Histogenesis of the deep cerebellar nuclei in the mouse: an autoradiographic study

Brain Research,95 (1975) 503-518 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

503

H I S T O G E N E S I S OF THE D E E P C E R E B E L L A R N U C L E I I N T H E M O U S E : AN AUTORADIOGRAPHIC STUDY

E L I Z A B E T H TABER PIERCE

Department of Anatomy, Harvard Medical School, Boston, Mass. (U.S.A.)

SUMMARY

In order to tag cells when they arise, pregnant mice were injected usually once or in some cases multiple times at a known time of gestation with tritiated thymidine. The offspring were killed and their brains prepared for autoradiography. Distribution o f labeled cells was plotted using a drawing apparatus. Neurons of the deep cerebellar nuclei arise on gestation days 10-17. (Later periods were not studied.) Most neurons arise on gestation day 11. Many medium and small sized neurons arise after gestation day 11 with a limited number of small neurons observed to arise through the 17th day. Neurons for all parts of the complex arise at the same time, thus no gradients could be established.

INTRODUCTION

The histogenesis of the cerebellum has attracted the attention of numerous investigators, who have used a number of experimental techniques on a variety of species in attempts to understand the origin and development of this structure. Insight into the origin of the cerebellum from phylogenetic studies has resulted in a vast literature 2,4,5,8-17,19-22,27 implying that the cerebellum evolves as a bilateral organ from the alar plate of the medulla oblongata. Larsell (ref. 14, p. 297) states that the cerebellum has its 'inception as a specialization of the cutaneous centers in the rostrolateral region of the medulla oblongata'. Jansen lz points out that for a long time the cerebellum was considered a superstructure of the vestibular area. Earlier investigators believed that in Petromyzon the primitive cerebellum was a rostromedial continuation of the acousticolateral area. However, Larsell is and Jansen 1~ advocate that the primitive cerebellum in Petromyzon is derived from at least two areas: the vestibularlateral lobe is a direct continuation of the special somatic afferent zone and the corpus cerebelli is derived from the general somatic zone. Jansen further questions whether

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505 the visceral afferent and intermediate (reticular) zones also contribute to the cerebellar anlage. He cites the need for further investigations in this area. In order to determine the site of origin of neurons using the autoradiographic technique, it is desirable to determine first the time of origin of neurons. This study reports data on temporal and spatial patterns of neuron formation in the deep cerebellar nuclei of the mouse. It is part of an ongoing research program to study neurogenesis using the mouse as the experimental animal. The observations in this paper complement those of Miale and Sidman 18. However, utilizing pregnancies timed to the hour of conception, it has been determined that neurons arise on the 10th day of gestation, a day earlier than that reported by Miale and Sidman, who used a different method to time pregnancies. It has also been determined, using the plotting method described by AngevinO, that within 3 nuclei - - medialis, interpositus and lateralis - no spatial gradients exist; neurons destined to form the three nuclei undergo final D N A division at similar times.

MATERIALS AND METHODS

The methods used in this study have been detailed elsewhere 24-26. To obtain cells labeled at the time of their birth (last mitotic division) female BALB/c mice mated to SJL males were given one injection subcutaneously at 9 a.m. on a known day of gestation; the dose was 5/zCi/g body weight. In a recent series, pregnancies were timed to the hour and the time of the injection recorded. The offspring were killed 2-3 months after birth. To obtain offspring labeled on the ninth day of gestation, a laporotomy was performed under ether anesthesia and 10/~Ci of tritiated thymidine in 10 #1 of water were injected into each embryo in utero with a microliter syringe and a 30gauge needle. The brains of all animals were sectioned serially at 10/~m in one of the cardinal planes and subsequently prepared for autoradiography. For 5 selected series, one for each of gestation days 10, 12 and 13, and two for gestation days 11, every tenth transverse section was examined for labeled cells; the sites of the labeled cells were recorded using a drawing tube as described by AngevinO. Eight sections were selected for illustration from each of the series plotted. In the reproductions of the autoradiograms, heavily labeled neurons were drawn black and lightly labeled neurons were drawn as open circles. In preparing all illustrations the outline of the cell body was drawn in order to show relative cell sizes. The heavily labeled cells were recorded as forming (born) on the day the tritiated thymidine was injected. Lightly labeled cells were recorded as forming at a later time. Forty-eight brains were examined, 10 of which recorded the birth of neurons from pregnancies timed to the hour; the rest were timed using the method of Miale and Sidman is. The cytoarchitecture of the deep cerebellar nuclei was studied in serial, Nissl stained sections fixed in acrolein, embedded in celloidin and cut at 20 # m in either the sagittal, horizontal or transverse plane. The transverse series was plotted using the technique described above. Every fourth section is illustrated (Fig. 1). Photographs of these sections were published in an atlas 23.

