Granule cell behavior on laminin in cerebellar microexplant cultures

Developmental Brain Research, 52 (1990) 63-73

63

Elsevier BRESD51~2

Granule cell behavior on laminin in cerebellar microexplant cultures Isao Nagata 1 and Norio Nakatsuji 2 1Department of Genetics, Tokyo Metropolitan Institute for Neurosciences, Fuchu, Tokyo (Japan) and 2Division of Developmental Biology, Meiji Institute of Health Science, Naruda, Odawara (Japan) (Accepted 5 September 1989)

Key words: Mouse cerebellum; Microexplant culture; Granule cell; Cell migration; Perpendicular orientation; Laminin

In order to study roles of the extracellular matrix (ECM) in the cerebellar granule cell migration, cerebellar microexplants of neonatal to postnatal ll-day-oid mice were cultured on 3 kinds of substrata, poly-L-lysine (PL), PL/fibronectin and PL/laminin. A prominent outgrowth of small granule cells, which did not uptake GABA, was observed only on the PL/laminin substratum. The granule cells showed the following sequence of events: (1) Many polygonal undifferentiated cells migrated out from the microexplants. These blast cells differentiated into small bipolar neurons with long fine neurites which extended radially from the explants. (2) These cells then changed their orientation perpendicular to their radial neurites, by protruding a short process from the cell body at right angles. (3) Finally, cell bodies of these granule cells adhered to each other to form cell aggregates. Quantitative labelings by bromodeoxyuridine revealed that there were less mitotic cells in explants from the later postnatal cerebella compared to the earlier postnatal ones. Anti-MAP2 immunoreactivity was localized in short perpendicular processes of the aggregated granule cells. Thus, this unique cell behavior exhibited on the PL/laminin substratum provides the first defined experimental system for studying the granule cell differentiation in vitro.

INTRODUCTION E m b r y o n i c neural tissues contain extracellular matrix ( E C M ) in extracellular spaces 1°'23'29, but little is k n o w n about its roles in neurogenesis. In recent years, various c o m p o n e n t s of the E C M were purified, and they are considered to be i m p o r t a n t for adhesion and migration of neural cells 3'5'11'12. In the mouse cerebellum, H a t t e n et al. 12 reported that the fibronectin-immunoreactivity was present along the external germinal layer and that fibronectin p r o m o t e d the granule cell migration in vitro, although this observation has not been confirmed 16. L a m i n i n is well k n o w n to p r o m o t e neurite outgrowth of m a n y kinds of brain n e u r o n s in vitro 4'6'11A4. It appears temporarily in the external granular layer ( E G L ) and along the B e r g m a n n glial processes 17. Selack et al. 31 showed that laminin p r o m o t e d granule cell migration in the cerebellar explant culture from n e w b o r n rats. We extended such observations by using a serum-free, h o r m o n a l l y defined m e d i u m and substrata coated with various E C M components. We found a peculiar sequential event of n e u r o n a l cell m o v e m e n t on the polyL-lysine/laminin substratum in microexplant cultures of the mouse cerebellum of various neonatal ages. Preliminary observations have been reported 19.

MATERIALS AND METHODS

Substrata Coating with poly-L-lysinewas made on glass coverslips (16 mm in diameter) as described by Schnitzer and Schachnera°. Sterile coverslips, immersed in a 100/~g/ml poly-L-lysine hydrobromide (PL, Sigma) solution, were dried under a sterile air flow, rinsed with distilled water and used as the PL substratum. About 50/~1 of a solution of bovine plasma fibronectin (20/~g/ml or 1 mg/ml, Sigma) or that of EHS tumor laminin (20 /~g/ml, E-Y laboratories or Collaborative Research) were applied to the dried PL substratum for 1-2 h at 37 °C, rinsed twice with the culture medium, and used immediately as the PL/fibronectin or PL/laminin substrata, respectively.

Culture medium A serum-free, hormone-supplemented medium was prepared according to Fischer7. Briefly, it contained bovine serum albumin (1 mg/ml, Biomedical technology), insulin (10/~g/ml, Sigma), transferrin (100/~g/ml, Sigma), aprotinin (1 ktg/ml, Sigma), Na-selenite (30 nM, Sigma) and L-thyroxine (T4, 0.1 nM, Sigma). Glucose, glutamine and antibiotics (penicillin and streptomycin) were added as described in the previous paper 3°.

