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Ultrastructural changes during development in the proliferating cells of the external granular layer and the granule cells of the internal granular layer of the rat cerebellar cortex were examined. Changes in the number of free ribosomes, development of membrane systems, nuclear appearance and the number of cristae and matrix density of mitochondria suggested that both the round and elongated cells of the external granular layer and the granule cells of the internal granular layer were at same stage of maturation at any given age. Quantitatively, the relative volumes of cytoplasm and of mitochondria in the granule cells of internal granular layer were significantly higher than in the proliferating external granular layer cells; however, mitochondrial density and its increase during development were not significantly different in the two cell populations. The activity of the mitochondrial enzyme, succinate dehydrogenase, was also similar in ultrastructurally preserved and metabolically competent perikaryal fractions enriched in replicating external granular layer cells, granule cells and Purkinje cells. These findings emphasize the similarities between proliferating external granular layer cells and granule cells of internal granular layer. They lend support to the view that these cell types at the ages examined, represent a continuum of maturation towards full neuronal differentiation.

Cerebellar histogenesis was studied in hamsters infected at birth with a parvovirus, rat virus strain PRE 308. Cerebellar granule cell precursors in these animals were selectively infected and lysed in the external germinal layer before their migration to form the internal granular layer. The effects of the absence of granule cells on cerebellar development and especially on the development of the Purkinje cells and their dendrites was analyzed using fluorescent antibody. Golgi, conventional paraffin, and electron microscopic methods. This study represents the first Golgi and ultrastructural study of the pathogenesis of rat virus infections in the cerebellum. The destruction of the granule cell precursors resulted in a dysplastic cerebellar hypoplasia with total disruption of normal cerebellar stratification and cytoarchitectonics. The Purkinje cells developed misshapen, progressively disoriented dendritic stems lacking tertiary dendrites and studded with numerous spines, devoid of afferent synaptic contacts (naked spines) and encased by glial processes. These developmental studies, together with the mouse mutant studies, demonstrated that the spines of the Purkinje cells were elaborated in the absence of both tertiary dendrites and afferent parallel fiber contacts. Such data suggested that spine formation, once triggered, was intrinsically programmed rather than being dependent on the development of parallel fiber contacts. Despite the loss of a major interneuronal component and disintegration of normal cytoarchitectonic relationships, synapses in the cerebellar cortex developed normally as long as both the pre- and post-synaptic elements were present. Thus synaptic specificity is maintained in the face of gross disruption of cytoarchitectonic relationships. If either the pre- or post-synaptic portion of a contact was absent, then glial processes isolated the persisting element or aberrant contacts formed. In addition to glial encasement of naked spines, there were dendrodendritic articulations between Purkinje cell dendrites, some of which were joined by septate, plaque-like junctions. Aberrant synaptic contacts between mossy and climbing fiber glomeruli and the smooth surface of the Purkinje cell somata were found rarely. In addition to these contacts which also occur in the hypoplastic cerebella produced by other methods, previously undescribed non-synaptic spine-articulations between Purkinje cell dendrites were seen. The role played by granule cells and their axons in Purkinje cell development appeared to be two-fold. First, the development of the orderly array of parallel fibers in the normal animal played a role in orienting and flattening the dendritic trees of Purkinje cells. Second, the formation of tertiary dendritic branches appeared to depend primarily upon the presence of an external germinal layer throughout this stage of Purkinje cell development. By contrast, dendritic spines developed and persisted in the absence of granule cells.

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramon y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.

Cell interactions must play an essential role in the implementation of the genetic programme. Cell signals during ontogenesis could modulate and, in some cases, determine choices in the processes of cell multiplication, differentiation, maturation, migration, and establishment of connections. This implies that, at molecular levels, the expression of each genetically coded signal molecule, giver (effector) and receiver (receptor), is also modulated by the preceding and the following cell interaction. Thus, in many cases the presence of signal effectors and/or receptors in a given cell should be necessary only for the required time.

