horn and is seen in laminae V, VI, VII. and VIH : at upper cervical levels the density of preterminat degeneration in the intermediate gray rivals that seen in the reticular formation in the medulla (Figs. 5 and 7A, C). Fragments of preterminal degenerating fibers are seen in close relation to neuronal somata in the intermediate gray and ventral horn (Figs. 10. 11 and 12): argyrophilic particles appear both in the neuropil and over neuronal somata. ROSTRAL
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i"
VH
t
:o AF " [G
:
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AUDAI_ CERVICAL I
Imm
I
THORACIC TS
LUMOAR
141
Fig. 6. Drawing of degeneration in TS 141 in representative horizontal sections through cervical, thoracic, and lumbar spinal segments. The enlargement of the area outlined in the lateral funiculus of the cervical section is reproduced as a photomicrograph in Fig. 10A.
391
Fig. 7. P h o t o m i c r o g r a p h s o f degeneration ( F i n k - H e i m e r ) in TS 170. A: low power picture o f fibers e n t e r i n g the lateral funiculus a n d intermediate gray. Outlined areas are reproduced at a higher m a g n i fication in 7B a n d 7C. x 25. B: degenerating fibers are seen entering the lateral funiculus a n d intermediate gray. Fibers coursing nearer to or within the intermediate gray are o f relatively smaller diameter t h a n those travelling in the lateral funiculus, x 100. C: degenerating fibers within the intermediate gray of the cervical spinal cord. × 100.
392
Fig. 8. Photomicrographs of degeneration (Fink-Heimer) in a transverse section through the lumbar spinal cord contralateral to the lesion in TS 115. A: montage of low power photomicrographs showing a hemisection of the spinal cord of TS ! 15 contralateral to the lesion for orientation. The dorsal horn is free of fragments of degenerated fibers such as those which appear in the ventral h o r n . . 25. Area outlined is reproduced at a higher magnification in Fig. 9B. B: degenerating fibers a n d preterminal degeneration in the lumbar spinal cord, TS 115. ~ 250.
393
Fig. 9. Photomicrographs of degenerating fibers (Fink Heimer) in the lateral funiculus contralateral to the lesion as seen in horizontal sections through cervical and thoracic spinal cord segments. A: degenerating fibers in the cervical cord, TS 170. Illustration is a montage of two photomicrographs taken at different planes of focus. ~ 125 B : degenerating fibers in the thoracic spinal cord, TX 138. x 125. C: degenerating fibers in the thoracic spinal cord, TS 170. x 250.
394 Following unilateral lesions of the medial cerebellar nucleus, degeneration necessarily appears bilaterally, since the fibers decussating within the cerebellum are so intimately related to all surfaces of the nucleus that injury of one medial cerebellar nucleus cannot be produced independently of damage to fibers originating from the other. In TS 170 the lesion, while large, is closely confined to the limits of the nucleus itself (Fig. 2). Degeneration seen following this lesion is predominantly contralateral (Figs. 5 and 6). In contrast, in TS 141 the damage to the nucleus itself is relatively minor; fibers crossing rostral to the nucleus are, however, significantly involved (Fig. 4). The pattern of degeneration in the spinal cord following this lesion is bilaterally symmetrical (Fig. 6). Since the ipsilateral degenerating axons seem to b e related to interruption of fibers crossing from the contralateral nucleus, the projection apparently is predominantly crossed.
Appearance of control mater&l In the spinal cord TS 144 in which a lesion was placed in the olfactory bulb sporadic argyrophilic fragmented fibers are seen in widely separated sections. These occur with no particular bias toward one or the other side and are equally rare in cervical, tholacic and lumbar segments. Except for these randomly occurring fragments and occasional cells with particulate artifact as described previously, the spinal cord sections in control material are clear of argyrophilic particulate aggregation.
Fig. 10. Photomicrograph of degenerated fibers and preterminal degeneration (Fink-Heimer) associated with neuronal soma and processesin transverse section through the cervical spinal cord, TS 170. Degenerating fibers and panicles of preterminal degeneration associated with two neuronal somata and their processes, x 500.
395
Fig. 11. Horizontal section through the thoracic spinal cord in TS 138. A degenerating fiber turns around the neuronal soma (Fink-Heimer). A montage of four photomicrographs taken at different planes of focus, x 500.
