EXPERIMENTAL

Prolonged

NEUROLOGY

Gestation

46, 69-77 (1975)

and Cerebellar

Development

in the Rat

IAN S. ZAGONl Departnzent of Biological Structure, University of Miami Medical School, Miarpzi, Florida 33152 and Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvaka State University, Hershey, Pennsylzlania 17033 Received

August

22,1974

The effect of prolonged gestation on the temporal sequence of cerebellar morphogenesis was studied in rat. Pregnant Holtzman albino rats received daily intramuscular injections of 5 mg progesterone dissolved in sesame oil beginning 1 day before birth (day 21). A second group of pregnant animals received injections of sesame oil, while a third group was untreated. Examination of gross specimens and midsagittal sections of day 25 cerebella (prolonged gestation) from fetuses of progesterone-treated females revealed that folial and fissure development proceeded in a normal fashion when compared to 3 day postpartum controls (groups 2 and 3). Examination of histological sections showed equivalent development with proliferation of external germinal cells, differentiation of basket and internal granule cells, migration of granule cells, elaboration and definition of the molecular layer, and the topographic positioning and growth of Purkinje cells. These results indicate that the temporal sequence involved in the postnatal portion of cerebellar production of microneurons (granule, basket, neurogenesis, particularly stellate), is not triggered by birth. In addition, morphogenetic processes such as cellular proliferation and differentiation, migration of neurons, and the organization as stratification of cerebellar components follows a defined schedule which appears to be independent of the changes accompanying parturition.

INTRODUCTION Neurogenesis in the central nervous system proceeds on schedule (reviews : 8, 16,21,22). Superimposed on this temporal one finds that the type of neuron(s) produced bears an important

a precise sequence, relation-

1 This work was supported by Florida American Cancer Society Grant F74UM-3 and American Cancer Society Grant DT-30. The assistance of Carol Clingan, Jeff Gillingham, and Eileen Zagon is gratefully acknowledged. The author’s present address is: Department of Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033. 69 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

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IAN

S. ZAGON

ship to size or connectivity, or both. Cerebellar development in rat is comprised of prenatal and postnatal stages, with large (Purkinje) and medium (Golgi, interstitial, marginal) neurons arising prenatally and small or microneurons (granule, basket, stellate) primarily originating postnatally (2, 47, 13, 14). Altman and Das (7) have called attention to the importance of birth in neuronal morphogenesis and suggested “. . . that postnatally formed microneurons, which are interposed among the prenatally-formed input, relay, and output elements, are the modulatory and plastic elements of the central nervous system whose structural and functional maturation may be dependent on the animal’s interaction with its external environment” (7, p. 358). Altman (2) has also suggested that postnatal proliferation may be synchronized or actually triggered by parturition. Recently, Das and Nornes (13) have shown that the rat cerebellum fails to incorporate [SH] thymidine one day before birth, even though labelling in the hippocampus and olfactory bulb was observed. These results could be interpreted to mean that birth may serve as a significant landmark between prenatal and postnatal phases of cerebellar morphogenesis, perhaps even functioning in a regulative capacity. The present investigation was undertaken to delineate the role of birth in cerebellar development. Pregnant female rats were injected with progesterone so that parturition was prolonged, and fetal cerebella were evaluated by morphological observation of (a) gross specimens, (b) midsagittal sections for determination of size, lobule, and fissure development, and (c) histological analysis of cellular proliferation and differentiation. MATERIALS

AND

METHODS

Sperm-positive albino rats were obtained from Holtzman (Madison, Wis.) . The day of sperm-positivity was considered the first day of gestation ; delivery normally occurred on day 22 of pregnancy. Animals were divided into three groups. The first group was briefly anesthetized with ether and injected daily with 5 mg of progesterone (Sigma, St. Louis, Mo.) intramuscularly on a daily basis beginning 1 day before birth (day 21) and continuing through the twenty-fourth day of gestation. The solution of progesterone was prepared by dissolving 5 mg of progesterone in 60 pl methylene chloride and adding 0.5 ml sesame oil. The second and third groups served as controls. Animals in the second group were treated as those in group 1 but progesterone was omitted from the solution ; group 3 females were untreated. Cesarean section was performed on group 1 females at day 24 and 25 under pentobarbital anesthesia (5 mg per 100 gm of body weight). Animals were removed with placenta intact, anesthetized by immersion in ice water

oil-injected deviation.

