Brain Research, 170 (1979) 443-457

443

© Elsevier/North-HollandBiomedicalPress

MORPHOLOGICAL IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF ISOLATED BRAIN CELL NUCLEI FROM THE DEVELOPING RAT CEREBELLUM

IAN S. ZAGON and PATRICIA J. McLAUGHLIN Department of Anatomy and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033 (U.S.A.)

(Accepted November16th, 1978)

SUMMARY Cell nuclei from developing rat cerebellum were isolated and the various types of nuclei were characterized and quantified. Nuclear pellets appeared to be both quantitatively and qualitatively representative of the entire cerebellum, and of sufficient purity to perform biochemical studies as well as morphological comparison with histological sections. Isolated nuclei were classified into 6 groups based on nuclear size and shape, heterochromatin aggregations, and nucleoplasmic density. The total population of cerebellar cells primarily consisted of two types of nuclei after day 10. One group of nuclei, resembling those of internal granule neurons or external germinal cells, contributed at least 70 ~ of the total isolated cell nuclei from day 10 to day 90, whereas another nuclear group that was identified as dark oligodendrocytes constituted 8-9 ~o of the total population on days 45 and 90. Nuclear DNA, RNA, and protein content of the cerebellum also were determined throughout postnatal development. DNA concentration markedly declined after day 15, while the RNA/ DNA ratio increased until day 3 and remained constant to day 90. The nuclear protein/DNA ratio increased from birth to day 3, decreased to its lowest value on day 10, and increased to day 90. Utilizing DNA values, the total cell population as well as contributions of different cell types were calculated. At birth the cerebellum was estimated to contain 5.9 million cells, increasing to 94 million by day 21. By day 90, 107 million cells were present, of which 8.6 million oligodendrocytes and 93.6 million internal granule cells were estimated.

INTRODUCTION The availability of techniques for the isolation and fractionation of brain cell nuclei in which nuclear structures and functional integrity are maintained, allows in

444 vitro investigations into the genomic activity of neurons and glia 11,21,22,25,36,38,46. Although some investigators have utilized this approach to assess nuclear activity during brain development6,10A2,17, TM, these studies have been limited, particularly in elucidating the biochemical and morphological properties of isolated cell nuclei from specific regions of the developing brain, and in integrating this information with previous in vivo morphogenetic investigations. In the course of experiments involving the effects of certain narcotic analgesics and chemical carcinogens on neuro-ontogeny 43-45, we have been interested in defining the in vivo and in vitro interactions of these agents on the biological activities of brain cell nuclei, particularly those in the developing cerebellum. Although cerebellar development has been extensively investigated, relatively little is known about the morphological and biochemical properties of cerebellar cell nuclei during this period12, is. In the present study, nuclear isolation procedures have been extended to the developing rat cerebellum in order to isolate, characterize, and quantify the various types of nuclei found during postnatal development, and to identify these different classes of cell nuclei by morphological comparison with in vivo preparations. In addition, total nucleic acid and protein contents during cerebellar ontogeny were determined and, utilizing DNA values, the total cell populations as well as the contributions of the individual cell types at various postnatal ages were ascertained. This study is intended to serve as the basis for further inquiries into the maturationdependent changes in nuclear activity that occur during normal and abnormal cerebellar development. MATERIALS AND METHODS

Animals Female (180-200 g) and male (250-300 g) Sprague-Dawley rats were obtained from Charles River Labs (Wilmington, Mass.) and mated. Animals were housed in solid bottom cages containing Easi-Litter (Westminster Scientific Company, Westminster, Md.) in an environment at 21 ± 0.5 °C with a relative humidity of 50 ± 10 ~. The room had a complete exchange of air 15-18 times per hour and a 12-h light-dark cycle; water and Purina Laboratory Chow were continuously available. At birth, litters were culled to 8 pups per mother. The cerebella from animals of postnatal days 0 (birth), 3, 6, 10, 15, 21, 45 and 90 were utilized for in vivo and in vitro studies. Isolation of nuclei With slight modifications, the isolation of brain cell nuclei essentially followed the methodology of Smith et al. 36 and Zagon et al. 46. Rats were decapitated, brains removed, and cerebella transected at the peduncles (as close as possible to the cerebellum). Cerebella were cleaned of meninges and immediately placed in ice-cold 0.25 M sucrose. All of the following procedures were performed at 0-4 °C. Minced cerebellar tissue (1.5 g, wet weight) was homogenized (1:10 w/v) with a loose fitting Teflon pestle (686 #m clearance) in a glass homogenizer with 1.4 M sucrose containing 5 mM MgC12 and 0.15 mM spermine at pH 6.5; the homogenate was centrifuged at

