Developmental studies on the cerebellum from reeler mutant mouse in vivo and in vitro

DEVELOPMENTAL

BIOLOGY

Developmental

79,64-80

(1980)

Studies on the Cerebellum from reeler Mutant Mouse in Vivo and in Vitro

KATSUHIKO MIKOSHIBA, KAZUHIRO NAGAIKE, SHINICHI KOHSAKA, KEN TAKAMATSU, EMIKO AOKI, AND YASUZO TSUKADA Department

of

Physiology Keio University School of Medicine, 35 Shinano-machi, Shinjuku-Ku, Tokyo, Japan Received April 4,1979; accepted January 8, 1980

The influence of the reeler mutation on the development of the cerebellum was examined morphologically and biochemically both in vivo and in vitro. SDS-polyacrylamide gel electrophoresis revealed that all cerebellar proteins which increase during development are found in the same amounts in reeler and control. The time schedule for migration of granule cells and formation of the granular layer in the reeler shows no significant difference from the control. Immunohistochemical methods using antisera against S-100 and glial fibrillary acidic (GFA) proteins reveal that most of the Bergmann cells are scattered around the molecular and granular layers. But in some part, the cells were aligned like those of the control though independently of the position of Purkinje cells. Proliferated astrocytes with fmely arborixed processes were observed in the central mass of the large neuron groups in the cerebellum from the reeler. High CNPase activity in the reeler cerebellum was suggested to be due to a decrease in granule cells. Autoradiography of the sections from the control cerebellum after intraperitoneal injection of 2-deoxy[‘%]glucose revealed that the incorporation of 2-deoxyglucose was maximum in the granular layer. Little was incorporated in the white matter. In the reeler, incorporation of 2-deoxyglucose was found not only in the granular layer but also in the white matter. The primary cultures from the reeler cerebellum were generally comparable to those of the normal control in terms of neuritic outgrowth, schedule of general development, and quantity of myelin formation, except for the lack of laminar structure. INTRODUCTION

Synaptogenesis is a long process which begins with recognition between cell surfaces and requires subsequent selective stabilization of labile contacts (Changeux and Danchin, 1976; Changeux and Mikoshiba, 1978). The cerebellum offers a useful model in which to study synaptogenesis in the central nervous system. The anatomy of the cerebellum is relatively well understood in both the adult (Palay and Chan-Palay, 1973) and the developing young rat (Altman, 1972a,b,c). Unitary activity of single neurons can be recorded by electrophysiological techniques, and much is known of cerebellar circuitry (Eccles et al., 1967). Several genetic mutations in mice are known to affect cerebellar structure at welldefined neuronal or synaptic loci (Sidman et al., 1965; Sidman, 1972, 1974; Sotelo, 0012-1606/60/110064-17$02.00/0 Copyright 0 1990 by Academic Press, AII rights of reproduction in any form

1973; Rakic, 1976; Changeux and Mikoshiba, 1978). The reeler is an autosomal recessive mutation occurring in mice (Falconer, 1951; Hamburgh, 1960,1963; Sidman, 1972, 1974; Mariani et al., 1977). The cerebellum from the reeler (rl/rl) is characterized by malposition of Purkinje cells in the central mass of the cerebellum. The majority of granule cells migrate to form a layer superficial to the Purkinje cells. It is known that synaptic contact is much altered in association with this inversion of laminar structure (Mariani et al., 1977). The reeler cerebellum thus offers a good model for investigation of the development of the nervous system when laminar structure is altered. We here report the biochemical and morphological analyses on the reeler cerebel64

Inc. reserved.

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Development of Reeler Mutant Cerebellum in Vivo and in Vitro

lum in viva and in vitro from the developmental point of view.

