Arguments in favour of endocytosis of glycoprotein components of the membranes of parallel fibers by Purkinje cells during the development of the rat cerebellum

Developmental Brain Research, 10 (1983) 287-299 287 Elsevier

Arguments in Favour of Endocytosis of Glycoprotein Components of the Membranes of Parallel Fibers by Purkinje Cells During the Development of the Rat Cerebellum

M. DONTENWlLL, G. DEVILLIERS, O. K. LANGLEY, G. ROUSSEL, P. HUBERT, A. REEBER, G. VINCENDON and J. P. ZANETI'A

Centre de Neurochimie du CNRS, 5 rue Blaise Pascal, 67084 Strasbourg Cedex (France)

(Accepted June 14th, 1983)

Key words: cerebellum - - synaptogenesis - - degradation - - chloroquine - - endocytosis

Chloroquine (a drug known to induce a dysfunction of lysosomes) was used to study the behavior of Concanavalin A binding glycoproteins located on the axolemma of parallel fibers in young rat cerebella, and abundant on these membranes at a period preceding synaptogenesis with the dendrites of Purkinje cells. Chloroquine induces in Purkinje ceils a large accumulation of grains consisting of membrane whorls in lysosomes. These grains stain for Concanavalin A, and do not stain either for a mitochondrial marker (aspartate aminotransferase mitochondrial isoenzyme) or for a marker of the Purkinje cell internal membrane (PSG). It is suggested that the material accumulating in the Purkinje cells under the effect of chloroquine comes from the parallel fibers. Together with the observation that a-D-mannosidase (involved in the degradation of these glycoproteins) is exclusively located inside Purkinje cells, these results provide a firm indication that this material enters the Purkinje cells through pinocytosis. The absence of ATPase activity (ATP- ase is a glycoprotein plasma membrane marker highly concentrated on parallel fibers) within these grains suggested that not all the components of these membranes are pinocytosed, but that the process is specific for certain molecules.

These results are compatible with the ultrastructural observations of others, and support the arguments in favour of the pinocytosis phenomenon being one of the first steps of synapse formation. The observed specificity of pinocytosis for certain molecules suggests that a receptor-mediated recognition of some glycans of glycoproteins is the preliminary event in the establishment of synapses.

INTRODUCTION

The search for mechanisms concerned with cell

recognit ion and synapse format ion during the onto-

genesis of the central nervous system is fundamental

in neurobiology. These studies are part icularly diffi-

cult due to the ext reme he terogenei ty of most of the

parts of the central nervous system. A n exception is

the mammal ian cerebel lum where only a few neuro-

nal types are present in the adult with s te reotyped

circuits and connectionslO,22,29, 31. Fur the rmore , one

neuronal cell type (the granule cell) is by far p redom-

inant and in the rat cerebel lum granule cells repre-

sent about 90% of the total cell populationS,8. Since

the deve lopment of the rat cerebel lum is largely post-

natal2-5,8, 37,it is possible within a short per iod to study

the various steps of the ontogenesis (i.e. cell multipli-

cation and migrat ion, synapse formation) . One other

advantage of the cerebel lum is that synapses involv-

ing the axons of granule cells (i.e. the paral lel fibers)

located in the cerebel lar molecular layer are predom-

inant. The postsynapt ic cells innervated by these ax-

ons are stellate cells, basket cells, dendri tes of Golgi

neurons, but mostly the Purkinje cells and their dendrites in the molecular layer2-5,S,lO,22,29,31.

An initial observat ion repor ted that the membrane

of newly formed paral le l fibers was rich in a specific

class of glycoproteins that binds to Concanavalin A 49

and which d isappeared in the per iod when synapto-

genesis was taking place in the cerebel lar molecular

layer2-4. Fur the rmore , the study of the lifetime of the

glycan part of these glycoproteins 32 indicated that at

least the mannose-r ich glycans of these glycoproteins

were degraded. A concomitant and transient in-

crease of a-D-mannosidase activity was also de-

tected 50 during the same per iod suggesting a close

correlation. These studies were confirmed by im-

munohistochemical localization of a-D-mannosidase 51,52 in the molecular layer, but surprisingly52,

this enzyme was located exclusively in the postsynap-

tic cells, and there was no evidence for the release of

enzyme into the extraceilular space (i.e. close to the

mannose-rich glycoprotein of the parallel f iber axo-

lemma). The only possible explanat ion of the degra-

288

dation of the glycans of these glycoproteins was the postulation that these components penetrate into the Purkinje cell (possible by a pinocytotic process).