445 27,000 x g in a Sorvall centrifuge with an SS-34 rotor for 15 min. Myelin was removed and the precipitate was resuspended in 30 ml of the initial 1.4 M sucrose solution. This suspension was brought to a 30 ml volume and layered manually over a discontinuous gradient consisting of 3 ml each of 1.8 M, 2.1 M, and 2.4 M sucrose that contained 5 mM MgC12 and 0.15 mM spermine, and centrifuged in a Beckman SW 27 swinging bucket rotor at 127,000 × g for 10 min; this yielded a nuclear pellet (2.4P). This 2.1/2.4 M interface and the 2.1 M and 2.4 M gradient layers were collected and resuspended without rehomogenization in 1.4 M sucrose to give a final concentration of approximately 1.5 M sucrose. Thirty milliliters of the resuspended solution were layered over a discontinuous gradient consisting of 4 ml each of 1.8 M and 2.2 M sucrose that contained 5 mM MgClz and 0.15 mM spermine. Following centrifugation with a Beckman SW 27 rotor at 72,000 × g for 10 min, a precipitate of nuclei (2.2P) was obtained. Isolated nuclei from both pellets (2.4P and 2.2P) were pooled and washed once for biochemical investigations, or twice for morphological studies. This washing procedure consisted of resuspending the nuclear precipitates in 0.25 M sucrose, shaking gently on a Vortex mixer, and centrifuging at 2000 x g for 15 min. For nuclei treated with a second wash, the sucrose supernatant was discarded and 4 ml of 0.25 M sucrose were added to the tube; this solution was resuspended using a Vortex mixer and respun as in the first wash.

Nuclear counts and size distribution Unstained nuclear preparations were examined with phase (Zeiss) and anoptral (Reichert) contrast microscopy at 400 x and 1000 x . Although the types of isolated cell nuclei differed with age, nuclei were classified according to the following criteria: (a) nuclear diameter, (b) the size, location, and number of heterochromatin aggregations and/or nucleoli, and (c) nucleoplasmic density. Nuclear counts were based on at least 1000 nuclei chosen from at least 10 random fields and performed 'blind' by the same person at 4 0 0 x . Nuclear diameter was measured with a calibrated ocular micrometer. Histological preparation Animals were anesthetized by immersion in ice water (days 0, 3, 6, 10) or etherization (days 15, 21, 45, 90) and fixed by cardiac perfusion with 1 0 ~ neutral buffered formalin at an air pressure of 120 mm Hg 42. Brains were removed, fixed by immersion for at least 3 days, and processed for light microscopy in polyester wax3L Cerebella were sectioned in sagittal and transverse (coronal) planes and 9/zm sections were stained with Harris' hematoxylin. Chemical and analytical procedures Total tissue D N A content was measured using the methods of Schneider 34; lipids were extracted successively with chloroform-methanol (3:1, v/v), 95 ~ ethanol, and 5 trichloroacetic acid. Homogenate samples were hydrolyzed for 2 h with 0.6 N KOH, and R N A was removed by precipitation with 1.2 M perchloric acid (PCA). D N A was determined by a modification of the diphenylamine reaction of Burton 13. Color development was measured at 610 and 650 nm as described by Zamenhof et al. 47.

446

Nuclear DNA and RNA. Acid-soluble components and lipids were removed from whole homogenates as described by Schmidt and Thannhauser 3z and Schneider 34. The content of DNA and RNA was measured by adding PCA (final concentration of 0.2 N) to a nuclear suspension. Hydrolysis of the residue was carried out in a 37 °C shaking waterbath for 2 h using 2.0 ml of 0.6 N KOH. The hydrolyzed supernatant was cooled on ice and the addition of 2.5 ml of 1.2 N PCA precipitated the DNA and protein; the supernatant was collected for RNA absorbancy determination at 260 nm on a Zeiss spectrophotometer. The residue was hydrolyzed at 80 °C for 20 min in 2 ml of 1 0 ~ PCA, centrifuged, and the pellet washed 3 times in 0.3 N PCA. Aqueous acetaldehyde-diphenylamine reagent 16was added to the DNA solution and the mixture incubated for 18 h at 37 °C 13. Color development was measured at 610 and 650 nm as described above; calf thymus DNA (A grade; CalBiochem, San Diego, Calif.) served as standard. Nuclear protein. The residue obtained after DNA extraction was assayed for protein using the Lowry et al. method ~6. Spectrophotometric readings were taken at 720 nm using a Zeiss spectrophotometer with tungsten lamp source. RESULTS