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Bornstein and Murray, 1958; Silberberg, 1972). The nutrient medium was composed of equal parts of Gey’s BSS (Gey and Gey, MATERIALS AND METHODS 1936), Eagle’s minimum essential medium, Animals. The reeler mutation (rlor’) on fetal calf serum, and chick embryo extract. inbred strains of mice with the BALB/c Glucose was added to produce a final conbackground is maintained in our laboracentration of 600 mg%. The explants were tory. The strain originated from the laboincubated at 35.5”C and the medium was ratory of the CNRS, Orleans La Source changed every 3 days. The development of (Dr. Moutier). Homozygous reelers (rl/rl) the cultured tissue was observed under the were generated by intercrossing rl/rl males phase-contrast microscope. and rl/+ or rl/rl females. Slab gel electrophoresis. The samples for Dissection of molecular layer, granular SDS-polyacrylamide gel electrophoresis layer, and white matter. Bovine cerebelwere homogenized with a motor-driven lum, obtained from the slaughterhouse, was Teflon glass Potter homogenizer in 10 vol dissected out on an ice-cooled plate. The of ice-cold deionized water. The homogecortex was cut sagittally with a razor blade nate was centrifuged at 45,000 rpm for 60 and the slices were dissected in a 0.32 M min with a Hitachi RP 65 rotor. The pellet sucrose solution with the aid of a binocular and supernatant were dissolved in 6% somicroscope. The dissected layers were frodium dodecyl sulfate supplemented with 2% zen at -80°C and stored. The detailed P-mercaptoethanol, and boiled for 3-5 min. method and the morphological observaSlab gel electrophoresis in linear gradients tions were described in previous papers of 7.5-20% polyacrylamide was performed (Mikoshiba and Changeux, 1978). in 0.1% SDS using the modified method Measurement of cyclic B’J-nucleotide (Mikoshiba and Changeux, 1978; Miko3’-phosphohydrolase (CNPase). CNPase shiba et al., 1979a) of Laemmli (1970). activity was assayed by the method of TsuImmunohistochemical reaction. Antikada et al. (1977, 1978). An assay mixture bodies against glial fibrillary acidic protein containing 50 ~1 of 0.5% Triton X-100 and (GFA) and S-100 protein were kindly do50 ~1 of cyclic 2’,3’-AMP was added as a nated by Dr. L. F. Eng and Dr. L. Levine. substrate to give a final concentration of Mice were sacrificed by vascular perfusion 7.5 n-&I in a total volume of 200 ~1. After with 10% formaldehyde containing 0.9% incubation for 20 min, 0.6 ml of ice-cold NaCl, and the brains were removed and ethanol was added and mixed well to stop posttixed with 10% formaldehyde in 0.9% the enzyme reaction. Then 0.6 ml of chloNaCl for 2-6 days at room temperature. roform was added and mixed well and cenParaffin-embedded samples were cut into trifuged at 1OOOgfor 20 min. One microliter sections 6 pm thick, which were then deof the water phase was applied to highparaffinized and equilibrated in Trisperformance liquid microchromatography buffered saline (0.15 M NaCl containing (Familic-100 Japan Spectroscopic Co., 0.05 M Tris-HCl buffer (pH 7.6)). Sections Ltd.), and the concentration of 2’-AMP were incubated with 3% normal goat serum ured at 254 nm, Protein was measured acin Tris-buffered saline for 30 min at room cording to the method of Lowry et al. temperature. Antisera against GFA and S(1951). 100 (1:lOOO dilution) were added and incuTissue culture of the cerebellum. Cere- bated for 48 hr at 4°C. Sections were bellar tissues from newborn reeler mice, washed in Tris-buffered saline (three times obtained by mating rl/rl males and rl/rl every 2 min). Goat anti-rabbit immunoglobfemales were cultured using Maximow’s ulin (diluted to 1:lO) was added and incudouble-coverslip method (Bornstein, 1958; bated for 30 min followed by washing with

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12th-day reeler cerebellum. The molecular layer of the reeler cerebellum was thinner than that of the control, and the cell density of the granular layer of the reeler was also lower than that of the control throughout development. The increase in thickness of the molecular layer became prominent from 9 to 12 days of age in the control cerebellum, while the reeler cerebella had a very thin molecular layer. The time schedule of migration of granule cells and the formation of the granular layer are thought to be good indicators of cerebellar development. No difference in migration was observed between the control and the reeler (Figs. 2 and 3). Both finish their cell migrations before the 18th day after birth. However, the cell density of the granular layer of the reeler was greatly diminished. The Purkinje cells were found mostly in the central mass of the cerebellum and a few of the Purkinje cells were distributed between the molecular and granular layers. Protein profiles determined by SDSpolyacrylamide gel electrophoresis of particulate fractions from the normal cerebella changed developmentally. In order to compare the quantitative changes of cerebellar proteins between the reeler and the control developmentally, the total pellet of the cerebellar homogenate was used. There were RESULTS several protein bands which increased in In Vivo Development of Cerebellum from amount developmentally in the cerebella reeler Mutant and are indicated as arrowheads in Fig. 6. The postnatal development of the cere- P400protein which is a protein characteristic bellum of the rodent is remarkable. The of the Purkinje cell (Mikoshiba et al., increase of wet weight is extremely rapid 1977a,b; Mikoshiba et al., 1979a) increased from the 10th to the 20th day after birth remarkably as the cerebellum developed. (Altman, 1972a,b,c). Figure 1 shows, in sag- The histone bands in the adult reeler cerittal section, the developing cerebella of ebella decreased compared to the control, both normal mice and the reeler mutant and this result confirmed previous results mice. The cerebellum was reduced in size that DNA content relative to protein dein the reeler with little enlargement beyond creased in the cerebellum of reeler (Mariani the 6th day after birth. It was difficult to et al., 1977). Proteins which increased dedistinguish the adult reeler cerebellum velopmentally (indicated by arrowheads in from that of the 12th-day reeler cerebellum Fig. 6) are included in the adult reeler cerexcept that migrating granule cells at the ebella in amounts to equal those in adult external granule layer were observed in the control cerebella. The present results sugT&buffered saline (three times every 2 min). Soluble antigen-antibody complex of horseradish peroxidase rabbit anti-horseradish peroxidase (diluted to 1:20) was then added and incubated for 30 min at room temperature. The sections were incubated with’ mixture containing 0.05% diaminobe&dine-4HC1 and 0.01% hydrogen peroxide in either 0.05 M Tris-HCl buffer (pH 7.6) or T&buffered saline and the peroxidase bound to the tissue forming the brown insoluble reaction product was observed. Uptake of 2-deoxyf4C]glucose into the brains from reeler and control mice. Ten microcuries of 2-deoxy[‘4C]glucose (45-55 mCi/mmole, New England Nuclear) was injected intraperitoneally into a mouse at room temperature in a quiet room with moderate illumination. After 45 min, the mouse was killed by decapitation and the skull was removed carefully in order not to harm the surface of the cortex. The removed brain was frozen in Freon-XII (about -70%) chilled in liquid nitrogen on a stainless steel plate. Sections of the brain were cut every 20 pm by a cryostat at -10°C. They were immediately dried on a hot plate at 80°C at least 5 min. X-Bay fihn (Fuji Industrial X-ray film No. 150) was exposed on the coverslips for 7 days.