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RESULTS

Normal topography and eytoarehitecture of the deep eerebellar nuclei In the mouse the cells of the deep cerebellar nuclei form a continuous complex which, however, can be divided into 3 nuclei in each hemisphere, the medialis, interpositus and lateralis. Cells ranging in size from large to small are found throughout the complex; small ceils, however, form prominent clusters ventrally and these might be delineated as subgroups. The 3 nuclei have approximately an equal caudal-rostra] length, but the medial nucleus in transverse sections appears most caudally, followed by the interpositus and then the lateral. The two medial nuclei are widely separated. The interpositus nucleus merges ventrally with the lateral and superior vestibular nuclei from which its ventral border in Niss] preparations is arbitrarily delineated.

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Likewise, the ventral-lateral border of the medial nucleus connects via cell bridges to the same nuclei. Caudally the interpositus nucleus can be subdivided into ventral and dorsal parts, the latter extending farther rostrally. The lateral nucleus has a poorly defined medial border, which opens rostrally into a hilus into which the axons o f its neurons project. In Nissl preparations the cells can be classified as large, medium and small. The large and medium sized neurons are multipolar and are similar in appearance with scattered Nissl substance and a centrally located nucleus. Two types o f small cells are recognized; one is fusiform and stains darkly, the other appears pale and has a poorly defined outline. Many o f the first type lie near the fourth ventricle.

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Time of origin of neurons Examination of autoradiograms of offspring labeled on the ninth day of gestation (Fig. 12) disclosed a large population of lightly labeled neurons, large, medium sized and small, in all areas of the complex. Specimens labeled on the 10th day (Figs. 2, 3 and 13) showed an increased number of lightly labeled neurons and a small number of heavily labeled neurons varying in size and location. Most of the neurons, in all areas o f the nuclear complex, arise on the 1 lth day (Figs. 4-7 and 14) and vary in size and type. Only a minimal number of neurons are labeled heavily on the 12th day (Figs. 8, 9 and 15); most of these are medium sized or small. A few large neurons are heavily labeled. Limited numbers of small and medium sized neurons are heavily

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labeled on the 13th (Figs. 10 and 11) and 14th days of gestation, but no large cells appear labeled. Small cells are heavily labeled through the 17th day of gestation. Data is presently not available for later timed series. DISCUSSION

The description of the cytoarchitecture presented in this study follows the terminology of Brunner 2, who divided the deep cerebellar nuclear complex of the mouse into 3 nuclei: medialis, interpositus and lateralis. The interpositus in Brunner's opinion did not warrant further subdivision. However, one might consider further subdivisions of all the nuclei, since within the medial and lateral nuclei small cells are

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510 g r o u p e d ventrally a n d the interpositus nucleus contains d o r s a l a n d ventral groups o f cells s e p a r a t e d by bundles o f axons p r o j e c t i n g r o s t r a l l y into the superior cerebellar peduncle. F l o o d and Jansen 6 i n d i c a t e d similar subdivisions for the 3 nuclei in the cat. The mouse differs f r o m the cat in t h a t its medial nucleus has a shorter rostral extent. The lateral nucleus extends rostral to the interpositus, a l t h o u g h its c a u d a l - r o s t r a l extent is similar but slightly shifted. A few large mesencephalic trigeminal neurons recognized in the cat were also observed in the mouse. In a recent Nissl a n d G o l g i study o f the lateral nucleus o f the rat 3, large, m e d i u m sized a n d small neurons were described. In G o l g i p r e p a r a t i o n s the small n e u r o n s a p p e a r e d m u l t i p o l a r or fusiform. M o s t were neurons with short axons; some had longer

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511 axons which could be traced to the hilus. The large neurons were of two types, one with smooth perikaryon and the other with an irregular outline and some spinous processes. Other observations indicated a more definitive arrangement o f the neurons then recognized from Nissl preparations. It would be of interest to be able to relate the time of origin of neurons to the types recognized in Golgi preparations. The autoradiographi technique as used in this study unfortunately does not permit this correlation. It is probable that all types arise at the same time, but that the Golgi I! neurons have an extended period of formation. The period of birth of neurons (days 10-17 of gestation) of the deep cerebellar nuclei observed in this study agrees with the observations of Miale and Sidman is, who further observed small neurons arising through the first postnatal week, material