A quantitative assessment has been carried out on the cell populations and DNA content of the cells in the internal granular layer of the central lobes of the cerebellum from 100 children with myelomeningocele and from 120 normal controls. The method used was a computerised analysis of a television image of the neurones in standardised sections. The cell and DNA complement of the declive and central lobe are only slightly reduced in the cases of myelomeningocele, but there is frequently cellular and DNA loss throughout the pyramid and uvula to the nodule. These findings suggest that the central lobes of the cerebellum are probably genetically normal but have irregular degeneration and arrest of growth, probably as an effect of pressure at the foramen magnum.

Lead encephalopathy was produced in neonatal Long-Evans rats by administering daily doses of lead acetate (600 milligrams of lead acetate/kilogram of body weight) through an esophageal catheter. Experimental rat pups showed behavioral changes, failed to gain weight at the same rate as controls, developed a paraplegia and died by 15 days of age. Lead analysis showed very high blood and tissue lead levels. Sequential histopathologic changes were studied in the cerebellum with observations also made in the choroid plexus, cerebral cortex and corpus striatum. Emphasis was placed on the cerebellum because this region of the brain was most severely altered. Petechial hemorrhages were evident in the cerebellum at three days and two days later the hemorrhagic lesions were almost confluent. The molecular and Purkinje cell layers were most extensively damaged by the hemorrhage. At eight, nine and ten days hemorrhages were fewer and massive amounts of edema fluid accumulated in the internal granular layer. Vascular anomalies of developing lead poisoned rats were examined with electron microscopy and with Golgi preparations. The evidence indicates that growing capillaries are the primary structure of the central nervous system (CNS) damaged by lead intoxication. The endothelial bud (or angioblast) appears to be a structure sensitive to lead poisoning and the encephalopathy probably results from the death of many of these buds.

The ultracytochemical acridine orange(AO) method has been employed to demonstrate DNA template activity in the developing cerebellar cortex in rats and its alteration after transplacental administration of ethylnitrosourea (ENU). Electron microscopic studies revealed that AO binds to DNA exclusively within the active extended euchromatin portion of the cell nucleus of the proliferating external granular layer. No or only a few AO reaction products were visible in the non-proliferating cell nuclei of the internal granular layer and of Purkinje cells. With increasing cellular differentiation of granule cells in the cerebellar cortex the number of AO positive cells as well as the number of AO reaction products per cell showed a progressive decrease. A single i.p. injection of 50 mg/kg of ENU in rats on day 15 to 21 of gestation resulted in occasional cell damage to the proliferating external granule cells of developing cerebellar cortex at 4 and 7 days after birth. Damaged cells of the external granular layer were found to have no or only a small number of AO reaction products in their nuclei. In further development from 14 to 28 days the cerebellar cortex of offspring from ENU-treated mother showed no abnormality and exhibited no definite alteration in distribution pattern of AO reaction products. ACTA PATHOL JPN. 34: 489–497, 1984.

Maturational changes of the cerebellum of frog tadpoles were studied with the electron microscope. In the premetamorphic tadpole, parallel fiber-like processes (PFP) were present in the incipient molecular layer, long before the appearance of the external granular layer (EGL). These PFP showed synaptic contacts with the precociously developed Purkinje cell dendrites. It appears that these PFP may be responsible for inducing the precocious elaboration of the Purkinje cell dendritic arborization. In the metamorphosing tadpoles, the EGL cells migrating into the internal granular layer were frequently seen in close association with the ependymoglial cell processes, which extend from the pia down toward the ependymal surface. This observation lends support to the hypothesis that glial processes guide the migrating EGL cells.

Newborn mice of four inbred strains were injected with a single dose of N-ethyl-N-nitrosourea. The wide range of tumours induced included a small number in the central and peripheral nervous systems. The 4 brain tumours all arose in the cerebellum. Three in one strain were medulloblastomas showing continuity with the internal granular layer. All three tumours showed diffuse infiltration through the molecular layer and continuity with densely-packed islets of cells that marginated immediately beneath the pia and closely resembled remnants of a persistent fetal external granular layer. The medulloblastomas are discussed with special relevance to the histogenesis of the equivalent tumour in man.