FIG. 1. Slagit ;tal sections of the developing cerebellum from normal and reeler mutant mice, new rhom and staining. Left-hand side: developing cerebella fro1 m control aged 3, 6, 9, 1%15, and 34 days. Hematoxylin-eosin mice. Right -bar id side: developing cerebella from reeler mutant mice. Numbers in figures indic :ate the days 20 X. postnatally. 07 n,ewbom mouse. Magnification: 67

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gest that the reeler cerebellum developed normally to the adult level as far as protein profile is concerned. In Vitro Development of Cerebellum from reeler Mutant The development of the cerebellum in culture was observed by phase-contrast microscopy and by the Bodian silver impregnation technique. The cultures from the reeler cerebellum were generally comparable to those of the normal control in terms of neuritic outgrowth. The quantity of myelin formation in the reeler seemed to be normal. The lack of laminar organization appeared to be observed in the reeler mutant and the groups of Purkinje cells were often observed randomly interspersed among the granule cells with no recognizable order (Fig. 7). In conclusion, from the culture study, the development of the reeler cerebellum appeared to be very similar to that of the control except for the lack of laminar structure. Glial Cell Markers (S-100, GFA Protein, and CNPase) in the reeler Cerebellum S-109 protein is considered to be a marker of glial cells in general (Moore, 1965; Moore and McGregor, 1965; Nagata et al., 1974) and GFA protein is considered to be a marker of astrocytes (Bigbee et al., 1977; Bignami and Dahl, 1973, 1974a,b; Bignami

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et al., 1972; Dahl and Bignami, 1973, 1976; Eng et al., 1971; Ludwin et al., 1976). There were many cells positive for the reaction with S-100 antiserum lying at the layer of the Purkinje cell somata in the cerebellum of the control (Fig. 4). In the white matter of the cerebellum, S-100 positive cells were also observed (Fig. 4A). The degree of staining intensity of the cells varied slightly from cell to cell. Purkinje cells and granule cells were devoid of staining (Figs. 4B and C). The staining by S-100 antiserum was the highest in the cell bodies of the Bergmann glia which were localized between the unstained Purkinje cells and the granular layer. The cell processes were not usually stained. The Purkinje cells, the granule cells, and the deep nuclear cells did not react with anti-S-100 serum. In the reeler cerebellum, the cells reacting with S-100 antiserum were scattered through the molecular layer rather than lined up at the interface between the molecular and granular layers. In a very few cases, the S-106 positive cells were aligned at the interface of molecular and granular layers (Fig. 4E). GFA protein was found in the control cerebellum (Fig. 5) in a distribution similar to that described before by the group of Eng, Bignami, and Dahl (Ludwin et al., 1976; Bignami and Dahl, 1973,1974a). With GFA protein antiserum, the most prominently stained parts of the cells were the

FIG. 2. Photomicrographs of a portion of the cerebellar cortex from control and reeler mutant mice of different ages (from newborn to 9 days old). Hematoxylin-eosin staining. Anterior part of the cerebellum was photographed in the reeler. Dorsal part of centralis was photographed in the control cerebellum. Magnification: 600 x. FIG. 3. Photomicrographs of portion of the cerebellar cortex from control and reeler mutant mice of different ages (from 12 to 34 days old). Hematoxylin-eosin staining. Anterior part of the cerebellum was photographed in reeler and dorsal part of centralis was photographed in the control cerebellum. Magnification: 600 X. FIG. 4. Peroxidase staining for S-106 protein of cerebellum from the control and reeler mutant mouse. A, B, and C: control cerebellum with different magnification (A, 109 x; B, 400 X; C, 200 x). D and E: cerebellum from reeler mutant mouse (D and E, 209 x). ML, molecular layer; CL, granular layer; WM, white matter. Arrows indicate Purkinje cell body. Purkinje cells and granule cells did not react with the antiserum against S-106 protein. Many of the S-100 positive cells were aligned at the interphase between the molecular and granular layers as Bergmann cells in the control (A, B, and C). Positive cells were also found in the white matter and a few cells were found in the granular layer, and much less in the molecular layer (A and B). In the reeler cerebellum, S-166 positive cells, probably Bergmann cells, were usually found scattered around the interface between the molecular layer and the granular layer (D) but in some cases they were aligned at the interphase (E) independently of the position of Purkinje cells.

FIG. 2. 69

FIG. 3. 70

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Development of Reeler Mutant Cerebellum in Vivo and in Vitro

FIG. 4.