Fig. 12. Degenerating fibers and preterminal degeneration (Fink Heimer) seen in the lumbar spinal cord. TS 138. Degeneration in the lumbar spinal cord. × 125.
396 DISCUSSION
The weight of the evidence supports the existence of projections trom the medial cerebeltar nucleus to at least cervical levels or the spinal cord in cataa,l'a,~.:~a,36, 41, monkey 4,~a, hedgehog 1~, opossum17, 28 and pangolinn; but assertions of fiber connections from cerebellar nuclei to thoracic and lumbar spinal segments have been few and widely separatedl,ZT,30,3~, 4o. Projections have been reported in the lumbar cord based on both Marchi z7,4° and Nauta I techniques, yet in other studies such connections were not seen. Recently, Earle and MatzkO 5, using Fink-Heimer and Nauta methods in hedgehog, specifically denied the existence of cerebellar projections to lower spinal cord levels. Before addressing the significance of spinal cord projections from the cerebellum in the tree shrew, the possibility that some of these degenerating fibers originate from areas other than the medial cerebellar nucleus must be excluded. Sporadic cell death has been reported in the lateral cerebeltar nucleus of the cat 14 and the lateral vestibular nucleus of the rat 39. Indeed, an occasional fragmented argyrophilic fiber can be seen in spinal cord sections of control material (TS 144). These did not, however, occur with the frequency, regularity of distribution, laterality or consistency of the degenerated fibers which were evident following medial cerebellar nucleus lesions. The likelihood of inadvertent damage to the vestibular nuclei in the course of placing lesions in the cerebellar nuclei has long been recognized7, 40,4~.44 In the present material, lesions made electrolytically are discrete and relatively small. The extent of tissue damage, as seen both in cresyl violet-stained and Fink-Heimer prepared sections, does not involve cells of the vestibular nuclei which appear to be normal upon light microscopic examination. No observable differences are seen between the vestibular nuclei ipsilateral and contralateral to the lesion. Although direct damage to the vestibular nuclei can be excluded, the possibility of retrograde or transneuronal reaction to the cerebellar lesion must be considered 9. Retrograde cell death in the vestibular nuclei conceivably could result from destruction of the medial cerebellar nucleus, a major target for vestibular projections. Such retrograde degeneration, undetectable at the short survival times optimal for FinkHeimer preparations, might produce changes in axon terminals. Alternatively, loss of the profuse connections supplied to the vestibular nuclei by the medial cerebellar nucleus could precipitate transneuronal changes in the vestibular nuclei which also might be reflected in degeneration of their axon terminals. The observed distribution of degeneration seen in this tree shrew material mitigates against the vestibular nuclei as the source of the argyrophilic fibers. Well over half the nucleus is destroyed in TS 170: degenerated axons seen entering the vestibular nuclei are dense bilaterally. Since the afferent fibers entering the vestibular nuclei are. apparently, equally dense on both sides, any resulting transneuronal changes should be proportionately distributed bilaterally. The degenerauon seen m TS 170 was. however, clearly heavier contralateral to the lesion. In TS 141 and TS 115. damage to the medial cerebellar nucleus is less extensive. It seems unlikely that this reduction in either vestibular axon terminals in the medial cerebetlar nucleus or loss of cerebellar affe-
397 rents to the vestibular nucleus could be significant enough to produce alterations in the vestibular cells sufficient to account for the observed anterograde changes in the spinal cord. Furthermore, in horizontal sections of the spinal cord the degenerating fibers appear in the lateral funiculus in a relatively dorsal position, whereas the fibers of the lateral vestibulospinal tract would be expected to course in the anterolateral fiber bundle if they follow the trajectory reported for other mammals 7,31,33. In this material the number of degenerating fibers does not diminish appreciably as they pass from the brain stem into the cervical spinal cord (Fig. 5). This finding is in contrast to several reports in which others, who were unable to follow cerebellar efferent fibers beyond cervical levels, stress the paucity of the degeneration observed as the projection enters the cordl~,lS, 36. Many of the fibers we observe entering the spinal cord seemed to be of relatively small diameter. Heimer 21 has suggested that axons