Brain src protooncogene is expressed in two forms, one identical to message in other tissues, and one containing an 18-nucleotide insert specific to brain. We have mapped mRNA for the two forms of src in rat brain with selective antisense oligonucleotide probes to the brain (src+) and peripheral (src-) forms. Fetal rat src mRNA levels were much higher in the central nervous system than any peripheral organ. In adult brain, src+ mRNA level was highest in the internal granular layer of the olfactory bulb, pyramidal cells of the hippocampus, granule cells of the dentate gyrus, and cerebellar granule cells. src+ and src- levels were similar in hindbrain, but src+ levels were higher than those of src- in forebrain. These distributions suggest that src+ may play roles in a number of neural processes, possibly including neuronal plasticity.

The distribution of GM1 ganglioside in developing mouse cerebellum was monitored by indirect immunofluorescent detection of choleragenoid receptors. In frozen sections of cerebellum from mice 5 to 10 days old, fluorescence is observed on granule cells in the inner rows of the external granular layer, in the growing molecular layer, the Purkinje cell layer, and the internal granular layer. In sections of adult mice, fluorescence is restricted to the bodies of Purkinje and internal granule neurons. The percentage of fluorescent dissociated or cultured cerebellar cells increases with the postnatal age of the mouse or the duration of time in vitro. No fluorescence is observed in the absence of choleragenoid or if the test material is extracted with chloroform:methanol. To determine whether the expression of surface GM1 ganglioside in culture is a reflection of a developmental program, mice are injected at particular times with [3H]thymidine and cerebellar cultures processed for simultaneous autoradiography and immunofluorescence. Granule cells from 8-day-old mice having cholera toxin receptors at 20 hr in vitro are a distinct population born 1 day or earlier prior to sacrifice. Cells synthesizing DNA on the day of sacrifice are not fluorescent at 20 hr in vitro. This observation correlates well with immunohistological results showing a lack of fluorescence in the outer proliferative rows of the external granular layer. Therefore GM1 ganglioside is not present on granule cell precursors but is expressed at some time after the cells become postmitotic. GM1 ganglioside is detected on growing parallel fibers in situ and neurites in vitro but not on adult axons, suggesting differential localization at a later stage of development.

Sheep have been used to study the effect of dietary iodine deficiency on the development of the fetal brain. Severe iodine deficiency caused reduction in fetal brain and body weights and in brain DNA and protein from 70 days gestation to parturition. The lowered brain weight and brain DNA at 70 days gestation indicates a reduced number of cells, probably due to slower neuroblast multiplication which normally occurs from 40-80 days in the sheep, and the reduction in DNA and protein after 80 days implies that the development of neuroglia could be slowed also in iodine deficiency. Morphological changes were observed in both the cerebral hemispheres and the cerebellum. In the cerebral hemispheres of the iodine-deficient fetuses an increased density of neurons was apparent histologically in the motor cortex and visual cortex and in the CA1 and CA4 areas of the hippocampus in comparison with controls. In the cerebellum there was delayed migration of cells from the external granular layer to the internal granular layer and increased density of Purkinje cells in the iodine-deficient fetal brains. In addition, the molecular area was increased and the medullary area reduced in comparison with controls. These change are indicative of delayed brain maturation. Evidence of fetal hypothyroidism was provided by low fetal thyroid iodine and plasma T4 values, thyroid hyperplasia from 70 days gestation, significant reduction in body weight at the same time as the brain retardation, and absence of wool growth and delayed skeletal maturation near parturition. It is apparent from the biochemical and histological changes observed during iodine deficiency that iodine is an essential element for normal fetal brain and physical development in the sheep.