In fact, such pinocytotic figures have been observed previously1, 28 at the electron microscopic lev-

el during the period of large-scale synaptognesis in the molecular layer of the cerebellum. Counting of the pinocytotic figures during development 28 indi-

cated a close correlation with synapse formation. La- beling of the extracellular space with electron-dense

material has demonstrated that material adhering to the external face of Purkinje cell is pinocytosed and degraded within these cells 28.

Due to the great quantity of parallel fiber glycoprotein to be degraded in the Purkinje cell during a

short period, a blockage of lysosomal function would be expected to induce a drastic accumulation of par-

allel fiber material in these cells. This paper reports the results of the action of chloroquine (which is known to impair lysosomal function) 12.21.23,26,36,38, 39,42,44,45 and discusses the possible mechanisms of

synapse formation in the molecular layer of the rat

cerebellum.

MATERIALS AND METHODS

Chemicals Chloroquine diphosphate salt, adenosine triphos-

phate disodium salt, ouabain, Concavanalin A (Con A) and horseradish peroxidase (HRP) were supplied by Sigma (St. Louis, MO, U.S.A.), horseradish peroxidase labeled sheep antirabbit immunoglobulins by

Institut Pasteur Production (Paris, France), diamino- benzidine tetrahydrochloride by Aldrich Europe (Beerse, Belgium) and glutaraldehyde for electron microscopy by Fluka (Buchs, Switzerland).

Animal treatment Unless otherwise specified, rats (Wistar albinos)

were fed from the seventh to the fourteenth postnatal day with a daily dose of 1 mg chloroquine (CQ) in 25 /A water, using a plastic pipette. Control rats were

treated in the same way with water only.

Histochemical methods Rats were anesthetized with ether and perfused in-

tracardially under constant pressure with a 4% solution of paraformaldehyde in phosphate buffer saline

(PBS) for 30 min at room temperature. After removal of the cerebellum, slices (100/xm thick) were cut either with a Sorvall TC2 sectionner after embedding in agarose 34,49 or with a model 0 Oxford Vibratome.

The slices were rinsed for at least 6 h in PBS. Subse- quent operations were performed at 4 °C unless otherwise specified.

Concanavalin A-horseradish peroxidase staining Slices were incubated for 1 h in a 100/~g/ml solution

of metallized Con A in PBS and rinsed for 6 h. The slices were then incubated in a 100 #g/ml solution of HRP in PBS, rinsed as above and revealed by the di-

aminobenzidine method ~3.

Na +, K +-dependent, ouabain-inhibited A TPase The histochemical method was adapted from that

proposed by Lewis 20 with the difference that MgSO 4 was replaced by MgCI 2. Adequate concentrations of Na ÷ (80 mM) and K ÷ (30 mM) were used as indicated 20. The slices were rinsed 5 times in water and incubated for 1 h at 37 °C in Tris-maleate buffer 20

containing or not containing 1 mM ouabain. After several rinses with cold water, the slices were incubated in a 1/1000 solution of ammonium sulphide for

5 rain then rinsed in PBS.

Immunohistochemistry Three antisera of different specificities were used

in this study. One was raised against a Purkinje cell specific glycoprotein subunit (PGS) as previously described 19,34 and was used as a marker of the intracel-

lular non-mitochondrial membranes of the Purkinje cell 34. The second was directed against aspartate aminotransferase mitochondrial isoenzyme (m- AAT) from liver mitochondria 16 and was used as a marker of mitochondrial material. The third was directed against rat brain a-D-mannosidase 5~, an enzyme involved in the degradation of the glycans of glycoproteins. The techniques were essentially the same as those previously described 39. Blanks were performed using normal rabbit serum instead of the specific antiserum. Slices were examined with an Or- thoplan Leitz microscope.

Electron microscopy After examination, the tissue slices were postfixed

in osmium tetroxide (1% in 0.1 M sodium phosphate

289

Fig. 1. Electron micrographs of the effect of chioroquine in Purkinje cells of cerebella of 14-day-old rats. A: magnification x 9800. Nu- merous non-functional lysosomes (arrowheads) are present in the cell body and dendrites. B: magnification x 31,000. The non-functional lysosome at the right edge clearly shows membraneous material surrounding granular material typical for lysosomes. Note the presence of 4 multivesicular bodies.