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FIG. 5. Peroxidase staining for glial fibrillary acidic (GFA) protein of cerebellum from the control and reeler mutant mice. A, cerebellum from control mouse; B-E, cerebellum from reeler mutant mouse. Bergmann fibers were observed as straight fibers independently (D) of the position of Purkinje cells. A fine mesh of Bergmann fibers was observed in the very thin molecular layer (C). Gliosis-like figures were found around the central mass of cells (B and C). Arrows indicate the Purkinje cells. Magnification: 400 x (A-E). ML, molecular layer; GL, granular layer.

MIKOSHIBA ET AL.

Development of Reeler Mutant Cerebellum in Vivo and in Vitro

processes. In contrast, the perikarya were not stained. The radial processes of Bergmann cells were well stained for GFA protein. In the cerebellum from reeler, very short GFA positive fibers were found in the molecular layer (Figs. 5B and C). In addition to the finding of Bignami and Dahl (1974a) three types of configurations of GFA positive cells were observed. The first was a fine mesh of GFA positive fibers in the thin molecular layer as has been well described by Bignami and Dahl (1974a). The second was Bergmann cells with straight fibers in the molecular layer. These were aligned like those of the control though independently of the position of the Purkinje cells (Fig. 5D). The third was proliferated astrocytes with finely arborized processes in the central part of the reeler cerebellum between the molecular layer and the central mass of large neuron groups (Figs. 5B and C) . CNPase is a marker enzyme for central myelin which is formed from the cytoplasmic membranes of oligodendroglia (Kurihara and Tsukada, 1967,1968; Kurihara et

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al., 1970; Tsukada et al., 1978). By microdissection technique, the molecular layer, the granular layer, and the subadjacent white matter were obtained from the cerebellum for the CNPase assay (Mikoshiba and Changeux, 1978). The CNPase activity of the dissected granular layer was about 5.6 times higher than that of the dissected molecular layer (Table 1). This result indicates that most of the myelinated mossy fibers radiate into the granular layer. Table 1B shows the CNPase activity of three parts of the brain from the reeler: brain stem, spinal cord, and cerebellum. CNPase activity of the total homogenate of the cerebellum from reeler showed higher values compared to those of the control although the CNPase activities of the brain stem and spinal cord showed no significant difference between the reeler and the control. Metabolic Mapping of the Cerebellum by Deoxyglucose Technique Figure 8 shows the autoradiograph of the cerebellum after intraperitoneal injection of 2-deoxy[14C]glucose. The autoradi-

FIG. 6. Protein profiles of the total particulate fraction of the developing control cerebellum and the adult AD, adult cerebellum (3 months old). Numbers indicate tht e days after birth. H, histone bands; F1, F, histone, The molecular weight markers were immunoglobulins IgG (MW ISO,OOO),phosphorylase b (94,000), ovalbumin (43,000), and chymotrypsinogen (25,000).

reeler cerebellum. NB, newborn cerebellum;

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FIG. 7. In vitro development of the explants of nelNborn reeler cerebellum. A: low magnification (40 X) the explant. B: Bodian’s impregnation showed numercUS fibers among the small cells (probably granule cell d. C: A large cell (probably Purkinje cell) with arbor&d processes was observed by Bodian’s impregnation. D: A group of large neurons (probably Purkinje cells) were often observed under the phase-contrast optical micr ‘Oscope. Myelin formation was also observed around the cell bodies. Magnification: A, 40 X; B, C, and D, 400 :<.

Development

MIKOSHIBA ET AL. TABLE

of Reeler Mutant Cerebellum in Vivo and in Vitro

1

CYCLIC 2',3'-NUCLEOTIDE 8'-PHOSPHOIIYDROLASE (CNPase) ACTIVITIES OF VARIOUS PARTS OF THE BRAIN (A) FROM HOMOZYGOUS reeler AND WILDTYPE CONTROL MICE AND THE SEPARATED LAYERS (B) FROM BOVINE CEREBELLUMS A

+/+ rl/rl

Cerebellum

Brain stem

Spinal cord

2.49 k 0.24 (4) 3.38 f 0.39* (4)

4.10 f 0.32 (4) 4.34 + 0.25 (4)

6.12 f 0.11 (5) 5.91 + 0.50 (3)

B Molecular layer Granular layer White matter

0.63 -+ 0.08 (5) 3.64 f 0.78 (5) 7.42 + 1.01 (4)

Whole lobe

1.92

(2)

a The mice were 13-15 weeks of age. CNPase activities are expressed as pmole/min/mg protein. Values are expressed as the mean rt standard deviation. Numbers in parentheses are numbers of animals examined. Separation of layers of the cerebellum was performed according to the method of Mikoshiba and Changeux (1978). Wet weight of the cerebellum was 65.6 mg (+/+) and 24.3 mg (rl/rl) in average. * P < 0.01.

ographs revealed that the deoxyglucose was intensely incorporated into the granular layer of the control cerebellum but not into white matter. In the reeler mutant, on the other hand, the incorporation of deoxyglucase was found not only in the granular layer but also in the white matter region where many Purkinje cells were located. This was easily detected by comparing the thickness of the distrjbution of deoxyglucase positive part in the section (Figs. 8A and C). DISCUSSION