Isolation procedure The nuclear isolation procedures utilized in this study resulted in final nuclear preparations for each age that were of a high degree of purity, with negligible cytoplasmic and capillary contamination as determined by phase contrast microscopy (Fig. 1). Cell nuclei were morphologically identifiable, nuclear membranes appeared to be intact, and internal structures were visible. Few damaged nuclei were observed. Examination of the myelin, supernatant, and various interfaces revealed some cell nuclei; however, these nuclear types resembled those found in the pellets. Nuclear yields were of sufficient size at each age to permit biochemical investigations. Morphology and distribution of isolated nuclei Examination with the phase contrast microscope revealed that the majority of isolated cell nuclei appeared to be either round or elliptical in shape (Fig. 1). Six groups of nuclei were established. For all ages examined, the distribution of cell nuclei and the percentage of each nuclear type are presented in Fig. 2. Cell nuclei of group I (Fig. l a, c) were 5-8 # m in diameter and had a dark nucleoplasm with 3 or more heterochromatin aggregations; their appearance increased 85-fold from days 0 to 21, constituting 8-9 ~ of the total nuclear populations on days 45 and 90. Group II cell nuclei (Fig. la, c-e) were 7-9 #m in diameter and had a light nucleoplasm containing 6 or more heterochromatin aggregations. Although this nuclear type contributed only 1.5 ~ of the cell nuclei at birth, it comprised at least 70 ~ of the isolated cell nuclei in preparations of 10 days and older. After weaning, isolated pellets primarily consisted of only these two types. Nuclear groups III and IV were prominent at younger ages, but disappeared (group III) or were present in small numbers (group IV) by day 45. Group III nuclei (Fig. lb) were 8-12/zm in diameter with 1 4 heterochromatin clumps

447

Fig. 1 Phase-contrast micrographs of isolated cell nuclei from developing rat cerebellum. All micrographs are x 800. a: 90-day°oldcerebellum. Nuclei from group I (arrow) and group II (cross-hatched arrow) can be observed, b: 3-day-oldcerebellum. Note group III nuclei (arrows) with light nucleoplasm and prominent heterochromatin aggregations,c: 15-day-oldcerebellum. Nuclei of group I (arrowhead), group II (cross-hatched arrow) and group IV (arrows) are indicated, d: 15-day-oldcerebellum. Note nuclei of group II (cross-hatched arrows), group IV (arrowheads), and group V (arrow). e: 90-day-old cerebellum. Arrow indicates a large nucleus from group VI surrounded by smaller nuclei of group II (cross-hatched arrow). and a light nucleoplasm, whereas group IV (Fig. lc, d) had diameters of 8-13 # m and a moderately dense nucleoplasm with 1-6 heterochromatin aggregations. Cell nuclei of groups V and VI were the largest nuclei observed, but neither group contributed more than 12 % to the total cell population at any age. Group V cell nuclei (Fig. ld) were 10-15 # m in diameter, had many heterochromatin aggregations and a light nucleoplasm, and were present on day 0, 15, 21, 45 and 90; their highest incidence (2.54 %) was recorded on day 15. The sixth nuclear group (VI) was 12/~m or greater in diameter with 1 or 2 large heterochromatin aggregations present in a light nucleoplasm (Fig. le); the percentage of these nuclei declined from day 0 to 15 (11.44 % to 0.98 %), with 2-3 % being found on days 45 and 90.

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Fig. 2. Distribution of nuclear types in the developing rat cerebellum. Data are presented as the percentage of nuclei in each group at different ages; values are based on a total of 5000 nuclei counted from 10 random fields at 400 ×. * = less than 1 ~.

Morphological comparison of in vitro and in vivo nuclei In order to define the cellular origin(s) of the 6 groups of isolated nuclei, neural cell nuclei in histological sections of developing cerebellum were examined and compared to in vitro nuclei. The small nuclei with dark nucleoplasm of group I resembled nuclei of dark oligodendrocytes (Fig. 3a) while nuclei of group 1I possessed morphological similarities to internal granule neurons (Fig. 3b, h) and their precursors, the external germinal cells. Unlike groups I and 1I, nuclei in group III were heterogeneous in appearance and resembled those observed in large and medium sized neurons such as 'young' Purkinje cells (Fig. 3c), deep cerebellar neurons (Fig. 3d) and Golgi cells, as well as interstitial neurons and marginal cells. Nuclei of group IV, which were prominent during early postnatal life, resembled those of cells associated with the ventricular area, namely ependymal and subventricular cells (Fig. 3e). Nuclei of group V were morphologically similar to two different types of cells: dividing cells in prophase (Fig. 3f) and cerebellar astrocytes (Fig. 3g). G r o u p VI nuclei resembled those of large neurons such as Purkinje cells (Fig. 3h), Golgi cells, and deep cerebellar neurons; depending on the stage of morphological maturation (e.g. nuclear diameter), these nuclei may be classified into group,rill.

