- Email: [email protected]
Differentiation of the cerebellar granule cell: Expression of a synaptic vesicle protein and the microtubule-associated protein MAP1A
Developmental Brain Research, 34 (1987) 1-7
1
Elsevier BRD 50564
Research Reports
Differentiation of the cerebellar granule cell: expression of a synaptic vesicle protein and the microtubule-associated protein MAP1A Martin A. Cambray-Deakin, Kathryn-Marie Norman and Robert D. Burgoyne The Physiological Laboratory, University of Liverpool, Liverpool ( U.K.) (Accepted 16 December 1986)
Key words." Synaptic vesicle; Cerebellar granule cell; Microtubule-associated protein; Cytoskeleton; Axon
The expression of the microtubule-associated protein MAPIA and the synaptic vesicle protein p65 by rat cerebellar granule cells developing in vivo and in vitro was examined. MAP1A in common with a series of previously described cytoskeletal proteins was present in immature parallel fibres and began to disappear from parallel fibres around P15. From the earliest age (P6) at which the molecular layer could be identified parallel fibres throughout the molecular layer expressed p65 with no evidence of any gradient of expression from young to mature axons, Granule cells in culture did not express p65 until later than 1 day in vitro. By 7 days in vitro numerous p65 containing varicosities were present on granule cell processes though MAP1A was still expressed. The results show that transport of synaptic vesicles and formation of varicosities is an early event in granule cell differentiation and precedes a major reorganisation of the axonal cystoskeleton.
INTRODUCTION During the first 3 weeks postnatally rat cerebeUar granule cells are produced in the external germinal layer and migrate through the molecular and Purkinje cell layers to reside in the granular layer 1-3. As granule cells migrate, their axons (parallel fibres) remain within the molecular layer and stack on previously formed axons. Thus, there is a gradation of maturation of parallel fibres through the molecular layer during cerebellar development. As Purkinje cell dendrites grow up into the molecular layer the parallel fibres form synapses on the Purkinje cell dendritic spines beginning in the internal, mature, region of the molecular layer at P12 and finishing the external, youngest, region of the molecular layer shortly after P21 (ref. 2). Following parallel fibre formation a number of
changes in both axolemmal components 17,2° and cytoskeletal composition 5,9-11 occurs in a wave through the molecular layer beginning in the oldest region from P10-15 and is complete throughout the molecular layer by P18-20. These developmental changes must be related to aspects of parallel fibre maturation and in particular could be related to the onset of synaptogenesis. One possible explanation for the marked switch in cytoskeletal composition could be that the new cytoskeletal structures are required for transport of synaptic vesicles in the differentiating axons. To examine this possibility we have examined and compared the time course of expression of the microtubule-associated protein M A P 1 A (ref. 6) and a synaptic vesicle protein p65 (mol. wt. = 65,000 Da), recognised by the monoclonal antibody Ab48 (ref. 16), by granule cells developing both in vivo and in monolayer culture.
Correspondence: R.D. Burgoyne, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Brownlow Hill, Liverpool,
L69 3BX, U.K.
0165-3806/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
MATERIALSAND METHODS
Immunocytochemistry Vibratome (50-100/~m) sections were prepared from rat cerebella from postnatal days 4, 6, 9, 11 and 18 (P4-18) and adult animals after fixation of cerebella in 4% formaldehyde in phosphate-buffered saline (PBS) for 2-3 days. The sections were washed in PBS followed by incubation in 0.1% Triton X-100, 0.3% bovine serum albumin in PBS (PBT) for 30 rain. The sections were then incubated overnight at 4 °C with Ab48 (ascites fluid diluted 1:20,000 in PBT) anti-MAP2 or anti-MAP1A (ascites fluid, 1:50,000). The sections were washed in PBT, incubated with anti-mouse biotin (1:100 in PBT; Amersham) for 60 min, washed in PBT and incubated with streptavidin-horseradish peroxidase (1:150 in PBT; Amersham) for 30 min. After washing, the bound peroxidase was visualised with diaminobenzidine. Granule cell-enriched cultures For granule cell-enriched cultures cerebella of 7day-old rats were dissociated