The analysis of the cerebellum from reeler mutant provides an insight into the mechanisms by which the cellular environment of neurons may affect their development and their afferent synapses. One of the obvious features of the reeler cerebellum is that the development in uiuo was greatly retarded and the wet weight of the cerebellum was about one-third of that of the control. Loss of granule cells was

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expected from the decrease of DNA content per wet weight which was about half of the control (Mariani et al., 1977). By the analysis of protein profiles, it is possible to investigate whether the protein components of mutants are normal or not. In the Purkinje cell degeneration mutant, for instance, which is characterized by the degeneration of Purkinje cells, one protein band (named as P400protein) clearly disappeared (Mikoshiba et aZ., 1977b, 1979a). We have analyzed by electrophoresis to know how the reeler mutation affects the development of the cerebellum. The reeler cerebellum contained the same protein profile as controls and in an equal amount. Proteins in reeler increase developmentally in normal fashion except for histones. It is therefore speculated that the reduced size of the reeler cerebellum is not due to an arrest of maturation as far as protein profiles are concerned. There is a loss of cells (granule cells) in uiuo in the reeler and this might occur due to the absence of target (Purkinje cell dendrites) in the molecular layer. If the loss of granule cells in the reeler cerebellum results from the absence of normal synaptic contact in uiuo, one would expect that the in vitro culture system may promote the growth of the granule cells where the cellular or fiber contacts are easily performed (Mikoshiba et al., 1979c). Mutations affecting the cerebellum have offered difficulties for study of development in uitro, in part because of the uncertainty of identifying the animal as a homozygote for the mutation on the day of birth. Homozygote reeler as welI as other cerebellar mutants such as weauer or staggerer can only be identified 1 week postnatally judging from the abnormal behavior (Sidman et aZ., 1965; Sidman, 1974). Therefore, monolayer culture or aggregation culture of the mutant was performed later than 7 days after birth, an age which is less favorable for culture (Messer, 1977; Messer and Smith, 1977; DeLong and Sidman, 1970). Wolf (1970) has tried to distinguish ho-

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mozygote and heterozygote retrospectively by morphological examination of part of sections used for culture. We have obtained 100% yield of reeler homozygotes by mating rl/rl males and females. Thus, we have begun a study of in vitro development of reeler cerebellum in primary culture from the day of birth. In vitro studies on the organized structure revealed little difference between the reeler and the normal cultures in spite of the disorganization of the laminar structure in the normal cerebellum in vivo. Although most of the neurons in the cerebrum divide before birth, granule cells in the cerebellum divide after birth. Even if the six layers are inverted in the reeler cerebrum, cell numbers, wet weight, and some other enzymes did not vary much from those of the control (Mikoshiba et al., 1979b, 1980), but those in the cerebellum showed a marked difference compared to those in the control. It is therefore speculated that inversion of brain cell layers causes some damage to the development of dividing cells in vivo. The discrepancy between cerebellar development in vivo and in vitro in the reeler mutant may be a significant point for elucidating the factors controlling the development of the cerebellum. The nervous system consists mainly of two types of cells, neurons and glia. The role of glia to support the function of neurons has been stressed recently. We have investigated whether the reeler mutation affects the glial cells or not. Glial cells are subgrouped into astrocytes, oligodendrocytes, and microglia. Astrocytes have been considered to play the role of modifying the local concentration of ions and neurotransmitters by active uptake mechanisms (Bowery and Brown, 1972; Iversen and Kelly, 1975; Schon and Kelly, 1974). They have also been postulated to play a nutritive and supporting role (Patterson and Chun, 1974). In addition, Rakic (1971, 1972) and Sidman (1974) have proposed that Bergmann astroglia in the cerebellum may act as a guide for migrating

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granule cells and have also proposed that the defective neuronal migration in the weaver (WV/WV) mutant is secondary to maldevelopment of Bergmann glia (Rakic, 1971; Rakic and Sidman, 1973). Genetical analysis has become available by making chimera mice to reveal the real target of the mutation (Mintz, 1962, 1965; Muller, 1977; Tarkowski, 1961; Whitten, 1956). Mice that are homozygous for the Gush allele at the glucuronidase locus have normal or high P-glucuronidase enzyme activity, whereas mice that are homozygous for the Gush allele have low enzyme activity. Because &glucuronidase is an independent locus with the reeler mutation, it is possible to make both chimeras in which the genetically mutant cells have high glucuronidase activity. Namely, the genetically reeler Purkinje cells are of the high P-glucuronidase genotype (Gush), and the genetically normal cells are of the low genotype (i.e., rl/rl, Gusb/Gusb cs +/+, Gush/ Gush). The presence of the genetically normal cells in an abnormal position suggest that the Purkinje cells are not being positioned according to their own genetic information, but rather by factors extrinsic to the Purkinje cells (Mullen, 1977). Thus, there arises the question of whether the reeler mutation is related to the abnormal arrangement of Bergmann cells. The present immunohistochemical analysis disclosed that Bergmann fibers exist in the molecular layer of the reeler and the hematoxylin-eosin staining showed that the time schedule of granule cell migration in reeler was the same as that of the control. Furthermore, the Bergmann cell bodies were regularly positioned in a way that was independent of the position of the Purkinje cells in some part of the reeler cerebellum. Thus reeler mutation does not appear to affect Bergmann cells. We may infer, further, that Purkinje cells have not followed Bergmann glia to their incorrect positions. Cyclic 2’,3’-nucleotide 3’-phosphohydrolase (CNPase) is believed to be a marker