449

Fig. 3. Light micrographs of developing rat cerebellum stained with hematoxylin. Fig. 3a, b, g, h are from 50-day-old rat cerebellum and Fig. 3c-f are from cerebella of newborn animals. Note that histologically processed nuclei are smaller than those observed in in vitro preparations. These differences probably reflect a combination of tissue shrinkage caused by histological procedures in the former and nuclear expansion association with in vitro methodology in the latter, a: the nucleus of an oligodendrocyte (arrow) and an astrocyte (arrowhead) located in the medullary layer. × 1250. b: internal granule neurons (arrows) in the internal granule layer, x 1250. c: the nuclei of Purkinje cells (arrows) located in the cerebellar cortex, x 800. d: deep cerebellar neurons in a newborn animal. Their nuclei (arrows) have a light nucleoplasm and prominent nucleoli, x 800. e: nuclei ofependymal cells and subventricular cells (arrows) of the fourth ventricle, x 800. f: an unidentified cell type (arrow) that appears to be in mitosis (prophase); note numerous heterochromatin aggregations. Although this cell was found in the internal granule layer, they were also seen in the molecular layer and the external granule layer. x 800. g: an astrocyte (arrow) located in the medullary layer, x 1250. h: Purkinje cell (arrow) in the cerebellar cortex. Note the internal granule neurons on the right side of the micrograph, x 800.

3.11 3.45 3.46 3.43 3.46 2.94 2.82 2.89

± 55± 5± ± ±

0.03 0.01 0.02 0.01 0.01 0.01 0.01 0.01

Tissue D N A (pg/mg)

2.08 1.27 1.33 1.35 1.27 0.93 0.81 0.83

5± ± 555± 5-

0.09 0.01 0.02 0.15 0.12 0.01 0.08 0.07

Nuclear D N A (Itg/mg)

66.8 36.8 38.5 39.4 36.8 31.5 28.5 28.9

D N A yield (%)

0.33 0.39 0.35 0.45 0.40 0.26 0.28 0.28

± ± ± 55± 5±

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

Nuclear R N A (pg/mg)

0.16 0.31 0.26 0.33 0.31 0.28 0.35 0.33

DNA

RNA

3.95 3.72 3.58 2.05 3.81 3.68 5.64 6.62

555± 5555-

0.02 0.10 0.08 0.01 0.02 0.14 0.27 0.34

Nuclear protein (mg/mg)

1.90 2.93 2.69 1.52 2.99 3.99 7.01 7.92

DNA

Protein

* Values ( # g / m g wet weight o f whole cerebellum) represent m e a n s ± S.E. for 6 10 d e t e r m i n a t i o n s at each age. D N A yield refers to t h e percentage o f nuclear D N A recovered f r o m the total h o m o g e n a t e D N A . See text for details o f isolation a n d analytical procedures.

0.82* 1.12 1.49 4.30 6.06 6.87 5.87 24.31

12 23 40 94 115 200 228 234

0 3 6 10 15 21 45 90

-k 5± ± 55-k ±

Cerebellar wet weight (rag)

Age (days)

DNA, R N A and protein concentrations in the developing rat cerebellum

TABLE I

451 Nucleic acid and protein content Nucleic acid and protein concentrations for developing rat cerebella are presented in Table I. Cerebellar cell density, estimated from the DNA content per unit tissue wet weight, increased between birth to day 3, remained unchanged from days 3 through 15, and decreased after day 15 to a fairly constant value. Calculation of the total DNA per cerebellum revealed that the greatest increases in DNA content occurred between days 0-3 and 6-10, with relatively small increases occurring after day 21. Based on whole tissue homogenate values, nuclear DNA recovery ranged between 28.5 ~ (day 45) and 66.8 ~ (day 0). The ratio of nuclear RNA to DNA increased from birth to day 3, reflecting a major increase in RNA content; after day 3, this ratio appeared to be relatively unchanged. The ratio of nuclear protein to DNA increased from birth to day 3, and decreased from days 3 to 10 because of respective increases and reductions in nuclear protein content.

DISCUSSION Although cerebellar ontogeny has been extensively investigated, very little is known about the biological activity of cerebellar cell nuclei during this period. Studies on preparations of isolated cell nuclei from the developing cerebellum are limited to Giuffrida et al.'s is examination of RNA polymerase activity in 5-30-day-old rats and a report by Burdman 1° on the relationship between DNA synthesis and nuclear protein in the cerebellum of 8-day-old rats. The present investigation was undertaken in order to provide basic information about genomic expression in the developing cerebellum, and our results furnish a morphological and biochemical profile of nuclear events occurring during both the preweaning and postweaning periods. In addition, this study reveals that nuclear isolation methodology can be utilized in estimating cell populations in developing neural tissues. Previously, investigations on cell quantification have been conducted largely on histological sections. However, not only must technical difficulties (e.g. size of tissues, variability in the density of cell distribution, tissue processing) be taken into consideration23, but developing tissues place added constraints on quantitative histology. In the case of the cerebellum, for example, cell proliferation and differentiation have a specific tempo for each lobule, as well as for the vermis and hemispheres2,3, and comprehensive analysis of this material would require a large number of samples. Furthermore, areal analysis and identification of cell types in the developing cerebellum are complicated at early stages of morphogenesis because its laminated architecture is often indistinct and neural cells are still proliferating and migrating from the ventricular and/or external germinal layers. Understandably, most investigations of normal and pathologic cerebellar tissues have been restricted to quantitative assessment of sagittal sections from one lobule of the vermis ~,4,7,19,81. Thus, nuclear isolation techniques provide a means to circumvent these problems and, as demonstrated in the present report, allow the estimation of individual and total cell populations from the entire developing cerebellum. It is evident that this technique could also be applied in studies concerned with other regions