290

buffer, pH 7.4), further dehydrated in ascending eth- anol concentration and embedded in Spurr resin. Ul- trathin sections were cut tangentially to the surface of

the slices and observed in a Philips EM 300 electron microscope with or without staining in uranyl acetate and lead citrate.

RESULTS

General effect of chloroquine on animals The daily dose of chloroquine used in this study (1

mg) was chosen in order to avoid considerable dam- age to the animals. Severe alterations observed by others TM (loss of hair, reduction of body weight) oc-

curred at doses of 10-20 mg/day. Under our experimental conditions, these effects of chloroquine were not discernible.

Morphological effects of chloroquine in the rat cerebellum

The main morphological effect of CQ observed at

the optical microscope level is the presence of bire- fringent grains, darkly stained with toluidine blue in some cells of the rat cerebellum. These grains are particularly concentrated in the Purkinje cell body and dendrites, and some of them are found in the basket and Golgi neurons. Deep nuclei neurons as well as granule cells and glial cells are virtually devoid of these structures.

At the level of electron microscopy, these grains appear to be non-functional lysosomes which accu- mulate membranes (Figs. 1A and B). This conclu- sion is based on the observation that together with membrane whorls, these structures consist in part of electron-dense granular material identical to that found in normal lysosomes. This granular material is found in the center or at the border of these structures. These grains which have been described differ-

ently in the literature as 'myelin-like bodies', 'autophagic bodies' and 'membranous cytoplasmic bodies', appeared in this study, with well preserved morphology to be 'non-functional lysosomes'.

These non-functional lysosomes are very often sit- uated in the vicinity of multivesicular bodies (Fig. 1B) which seem to be much more abundant than in untreated rats.

Characterization of" the membraneous material found in non-functional lysosomes

In order to further characterize the protein constit- uents of the membraneous material found in these

non-functional lysosomes, and thus the structure from which it originates, affinity and immunohistochemistry were performed with specific ligands. We have used 4 different markers: (i) Concanavalin A

associated to horseradish peroxidase for labeling glycans with terminal mannose and/or glucose residues; (ii) antibodies directed against a-D-mannosidase (an enzyme concentrated in Purkinje cells)52; (iii) anti-

bodies against PSG (Purkinje cell specific glycoprotein subunit)19, 34, which is a very minor Con A-binding

glycoprotein from the rat cerebellum present only in Purkinje cells in the cerebellum and localized on the intracellular membranes of these cellslg; (iv) antibodies against aspartate-aminotransferase (m-AAT) isolated from rat liver mitochondriaF6.

Staining with Con A-HRP As previously described 49, Concanavalin A binds in-

tensively to the membranes of newly formed parallel fibers. This binding is responsible for the dark staining by the Con A-HRP method of the molecular layer in young untreated rats (Fig. 2A). The dendrites and some of the Purkinje cells appeared to be virtually devoid of label and are clearly depicted as pale structures (Fig. 2A). This is not the case in rats treated with chloroquine. Purkinje cell perikarya and dendrites are strongly stained (Fig. 2B) and the staining is punctate (Fig. 2D). At low magnification (Fig. 2B), the staining seems to demonstrate a pref- erential accumulation of the Con A-positive grains in Purkinje cells compared with other structures of the cerebellum.

At the electron microscopical level (Fig. 3A), the staining in Purkinje cells is essentially localized on the membranes found in these grains. The lysosomal part is not labeled, nor apparently is the membrane surrounding these structures. The label in these structures is more intense by far than that of the endoplasmic reticulum of the Purkinje cell (Fig. 3A).

Immunohistochemistry using anti-PSG As shown in Figs. 4A and C, anti-PSG labeled the

whole Purkinje cell membrane of untreated rats. However, in rats treated with the drug, the grains

291

Fig. 2. Optical microscopy of cerebellar section~ of 14-day-old rats treated (B, D, F) or not (A, C, E) with chloroquine and stained with Con A-HRP (A, B, C, D) or Na ÷ + K ÷ ATPase (E, F). The bars represent 100/~m in A and B and 50/~m in C-F. In B and D, note the strong staining of Purkinje cell bodies and dendrites in experimental rats compared to controls (A and C). E and F: the staining for AT- Pase is strong in the molecular layer, whereas the Purkinje cell bodies are unstained both in control (E) and experimental (F) rats. Ab- breviations: M, molecular layer; P, purkinje cells; G, granular layer.