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Figs. 8 a-d and 9 e-h. Camera lucida drawings of 8 selected frontal sections through the deep cerebellar nuclei of a mouse labeled on the 12th day of gestation. which was not examined in the present study. The peak neuron formation is on day 11 with all sizes o f neurons forming for all areas of the complex. N o large neurons form after day 12, whereas small neurons continue to arise after this time. However, after day 13 their number is not impressive. These observations consolidate those o f Miale and Sidman TM and adduce evidence that a small population of neurons arises even earlier, on day 10 o f gestation. Since the observations presented in this paper were obtained from material in which the pregnancies were timed to the hour, the results suggest that the timing of the material of Miale and Sidman 18 for their 10-day period was earlier than that studied here. One of the specimens in this timed study came from an animal injected 243 h after copulation (day 0), clearly an early 10-day animal. Pregnant females injected at earlier times produce unlabeled embryos or offspring. It

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has been suggested that embryos younger than 10 days have vascular systems too poorly developed to transport the label ~6. Direct injection of the label into the embryo produces labeling. The simultaneity of neuron origin for all 3 nuclei, with no nucleus showing a time pattern differing from the others, or any part of one nucleus showing a gradient, are noticeable features of the mouse. In the chick, Fujita 7 described large neurons within the medial nucleus, the largest in the complex arising first, and neurons located nearest to the ependyma last. He did not, however, mention the size of the latter neurons. Examination of this region in a 60 mm chick (Minot collection, Harvard Medical School) showed mostly small neurons. It appears that in both chick and mouse, large cells form generally before small cells have completed their numbers.

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Phylogenetically deep cerebellar nuclei first appear definitely incorporated within the cerebellum in birds. The complex is recognized in Petromyzon in the dorsolateral area of the hindbrain. Further differentiation occurs in reptiles in which the complex can be divided into medial and lateral parts and is shifted dorsally. The medial component probably represents the r o o f nucleus in mammals, while the lateral part may represent the oldest part of the dentate nucleus. With the development of the lateral hemispheres in mammals there is a parallel development of the lateral nucleus with the appearance of a lateral segment. One might expect that the acquisition of newer parts would be recognized with some sort of a pattern in the birth of neurons, older parts forming before newer parts.

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Apparently this is not the case as no spatial or temporal differences were recognized. If the time of origin of neurons forming the deep cerebellar nuclei is compared with that for the vestibular nuclei and reticular formation, areas attributed to be the sources of the deep cerebellar neurons, the latter in general has a later time of origin. Although it is interesting to compare time of origin of neurons with accepted ideas concerning their phylogenetic appearance and/or augmentation, parallels and conflicts are equally abundant. It is hoped that by utilizing both the autoradiographic and Golgi techniques information will be forthcoming pertaining to the sites of origin of the deep cerebellar nuclei in the mouse, which in turn will relate to their phylogenetic origin.

516

Fig. 12. Deep cerebellar nuclei neurons labeled on the 9th day of gestation, x 240. Fig. 13. Deep cerebellar nuclei neurons labeled late on the 10th day of gestation, section. Fig. 14. Deep cerebellar nuclei neurons labeled on the 1 lth day of gestation. , 240. Fig. 15. Deep cerebellar nuclei neurons labeled on the 12th day of gestation. ~ 240.

Frontal section. x 240. Frontal Frontal section. Frontal section.

517 ACKNOWLEDGEMENTS This investigation was s u p p o r t e d by U . S . P . H . S . g r a n t NB-03756, Th e Wellingt o n F u n d , a n d the N a t i o n a l A s s o c i a t i o n o f M e n t a l H e a l t h , Inc.

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518 25 TABER PIERCE, E., Histogenesis of the dorsal and ventral cochlear nuclei in the mouse. An autoradiographic study, J. comp. Neurok, 130 (1967) 1-25. 26 TABERPIERCE, E., The time of origin of neurons in the brain stem of the mouse. In D. FORD (Ed.~, Neurobiological Aspects of Maturation and Aging, Progress in Brain Research, Vol. 40, Elsevier, Amsterdam, 1973, pp. 53-66. 27 WESTON, J. K., The reptilian vestibular and cerebellar gray with fiber connections, J. comp. Neurol., 65 (1936) 93-199.