452 of the brain, as well as in investigations defining alterations in populations of neural cells from abnormal tissues. The nuclear isolation technique utilized in the present study for developing rat cerebellum was similar to that used previously in studies on adult human 46 and rat 36 neural tissues. Essentially, this procedure involved homogenization of neural tissues in hypertonic sucrose solution, centrifugation through two discontinuous gradients, and two final washes in 0.25 M isotonic sucrose. Examination with phase contrast optics revealed that isolated cell nuclei did not appear damaged and had little cytoplasmic contamination; in addition, internal structural details were visible. Although the recovery values for nuclear DNA ranged from 28.5 to 66.8 ~o, and were comparable to those reported in other investigations on adult 21,22,25,27 and developing17,18 brain tissues, these limited yields raise the question of whether or not the nuclear populations isolated in our in vitro study accurately represent the population of nuclei found in cerebellar tissue at each age. A number of observations, however, suggest that there are qualitative and quantitative similarities between the neural cell populations reported in our in vitro investigation and those found in vivo. Both large and small isolated cerebellar cell nuclei were morphologically similar to those cell nuclei observed in histological preparations of developing cerebellum. Moreover, it does not appear that certain types of nuclei were excluded or disproportionately represented by our methodology, since examination of the myelin and various interfaces revealed that the types and estimated proportions of nuclei were compatible with those found in nuclear pellets. Finally, although quantitative histological studies on the cell populations of the entire developing rat cerebellum are not available, Smith et al. 36 have reported a favorable comparison between in vitro and in vivo estimates of the internal granule neuron populations from the adult rat cerebellum. Further evaluation of the cellular composition and proportion of glia and neurons from the developing cerebellum are warranted in order to test the validity of our results. Interpretation of the cellular origin of isolated cell nuclei by comparison with nuclei found in histological preparations is often attended by a number of difficulties that should be mentioned in view of the present study. First, isolation procedures (as well as histological methodology) may introduce artifacts such as alterations in nuclear size and shape, nucleoplasmic density, and distribution of heterochromatin aggregations and/or nucleoli that could limit morphological correlations. It should be noted that preliminary examination of isolated nuclei embedded in polyester wax and stained with hematoxylin revealed that these nuclei appeared similar to those in vitro nuclei observed with phase contrast microscopy and therefore were not altered by histological procedures. Secondly, the task of defining the cellular derivation of isolated nuclei from developing brain is even more complicated because of the proliferation and maturation of different cellular components. Conversely, however, knowledge of the neuro-ontogenic development, particularly as to the time of origin of each cell type, may be an important asset in interpreting the cellular origin of isolated cell nuclei. In this study, autoradiographic and light and electron microscopic findings concerned with cerebellar morphogenesis can be integrated with the present data in

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Fig. 4. Total number of cell nuclei in the developing rat cerebellum. Values at different ages are based on total DNA content (Table I) divided by the amount of DNA per nucleus(6.28pg/nucleus).Stippling represents the percentage of group 1I cell nuclei. order to provide verification of our morphological identifications. The rat cerebellum develops on a precise timetable that extends over both the prenatal and postnatal periods2-5,15,4L Prenatally, the subventricular region (group IV cells, and possibly mitotic cells in group V) of the 4th ventricle is the primary germinative matrix, with large and medium size neurons (groups III and V|) mainly arising before birth 15. External germinal cells (group II) that migrate to the subpial area of the cerebellar cortex and form a secondary germinative matrix (the external germinal layer) also originate prenatally from this area. Thus our findings of a high frequency of nuclei from groups II|, IV, and VI during early postnatal development suggest that these cells constitute the largest proportion of cerebellar cells arising prenatally, with only a small population of external germinal cells being derived before birth. Postnatally, prolifera-