0 1 2 3

22 23 24 25

0 Included b Standard

Days postpartum

Gestation day

and

untreated

animals.

4

No. litters

Controlsa

48

No. fetuses

COMPARISONOFLITTERNUMBERSANDFETALWEIGHTSIN

SD2 0.22 0.46 0.33 0.80

Mean 6.21 7.07 8.30 9.20

f

w

f f f f

1

5 5

No. litters

PROGESTERONE-TREATED AND

wt

Fetal

TABLE GROUPS

38 13

No. fetuses

Progesterone-treated

CONTROL

7.01 f 7.70 f

f

Fetal

Mean

wt

0.51 0.62

SDb

k)

-3

72

IAN

S. ‘ZAGON

0.5mm

A

6

B’

-

0.5mm

C’

FIG. 1. Dorsal views of the fixed brains from (A) newborn control, (B) 3-day-old control, and (C) %-day fetus. A’ to C’ are outlines of midsagittal sections of the cerebellum. Note the increased number of folia and fissures between A, A’ and B, B’ and the similarity between 3-day-old control and Z-day fetus.

and fixed with 10% neutral buffered formalin by cardiac perfusion using a 25gauge syringe and an air pressure of 120 mm Hg; five fetuses were studied each day. Three animals from each of the control groups were examined on postnatal days 0, 1, 2, 3, and 4 using similar anesthetic and perfusion techniques except that a 27-gauge syringe was employed for animals of days 0 and 1. Brains were removed, fixed 10% neutral buffered formalin for at least 3 days, and processedfor light microscopy after embed-

CEREBELLAR

DEVELOPMENT

73

ding in polyester wax (23). The cerebellum was sectioned in sagittal and transverse (coronal) planes and 9 pm sections were stained with Harris’ hematoxylin. Midsagittal sections of the vermis were projected and drawn at 60X with a Bausch and Lomb microprojector. Drawings of whole brains were traced directly from photomicrographs of the specimens immersed in fixative. To determine whether the means of the weights measured in the various groups differed significantly, the t test was applied (15). RESULTS Untreated and oil-injected females delivered within an 18 hr span of expected parturition time ; the oil injected females generally gave birth later than untreated females. No differences in litter size, fetal weights, or gross morphology and histology of the brain could be detected between members of the groups. Intrauterine increase in fetal weight did continue during the prolonged gestation (Table 1). However, 24-day and 25-day fetuses weighed significantly (P < 0.001 and P < 0.01 respectively) less than their normal postnatal counterparts. Cerebella from experimental and control groups were carefully inspected and evaluated relative to Larsell’s (18) description. The major distinguishing features of cerebellar morphogenesis between newborn and 3-day control animals were: increase in extent of the vermis and hemispheres (compare Fig. 1 A, B) ; elaboration, definition, and in some cases subdivision of

FIG. 2. Comparison of sagittal sections of the cerebellar cortex from (A) newborn control, (B) 3-day-old control, and (C) Z-day fetus. By day 3 the cortex has become stratified, a distinct molecular layer can be observed, and Purkinje cells have grown and become ordered into a single row (compare A and B) ; 25-day fetus (C) resembles day-3 control (B). X 350.