Microexplant culture We modified the methods of cerebellar microexplant cultures which have been reported by Nagata et al.21 and Fischer et al. 8. Vermal regions of cerebellar tissues were dissected out from neonatal to P14 mice (BALB/c) and freed from meninges and choroid plexus. Slices were then made with a razor blade, from which white matter and deep cerebellar nuclei were removed. Rectangular pieces (300-400 /tm) were dissected out from the remaining tissue, which mainly consisted of the cerebellar gray

Correspondence: I. Nagata, Department of Genetics, Tokyo Metropolitan Institute for Neurosciences, 2-6 Masushidai, Fuchu, Tokyo 183, Japan. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Phase-contrast micrographs showing outgrowth of neurites and neurons in the cerebellar microexplant cultmcs from P3 mice. Microexplants were cultured for 4 days on 3 kinds of substrata: PL (a), PL/fibronectin (b) or PL/laminin (c) Bars -. 51) ,'~m

matter, using a razor blade. Such prepared microexplants were rinsed with the culture medium and placed evenly on the PL-, PL/fibronectin- or PL/laminin-coated glass coverslips (10-20 microexplants/coverslip) with 50/d of the culture medium. One or two hours after the plating, 3 coverslips were transferred into a Petri dish (35 mm in diameter, Nuclon), added with 1 ml of the culture medium, and put in a CO 2 incubator (4% COz/96% air, 37 °C). For some explants, we used embryonic primordial cerebellar tissues on the 18th day of gestation.

Measurement of neurite and neuron outgrowth Distances of neurite tips or neuronal cell bodies from the margin of explants were measured directly under a phase contrast microscope (Olympus) using an ocular micrometer, or indirectly after taking photographs of the same field at intervals of 2-24 h. The outgrowth distance of neurites or cell bodies was measured on at least 20 explants to obtain mean values. In case of the uneven outgrowth of neurites or neurons, we measured their maximum distance in each explant.

Products) at 1:500 dilution and the HRP-conjugaied anti-rabbit IgG antibody (Cappel) as the second antibody. HRP was detected by a conventional diaminobenzidine (DAB) reaction. For MAP2 immunostaining, explants were fixed with formaline-saline (pH 7.0) for 15 rain at room temperature. Fixed samples were immersed in a permealizing solution, 0.1% Triton X-100 ill phosphate-buffered saline (PBS), for 10 rain. They were stained with an anti-MAP2 antibody solution (rabbit antiserum at 1:500 dilution, Bio-Yeda) overnight at 4 °C, rinsed with PBS, and treated with the HRP- or FITC-conjugated second antibody for 1 h at room temperature. The DAB deposits from HRP reaction were enhanced by silver enhancers (Amersham). Neuron-specific enolase immunostaining was carried out according to the same procedure as described above, except using anti-neuron specific enolase antibody solution (rabbit antiserum at 1:500 dilution, Funakoshi), L1 immunostaining was performed according to Nagata et al. 2~. lmmunofluorescence was examined by a fluorescence microscope (Olympus). [3H]GABA uptake was carried out according to Hekmat ct al. 13,

Cell proliferation Scanning electron microscopy Scanning electron microscopy was carried out as described by Nakatsuji and Nagata 22.

Immunocytochemistry and GABA uptake analysis Immunocytochemical staining for glial fibriltar acidic (GFA) protein was carried out as described previously2a, except that we used a commercially purchased rabbit antiserum (Bio-Science

DNA synthesis by cultured cells was monitored with the bromodeoxyuridine (BrDU)-antiBrdU antibody immunocytochemistry9. For continuous labeling, cerebellar microexplants were incubated with a 1 × 10 ~ M 5-bromodeoxyuridine (Sigma) solution for 6 days with a medium change on 3rd day. For pulse labelings, 0.5-1.0 × 10 3 M BrdU were added to the medium on the 2nd day of the culture for 4 h. Such labeled explant cultures were rinsed with calcium- and magnesium-free PBS (CMF-PBS), dried under an air

Fig. 2. Phase-contrast micrographs of the microexplant culture on the PL/laminin substratum, a: outgrowth of undifferentiated cells after 1 day of culture using a P2 mouse cerebellum, b: radially migrating bipolar neurons after 2 days of culture using a P2 cerebellum, c: onset of the orientation change after 3 days of culture using a P4 cerebellum, d: the perpendicular orientation is completed after 4 days of culturing a P4 cerebellum, e: aggregation of cell bodies whose short neurites orient perpendicular to the long radial neurites, after 5 days of culturing a P4 cerebellum. Bars = 20 ktm.