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for the myelin membrane of the oligodendrocyte (Kurihara and Tsukada, 1967; Tsukada et al., 1977). Therefore, the higher specific activity of CNPase of cerebella from reeler would be expected due to hyperplasia of oligodendrocyte or hyperlamella formation by the oligodendrocyte. But immunohistochemical observation by S-100 protein antiserum and electron microscopic observation denied these possibilities. The higher specific activity of CNPase of cerebella from reeler than from the control might result from a relative increase in myelinated fibers, due to the loss of granule cehs which have nonmyelinated parallel fibers. When we compared CNPase activity per cerebellum after correcting for the loss of granule cells by multiplying by the factor of decrease of wet weight in the reeler, the activity was actually about half of the control value. The loss of myelinated fibers might be due to the retrograde degeneration of mossy fiber which is the principal myelinated fiber input to the cerebellum. The degeneration of mossy fibers could be expected due to the loss of their target cells (granule cells) as has already been observed morphologically (Sotelo and Changeux, 1974). This indicates that the absence of synaptic contact influences the number of neuronal fibers. In the normal cerebellum, mossy fibers make synapses with the dendrites of granule cells and not with Purkinje cells (Eccles et al,, 1967; Palay and Chan-Palay, 1973). Deoxyglucose incorporation was maximum in the granular layer, where mossy fiber terminals make synapses with the dendrites of granule cells, and little incorporation was observed in the white matter from the normal cerebella. Even in the nervous cerebellum where Purkinje cells are absent, deoxyglucose incorporation was maximum in the granular layer (Mikoshiba et al., manuscript in preparation). It appears likely that mossy fiber rather than climbing fiber plays an important role for the incorporation of deoxyglucose in the cerebellum. In a previous paper, we reported that heter-

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ologous synapses between mossy fibers and Purkinje cell dendrites were identified in the reeler cerebellum by both morphological and electrophysiological but not metabolic criteria (Mariani et al., 1977). Deoxyglucose incorporation into the central celI mass of Purkinje cells in the white matter region of the reeler cerebellum supports the idea that the heterologous synapses (Purkinje cell-mossy fiber) are functional. Since heterologous synaptic contact was demonstrated to be functional by the 2-deoxyglucase technique, it is suggested that the cerebellar circuitry was reorganized as a result of the reeler mutation. This indicates that the “recognition” step between cell surfaces might not require a complementarity of structure as strict as that assumed to account for the specificity of synaptic contact (Sperry, 1963; Gaze, 1970). Therefore, the developing neuronal network determined genetically can be finally established by the cellular environment. We are grateful to Dr. Jean-Louis Guenet (Institut Pasteur) for providing reeler mutants, and Dr. L. F. Eng (Veterans Administration Hospital) for the hind gift of GFA antiserum. REFERENCES

ALTMAN, J. (1972a). Postnatal

development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145,353-398. ALTMAN, J. (1972b). Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Comp. Neurol. 145399-464. ALTMAN, J. (1972c). Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 146,465-514. BIGBEE, J. W., KOSEK, J. C., and ENG, L. F. (1977). Effects of primary antiserum dilution on staining of “antigen-rich” tissues with the peroxidase-antiperoxidase technique. J. Histochem. Cytochem. 26, 443-447. BIGNAMI, A., and DAHL, D. (1973). Differentiation of astrocytes in the cerebellar cortex and the pyramidal tracts of the newborn rat. An immunofluorescence study with antibodies to a protein specific to astro-

cytes. Bruin Res. 49,393-402. BIGNAMI, A., and DAHL, D. (1974a). The development of Bergmann glia in mutant mice with cerebellar

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malformation: Reeler, staggerer and weaver. Immunofluorescence study with antibodies to the glial fibrihary acidic protein. J. Comp. Neurol. 155,219230. BICNAMI, A., and DAHL, D. (197413).Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein. J. Comp. Neurol. 153,27-38. BIGNAMI, A., ENG, L. F., and UYEDA, C. T. (1972). Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43, 429-435. BORNSTEIN,M. B. (1958). Reconstituted rat-tail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes. Lab. Znvest. 7, 134-137. BORNSTEIN,M. B., and MURRAY, M. R. (1958). Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum. J. Biophys. Biothem. Cytol. 4,499~504. BOWERY,N. G., and BROWN,D. A. (1972). Gammaaminobutyric acid uptake by sympathetic ganglia. Nature New Biol. 238,89-91. CHANGEUX,J.-P., and DANCHIN, A. (1976). Selective stabilization of developing synapses, a mechanism for the specification of neuronal networks. Nature (London) 264,705-712. CHANGEUX,J.-P., and MIKOSHIBA, K. (1978). Genetic and “epigenetic” factors regulating synapse formation in vertebrate cerebellum and neuromuscular junction. In “Maturation of the Nervous System” (M. A. Corner, R. E. Baker, N. E. Van de Pol;D. F. Swaab, and H. B. M. Uylings, eds.), Progress in Brain Research, Vol. 48, pp. 43-66. Elsevier, Amsterdam/New York/Oxford. DAHL, D., and BIGNAMI, A. (1973). Immunochemical and immunofluorescence studies of the glial fibrillary acidic protein in vertebrates. Brain Res. 61, 279-293. DAHL, D., and BIGNAMI, A. (1976).Immunogenic properties of the glial fibrillary acidic protein. Brain Res. 116,150-157. DELONG, G. R., and SIDMAN, R. L. (1970). Alignment defect of reaggregating cells in cultures of developing brains of reeler mutant mice. Develop. Biol. 22, 584-600. ECCLES,J. C., ITO, M., and SZENTAGOTHAI,J. (1967). “The Cerebellum as a Neuronal Machine.” Springer-Verlag, New York. ENG, L. F., VANDERHAEGHEN,J. J., BIGNAMI, A., and GERSTL,B. (1971). An acidic protein isolated from fibrous astrocytes. Brain Res. 29,351-354. FALCONER,D. S. (1951).Two new mutants, “trembler” and “reeler,” with neurological actions in the house mouse (Mus musculus L.). J. Genet. 50,192-201. GAZE, R. M. (1970). “The Formation of Nerve Connections.” Academic Press, New York. GEY, G. O., and GEY, M. K. (1936). The maintenance