Thymidine-H3 was injected intraperitoneally into 6- and 13-day old rats and they lived afterwards for periods ranging from one hour to 60 days. Autoradiographic data obtained from animals surviving for short periods were used to estimate rates of regional cell proliferation. Animals with longer survival were used to deduce the movements of new cells from germinal sites, through migratory channels, to target areas, and to determine their mode of differentiation. The formation and differentiation of microneurons goes on during infancy, though in most structures at a declining rate. In the wall of the olfactory ventricle cell multiplication continued at a high rate at six days, with a decline at 13 days, and the new cells migrated to the layers of the olfactory bulb. The migration of labeled cells from the lateral ventricle was traced, by way of the fimbria, to the polymorph cell layer of the dentate gyrus, and from there to the granular layer. The “older” granule cells were located in the upper part of the granular layer, the “younger” cells at its base. Cell multiplication continued at a very high rate in the external granular layer of the cerebellar cortex, whence cells migrated to the molecular layer and internal granular layer. Speed of migration was approximately 50 μU/day in the olfactory bulb, and 60–70 μ/day in the cerebellum. The possible significance of these findings was discussed.

The changing pattern of ChE staining in the cerebellar cortex was examined in rats 0, 3, 7, 10, 15, 21, and 30 days old. For correlative purposes, Golgi-impregnated sections from rats of the same ages were also studied. The following conclusions were drawn about the mature cerebellum. (a) Most of the nerve cells of the cerebellar cortex (Purkinje, granule, basket, and stellate cells) are noncholinergic; the Golgi cells are cholinergic. (b) Of the noncholinergic neurons, the Purkinje cells are noncholinoceptive because the climbing fibers, parallel fibers, and the basket terminals are noncholinergic. (c) A large proportion of the mossy fibers that apparently originate directly or indirectly in the acousticovestibular system are cholinergic; the others are predominantly noncholinergic. (d) The granule cells are cholinoceptive. This is due to Golgi-cell terminals in the glomeruli in all lobules and to the additional cholinergic mossy fiber terminal in the lobules that receive acousticovestibular projection (nodulus, flocculus, part of uvula, tuber, folium, and lingula). (e) Available evidence in the literature about other neurochemical transmitters in the cerebellar cortex was considered. The following conclusions were drawn about the postnatal development of the “wiring pattern” of the cerebellar cortex. (a) In the newborn rat the only input to the Purkinje cell is by way of climbing fibers which contact their soma. (b) A few days after birth the cholinergic mossy fibers reach the zone of Purkinje cells. The transient ChE staining of Purkinje cells and the development of transient perisomatic processes suggest that mossy fibers may establish direct contact with the Purkinje cells during this period. (c) The cessation of Purkinje-cell staining during the second week, the descent of the precursors of granule cells from the external into the internal granular layer, and the increased staining of the glomeruli indicate that the mossy fibers establish contact with the granule cells.

To examine the formation and differentiation of the external granular layer, chick embryos ranging in age from two to 20 days were treated with tritiated thymidine and sacrificed after various time intervals. By studying the migration of the labeled cells at different stages of development, the following observations were made: (1) cells originating from the neuro-epithelial layer along the ventricular surface of the cerebellar plate migrate through previously formed cell layers of neuroblasts to the surface; (2) the development of the lateral portion of the cerebellar plate is approximately 24 hours more advanced than that of the medial region, thus explaining the appearance of the external granular layer first laterally and then medially; (3) at no time during development can a proliferation center be found in the ventrolateral angle of the fourth ventricle. Based on theses results, it is concluded that the cells of the external granular layer originate from the neuroepithelium bordering the ventricular surface of the cerebellar plate and migrate from this position straight to the surface. No evidence for a surface migration from the ventrolateral angle in a dorsomedial direction can be found. Shortly after the formation of the external granular layer at day six, its cells begin to proliferate. During this period, which lasts until day 15, relatively few cells leave the external granular layer to migrate inwards. After this day a massive inward migration occurs, and the cells of the external granular layer differentiate into the granular cells and perhaps the glia cells. In the internal granular layer they intermingle with the Purkinje cells and Golgi II neurons, which originate in the neuro-epithelial layer between days three and six of development. Shortly after hatching, when the external granular layer has fulfilled its function in cell production, it disappears.