by trypsin treatment 13,14, plated at a cell density of 1.4 × 103 per mm 2 on poly-L-lysine coated cover slips and mantained in Eagle's Minimal Essential Medium, 30/~g/ml insulin, 30 nM sodium selenite, 33 mM glucose, 290/~g/ml glutamine, 25 mM KC1, 25 units/ml penicillin, 25 ktg/ml streptomycin. Cultures were fixed at various times with 4% formaldehyde (30 min), permeabilised by incubation in PBT for 30 min and incubated with Ab48 (1:500) or anti-MAP1A (1:500) for 2 h. After washing in PBS, the cultures were incubated with anti-mouse Texas red (Amersham, 1:50) for 45 min. All reagents were diluted in PBT. The coverslips were washed with acid-alcohol at -10 °C and mounted in 0.25% 1,4-diazabicyclo-[2.2.2.]octane (Sigma), 0.002% p-phenylenediamine in glycerol/PBS (9:1). Immunofluorescence was examined using a × 63 oil immersion lens on a Zeiss Universal microscope with the appropriate filters for Texas red fluorescence. Control cultures incubated with nonimmune mouse antiserum were unstained. Antibodies The mouse monoclonal antibody Ab48 which recognises the synaptic vesicle protein p65 has been pre-
viously characterised ~6 and was a gift from Dr. L. Reichardt. Mouse monoclonal antibodies against MAP1A (anti-MAP1A. 1, ref.6) and MAP2 (MAP2.3) were supplied by Amersham. RESULTS
MA P1A The localisation of MAP1A in cerebellar sections was compared to that of MAP2 which is known not to be expressed in parallel fibres at any developmental stage 4'5,7~8. In sections of adult cerebellum antiMAP1A and anti-MAP2 stained Purkinje cell dendrites strongly as well as granule cell bodies; parallel fibres were unstained by anti-MAP1A and antiMAP1A (anti-MAP1A.1, ref. 6) and MAP2 anti-MAP2 which did not stain parallel fibres in sec-
Fig. 1. Vibratome sections of adult rat cerebellum stained with anti-MAP1A (a) of anti-MAP2 (b), Purkinje cell bodies (P) and dendrites in the molecular layer (ML) and granule cell bodies in the granular layer (GL) are stained by both antibodies but with differing intensities. Varicose fibres in the molecular layer, which are probably dendrites of Golgi cells in the granular layer, were stained by anti-MAP2. Parallel fibres between dendrites in the molecular layer are not stained by either antibody. Bar = 25/~m.
younger parallel fibres in the most external region of the molecular layer (Fig. 2b). The processes of granule cells after 8 days in vitro (8 D I V ) were all stained by a n t i - M A P 1 A (Fig. 3). M A P 1 A was still present in granule cell processes in culture even after 18 D I V (not shown).
Synaptic vesicleprotein p65
......
i ii
!ii!i
i!~!i
Ab48 staining of v i b r a t o m e sections from adult cerebellum was that which was expected of an antigen with a synaptic localisation 16. Fine punctate staining in the molecular layer was p r e s u m a b l y due to staining of the presynaptic varicosities of parallel fibres (Fig. 4e). Within the granular layer larger aggregates of staining due to the large mossy fibre terminals within the glomeruli was seen (Fig. 4e). F r o m early states of cerebellar d e v e l o p m e n t punctate staining due to
Fig. 2. Vibratome sections of P8 (a, c) and P15 (b, d) rat cerebellum stained with anti-MAP1A (a, b) or anti-MAP2 (c, d). Purkinje cell bodies (P) and dendrites and granule cell bodies in the granular layer (GL) are stained with both antibodies at both ages. Parallel fibres in the molecular layer (ML) which are cut in cross-section are stained by anti-MAP1A (arrows) but not anti-MAP2. In the section from P15 cerebellum antiMAPIA staining of parallel fibres is most intense in the outer(upper) region of the molecular layer. Cells in the external germinal layer (EGL) are unstained by the antibodies. Bar = 25/~m.
tions from P8 an P15 cerebella (Fig. 2c, d) antiM A P 1 A stained i m m a t u r e parallel fibres (Fig. 2a, b). In P8 cerebellar sections staining was present throughout the molecular layer indicative of staining of parallel fibres. By P15 a gradient of M A P I A expression through the molecular layer was a p p a r e n t with a n t i - M A P 1 A . Staining was most intense in the
Fig. 3. Cerebellar granule cells in culture at 8 DIV stained with anti-MAP1A (b). The distribution of granule cell processes in the culture is demonstrated by the corresponding phase-contrast micrograph (a). All processes in the culture are stained by anti-MAP1A. Flat non-neuronal cells were also stained by antiMAP1A. Bar = 25/~m.