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66 flow, immersed in 70% ethanol for a few minutes and rinsed with CMF-PBS. They were treated with 1 N HCI for 30 min, washed several times with CMF-PBS, and incubated with the monoclonal antibody against BrdU (Becton-Dickinson) overnight at 4 °C. followed by the treatment with the HRP-conjugated second antibody.

showed uptake of [3H]GABA which is a marker tor inhibitory neurons such as basket or stellate cells. At this early stage of the cultures most presumed granule cells migrated along radially oriented parallel bundles of their neurites (see also ref. 22), whereas the GABA-uptaking cells migrated in rather random orientations.

RESULTS

Explants on the PL- or PL/fibronectin-coated substrata Outgrowth of neurites and neuron-like small cells from the cerebellar explants started by 1 day on the PL substratum. However, only a small number of cell bodies migratedout from the explant. They remained within 300 /zm of distance even after 5 days of culture (Fig. la). The addition of fibronectin to the substratum gave only a weak promoting effect on both the neurite extension and migration of neuron-like cells from the microexplants (Fig. lb). Even a high concentration of fibronectin (1 mg/ml) failed to give more promoting effects. On the PL/fibronectin substratum, the neuron-like small cells remained in the vicinity of explants (within 300 p m of distance), forming a network of their neurites. Explants on the PL/laminin-coated substratum An extensive migration of many small neuron-like cells occurred on the PL/laminin substratum. They migrated out up to a 700-#m distance from the explants in 5 days (Fig. lc). Within several hours after plating, many phase-dark flat cells and some neurites started to migrate out from the explant margin (Figs. 2a and 8a). Most of such cells appeared undifferentiated: they were negative for L1, neuron-specific enolase or GFA protein immunostaining, and took up BrdU (described later). Thus, these cells are cerebellar neuronal blast cells from the EGL. By comparing individual cells through serially taken photographs, we found that most of the immature cells differentiated into bipolar spindle-shaped neuronal cells by 16 h of culture (Figs. 2b, 3 and 4a). These neuron-like cells migrated out radially from the microexplants, with a clockwise bending of neurites in all explants. During the radial migration, their cell bodies were frequently attached to each other, and their bipolar neurites were fasciculated. Such bipolar cells are presumed to be cerebellar granule cells and not basket or stellate cells because of the following reasons. (1) Each small cell had two long fine neurites in opposite directions (Fig. 3, see also ref. 22). This is a characteristic feature of granule cells in vivo L28 and in vitro 31. (2) In addition to the expression of neuron-specific enolase, most cells had L I antigen on their cell surfaces. This antigen is expressed by granule cells, but not by basket or stellate cells25. (3) Only less than 10% of cells in our microexplant culture

Neuron outgrowth and age of the explants The mean rate and standard deviation of the out-

Fig. 3. Phase-contrast micrograph of radially migrating granule cells in a marginal area of a cerebellar microexplant culture on the PL/laminin substratum. The microexplant was dissected out from a P4 mouse and cultured for 1.5 days. Arrows indicate growth cones at the distal tips. Bar = 20 pro.

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Fig. 5. Distance covered by the outgrowth of neurons on the PL (broken lines) or PL/laminin (solid lines) substratum. Microexplants were dissected out from cerebella of P0 (0). P2 ((~)), P4 (@), P7 ([--]) or P l l (I-o]) mice.

growth during the first 2 days on the PL/laminin substratum were approximately 140 + 20 pm/day for explants at all developmental stages used (Fig. 5). Explants obtained from P2-P5 mice gave the largest distance of the radial neuron outgrowth and also the largest number of the migrating neurons per explant. Microexplants from the embryonic and neonatal (earlier than P2) cerebellar explants gave extensive migration of the blast cells, but scarcely completed the perpendicular orientation change. Explants from mice older than P7 gave decreased distance and number of the neuron outgrowth. For example, microexplants from P l l mice showed only poor outgrowth, and those from P14 mice no longer attached to the substratum. Thus, migratory behavior of granule cells from the microexplants was clearly dependent on the age of the explants.