The cerebellum of the neonatal puppy was found to be incompletely differentiated. Its gross dimensions were less than half those of the normal adult, and although major lobules were recognizable, folial development was rudimentary. Microscopically, the cortex on the second postnatal day was distinguished by a thick subpial external granular layer of actively dividing cells. Beneath this transient layer, permanent cortical layers were largely undeveloped and myelination was not yet evident. During the first two postnatal weeks, the width of the external granular layer remained relatively constant (at slightly more than 50 μ), while other cortical layers underwent considerable growth, with histologic and cytologic differentiation. After the second postnatal week, the width of the external granular layer declined steadily, so that by the tenth week only a vestige of the layer remained. Between the second and seventy-second postnatal days, a tenfold increase occurred in the width of the molecular layer. Major growth and differentiation also occurred in the internal granular layer, which became almost tripled in its width during the first ten postnatal weeks. Myelination proceeded in a centrifugal direction and occurred earlier in the vermis than in the hemispheres. By the end of the first month, myelination of the vermis was practically complete, and by the tenth week, the arbor vitae of the hemispheres appeared fully myelinated.

Embryonic and young postnatal mice were exposed once to thymidine-H3, to label cells preparing for division. Histogenesis of cerebellum was studied in autoradiograms. Purkinje cells and neurons of roof nuclei form simultaneously in the primitive ependyma of the young embryo and migrate outward to reach their final positions. A transient external granular layer arises by proliferation of cells on the lateral caudal cerebellar surface lining the fourth ventricle. These cells migrate over the external cerebellar surface, and continue to proliferate abundantly until a few weeks after birth. The layer disappears in the third postnatal week. Most external granule cells divide a few times and then migrate inward past the Purkinje cells to become granule cell neurons. A smaller population of cells arises in the granule layer itself and is characterized by an unusually long interval between deoxyribonucleic acid (DNA) synthesis and mitosis. These cells migrate short distances and may assume positions around the perikarya of Purkinje cells; cells of this class are probably neuroglia. An hypothesis is presented which accounts for the extensive cell migrations during histogenesis as a means for attaining particular synaptic contacts.

The development of rat cerebellum was studied by the complete 3H-thymidine labelling method which provides a 100% labelling of cell nuclei at the time of birth. The decrease in labelling index and intensity after birth was studied for a total of 42 days in order to compare proliferative activity of the different cell populations. The results indicate that Purkinje cells and cells of the nucleus lateralis cease to divide after birth. Golgi cells showed a very slow proliferative activity up to day 20. A rapid cell turnover rate was found for basket cells and cells of the molecular layer up to day 8 and a slow proliferation at later times. A high proliferative activity of the external granular layer cells after day 3 to day 8 and a similar decline of labelling over internal granular cells was found. The latter finding was interpreted as being due to cell renewal of internal granular cells as well as cell migration from external granular cells into the internal granular layer. The transit time for the migratory process was calculated to be approximately 24 hours.

The neocortex of the armadillo, Dasypus novemcinctus, was explored by electrical stimulation and the motor area determined to be caudal to the supraorbital sulcus. Movements of the tongue, lips, pinna, scalp, shoulder, back, fore-limb and, to some extent, hind-limb could be elicited. The motor area for the lips and tongue was particularly large and it had the lowest threshold to stimulation. The motor cortex was characterized by deeply-staining pyramidal cells and the lack of a well-developed internal granular layer. The pyramidal cells were largest and most numerous in the limb area. Ablation of the motor cortex or part of it resulted in fiber degeneration within the contralateral ventral and lateral funiculi of the spinal cord. Degenerating fibers distributed extensively to the medial parts of laminae IV through VI and, to a much less extent, to the medial part of lamina III and the lateral parts of laminae V and VI. Degenerating fibers were not observed in the spinal cord after presupraorbital lesions or after ablations of the caudal or ventrocaudal neocortex.