Fig. 4. Vibratome sections of P6(a), P9(b), Pll(c), P18(d) and adult (e) cerebellar staining with Ab48. Cells of the external germinal layer (EGL), Purkinje cells (P) and granule cell bodies of the granular layer (GL) are unstained at all ages. Fine punctate staining is present throughout the molecular layer (ML) at all ages. From P9 until P18 there is a progressive increase in the size of stained aggregates in the granular layer which represent the synaptic endings of mossy fibres in the glomeruli. Bar - 25 !tm.
Ab48 was visible throughout the molecular layer (Fig. 4). This was seen as early as P6 (Fig. 4a). At earlier times examined (e.g. P4) diffuse staining with Ab48 was found in the nascent molecular layer (not shown). From P6 to P18 the size of the stained aggregates in the granular layer increased reflecting the increasing mossy fibre input to the glomeruli within the granular layer. While p65 appeared to be expressed in parallel fibres from early stages, granule cell bodies in either the external germinal layer or granular layer were unstained. In many cases Ab48 staining near the upper region of the molecular layer extended into the external germinal layer between the cell bodies of the premigratory granule cells (Fig. 4b), indicating the presence of synaptic vesicles in the early axonal outgrowths of the granule cells. In monolayer cultures enriched in granule cells no
specific staining with Ab48 could be detected up to 1 DIV despite the growth of processes (not shown), By 4 DIV marked Ab48 staining was present around cell bodies and in varicosities along granule cell process (Fig. 5a, b). By 7 DIV Ab48 staining had increased in intensity and the processes, many of which had now formed into bundles, all had punctate staining distributed along their length (Fig. 5c, d), a pattern that persisted during longer periods in culture ~2. DISCUSSION The finding of a transient expression of MAPIA in parallel fibres during the first two weeks postnatally is consistent with a previous study using a monoconal antibody which recognised uncharacterised MAP1 polypeptides 5. In the latter study, however, some
Fig. 5. Cerebellar granule cells in culture at 4 DIV (a, b) and 7 DIV (b, c) stained with Ab48 (b, d). Varicositiesstained by Ab48 (arrows) are visible in both 4 DIV and 7 DIV cultures. Flat, non-neuronal cellswere not stained by Ab48. Correspondingphase-contrast micrographs are shown in a, b. Bar = 25~m.
staining of parallel fibres persisted even in adult cerebella. It is not clear, therefore, whether or not the same polypeptide was recognised by the antibodies used in the present and the previous study. The time course of loss of M A P 1 A from parallel fibres was essentially the same as that reported for other developmental changes of cytoskeletal proteins in parallel fibres which includes loss of the 200-kDa neurofilament polypeptide t° and two microtubule-associated proteins M A P I ( x ) 9 and MAP35 and detyrosylation of parallel fibre a-tubulin 1~. It appears then that the cytoskeleton of the granule cell axon undergoes a marked alteration in composition between P10 and P20. In granule cells maintained in monolayer culture the switch in cytoskeletal composition is either delayed or absent. The loss of the 200-kDa neurofilament polypeptide (ref. 10 and our unpublished observations) and M A P I ( x ) 9 and a-tubulin detyrosylation 12 did not occur in granule cell processes even after 10 days in culture. A similar situation was found for M A P 1 A which persisted in granule cell process until at least 18 days in culture. It is clear from the results with Ab48 that the synaptic vesicle protein p65 is expressed within parallel fibres at early stages of granule cell development. Even at P6 parallel fibres throughout the molecular layer were stained by Ab48 and at no developmental stage was any indication of a gradation of immunostaining from the lower (oldest) to the upper (youngest) molecular layer seen. After dissociation from P7 cerebellum, granule
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 Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of molecular layer, J. Cornp. Neurol., 145 (1972) 399-464. 3 Altman, J. Morphological development of the rat cerebellum and some of its mechanisms, Exp. Brain Res., Suppl. 6 (1982) 8-46. 4 Bernhardt, R. and Matus, A., Initial phase of dendritic growth: evidence for the involvement of high molecular weight microtubule-associated proteins (HMWP) before the appearance of tubulin, J. Cell Biol., 92 (1982) 589-593. 5 Bernhardt, R., Huber, G. and Matus, A. Differences in the
cells in culture only begin to transport synaptic vesicles containing p65 out into their processes after 1 day in culture and the intensity of Ab48 staining increased between 4 and 7 days in vitro. It would appear that, like cytoskeletal and axolemma117-19 developmental changes, expression of p65-containing synaptic vesicles is delayed in culture. The punctate staining with Ab48 on processes in culture most probably represents staining of synaptic vesicle-filled varicosities which have been seen by electron microscopy15.18. The present results demonstrate, in agreement with previous studies on the developmental appearance of synaptic vesicles in parallel fibres in vivo ~'2 that granule cells in vivo and in vitro transport synaptic vesicles along their axons well before the developmental modifications in cytoskeletal proteins. Therefore, the developmental switch in axonal cytoskeletal composition is not required for the transport of synaptic vesicles and formation of presynaptic varicosities and must be related to some late events in granule cell differentiation. Whether the cytoskeletal reorganisation is related to the formation of postsynaptic contacts on parallel fibres, which occurs much later (from P12) than the formation of presynaptic structures, remains to be determined. ACKNOWLEDGEMENTS We wish to thank Dr. L.F. Reichardt for the gift of Ab48. This work was supported by a project grant to R.D.B. from The M.R.C.