292

corresponding to the non-functional lysosomes ap-

peared as white spots inside the Purkinje cell per ika-

ryon and dendri tes (Fig. 4D). As shown in Fig. 3B,

this in terpre ta t ion is verified at the electron micro-

scopical level, since the picture obta ined with anti-

PSG is exactly the reverse of that obta ined for Con-

canavalin A: the membranes inside the non-func-

tional lysosomes are unstained whereas the outer

side of the membrane surrounding these structures

and the internal membranes of the cell (except mito-

chondria) are labeled. This absence of label in non-

functional lysosomes is not due to a lack of penetra-

tion of the ant ibodies in these structures, since they

can be labeled (in similar condit ions) with an anti-

Fig. 3. Electron micrographs of Purkinje cells of cerebella of 14-day-old rats treated with chloroquine and stained for Con A-HRP (A) and anti-PSG (B). A: the membraneous material of non-functional lysosomes strongly binds Con A. Note the very low Con A-binding in the endoplasmic reticulum. Magnification; x 49,000. B: the membraneous material of non-functional lysosome is free of labeling for PSG. Note that endoplasmic reticulum and the cytoplasmic side of the membrane surrounding the non-functional lysosomes is also stained. Magnification: × 49,000.

293

Fig. 4. Indirect immunohistochemical (HRP) stain for PSG (A-D) and m-AAT (E, F) of cerebella of control (A, C, E) and experimental (B, D, F) 14-day-old rats. A and B: at low magnification, no differences can be detected between control and experimental animals due to the intense staining for PSG. C and D: note the presence of clear grains in Purkinje cell dendrites of the experimental animal (D) compared to the control (C). E and F: note the presence of clear grains in Purkinje cell bodies and dendrites in the experimental animal (F) compared to the control. Same abbreviations and scales as in Fig. 2 eg, external germinative layer.

294

body directed against a lectin-like molecule from the rat cerebellum (unpublished results).

Immunohistochemistry using anti-m-AA T As shown in Figs. 4E and F, m-AAT is particularly

concentrated in the Purkinje cells. These results can be easily explained by the high content of mitochondria within the perikarya and dendrites of these cells

observed by others using electron microscopy, either by classical morphology 2 4.29 or by histochemical

methods 2,3. As shown in Fig. 4F, the non-functional

lysosomes appeared as white structures, similar to results obtained with anti-PSG.

lmmunohistochemistry using anti-a-D-mannosidase The non-functional lysosomes appeared to be de-

void of stain. This is clearly seen by electron microscopical examination of the tissue sections (Fig. 5A and B). this enzyme is essentially a cytosolic protein

particularly concentrated in Purkinje cell dendrites 52. It is absent from the lysosomes of normal rats and from the non-functional lysosomes of rats

treated with chloroquine. Furthermore, the parallel fibers and the extracetlular space in between them are virtually free of this enzyme both in control and

experimental rats (see ref. 52 and Fig. 5).

Histochemistry of Na+-K+-A TPase Histochemical staining for Na+-K + ATPase is

shown in Fig. 2E and F. This enzyme which is a glycoprotein 30, possibly Con A-binding al, is particularly

concentrated in neuronal plasma membranes 25. Our histochemical results at the optical level are in agreement with such a localization since the molecular layer (mostly composed of parallel fibers) is heav- ily stained for this enzyme. The label is not present inside the Purkinje cells, both in control and experi-

mental cerebella.