79

of human normal cells and tumor cells in continuous culture. I. Preliminary report: Cultivation of mesoblastic tumors and notes on methods of cultivation. Amer. J. Cancer 27,45. HAMBURGH, M. (1960). Observations on the neuropathology of “reeler,” a neurobiological mutation in mice. Rxperientia 16,460-461. HAMBURGH, M. (1963). Analysis of the postnatal developmental effects of “reeler,” a neurological mutation in mice. A study in developmental genetics. Develop. Biol. 8, 165-185. IVERSEN,L. L., and KELLY, J. S. (1975). Uptake and metabolism of y-aminobutyric acid by neurons and glial cells. Biochem. Pharmacol. 24,933-938. KURIHARA, T., NUSSBAUM, J. L., and MANDEL, P. (1970). 2’,3’-Cyclic nucleotide 3’-phosphohydrolase in brains of mutant mice with deficient myelination. J. Neurochem. 17,993-997. KURIHARA, T., and TSUKADA, v. (1967). The regional and subcellular distribution of 2’,3’-cyclic nucleotide 3’-phosphohydrolase in the central nervous system. J. Neurochem. 14,1167-1174. KURIHARA, T., and TSUKADA, Y. (1968). 2’,3’-Cyclic nucleotide 3’-phosphohydrolase in the developing chick brain and spinal cord. J. Neurochem. 15,827832. LAEMMLI, U. K. (1970).Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LOWRY,0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin reagent. J. Biol. Chem. 193.265-275. LUDWIN, S. K., KOSEK, J. C., and ENG, L. F. (1976). The topographical distribution of S-100 and GFA proteins in the adult rat brain: An immunohistochemical study using horseradish peroxidase-labelled antibodies. J. Comp. Neurol. 165, 197-208. MARIANI, J., CREPEL,F., MIKOSHIBA, K., CHANGEUX, J.-P., and SOTELO,C. (1977). Anatomical, physiological and biochemical studies of the cerebellum from reeler mutant mouse. Phil. Trans. Royal Sot. London Ser. B 281, l-28. MESSER,A. (1977). The maintenance and identification of mouse cerebellar granule cells in monolayer culture. Brain Res. 130, I-12. MESSER,A., and SMITH, D. M. (1977). In vitro behavior of granule cells from staggerer and weaver mutants of mice. Brain Res. 130, 13-23. MIKOSHIBA, K., and CHANGEUX, J.-P. (1978). Morphological and biochemical studies on isolated molecular and granular layers from bovine cerebellum. Brain Res. 142,487-504. MIKOSHIBA, K., HUCHET, M., and CHANGEUX,J.-P. (1977a). Biochemical studies on the cerebellum of neurological mutants of mice with reference to separated molecular and granular layers of beef cerebellum. Proc. Znt. Union Physiol. Sci. 13,510. MIKOSHIBA, K., HUCHET, M., and CHANGEUX,J.-P. (1977b). Biochemical studies on P400 protein: A