Orientation change of granule cells After the outgrowth of radial neurites and cell bodies, granule cells started to change their orientation (Fig. 2c), and became perpendicular to their long radial neurites (Figs. 2d and 4b). This orientation change occurred in almost all cells of most explants. At an early stage of this transition, we frequently observed the granule cells with

Fig. 4. Scanning electron micrographs showing microexplants from P4 cerebella on the PL/laminin substratum, a: radial migration after 2 days of culture, b: perpendicular orientation after 3 days. c: aggregation after 5 days of culture. Bars = 10 ~m.

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Fig. 6. Onset time of the orientation change and cell aggregation in the mouse cerebellum microexplants which were dissected out from P2, P4, P5, P7, P10 or P l l mice. Number of days in culture until start of the orientation change (open square) or aggregation (solid square) is shown by means and standard deviations.

Fig. 7. Light microscopic autoradiogram of the cerebellar microexplant on the PL/laminin substratum. The microexplant was dissected out from a P4 mouse, cultured for 3 days and labeled with [3H]GABA for 20 min at 37 °C. An accumulation of the silver grains is seen over a larger neuron (arrowheadL Double-headed arrow indicates the direction of long neurites of granule cells. Bar = 2(1 /~m.

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Fig. 8. Schematic diagrams of the sequential neuronal cell behavior in mouse cerebellar mieroexptant culture on the PL/laminin substratum. First, the blast cells migrate out from the explants and differentiate into typical bipolar granule cell neurons (blank cells in a,b). They migrate out radially with a clockwise bending, thus making the radial neurite bundle (blank cells in c). Then they start to change their orientation by protruding new short processes perpendicular to the radial neurites (blank cells in d.e). Finally, they make aggregates of the cell body (blank cells in 0. A few larger-sized cells (filled) represent GABA-uptaking inhibitory neurons (see also ref. 131. which also orient perpendicular to the radial neurite bundles at later stages of the culture (filled cells in d,e,f).

69 a perpendicular short process (arrows in Fig. 2c and Fig. 8d) and other neurons still in radial orientation (Fig. 2c and arrow in Fig. 4b). The onset time of the orientation change was dependent on the developmental stage of cerebellar explants. Younger explants took a longer time in culture than older explants before the orientation change. For example, neurons from P2-P5 mice started to change their orientation on the 3rd or 4th day, and those from P 7 - P l l mice started on the 2nd day in culture (Fig. 6). These facts indicate that the orientation change may not be an artificial phenomenon but an inherent nature of granule cells related to the developmental process. At this stage of cultures, over 90% of the perpendicularly oriented cells did not incorporate [3H]GABA, and less than 10% of small cells took up GABA (arrowhead in Fig. 7). The latter cells were short bipolar or tripolar neurons in contrast to presumed granule cells having long neurites, and they also oriented perpendicular to the granule cell neurites (Fig. 8d).

Fig. 9. Immunostaining of the cerebellar microexplant for GFA protein. The microexplant was dissected out from a P5 mouse and cultured for 6 days. Bar = 50/~m.

Fig. 10. Labeling of microexplants with BrdU. The explants were dissected out from a P2 (a). P9 (b) or P1 (c) mouse, labeled continuously for 6 days (a,b) or in pulse for 4 h (c). A large arrow in b or c indicates labeled cells. Small arrows show unlabeled cell aggregates. Bars = 50/~m.

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Fig. 11. Percentage of labeled cells in pulse (open circle) or continuous (solid circle) labeling with BrdU. The percentage was calculated on the total population of cells visible outside the explant. For pulse labeling, explants were dissected out from P1, P3, P4, P7 or Pll mice, and labeled for 4 h after 1 day of culture. For continuous labeling, explants were dissected out from PI, P4, P7, P9, Pll or P12 mice, and labeled for 6 days.