DISCUSSION

Specificity of the action of chloroquine It is well known that chloroquine induces a

dysfunction of lysosomes, thereby producing an accumulation of membraneous cytoplasmic bodies in a great number of cells 14A8,38.47 and in a certain way mimicking lysosomal disorders. In contrast, with neural lysosomal disorders (where both neurons and

glial cells are affected) (for review, see refs. 6, 40), chloroquine induces a perturbation of certain cells within the central nervous system, mainly large neurons 1s,47. The main consequence is the accumulation of myelin-like bodies considered generally as 'autophagic vacuoles'. The mechanism by which chloroquine affects the lysosomal function is still unclear,

although some hypotheses have been put forward. According to d'Arcy Hart and Young 9, chloroquine

perturbs the fusion of pinocytotic vacuoles with lysosomes. From our pictures, this is not verified since most of the grains induced by the drug contained a membrane part and a lysosomal part as previously re- ported 14. Chloroquine appears to act at a later stage than the fusion of the two stuctures. More probable, from our data, is the proposal of several authors 12,26,39,42, that the drug acts by elevating the lyso-

somal pH (and thus by inhibiting lysosomal enzymes), and possibly by impairing the recycling of the receptors for the lysosomal enzymes 12,42.

The accumulation of grains within Purkinje cells is particularly significant during the period between the thirteenth and the eighteenth postnatal day, even though low doses of chloroquine were used. Grains were present both in the cell body and the dendrites of Purkinje cells, with some morphological differences, since in the latter they seemed to be smaller and darker than those of the perikaryon.

Origin of the material accumulating in non-functional lysosomes

The main question remained: where is this membraneous material coming from? Several possibilities have to be considered: Golgi apparatus, endoplasmic reticulum, mitochondria, and also plasma membrane (including plasma membranes of other cells).

The possibility that this material came from mitochondria could be ruled out, since aspartate aminotransferase mitochondrial isoenzyme (a mitochon-

drial marker) is absent from these structures and since we have previously shown 48 that rat brain mitochondria have practically no Con-A-binding glyco-

proteins. The further possibility of an ergastoplasmic origin

was also ruled out due to the absence of label for PSG (which labeled all Purkinje cell intracellular membranes except mitochondria) 19. These results are only partially in agreement with those of others 14, who

295

Fig. 5. Electron micrographs (magnification: x 31,000) of Purkinje cell dendrites of cerebella of 14-day-old rat treated (B) of not (A) with chloroquine and stained with anti-a-mannosidase antibody. Note the intense cytosolic stain of the Purkinje cell dendrites and spines and the absence of staining in and between bundle of parallel fibers. Non-functional lysosomes are free of label.

296

showed a labeling for glucose-6-phosphatase (a marker for endoplasmic reticulum) in 'autophagic va-

cuoles' isolated from rat liver. As mentioned by the authors 14, the precipitate was essentially localized on the membrane surrounding these structures, (al-

though discrete labeling was found in the membraneous material inside the vacuoles). However, due to

the bad preservation of these structures, this internal labeling remained speculative. Using anti-PSG, the inner part of the grains is completely unlabeled,

whereas the surrounding ER membranes are consis- tently labeled. Thus, from our results, there is no indication that the membraneous material is derived from the ER of Purkinje cells.

The last possibility is that this material originates

from plasma membranes either of the Purkinje cell it- self or of the parallel fibers, since both possess Con A-binding material at the period studied here.

One of the glycoproteins binding to Con A in the Purkinje cell is PSG. Earlier immunohistochemical studies19, 34 have shown that it is widely distributed on internal membranes of Purkinje cells (perinuclear

membrane, smooth and rough endoplasmic reticulum, synaptic vesicles, subaxolemmal cisternae and also the cytoplasmic side of the plasma membrane of Purkinje cells 19. Due to its uniform cell distribution, PSG is probably one of the major Con A-binding components of these cells. If the membraneous

material found in the grains is derived from the entire plasma membranes of Purkinje cells, PSG would be expected to be present within them. This is not the case.

Furthermore, for quantitative reasons, the hypothesis that the Con A-binding material found on the membraneous material of the grains originated from the membranes of parallel fibers is more probable. Before the fourteenth postnatal day, the axolemma of parallel fibers massively binds Con A (ref. 49

and this study). When rats were treated with chloroquine at the period where this material disappears from parallel fibers, a simultaneous increase of Con A-binding material is found in the non-functional lysosomes of Purkinje cells, suggesting a transfer of such a material from parallel fibers into the Purkinje cells. In control rats the accumulation of Con A-binding material in Purkinje cells simultafieous to the dis- appearance of that of parallel fibers is not detectable, probably due to a very rapid catabolism.