The development of the molecular layer was studied in rats made hypo- and hyperthyroid at birth. The width and area of the molecular layer, packing density of granule cells in the internal granular layer, and synaptogenesis in the outer and inner halves of the molecular layer were determined. Hypothyroidism caused: (1) transient developmental retardation in width and area of the molecular layer, with normal levels being approached by 30 days, (2) a slight, but not significant, increase in packing density of granule cells, probably reflecting a reduction in glomeruli or synaptogenesis in the internal granular layer, as well as crowding produced by increased numbers of granule cells 20, (3) retardation of synaptogenesis in the molecular layer, especially in the outer half, as late as 55 days. This retardation was manifested by a decrease in the synaptic content of the Purkinje cell dendritic domain, and decreased size of the parallel fiber synaptic domain, resulting (along with the increase in granule cells) in a greater packing density of parallel fibers in the molecular layer. Hyperthyroidism caused: (1) an accelerated increase in width of the molecular layer from day 10 on, such that by 30 days the molecular layer was wider than in controls. This was accompanied by only a small, transient increase in the area of the molecular layer and a sharp leveling off by 15 days such that the area was greatly decreased by 30 days compared to controls. This was interpreted to mean that the length of the molecular layer was decreased in the sagittal plane, resulting in taller and narrower Purkinje cell dendritic trees. Possible reasons for this are discussed, (2) a significant decrease in packing density of granule cells, probably reflecting both the decrease in the number of granule cells 20 and an accelerated development of glomeruli, (3) an acceleration of synaptogenesis in the inner and outer halves of the molecular layer and an increase in the number of synaptic profiles/cell at 30 days. This probably is the result of accelerated growth of the Purkinje cell dendritic tree and the deficit in granule cells and basket cells described previously 20. Total synaptic profiles were decreased, however, due to the decreased sagittal length and area of the molecular layer. Thus it was shown that early hypo- and hyperthyroidism both cause a terminal deficit in total synaptic profiles in the rat cerebellar cortex, but for different reasons.

Histochemical examination of the activity of naphthylamidase (LNAse) in the cerebellar cortex of 70 human autopsies consistantly revealed a marked activity mainly in the internal granular layer with pH optimum of 5.8. Slight enzyme activity was also localized in sites corresponding to lipofuscin deposits and areas of acid phosphatase activity in the Bergmann glial cells, Purkinje cells and in perivascular cells. The histochemical findings support the LNAse reaction as a lysosome marker. Differences in localization of LNAse and acid phosphatase could possibly be due to prior release of the latter enzyme from the internal granular layer. Significant correlation between demonstrable loss of granule cell nuclei (the so-called acute, selective necrosis of the granular layer) and low pH of the cerebellar tissue could be demonstrated in 21 cases. The present findings support the hypothesis that an enzymatic disintegration of the granule cells takes place in postmortem cerebella with low pH simulating a necrotic vital phenomenon.

The sequence of differentiation of the cerebellar granule cell in chick embryos from the eighth to the 15th days of incubation has been studied in Golgi-stained celloidin sections. In the germinal-cell phase, the presumptive granule cell sends out one or two horizontal processes which may originate either in the body of the cell or in the extension which attaches it to the pial surface. Thus the germinal cell may be converted into either a monopolar or a bipolar presumptive granular cell. Bipolar cells may have two processes of the same length (symmetrical cells) or of unequal length (asymmetrical cells). In the symmetrical as well as asymmetrical bipolar cells the leading process is formed, by means of which the perikaryon emigrates until it situates itself definitely in the internal granular layer. Thus, symmetrical and asymmetrical bipolar cells give rise to a granule cell with parallel fibers of equal or different lengths. The monopolar element may originate a second process or may remain in the monopolar phase until it reaches the internal granular layer. Once there, it completes the formation of the parallel fibers.

We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells. © 1996 John Wiley-Liss, Inc.

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsell's classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex.

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

The rejoining of the strand breaks formed by X- and -y-irradiation of the DNA in the internal granular layer neurons of the canine cerebellum was followed in vivo at the level of the 165S chromosomal subunit. Single-strand breaks were produced with an efficiency of 58 + 5 eV in the DNA of differentiated neurons, a value similar to that found for cultured dividingcell lines, e.g., Chinese hamster ovary cells and murine leukemic lymphoblasts. The expression of the strand-break rejoining in neuronal DNA in vivo differed from that observed in dividing cells in vitro because reconstituted 165S subunits were degraded into smaller molecules during the early repair period. Large molecular weight species similar to those observed in unirradiated neurons did not appear in significant quantities until 10 hr after irradiation. The exact mechanistic relationships of these events are not known at this time.