Aggregate formation by granule cells During and after the orientation change, cell bodies of granule cells adhered to each other to form aggregates consisting of 3-30 cells (Figs. 2e and 4c). The onset time of the aggregate formation was also dependent on the developmental stage of the explants (Fig. 6). At this later stage of cultures, we could discriminate

two types of small neurons, both of which oriented perpendicular to granule cell neurites, even under a phase contrast microscope. There were z~t least 3 clear differences between them. The first difference was in cell size and adhesiveness of cell bodies. Cell bodies of granule cells were round and the smallest (5-7 um in diameter), but those of G A B A - u p t a k i n g cells were elliptical and larger (7-12 a m ) than granule cells, as is so in the cerebellum 24. Granule cells frequently formed aggregates with each other, whereas the G A B A - u p t a k i n g cells had no tendency to form aggregates with each other or with granule cells and remained solitary (Fig. Be,f). The second difference was expression of antigenic cell markers. The neuronal cell surface molecule L I , which is expressed on granule cells but not on basket and stellate cells 2s, was expressed on the aggregated cells hut not on the larger solitary neurons. Cytoskeletal M A P 2 protein was expressed in both types of neurons: the aggregated small cells had 2-6 fine short processes (30-70 um) (Fig. 12b, short arrows in Fig. 12c), whereas the larger solitary neurons had 1-2 thick longer elaborated processes (200-500 a m ) (long arrows in Fig. 12c). The third difference was the number of the two types of cells. Although it is hard to count the exact number of cells at later stages of culture because of the granule celt's tendency to aggregate, the number of small cells which did not take up G A B A was much larger than the larger solitary cells which took up G A B A (Fig. 8e,f). The G A B A - u p t a k i n g cells survived longer even after the aggregated small cells degenerated.

Fig. 12. Immunostaining of the microexplant cultures for MAP2 protein. Explants were dissected out from P4 mice and cultured for 4 (a) or 9 (b,c) days. Bars = 50 ~m.

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Unique sequence of cell behavior on the PL/laminin substratum Fig. 8 summarizes the above-described sequences of behaviors, which were displayed by two types of cells, granule cells and GABA-uptaking inhibitory cells, on the PL/laminin substratum. In addition to the morphological and the antigenic characteristics, the sequential behaviors of the two types of cells also appeared to be different. Such sequential processes were sensitive to the conditions which would change the status of laminin coating. Firstly, no typical migration of the small granule cells was observed when the laminin solution was applied directly to glass coverslips. Secondly, outgrowth of the neurons was decreased to the level of outgrowth on the substratum containing PL alone without laminin, when the anti-laminin antibody was added to the culture medium or when the substratum was pre-treated with the antibody. We detected astrocytes immunocytologically with the anti-GFA protein antibody. They showed only limited outgrowth confined within 200 pm from the explant margin after 6 days in culture (Fig. 9). Since most of the small neuronal cell bodies migrated extensively up to 700 pm of distance (Fig. 5), the unique cell behavior observed in this study was not affected by the glial cells. Labeling by BrdU Some of the explants were labeled with BrdU, an analog of thymidine, in order to know whether the migrating neurons on the PL/laminin substratum maintained proliferative activity or already lost the activity. After continuous or pulse labeling, uptake of the analog was detected with an immunocytochemical staining with monoclonal antibodies to the analog. Fig. 10a shows a P2 cerebellar microexplant that was continuously labeled for 6 days. Most of the neurons which migrated out from the explant have positive labeling. In explants from mice at ages P5-P9, labeled cells were sparse and localized in the vicinity of the explants (Fig. 10b). Explants from mice older than P10 showed no labeling. Counting data (Fig. 11) indicates that the percentage of the labeled cells decreased from ca 100% in the case of neonatal mice to ca 0% in the case of P10 mice along a roughly sigmoidal curve. Pulse labeling (Fig. 10c) confirmed that some of the migrating blast cells on the PL/laminin substratum had the proliferative activity. The number of labeled cells, however, decreased swiftly as the cerebellar microexplants were taken from older mice (Fig. 11). These results indicate that the migrating cells in explants from neonatal to P9 mice contain both mitotic and postmitotic cells, whereas those in P10 explants are all postmitotic.

Anti-MAP2 immunocytochemistry Radially migrating bipolar neurons showed weak MAP2 staining on the distal leading neurite, but not in the growth cone and the opposite trailing neurite. After the orientation change, most granule cells had MAP2positive staining in the cell body and short processes both parallel and perpendicular to their radial neurite bundles, which were negative for the staining (Fig. 12a). After the aggregate formation, the MAP2 staining was detected in the cell bodies and short neurites around the aggregates (Fig. 12b). At later stages of the culture we found another type of cells which had a few thick, but elaborated MAP2-positive neurites in some regions (Fig. 12c). They had larger cell bodies (long arrows) than those of the aggregated granule cells (short arrows). Such cell types corresponded well to the GABA-uptaking cells. DISCUSSION