developmental patterns of three microtubule-associated proteins in the rat cerebellum, J. Neurosci., 5 (1985) 977-991. 6 Bloom, G.S., Schoenfeld, T.A. and Vallee, R.B., Widespread distribution of the major polypeptide component of MAP1 (microtubule-associated protein 1) in the nervous system, J. Cell Biol., 98 (1984) 320-330. 7 Burgoyne, R.D., Microtubule proteins in neuronal differentiation, Comp. Biochem. Physiol., 83 (1986) 1-8. 8 Burgoyne, R.D. and Cumming, R., Ontogeny of microtubule-associated protein 2 in rat cerebellum: differential expression of the doublet polypeptides, Neuroscience, 11 (1984) 157-167. 9 Calvert, R. and Anderton, B.H., A microtubule-associated protein (MAP1) which is expressed at elevated levels during development of the rat cerebellum, EMBO J., 4 (1985) 1171-1176.
10 Cambray-Deakin, M.A. and Burgoyne, R.D., Transient expression of neurofilament-like (RT 97) immunoreactivity in cerebellar granule cells, Dev. Brain Res., 28 (1986) 282-286. 11 Cumming, R., Burgoyne, R.D. and Lytton, N.A., Immunocytochemical demonstration of ct-tubulin modification during axonal maturation in the cerebellar cortex, J. Cell Biol., 98 (1984) 347-351. 12 Cumming, R., Burgoyne, R.D. and Lytton, N.A., Immunofluorescent distribution of a-tubulin, fl-tubulin and microtubule-associated protein 2 during in vitro maturation of cerebellar granule cell neurons, Neuroscience, 12 (1984) 775-782. 13 Currie, D.N., Dutton, G.R. and Cohen, J., Monolayer culture of perikarya isolated from postnatal rat cerebellum, Experientia, 35 (1979) 345-347. 14 Dutton, G.R., Currie, D.N. and Tear, K., An improved method for the bulk isolation of viable perikarya from postnatal cerebellum, J. Neurosci. Methods, 3 (1981)421-427. 15 Kingsbury, A.E., Gallo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985)
17-25. 16 Matthew, W.D., Tsavaler, L. and Reichardt, L.F., Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue, J. Cell Biol., 91 (1981) 257-269. 17 Pigott, R. and Kelly, L.S., Immonucytochemical and biochemical studies with the monoclonal antibody 69 A 1: similarities of the antigen with cell adhesion molecules L1, NILE and Ng-CAM, Dev. Brain Res., 2 (1986) 111-122. 18 Trenkner, E. and Sidman, R.L., Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation, J. Cell Biol., 75 (1977) 915-940. 19 Webb, M., Gallo, V., Schneider, A. and Balazs, R., The expression of concanavalin A binding glycoproteins during the development of cerebellar granule neurons in vitro, Int. J. Dev. Neurosci., 3 (1985) 199-208. 20 Zanetta, J.-P., Roussel, G., Ghandour, M.S., Vincendon, G. and Gombos, G., Postnatal development of rat cerebellum: massive and transient accumulation of concanavalin A binding glycoproteins in parallel fibre axolemma, Brain Research, 142 (1978) 301-319.