74

IAN

S. ZAGON

vermian lobules (compare Fig. 1 A’, B’) ; and the delineation of crus I from its analage, the ansoparamedian lobule of the hemisphere (compare Fig. 1 A, B). Gross examination of the cerebella from day 24 and 25 fetuses revealed a development identical to that of postpartum equivalents (Fig. 1 B, C). The histology of developing rat cerebellar cortex has been described elsewhere ( 1, 3-6, 25). At birth (Fig. 2 A), a sheet of three to seven cells termed the external germinal layer, extends over the surface of the cerebellum. The molecular layer is either absent or appears as a thin white band that includes some scattered cells. Purkinje cells, located deep to the molecular layer, are small and round with clear oval nuclei ; they are usually arranged in a layer of 2-5 cells. By the third postnatal day (Fig. 2 B), the external germinal layer is made up of six to eight rows of cells, and a welldefined molecular layer can be observed. Migrating granule cells and an occasional differentiating basket cell can be seen in the molecular layer. The Purkinje cells are larger, display the formation of a supranuclear cytoplasmic mitre or cone which is directed towards the surface of the cerebellar cortex, and are arranged in one or two irregular rows. Histological examination of cerebella from fetuses of days 24 and 25 (Fig. 2 C) revealed morphological features similar to 3-day controls. DISCUSSION Treatment with progesterone during the terminal stages of an otherwise normal pregnancy has been shown to delay parturition (10, 12, 17, 20). Barrow and Rowland (lo), using the criteria of fetal weight, calcification pattern, and skin pigmentation, concluded that intrauterine growth and maturation of postterm fetuses continues up to 2 days beyond birth in both progesterone-treated and uterus-ligated rats. Increases in fetal weight were recorded in the present study, but note that fetuses of prolonged gestation were significantly underweight when compared to normal postnatal equivalents. This weight loss in day-24 and day-25 fetuses could be due to a number of factors including undernutrition, stress, hormonal unbalance, or the unreliable nature of animal birthdates that could result in sk’ewing of statistical data. Despite such differences in weight, cerebellar development appeared uneffected. These results agree with some closely associated experiments reported by Barnes and Altman (9) who found that dietary deprivation of pregnant females resulted in underweight pups with normal brain weights ; only after the fifth postnatal day were brain weights significantly lower in these undernourished animals. The possible effects of progesterone on the developing nervous system has not been extensively investigated. Vernadakis (24) showed that addition of progesterone to explants of 16-day chick embryo cerebellum or g-day

CEREBELLAR

DEVELOPMENT

i3

and 16-day-old chick embryo spinal cord cultures has no effect on acetycholinesterase levels nor quantity of protein. The absence of a direct effect of progesterone on nervous system development is supported by the results of the present study, with no differences detected between cerebella from experimental and untreated animals. Observation by Das and Nornes (13) of a quiescent period with little uptake of [“HI thymidine in cerebellum 1 day before birth can be expanded upon in light of the present findings. Data from this study suggest that this “stationary” phase in cell proliferation has discrete temporal boundaries and that it is unaffected by the episode of birth. Although this period of proliferative inactivity has been reported only in rats (13), it may be of widespread occurrence in altricial animals and should be evaluated during future studies. The present investigation suggests that the temporal sequence of events during cerebellar neurogenesis in the rat is not obviously regulated by birth. Furthermore, the progression of development such as folial and fissure formation, stratification of cerebellar components, and topographical cell associations, does not appear to be altered by prolonged pregnancy. These findings indicate that (a) phenomena associated with parturition, such as maternal or fetal hormonal changes, or the influence of a new environment (or both), do not appear to trigger morphogenetic expression; and (b) although birth appears to be a possible landmark between genesis of specific neuronal types in the rat cerebellum, the date of parturition may only be incidental to the scheduled processing of genomic information. This is not surprising since cerebellar development in rat is one of the few examples of a synchrony between discrete phases of neuron production (macroneurons, microneurons, neuroglia) and major periods of neuron production (fetal, infantile, juvenile). In some animals for instance, the entire program of cerebellar morphogenesis is mainly prenatal (i.e., precocial types such as the chick, guinea pig, and ungulate), while in others (i.e., altricial types such as the mouse, rat, or man) postnatal development becomes an important feature. These categories may overlap so that in mice, for example, microneuron production occurs during both prenatal and postnatal stages ( 19). Based on the present evidence, it appears that the synchrony observed in rats is simply a consequence of coinciding timetables for neuronal maturation and the major developmental periods. Results from this study compare favorably with observations concerned with prolonged pregnancy in women. Postterm infants (those born after 42 wk or more of gestation) may be clinically indistinguishable from term infants, or they may appear and behave like infants of l-3 wk postnatal life (11). Thus it can be surmised that as in rat. the timetable for neuro-