454 tion of the external germinal cells (group II), and their differentiation into internal granule neurons (group II), as well as the much smaller populations of basket and stellate neurons (included in group III), is known to take place during the preweaning period 1-z. The results of the present study corroborate these findings, with only a small percentage of group II nuclei found at birth, and marked increases occurring during the first postnatal month. Finally, glial cells reported to be derived from the subventricular layer of the 4th ventricleZ2,39 and from dispersed stem cells 1,z arise principally during the postnatal period3, 39. Our observations correspond to this temporal sequence, with nuclei of oligodendrocytes (group I) first noted on day 6 and increasing thereafter, and nuclei of cerebellar astrocytes (group V) observed after day 16. DNA content has been considered a measure of cell number (40). In the present study, this value, based on counting the cells in an aliquot of cerebellar tissue and determining the DNA content of a similar volume, was found to be 6.28 × 10 a2 g DNA per average cerebellar cell. This value approximated the values reported by Mirsky 29 (6.1 × 10-1~ g DNA) for rat diploid chromosomes and McEwen et al. 2s (6.73 × 10-12 g DNA) for rat cerebellar cell nuclei. Thus, the total neural cell population of the rat cerebellum can be determined at each stage of development (Fig. 4); DNA content was assumed to be constant for all diploid nuclei and not affected by the small number of cells reported to be tetraploid s,9,24. From postnatal days 0 to 21, the number of cerebellar cells increased from 5.9 × 106 to 9.4 × 107, a 15.7-fold increase; thereafter, the acquisition of cells appeared to diminish and by day 90, 1.07 × 108 cells were present. The largest increases occurred from birth to day 3 (2. l-fold) and between days 6 and 10 (2.3-fold). Assuming that the nuclear population recovered in our procedure is representative of the nuclear population in the entire cerebellum, the contribution of each nuclear type to the total cell population can also be determined at each age. Thus, of the 5.9 million cerebellar cells present at birth, about 4 million are large and medium sized neurons, 1.75 million are ependymal and subventricular cells, and only 91,500 are external germinal cells and/or internal granule neurons. These latter cells exhibit a 1000-fold increase from birth to day 90 (Fig. 4), with a 12.4-fold increase observed between birth and day 3, a 7.3-fold increase between days 3 and 6, and a 4.3-fold increase between days 6 and 10; after day 10, smaller increases in cells were recorded. Another prominent population at day 90, also demonstrating marked increases throughout postnatal development, are the oligodendrocytes. Between birth and day 90, these cells increased from approximately 11 thousand to 8.6 million, an 800-fold increase. These data corroborate and extend earlier autoradiographic and light and electron microscopic findings2-4,15, 32 concerning cerebellar morphogenesis. The prenatal development of large and medium sized neurons from subventricular cells has been previously mentioned, and it appears that this cell population predominates early in postnatal development. By the beginning of the second week, the marked proliferation of the external germinal cells and their differentiation into internal granule neurons (as well as smaller numbers of basket and stellate neurons) are also evident in this report and have been extensively documented in in vivo investigations. Although very little is known about the quantity of total cells or individual cell types

455 during development, our estimates in the adult rat compare favorably with data gathered from other studies. Smolyaninova7 has reported that there are 250 internal granule neurons for every Purkinje cell, with estimates of Purkinje cells20,a7 varying from 3.2 x 105 to 5.0 x 105; 80-125 million cerebellar granule neurons can be calculated from this information. Furthermore, our in vitro glia (astrocytes and oligodendrocytes) to neuron ratio of 0.08 at day 90, is consistent with the 0.06 ratio reported by Closet al. 14 in histological preparations of the cerebellar cortex. In view of the close correlation between the in vitro cell estimates (based on tissue DNA values) presented in this study and those reported for in vivo investigations we felt confident in regard to the present procedures utilized for DNA determinations. Although our value of 2.89/xg/mg cerebellar tissue DNA is similar to the 2.99 #g/mg found by Yu 41, both our tissue and nuclear (0.83/~g/mg) DNA concentrations are markedly lower than the tissue (5.16 #g/mg) and nuclear (3.15 gg/mg) DNA content reported by McEwen and Zigmond 27 and McEwen et al. 2a. This discrepancy may be due to the inclusion of procedures to remove lipids and acid soluble phosphates prior to precipitation of DNA as described in the present study and by Yu 41, whereas this protocol is unspecified in reports by McEwen et al. 2s and McEwen and Zigmond27; the presence of lipids and acid soluble phosphates may introduce an error in DNA determination 3° (unpublished observations). Cerebellar cell nuclei isolated in the present study exhibited a high degree of purity, suggesting that much of the RNA and protein measured in nuclear pellets was nuclear and not cytoplasmic in origin. Although it is uncertain as to how much nuclear RNA and protein has been lost during the isolation procedures, our values for adult rat cerebellum are comparable to the estimates of nuclear RNA and protein content reported by McEwen and Zigmond 27. As might be expected, the ratios of nuclear RNA to DNA and nuclear protein to DNA are higher in the present investigation because of the 3.8-fold decrease in the concentration of nuclear DNA recorded in our study; however, our ratios are similar to those reported for other adult brain regions by McEwen and Zigmond 27. Although a low ratio of RNA to DNA in cerebellar cell nuclei was observed at birth, this ratio increased from birth to day 3, perhaps suggesting a correlation with the increasing nuclear activity of the abundant population of large and medium size neurons that were prenatally derived. The decrease in nuclear protein to DNA ratios from days 3 to 10, may be related to the extensive mitotic activity of the external germinal cells, while the subsequent increase in this ratio after day I0 probably reflects differentiation of cerebellar constituents.