Discrimination of granule cells from other neurons At least 3 kinds of small interneurons derive from the E G L in the cerebellar cortex t. The first kind of neurons are granule cells which constitute about 90% of neurons and have an excitatory transmitter. Another two kinds of neurons are basket and stellate cells, both of which have an inhibitory transmitter, GABA. Since we used the gray matter of the cerebella for microexplants, the possible candidates of neurons migrating out from the explants are either the 3 kinds of small neurons, Golgi type II cells or Purkinje cells. There are two reasons why the migrating neurons may not contain the latter two cells. One reason is the size of cells: the largest size of neurons outside explants was less than 12/~m in diameter, while Golgi type II cells and Purkinje cells are 9-16 pm and 20-25 pm in diameter, respectively24. Another reason is the non-detectability of Golgi type II cells and the non-survival of Purkinje cells under a similar culture condition (M. Schachner, personal communication). Thus, it is likely that possible neuronal cell types in our explant cultures are granule, basket or stellate cells. We could discriminate at least two different types of neurons by antigenic markers and the GABA-uptake ability. Presumed granule cells occupied more than 90% of the small cells in the present explant culture. They were smallest in size, had bipolar long fine neurites, and had a tendency to aggregate each other at later stages of cultures. They expressed L1 antigen always on their cell bodies and long radial neurites. The other type of neuron was GABA-uptaking inhibitory cells and had a size larger than granule cells. They were short bipolar neurons at earlier stages, and then differentiated into elliptical cells with thick elaborated neurites at later stages. They did not express L1 antigen on their surface and tended to

72 remain solitary. Very recently, Hekmat et al. ~~ showed that G A B A - u p t a k i n g inhibitory cells oriented perpendicular to the granule cell neurites in both cerebellar microexplant and dissociation-reaggregation cultures on a similar, but not the same, laminin substratum. Concerning the G A B A - u p t a k i n g cells, our data agree well with their report. Here we mainly observed another type of neurons, granule cells, which occupied over 90% of small neurons in our cultures. Radial outgrowth On the PL/laminin substratum, we observed radial outgrowth of the undifferentiated cells which were shown to have proliferative activity by the BrdU labeling. They transformed into massive radial migration of the bipolar spindle-shaped granule cells. Liesi ~7 showed that the immunological staining for laminin is present temporarily in the extracellular space of the E G L . The finding that granule cells migrate massively on the laminin substratum, but not on substrata of other E C M components supports the hypothesis that laminin plays a role in neuronal migration ~7'1s. Such radial migration on the PL/laminin substratum might be relevant to the movement of immature granule cells in situ parallel to the cerebellar surface. Perpendicular orientation From extensive light and electron microscopic observations, it is known that postmitotic granule cells migrate inward by apposing their leading processes intimately to the surface of Bergmann glial processes, which are oriented vertically and thus perpendicular to the granule cell axons in the cerebella ~°'26'27 (but see also ref. 32). In our microexplant cultures, there was no direct contact between the Bergmann glia and most of the granule cells; still the granule cells oriented at right-angles and migrated up to about 200/zm across their parallel neurite

REFERENCES 1 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. 2 A-Beuret, D. and Matus, A., Changes in the cytoplasmic distribution of microtubule-associated protein 2 during the differentiation of cultured cerebellar granule cells, Neuroscience, 14 (1985) 1103-1115. 3 Cohen, J., Burne, J.F., Winter, J. and Bartlett, P., Retinal ganglion cells lose response to laminin with maturation, Nature (Lond.), 322 (1986) 465-467. 4 Davis, G.E., Varon, S., Engvall, E. and Manthorpe, M., Substratum-binding neurite promoting facotrs: relationship to laminin, Trends Neurosci., 8 (1985) 528-532. 5 Dufour, S., Duband, J.-L., Humphries, M.J., Obara, M., Yamada, K.M. and Thiery, J.P., Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules, EMBO J., 7 (1988)