1
Elsevier BRD 50564
Research Reports
Differentiation of the cerebellar granule cell: expression of a synaptic vesicle protein and the microtubule-associated protein MAP1A Martin A. Cambray-Deakin, Kathryn-Marie Norman and Robert D. Burgoyne The Physiological Laboratory, University of Liverpool, Liverpool ( U.K.) (Accepted 16 December 1986)
Key words." Synaptic vesicle; Cerebellar granule cell; Microtubule-associated protein; Cytoskeleton; Axon
The expression of the microtubule-associated protein MAPIA and the synaptic vesicle protein p65 by rat cerebellar granule cells developing in vivo and in vitro was examined. MAP1A in common with a series of previously described cytoskeletal proteins was present in immature parallel fibres and began to disappear from parallel fibres around P15. From the earliest age (P6) at which the molecular layer could be identified parallel fibres throughout the molecular layer expressed p65 with no evidence of any gradient of expression from young to mature axons, Granule cells in culture did not express p65 until later than 1 day in vitro. By 7 days in vitro numerous p65 containing varicosities were present on granule cell processes though MAP1A was still expressed. The results show that transport of synaptic vesicles and formation of varicosities is an early event in granule cell differentiation and precedes a major reorganisation of the axonal cystoskeleton.
INTRODUCTION During the first 3 weeks postnatally rat cerebeUar granule cells are produced in the external germinal layer and migrate through the molecular and Purkinje cell layers to reside in the granular layer 1-3. As granule cells migrate, their axons (parallel fibres) remain within the molecular layer and stack on previously formed axons. Thus, there is a gradation of maturation of parallel fibres through the molecular layer during cerebellar development. As Purkinje cell dendrites grow up into the molecular layer the parallel fibres form synapses on the Purkinje cell dendritic spines beginning in the internal, mature, region of the molecular layer at P12 and finishing the external, youngest, region of the molecular layer shortly after P21 (ref. 2). Following parallel fibre formation a number of
changes in both axolemmal components 17,2° and cytoskeletal composition 5,9-11 occurs in a wave through the molecular layer beginning in the oldest region from P10-15 and is complete throughout the molecular layer by P18-20. These developmental changes must be related to aspects of parallel fibre maturation and in particular could be related to the onset of synaptogenesis. One possible explanation for the marked switch in cytoskeletal composition could be that the new cytoskeletal structures are required for transport of synaptic vesicles in the differentiating axons. To examine this possibility we have examined and compared the time course of expression of the microtubule-associated protein M A P 1 A (ref. 6) and a synaptic vesicle protein p65 (mol. wt. = 65,000 Da), recognised by the monoclonal antibody Ab48 (ref. 16), by granule cells developing both in vivo and in monolayer culture.
Correspondence: R.D. Burgoyne, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Brownlow Hill, Liverpool,
L69 3BX, U.K.
0165-3806/87/$03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
MATERIALSAND METHODS
Immunocytochemistry Vibratome (50-100/~m) sections were prepared from rat cerebella from postnatal days 4, 6, 9, 11 and 18 (P4-18) and adult animals after fixation of cerebella in 4% formaldehyde in phosphate-buffered saline (PBS) for 2-3 days. The sections were washed in PBS followed by incubation in 0.1% Triton X-100, 0.3% bovine serum albumin in PBS (PBT) for 30 rain. The sections were then incubated overnight at 4 °C with Ab48 (ascites fluid diluted 1:20,000 in PBT) anti-MAP2 or anti-MAP1A (ascites fluid, 1:50,000). The sections were washed in PBT, incubated with anti-mouse biotin (1:100 in PBT; Amersham) for 60 min, washed in PBT and incubated with streptavidin-horseradish peroxidase (1:150 in PBT; Amersham) for 30 min. After washing, the bound peroxidase was visualised with diaminobenzidine. Granule cell-enriched cultures For granule cell-enriched cultures cerebella of 7day-old rats were dissociated by trypsin treatment 13,14, plated at a cell density of 1.4 × 103 per mm 2 on poly-L-lysine coated