ACKNOWLEDGEMENTS This work was supported by Grants PDT-27A and PDT-27B from the American Cancer Society, Grants 1P30 CA18450 and CA22815 awarded by the National Cancer Institute, and NIDA Grant 01816. Special thanks are extended to Dr. Joseph Altman for his thoughtful review of this manuscript and to Eileen J. Zagon for assistance in this study.

456 REFERENCES 1 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis, lI. 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, J. comp. Neurol., 128 (1966) 431-474. 2 Altman, J., Autoradiographic and histological studies of postnatal neurogenesis. 111. Dating the time of production and onset of differentiation of cerebellar microneurons in rats, J. comp. Neurol., 136 (1969) 269-294. 3 Altman, J., Postnatal neurogenesis and the problem of neural plasticity. In W. A. Himwich (Ed.), Developmental Neurobiology, C. C. Thomas, Springfield, 1970, pp. 197-237. 4 Altman, J., Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer, J. comp. NeuroL, 145 (1972) 353-398. 5 Altman, J. and Das, G. D., 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. comp. Neurol., 126 (1966) 337-390. 6 Banks-Schlegel, S. P. and Johnson, T. C., R N A metabolism in isolated mouse brain nuclei during early postnatal development, J. Neurochem., 24 (1975) 947-952. 7 Barnes, D. and Altman, J., Effects of different schedules of early undernutrition on the preweaning growth of the rat cerebellum, Exp. Neurol., 38 (1973) 406-419. 8 Bohn, R. C. and Mitchell, R. B., Cytophotometric identification of tetraploid Purkinje ceils in young and aged rats, J. Neurobiol., 7 (1976) 255-258. 9 Bregnard, A., Knusel, A. and Kuenzle, C. C., Are all the neuronal nuclei polyploid ? Histochemistry, 43 (1975) 59-61. 10 Burdman, J. A., The relationship between D N A synthesis and the synthesis of nuclear proteins in rat brain during development, J. Neurochem., 19 (1972) 1459-1469. l I Burdman, J. A., Haglid, K. and Dravid, A. R., Protein synthesis in fractions from isolated brain cell nuclei, J. Neurochem., 17 (1970) 669-676. 12 Burdman, J. A., Szijan, I., Franzoni, L. and Garcia Argiz, C. A., The relationship between D N A synthesis and nuclear proteins in the cerebrum and cerebellum of 8-day-old rats, J. Neuroehem., 20 (1973) 1719-1725. 13 Burt•n• K.• A study •f the c•••rimetric estimati•n •f de•xyri b•nuc•eic acid• Bi•chem. J.• 62 ( • 956) 315-323. 14 Clos, J., Faire, C., Selme-Matrat, M. and Legrand, J., Effects of undernutrition on cell formation in the rat brain and specially on cellular Composition of the cerebellum, Brain Research, 123 (1977) 13 26. 15 Das, G. D. and Nornes, H. O., Neurogenesis in the cerebellum of the rat : an autoradiographic study, Z. Anat. Entwiekl.-Gesch., 138 (1972) 155-165. 16 Dische, Z., Color reactions of nucleic acid components. In E. Chargaff and J. N. Davidson (Eds.), Nucleic Acids, Vol. 1, Academic Press, New York, 1955, pp. 285-305. 17 Gomez, C. J., Garcia Argiz, C. A., Franzoni, L. and Krawiec, L., Ribonucleic acid synthesis in isolated cell nuclei of developing brain, Neurobiology, 1 (1971) 129-143. 18 Giuffrida, A. M., Cox, D. and Mathias, A. P., RNA polymerase activity in various classes of nuclei from different regions of rat brain during postnatal development, J. Neurochem., 24 (1975) 749-755. 19 Griffin, W. S. T., Woodward, D. J. and Chanda, R., Malnutrition and brain development: Cerebellar weight, DNA, RNA, protein and histological correlations, J. Neuroehem., 28 (1977) 1269-1279. 20 I nukai, T., On the loss of Purkinje cells, with advancing age, from the cerebellar cortex of t he albino rat, J. comp. Neurol., 45 (1928) 1-31. 21 Kato, T. and Kurokawa, M., Isolation of cell nuclei from the mammalian cerebral cortex and their assortment on a morphological basis, J. Cell Biol., 32 (1967) 649-662. 22 Knusel, A., Lehner, B., Kuenzle, C. C. and Kistler, G. S., Isolation of neuronal nuclei from rat brain cortex, J. Cell Biol., 59 (1973) 762-765. 23 Konigsmark, B. W., Methods for the counting of neurons. In W. J. H. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer-Verlag, New York, 1970, pp. 315-340. 24 Lentz, R. D. and Lapham, L. W., A quantitative cytochemical study of the D N A content of neurons of rat cerebellar cortex, J. Neurochem., 16 (1969) 379-384.