80

DEVELOPMENTAL BIOL.OCY

cerebelbrr protein. Proc. Znt. Sot. Neurochem. 6, 309. MIKOSHIBA, K., HUCHET, M., and CHANGEUX,J.-P. (1979a). Biochemical and immunological studies on the Pm protein, a protein characteristic of the Purkinje cell from mouse and rat cerebellum. Develop. Neurosci. 2,254-275. MIKOSHIBA, K., KOHSAKA,S., TAKAMATSU, K., AOKI, E., and TSUKADA, Y. (1979b). Neurochemical studies on the inverted laminar cerebral cortex of the reeler mutant mouse. Neurosci. Lett. Suppl. 2, S-46. MIKOSHIBA, K., KOHSAKA, S., TAKAMATSU, K., AOKI, E., and TSUKADA, Y. (1980). Morphological and biochemical studies on the cerebral cortex from reeler mutant mice: Development of cortical layer and metabolic mapping by deoxyglucose method. J. Neurochem. 34,835-844. MIKOSHIBA, K., NAGAIKE, K., AOKI, E., and TSUKADA, Y. (1979c). Biochemical and immunohistochemical studies on dysmyelination of quaking mutant mice in vivo and in vitro. Brain Res. 177,287-299. MINI, B. (1962). Formation of genotypically mosaic mouse embryos. Amer. 2001. 2,432 (Abstr.). MINT& B. (1965). Genetic mosaicism in adult mice of quadriparental lineage. Science 148,1232-1233. MOORE, B. W. (1965). A soluble protein characteristic of the nervous system. Biochem. Biophys. Res. Commun. 19,739-744. MOORE, B. W., and MCGREGOR, D. (1965). Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver. J. Biol. Chem. 240,16471653. MULLEN, R. J. (1977). Genetic dissection of the CNS with mutant-normal mouse and rat chimeras. In “Society for Neuroscience Symposia,” Vol. 2: “Approaches to the Cell Biology of Neurons” (W. M. Cowan and J. A. Ferrendelli, eds.), pp. 47-65. Sot. Neurosci., Bethesda, Md. NAGATA, Y., MIKOSHIBA, K., and TSUKADA, Y. (1974). Neuronal cell body enriched and glial cell enriched fractions from young and adult rat brains: Preparation and morphological and biochemical properties. J. Neurochem. 22,493~503. PALAY, S., and CHAN-PALAY, V. (1973). “Cerebellar Cortex, Cytology and Organisation.” Springer, Berlin. PAITERSON, P. H., and CHUN, L. L. Y. (1974). The influence of nonneuronal cells on catecholamine and acetylcholine synthesis and accumulation in cultures of dissociated sympathetic neurons. Proc. Nat. Acad. Sci. USA 71,3607-3610. RAKIC, P. (1971). Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electron-microscopic study in Macacus Rhesus. J. Comp. Neural. 141,283-312. RAKIC, P. (1972). Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol. 5,61-84. RAKIC, P. (1976). Synaptic specificity in the cerebellar

VOLUME 79,198O

cortex: Study of anomalous circuits induced by single gene mutations in mice. Cold Spring Harbor Symp. Quant. Biol. 40,333-346. RAKIC, P., and SIDMAN, R. L. (1973). Weaver mutant mouse cerebellum: Defective neuronal migration secondary to specific abnormality of Bergman glia Proc. Nat. Acad. Sci. USA 70,240~244. SCHON,F., and KELLY, J. S. (1974). The characterization of [3H]-GABA uptake into the satellite glial cells of rat sensory ganglia. Brain Res. 66, 289-399. SIDMAN, R. L. (1972). Cell interactions in developing mammalian central nervous system. In “Cell Interactions” (L. G. Silvestri, ed.), Proceedings, Third Lepetit Colloquium, pp. 1-13. North-Holland, Amsterdam. SIDMAN, R. L. (1974). Interaction among developing mammalian brain cells. In “The Cell Surface in Development” (A. A. Moscona, ed.), Proceedings, Int. Sot. Develop. Biologists, pp. 221-253. Wiley, New York. SIDMAN, R. L., GREEN, M. C., and APPEL, S. H. (1965). “Catalog of the Neurological Mutants of the Mouse.” Harvard Univ. Press, Cambridge, Mass. SILBERBERG,D. H. (1972). Cultivation of nerve tissue. In “Growth, Nutrition, and Metabolism of Cells in Culture” (G. H. Rothblat and V. J. Cristofalo, eds.), Vol. II, pp. 131-167. Academic Press, New York/ London. SOTELO, C. (1973). Permanence and fate of paramembranous synaptic specializations in “mutants” and experimental animals. Brain Res. 62.345-351. SOTELO,C., and CHANGEUX,J.-P. (1974). Trans-synaptic degeneration “en cascade” in the cerebelbu cortex of staggerer mutant mice. Brain Res. 67, 519-526. SPERRY, R. (1963). Chemoaffinity in the orderly growth nerve fiber patterns and connections. Proc. Nat. Acad. Sci. USA 50,703-710. TARKOWSKI, A. K. (1961). Mouse chimeras developed from fused eggs. Nature (London) 190,857-860. TSUKADA, Y., NAGAI, K., and SUDA, H. (1977). A new method for the measurement of 2’,3’-cyclic nucleotide 3’-phosphohydrolase (CNPase) by high performance liquid chromatography, and an attempt at partial purification of the enzyme from rat brain. Bull. Japan. Neurochem. Sot. 16, 85-88 (abstract in Engl. Neurochem. Res. 3,662-663,1978). TSUKADA, Y., and SUDA, H. (1978). Purification of 2’,3’-cyclic nucleotide 3’-phosphohydrolase (CNPase) from bovine cerebral white matter. In “Proceedings of the European Society for Neurochemistry” (V. Neuhoff, ed.), Vol. I, p. 122. Verlag Chemie, Weinheim/New York. WHIITEN, W. K. (1956). Culture of tubal mouse ova. Nature (London) 177.96. WOLF, M. K. (1970). Anatomy of cultured mouse cerebellum. II. Organotypic migration of granule cells demonstrated by silver impregnation of normal and mutant cultures. J. Comp. Neurol. 140,281-298.