ot very small caliber may fail to impregnate with silver to sufficient degree to be seen by the light microscope: only their preterminal enlargements are revealed. This phenomenon could account for at least some of the inconsistencies reported in various studies. The appearance of the fibers degenerating in the tree shrew spinal cord is similar to that of the degenerating fibers in the medulla and pons: to accept them as efferents of the medial cerebellar nucleus seems to be the most parsimonious interpretation of these results. This being so, the significance of this projection should be considered in relation to that of other mammals. The cerebellar nuclei project to the reticular formation, the vestibular nuclei, and the red nucleus (all of which have direct fiber connections to the spinal cord) in a large number of species studied 25, including the tree shrew 47. Recent reports have confirmed projections from cerebellar nuclei to the superior colliculus including those layers from which the tectospinal projections originate 3'~s. Medial, interpositus, and lateral nuclei of the cerebellum also project to the ventral anterior and ventrolateral nuclei of the thalamus from which efferent fibers are distributed to the motor cortex25, 27. Thus, the mammalian nervous system appears to be organized in such a way that the cerebellum maintains extensive and direct control over the nuclei which are involved in the initiation of motor behavior. It is entirely consistent with this pattern that cerebellar efferent fibers reach the spinal cord. A common pattern of organization, however, may also accomodate variation among species. The motor behavior of tree shrew may be more strongly influenced by the cerebellum than by the corticospinal system. Kornhuber 24 has proposed that the cerebellum is uniquely responsible for aiming ballistic movement, i.e. movement which occurs too rapidly to be directed continuously by sensory feedback. The direction and target localization of such a movement must be determined prior to initiation of the motor behavior. In fact, the typical locomotor behavior of tree shrews fits this description most precisely. Tupaia glis, the species used in this study, occupies a semi-arboreal econiche. Rapid, accurately directed short leaps are characteristic of the locomotor activity of these animals ')3. 29,38,43. Forelimb function is precise, fast, and stereotyped in nature. When a meal worm is offered beyond the mesh of the cage door, a tree shrew instantly projects a forelimb through the small opening to snatch the bait, continuing the activity for
398 dozens o f repetitions without missing the w o r m ( M a r t i n , p e r s o n a l c o m m u n i c a t i o n ) . The ballistic quality o f the m o t o r b e h a v i o r o f T u p a i a g l i s has obvious a d a p t i v e value in a s e m i - a r b o r e a l habitat. I n this context, it is interesting t h a t in recent r e p o r t s o f cerebellar projections in the h e d g e h o g 16 a n d o p o s s u m 2s, based on F i n k - H e i m e r procedures, d e g e n e r a t i n g axons were n o t seen c a u d a l to the cervical spinal segments. N e i t h e r o f these n o c t u r n a l a n i m a l s are c h a r a c t e r i z e d by rapid, c o o r d i n a t e d m o t o r activity. The s h o r t survival times which are o p t i m a l for d e m o n s t r a t i o n o f d e g e n e r a t i n g axons limit the o p p o r t u n i t y for behavioral observation, while the small lesions which are desirable for a n a t o m i c a l p u r p o s e s do not p r o d u c e m a r k e d changes ill c o o r d i n a tion. All the a n i m a l s showed some i n c o o r d i n a t i o n in the first days following surgery, b u t this could be related as much to e d e m a a n d other t r a n s i t o r y p o s t - o p e r a t i v e changes as to the direct cerebellar damage. F u r t h e r studies o f cerebellar c o n t r o l o f m o t o r b e h a v i o r in the tree shrew are n o w u n d e r w a y in our l a b o r a t o r y . ACKNOWLEDGEMENTS W e t h a n k Dr. M i m i H a l p e r n , Dr. Jacqueline J a k w a y , a n d Dr. W a l t e r R i s s for their suggestions; Dr. H e n r i Begleiter a n d Dr. F r a n k Scalia for p r o v i d i n g the stereotaxic e q u i p m e n t ; Mr. lrvin W i l s o n for technical assistance; Mrs. S u d h a Joshi, Miss C a r o l J a r y n o w s k i a n d Mrs. R o s e K r a u s for m a n u s c r i p t p r e p a r a t i o n s ; Mr. J a c k lllari for p h o t o g r a p h i c assistance; a n d Mrs. Tina M a g n o , w i t h o u t w h o m the project c o u l d n o t have been accomplished.
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