bundles 22. These results suggest that thert: is an intrinsic nature of granule cells to orient perpendicular to their axon bundles even in the absence of glial cells in vitr~,~, Formation o f cell aggregates The aggregate formation started at later particular stages of culture, during and after the orientation change of granule cells. A continuous labeling study by BrdU confirmed that cells which have finished a BrdU uptake migrated outside explants and then aggregated. Formation of cell aggregates on the PL/laminin substratum may be caused by loss or decrease of the adhesiveness of the cell body to the laminin substratum or by an increase of the cell-to-cell adhesiveness between neurons 3'2°. Hekmat et al.~3 showed that the embryonic type of N - C A M disappeared from the granule cells and neurites at later stages in an in vitro cerebellar microexplant culture. In contrast, L1 continued to be expressed on granule cell bodies even at later stages of our culture. It is, therefore, possible that the postmigratory neurons in vitro adhere to each other with the adult type of N - C A M , LI or other adhesion molecules. Immunocytochemical study using the anti-MAP2 antibody has shown that MAP2 protein localized in cell bodies and their dendrite-like short neurites after cell aggregation. There was little staining on the radially oriented granule cell neurite bundles. Such localization is in agreement with the previous reports that MAP2 is limited around granule cell bodies in the inner granular layer of the cerebellum ~5, and that its localization is restricted to the initial portion of the neurites of granule cells in dissociated cerebellar cultures 2. Acknowledgements. I.N. thanks Dr. Y. Ishihara for his encouragement, and Dr. M. Schachner for giving the opportunity to do related experiments in her laboratory. We are grateful to Dr. M. Schachner for the gift of LI antibody, and Dr. H. Shimazu for critical reading of the manuscript.

2661-2671. 6 Edgar, D., Timpl, R. and Thoenen, H., Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies J. Cell Biol., 106 (1988) 1299-1306. 7 Fischer, G., Cultivation of mouse cerebellar cells in serum-free, hormonally defined media: survival of neurons, Neurosci. Len, 28 (1982) 325-329. 8 Fischer, G., Kiinemund, V. and Schachner, M., Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surfaces glycoprotein L1, J. Neurosci., 6 (1986) 605-612. 9 Gratzner, H.G., Monoclonal antibody to 5"-brom0, and 5iododeoxyuridine: a new reagent for detection of DNA replication, Science, 218 (1982) 474-475. 10 Grovas, A.C. and O'Shea, K.S., An SEM examination of granule cell migration in the mouse cerebellum, J. Neurosci. Res., 12 (1984) 1-14. 11 Gundersen, R.W., Response of sensory neurites and growth

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cones to patterned substrata of laminin and fibronectin in vitro, Dev. Biol., 121 (1987) 423-431. Hatten, M.E., Furie, M.B. and Rifkin, D.B., Binding of developing mouse cerebellar cells to fibronectin: a possible mechanism for the formation of the external granular layer, J. Neurosci., 2 (1982) 1195-1206. Hekmat, A., Kfinemund, V., Fischer, G. and Schachner, M., Small inhibitory cerebellar interneurons grow in a perpendicular orientation to granule cell neurites in culture, Neuron, 2 (1989) 1113-1122. Hopkins, J.M., F-Holevinski, T.S., McCoy, J.P. and Agranoff, B.W., Laminin and optic nerve regeneration in the goldfish, J. Neurosci., 5 (1985) 3030-3038. Huber, G. and Matus, A., Differences in the cellular distribution of two microtubule-associated proteins, MAP1 and MAP2, in rat brain, J. Neurosci., 4 (1984) 151-160. Hynes, R.O., Patel, R. and Miller, R.H., Migration of neuroblasts along preexisting axonal tracts during prenatal cerebellar development, J. Neurosci., 6 (1986) 867-876. Liesi, P., Do neurons in the vertebrate CNS migrate on laminin? E M B O J., 5 (1985) 1163-1170. Liesi, P. and Risteli, L., Glial cells of mammalian brain produce a variant form of laminin, Exp. Neurol., 105 (1989) 86-92. Nagata, I., Movement of small neurons from mouse cerebellar microexplants on extracellular matrix components, Cell Struct. Funct., 12 (1987) 648. Nagata, I. and Schachner, M., Conversion of embryonic to adult form of the neural cell adhesion molecule (N-CAM) does not correlate with pre- and postmigratory states of mouse cerebellar granule neurons, Neurosci. Lett., 63 (1986) 153-158. Nagata, I., Keilhauer, G. and Schachner, M., Neuronal influence on antigenic marker profile, cell shape and proliferation of cultured astrocytes obtained by microdissection of distinct layers from the early postnatal mouse cerebellum, Dev. Brain Res., 24 (1986) 217-232. Nakatsuji, N. and Nagata, I., Paradoxical perpendicular contact

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