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genesis as well as establishment of behavioral patterns in man does not totally rely on birth for impetus. REFERENCES 1. ADDISON, W. H. F. 1911. The development of the Purkinje cells and of the cortical layers in the cerebellum of the albino rat. J. Comb. Nezbrol. 21: 459-488. 2. ALTMAN, J. 1966. Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. I. Camp. Neural. 128 : 43 l-474. 3. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Co+@. Nezlrol. 136: 269-294. 4. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Camp. Neural. 145 : 353-398. 5. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Camp. Neural. 145 : 399-464. 6. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Conzp. Neural. 145: 465-514. 7. ALTMAN, J., and G. P. DAS. 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions. J. Comb. Neurol. 126 : 337-390. 8. ANGEVINE, J. B., JR. 1970. Critical cellular events in the shaping of neural centers, pp. 62-72. In “The Neurosciences. Second Study Program.” F. 0. Schmitt [Ed.]. Rockefeller University Press, New York. 9. BARNES, D., and J. ALTMAN. 1973. Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neural. 38: 406419.

10. BARROW, M. V., and C. A. ROWLAND. 1972. Prolonged gestation in the rat and its usefulness in experimental teratology. Teratology 2 : 125-136. 11. BEHRMAN, R. E. 1973. The high-risk infant, pp. 45-49. Is “Neonatology.” R. E. Behrman [Ed.]. C. V. Mosby, St. Louis. 12. B@E, F. 1938. Prolonged pregnancy in rats. Acta Path. Stand. Su~pl., 36: 1-146. 13. DAS, G. D., and H. 0. NORNES. 1972. Neurogenesis in the cerebellum of the rat : an autoradiographic study. 2. An&. Enfwk%l.-Gem%. 138 : 155-165. 14. HAAS, R. J., and J. WERNER. 1973. Cytokinetics of neonatal cerebellar neurons in rats as studied by the “complete H3-thymidine labelling” method. Exp. Brain Res. 18 : 59-68. 15. HILL, A. B. 1966. “Principles of Medical Statistics.” Oxford University Press, New York. 16. JACOBSON, M. 1970. “Development Neurobiology.” Holt, Rinehart and Winston, New York. 17. JOLLIE, W. P. 1962. Action of progesterone in determining persistence of placental elements during parturitional delay in the rat. Endocrinology 71: 573-579.

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0. 1952. The morphogenesis and adult pattern of the lobules and fissurrs of the cerebellum of the white rat. J. Co~zp. Nrllrol. 97: 281-356. 19. MIALE, I. L., and R. L. SIDMAN. 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. NEZIYUI. 4: 277-296. 20. NELSON, W. O., J. J. PFIFFNER, and H. 0. HATERIUS. 1930. The prolongation of pregnancy by extracts of corpus luteum. Anzrr. J. Pkysiol. 91 : 690-695. 21. SIDI\IAN, R. L. 1970. Autoradiographic methods and principles for study of the nervous system with thymidine - H3, pp. 252-274. In “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer, Berlin. 22. SIDMAN, R. L. 1970. Cell proliferation, migration and interaction in the developing mammalian central nervous system, pp. 100-107. In “The Neurosciences. Second Study Program.” F. 0. Schmitt [Ed.]. Rockefeller University Press, New York. 23. SIDMAN, R. L., P. A. MOTTLA, and N. FEDER. 1961. Improved polyester wax embedding for histology. Stain Tcrkvol. 36: 279-284. 24. VERXADAKIS, A. 1971. Hormonal dependence of embryonic neural tissue in culture, pp. 67-74. I+t “Hormones in Development.” M. Hamburgh and E. J. W. Barrington [Eds.]. Appleton-Century-Crofts, New York. 2.5. ZAGOK, I. S. 1972. Light and electron microscope observations on the postnatal development of the rat cerebellum. Doctoral Dissertation, University of Colorado llcdical Center, Denver, Colorado. 18. TARSELL,