cover slips and mantained in Eagle's Minimal Essential Medium, 30/~g/ml insulin, 30 nM sodium selenite, 33 mM glucose, 290/~g/ml glutamine, 25 mM KC1, 25 units/ml penicillin, 25 ktg/ml streptomycin. Cultures were fixed at various times with 4% formaldehyde (30 min), permeabilised by incubation in PBT for 30 min and incubated with Ab48 (1:500) or anti-MAP1A (1:500) for 2 h. After washing in PBS, the cultures were incubated with anti-mouse Texas red (Amersham, 1:50) for 45 min. All reagents were diluted in PBT. The coverslips were washed with acid-alcohol at -10 °C and mounted in 0.25% 1,4-diazabicyclo-[2.2.2.]octane (Sigma), 0.002% p-phenylenediamine in glycerol/PBS (9:1). Immunofluorescence was examined using a × 63 oil immersion lens on a Zeiss Universal microscope with the appropriate filters for Texas red fluorescence. Control cultures incubated with nonimmune mouse antiserum were unstained. Antibodies The mouse monoclonal antibody Ab48 which recognises the synaptic vesicle protein p65 has been pre-
viously characterised ~6 and was a gift from Dr. L. Reichardt. Mouse monoclonal antibodies against MAP1A (anti-MAP1A. 1, ref.6) and MAP2 (MAP2.3) were supplied by Amersham. RESULTS
MA P1A The localisation of MAP1A in cerebellar sections was compared to that of MAP2 which is known not to be expressed in parallel fibres at any developmental stage 4'5,7~8. In sections of adult cerebellum antiMAP1A and anti-MAP2 stained Purkinje cell dendrites strongly as well as granule cell bodies; parallel fibres were unstained by anti-MAP1A and antiMAP1A (anti-MAP1A.1, ref. 6) and MAP2 anti-MAP2 which did not stain parallel fibres in sec-
Fig. 1. Vibratome sections of adult rat cerebellum stained with anti-MAP1A (a) of anti-MAP2 (b), Purkinje cell bodies (P) and dendrites in the molecular layer (ML) and granule cell bodies in the granular layer (GL) are stained by both antibodies but with differing intensities. Varicose fibres in the molecular layer, which are probably dendrites of Golgi cells in the granular layer, were stained by anti-MAP2. Parallel fibres between dendrites in the molecular layer are not stained by either antibody. Bar = 25/~m.
younger parallel fibres in the most external region of the molecular layer (Fig. 2b). The processes of granule cells after 8 days in vitro (8 D I V ) were all stained by a n t i - M A P 1 A (Fig. 3). M A P 1 A was still present in granule cell processes in culture even after 18 D I V (not shown).
Synaptic vesicleprotein p65
......
i ii
!ii!i
i!~!i
Ab48 staining of v i b r a t o m e sections from adult cerebellum was that which was expected of an antigen with a synaptic localisation 16. Fine punctate staining in the molecular layer was p r e s u m a b l y due to staining of the presynaptic varicosities of parallel fibres (Fig. 4e). Within the granular layer larger aggregates of staining due to the large mossy fibre terminals within the glomeruli was seen (Fig. 4e). F r o m early states of cerebellar d e v e l o p m e n t punctate staining due to
Fig. 2. Vibratome sections of P8 (a, c) and P15 (b, d) rat cerebellum stained with anti-MAP1A (a, b) or anti-MAP2 (c, d). Purkinje cell bodies (P) and dendrites and granule cell bodies in the granular layer (GL) are stained with both antibodies at both ages. Parallel fibres in the molecular layer (ML) which are cut in cross-section are stained by anti-MAP1A (arrows) but not anti-MAP2. In the section from P15 cerebellum antiMAPIA staining of parallel fibres is most intense in the outer(upper) region of the molecular layer. Cells in the external germinal layer (EGL) are unstained by the antibodies. Bar = 25/~m.
tions from P8 an P15 cerebella (Fig. 2c, d) antiM A P 1 A stained i m m a t u r e parallel fibres (Fig. 2a, b). In P8 cerebellar sections staining was present throughout the molecular layer indicative of staining of parallel fibres. By P15 a gradient of M A P I A expression through the molecular layer was a p p a r e n t with a n t i - M A P 1 A . Staining was most intense in the
Fig. 3. Cerebellar granule cells in culture at 8 DIV stained with anti-MAP1A (b). The distribution of granule cell processes in the culture is demonstrated by the corresponding phase-contrast micrograph (a). All processes in the culture are stained by anti-MAP1A. Flat non-neuronal cells were also stained by antiMAP1A. Bar = 25/~m.