Mechanism of degradation of the glycans of the ('on A-binding glycoproteins from parallel fibers

Life-time studies of the glycans of the Con A-binding glycoproteins from parallel fibers 32 indicated that they are actually degraded, a-D-mannosidase (nec- essarily involved in this degradation) is localized in the target cells of parallel fibers 52, both in control and

treated rats. The hypothesis of in situ degradation has to be eliminated since immunohistochemical studies at the electron microscopical level do not show any staining for this enzyme in the vicinity of the parallel fibers. The observations that a-D-mannosidase is absent from lysosomes do not permit clear conclusions on the way in which these glycans can be degraded. According to Montreui124, the first step of degradation of N-glycans could be the action of an

endo-fl-D-N-acetyl-glucosaminidase. However, it has been shown 27 that this enzyme is cytosolic. Prelimi-

nary results in our laboratory confirm its cytosolic localization in the rat cerebellum. The only possible ex- planation of our results is a two-step degradation: one lysosomal, catabolizing the protein part, and the other cytosolic, catabolizing the remaining glycan. However, this possibility remains essentially specu-

lative. Nevertheless, the exclusive localization of a-D-mannosidase in target cells is another argument for a transfer of Con A-binding glycoprotein from the parallel fibers into the target cells.

Mechanism of transfer of glycoproteins j?om parallel fiber,; into their target cells

The transient Con A-binding glycoproteins of parallel fibers behave as intrinsic membrane proteins since they are insoluble in neutral detergent -~4. Un-

less some proteases acting in situ liberate the carbohydrate bearing part of these membrane glycoproteins, we have to assume that part of the membrane of parallel fibers is endocytosed by the target cells. Since

the material accumulating in non-functional lysosomes is membrane-bound, it is reasonable to elimi- nate the protease hypothesis. In this case, pinocytotic figures of the membrane of parallel fibers by the target cells should be morphologically detected both in control and chloroquine-treated animals. This is effect- ively the case in normal animalsl,28, both in rat cere-

bellum and chick embryo cerebellum, as the period of formation of the synapses between these structures. Quantitative data on the number of these pino-

297

cytotic figures are available from Palacios-Prii et al. 28

which show a good correlation with the formation of

synapses. These last authors also demonstrate the

relationship between these pinocytotic figures and the lysosomes through coated vesicles and ergasto-

plasm. Such a phenomenon could involve either a com-

plete degradation of the components of the parallel

fiber plasma membrane or a selective degradation of

only some components. Our histochemical results on

Na ÷, K ÷ ATPase (although not definitely conclu- sive) suggest that some specificity exists.

We have presented elsewhere 33 histochemical re-

suits indicating that lectin-like molecules susceptible

to interact with the Con A-binding glycoprotein of

the membrane of the parallel fibers are actually pre-

sent in Purkinje, Golgi and basket cells. Further-

more, we have recently (Dontenwill et al., J. Neuro-

chem., in press) obtained monospecific antibodies against a lectin-like molecule, which is very concen-

trated in the target cells of the parallel fibers.

It is thus reasonable to assume that the endocytotic

mechanism observed here results from a specific rec-

ognition phenomenon: the glycans of the Con

A-binding glycoproteins from parallel fibers interact first with specific receptor molecules found on the

membrane of their target cells. This recognition is

followed by endocytosis of the glycoprotein-receptor

complexes into the target cells followed by migration

towards the lysosomes where the glycoproteins are at

least partially degraded. Chloroquine inhibits this de-

gradation by elevation of the lysosomal pH and thus

provokes an accumulation of non-degraded material.

This hypothesis is entirely compatible with the results of others 28.

Of particular interest is the period where such phe- nomena are observed: the period of maximum syn-

apse formation in the molecular layer of the ratt or

chick embryo 2s cerebellum. The observation that pi-

nocytotic pictures, very similar to those observed in

many cases of receptor-mediated pinocytosis (15, 35,

46), are very often encountered in maturing nervous

tissues just before the period where mature synapses are detected1,7,1:,lT,28, 43 could indicate that the phe-

nomenon observed is much more general (possibly

with different molecules) and could be related to the process of synapse formation.

ACKNOWLEDGEMENTS

The authors wish to thank Ms. F. Herth and F. Schohn for typing and N. J. Cook for reviewing the

manuscript. M. D. is in receipt of a bursary from the DGRST.

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Arguments in favour of endocytosis of glycoprotein components of the membranes of parallel fibers by Purkinje cells during the development of the rat cerebellum