457 25 Lovtrup-Rein, H. and McEwen, B. S., Isolation and fractionation of rat brain nuclei, 3. CellBiol., 30 (1966) 405-415. 26 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 27 McEwen, B. S. and Zigmond, R. E., Isolation of brain cell nuclei. In J. Marks and R. Rodnight (Eds.), Research Methods in Neurochemistry, l/oL 1, Plenum Press, New York, 1972, pp. 139-170. 28 McEwen, B. S., Plapinger, L., Wallach, G. and Magnus, C., Properties of cell nuclei isolated from various regions of rat brain: divergent characteristics of cerebellar cell nuclei, J. Neurochem., 19 (1972) 1159-1170. 29 Mirsky, A. E., Harvey Lectures 1950-1951, 47 (1952) 98. 30 Munro, H. N. and Fleck, A., The determination of nucleic acids, Meth. Biochem. AnaL, 14 (1966) 113-176. 31 Nicholson, J. L. and Altman, J., The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. I. Cell proliferation and differentiation, Brain Research, 44 (1972) 13-23. 32 Ram6n y Cajal, S., Histologie du Systeme Nerveux de l'Homme et des Vertebres, 2 vols., L. Azoulay, (Trans.), Madrid, 1909-1911, pp. 80-119. 33 Schmidt, G. and Thannhauser, S. J., A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues, J. biol. Chem., 161 (1945) 83-89. 34 Schneider, W. C., Phosphorus compounds in animal tissues. I. Extraction and estimation of deoxypentose nucleic acid and of pentose nucleic acid, J. biol. Chem., 161 (1945) 293-303. 35 Sidman, R. L., Mottla, P. A. and Feder, N., Improved polyester wax embedding for histology, Stain Technol., 36 (1961) 279-284. 36 Smith, S. J., McLaughlin, P. J. and Zagon, I. S., Granule neurons and their significance in preparations of isolated brain cell nuclei, Brain Research, 103 (1976) 345-349. 37 Smolyaninov, V. V., Some special features of organization of the cerebellar cortex. In I. M. Gelfand, V. A. Gurfinkel, S. V. Formin and M. L. Tsetlin (Eds.), MIT Press, Cambridge, 1971, pp. 250--423. 38 Sporn, M. B., Wanko, T. and Dingman, W., The isolation of cell nuclei from rat brain, J. CellBioL, 15 (1962) 109-120. 39 Swarz, J. R. and del Cerro, M., Lack of evidence for glial cells originating from the external granular layer in mouse cerebellum, J. Neurocytol., 6 (1977) 241-250. 40 Venderly, R., The deoxyribonucleic acid content of the nucleus. In E. Chargaff and J. Davidson (Eds.), The Nucleic Acids, I/ol. 2, Academic Press, New York, 1955, pp. 157-165. 41 Yu, W. A., The effect of 5-bromodeoxyuridine on the postnatal development of the rat cerebellum: a biochemical study, Brain Research, 118 (1976) 281-291. 42 Zagon, I. S., Prolonged gestation and cerebellar development in the rat, Exp. NeuroL, 46 (1975) 69-77. 43 Zagon, I. S. and McLaughlin, P. J., The effects of different schedules of methadone treatment on rat brain development, Exp. NeuroL, 56 (1977) 538-552. 44 Zagon, I. S. and McLaughlin, P. J., Perinatal methadone exposure and brain development: a biochemical study, J. Neurochem., 31 (1978) 49-54. 45 Zagon, I. S. and McLaughlin, P. J., Transplacental exposure to ethylnitrosourea and its effects on body, brain, and cerebellar development, Cancer Res., 38 (1978) 1263-1268. 46 Zagon, I. S., McLaughlin, P. J. and Smith, S., Neural populations in the human cerebellum: estimations from isolated cell nuclei, Brain Research, 121 (1977) 279-282. 47 Zamenhof, S., Bursztyn, H., Rich, K. and Zamenhof, P. J., The determination of deoxyribonucleic acid and ofceU number in brain, J. Neurochem., 11 (1964) 505-509.