Fig. 4. Vibratome sections of P6(a), P9(b), Pll(c), P18(d) and adult (e) cerebellar staining with Ab48. Cells of the external germinal layer (EGL), Purkinje cells (P) and granule cell bodies of the granular layer (GL) are unstained at all ages. Fine punctate staining is present throughout the molecular layer (ML) at all ages. From P9 until P18 there is a progressive increase in the size of stained aggregates in the granular layer which represent the synaptic endings of mossy fibres in the glomeruli. Bar - 25 !tm.
Ab48 was visible throughout the molecular layer (Fig. 4). This was seen as early as P6 (Fig. 4a). At earlier times examined (e.g. P4) diffuse staining with Ab48 was found in the nascent molecular layer (not shown). From P6 to P18 the size of the stained aggregates in the granular layer increased reflecting the increasing mossy fibre input to the glomeruli within the granular layer. While p65 appeared to be expressed in parallel fibres from early stages, granule cell bodies in either the external germinal layer or granular layer were unstained. In many cases Ab48 staining near the upper region of the molecular layer extended into the external germinal layer between the cell bodies of the premigratory granule cells (Fig. 4b), indicating the presence of synaptic vesicles in the early axonal outgrowths of the granule cells. In monolayer cultures enriched in granule cells no
specific staining with Ab48 could be detected up to 1 DIV despite the growth of processes (not shown), By 4 DIV marked Ab48 staining was present around cell bodies and in varicosities along granule cell process (Fig. 5a, b). By 7 DIV Ab48 staining had increased in intensity and the processes, many of which had now formed into bundles, all had punctate staining distributed along their length (Fig. 5c, d), a pattern that persisted during longer periods in culture ~2. DISCUSSION The finding of a transient expression of MAPIA in parallel fibres during the first two weeks postnatally is consistent with a previous study using a monoconal antibody which recognised uncharacterised MAP1 polypeptides 5. In the latter study, however, some
Fig. 5. Cerebellar granule cells in culture at 4 DIV (a, b) and 7 DIV (b, c) stained with Ab48 (b, d). Varicositiesstained by Ab48 (arrows) are visible in both 4 DIV and 7 DIV cultures. Flat, non-neuronal cellswere not stained by Ab48. Correspondingphase-contrast micrographs are shown in a, b. Bar = 25~m.
staining of parallel fibres persisted even in adult cerebella. It is not clear, therefore, whether or not the same polypeptide was recognised by the antibodies used in the present and the previous study. The time course of loss of M A P 1 A from parallel fibres was essentially the same as that reported for other developmental changes of cytoskeletal proteins in parallel fibres which includes loss of the 200-kDa neurofilament polypeptide t° and two microtubule-associated proteins M A P I ( x ) 9 and MAP35 and detyrosylation of parallel fibre a-tubulin 1~. It appears then that the cytoskeleton of the granule cell axon undergoes a marked alteration in composition between P10 and P20. In granule cells maintained in monolayer culture the switch in cytoskeletal composition is either delayed or absent. The loss of the 200-kDa neurofilament polypeptide (ref. 10 and our unpublished observations) and M A P I ( x ) 9 and a-tubulin detyrosylation 12 did not occur in granule cell processes even after 10 days in culture. A similar situation was found for M A P 1 A which persisted in granule cell process until at least 18 days in culture. It is clear from the results with Ab48 that the synaptic vesicle protein p65 is expressed within parallel fibres at early stages of granule cell development. Even at P6 parallel fibres throughout the molecular layer were stained by Ab48 and at no developmental stage was any indication of a gradation of immunostaining from the lower (oldest) to the upper (youngest) molecular layer seen. After dissociation from P7 cerebellum, granule
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 Altman, J., Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of molecular layer, J. Cornp. Neurol., 145 (1972) 399-464. 3 Altman, J. Morphological development of the rat cerebellum and some of its mechanisms, Exp. Brain Res., Suppl. 6 (1982) 8-46. 4 Bernhardt, R. and Matus, A., Initial phase of dendritic growth: evidence for the involvement of high molecular weight microtubule-associated proteins (HMWP) before the appearance of tubulin, J. Cell Biol., 92 (1982) 589-593. 5 Bernhardt, R., Huber, G. and Matus, A. Differences in the
cells in culture only begin to transport synaptic vesicles containing p65 out into their processes after 1 day in culture and the intensity of Ab48 staining increased between 4 and 7 days in vitro. It would appear that, like cytoskeletal and axolemma117-19 developmental changes, expression of p65-containing synaptic vesicles is delayed in culture. The punctate staining with Ab48 on processes in culture most probably represents staining of synaptic vesicle-filled varicosities which have been seen by electron microscopy15.18. The present results demonstrate, in agreement with previous studies on the developmental appearance of synaptic vesicles in parallel fibres in vivo ~'2 that granule cells in vivo and in vitro transport synaptic vesicles along their axons well before the developmental modifications in cytoskeletal proteins. Therefore, the developmental switch in axonal cytoskeletal composition is not required for the transport of synaptic vesicles and formation of presynaptic varicosities and must be related to some late events in granule cell differentiation. Whether the cytoskeletal reorganisation is related to the formation of postsynaptic contacts on parallel fibres, which occurs much later (from P12) than the formation of presynaptic structures, remains to be determined. ACKNOWLEDGEMENTS We wish to thank Dr. L.F. Reichardt for the gift of Ab48. This work was supported by a project grant to R.D.B. from The M.R.C.
developmental patterns of three microtubule-associated proteins in the rat cerebellum, J. Neurosci., 5 (1985) 977-991. 6 Bloom, G.S., Schoenfeld, T.A. and Vallee, R.B., Widespread distribution of the major polypeptide component of MAP1 (microtubule-associated protein 1) in the nervous system, J. Cell Biol., 98 (1984) 320-330. 7 Burgoyne, R.D., Microtubule proteins in neuronal differentiation, Comp. Biochem. Physiol., 83 (1986) 1-8. 8 Burgoyne, R.D. and Cumming, R., Ontogeny of microtubule-associated protein 2 in rat cerebellum: differential expression of the doublet polypeptides, Neuroscience, 11 (1984) 157-167. 9 Calvert, R. and Anderton, B.H., A microtubule-associated protein (MAP1) which is expressed at elevated levels during development of the rat cerebellum, EMBO J., 4 (1985) 1171-1176.
10 Cambray-Deakin, M.A. and Burgoyne, R.D., Transient expression of neurofilament-like (RT 97) immunoreactivity in cerebellar granule cells, Dev. Brain Res., 28 (1986) 282-286. 11 Cumming, R., Burgoyne, R.D. and Lytton, N.A., Immunocytochemical demonstration of ct-tubulin modification during axonal maturation in the cerebellar cortex, J. Cell Biol., 98 (1984) 347-351. 12 Cumming, R., Burgoyne, R.D. and Lytton, N.A., Immunofluorescent distribution of a-tubulin, fl-tubulin and microtubule-associated protein 2 during in vitro maturation of cerebellar granule cell neurons, Neuroscience, 12 (1984) 775-782. 13 Currie, D.N., Dutton, G.R. and Cohen, J., Monolayer culture of perikarya isolated from postnatal rat cerebellum, Experientia, 35 (1979) 345-347. 14 Dutton, G.R., Currie, D.N. and Tear, K., An improved method for the bulk isolation of viable perikarya from postnatal cerebellum, J. Neurosci. Methods, 3 (1981)421-427. 15 Kingsbury, A.E., Gallo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985)
17-25. 16 Matthew, W.D., Tsavaler, L. and Reichardt, L.F., Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue, J. Cell Biol., 91 (1981) 257-269. 17 Pigott, R. and Kelly, L.S., Immonucytochemical and biochemical studies with the monoclonal antibody 69 A 1: similarities of the antigen with cell adhesion molecules L1, NILE and Ng-CAM, Dev. Brain Res., 2 (1986) 111-122. 18 Trenkner, E. and Sidman, R.L., Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation, J. Cell Biol., 75 (1977) 915-940. 19 Webb, M., Gallo, V., Schneider, A. and Balazs, R., The expression of concanavalin A binding glycoproteins during the development of cerebellar granule neurons in vitro, Int. J. Dev. Neurosci., 3 (1985) 199-208. 20 Zanetta, J.-P., Roussel, G., Ghandour, M.S., Vincendon, G. and Gombos, G., Postnatal development of rat cerebellum: massive and transient accumulation of concanavalin A binding glycoproteins in parallel fibre axolemma, Brain Research, 142 (1978) 301-319.