External Granular Layer

Abstract: Patterns of lamination during development of the fetal human cerebellar cortex were analyzed in Nissl- and H & E-stained serial sections, rapid Golgi preparations, reduced silver impregnations, electron micrographs, and autoradiograms. The layering pattern changed dramatically with time, as analyzed in detail for the culmen, the earliest region to differentiate. Up to about 10 weeks of gestation, cells proliferated only at or near the ventricular surface and migrated radially outward to occupy the full thickness of the cerebellar primordium except for an outermost cell-sparse marginal layer (2-layer stage). The external granular layer first appeared at 10-11 weeks while another group of cells became concentrated beneath the marginal layer (3-layer stage). At 20–21 weeks the lamina dissecans first became evident as a relatively acellular band in the midst of the zone of compact cells below the marginal (now molecular) layer, and for the next ten weeks the cerebellar cortex displayed this 5-layered form. At about 32 weeks the lamina dissecans disappeared (4-layer stage) and postnatally the external granular layer in turn disappeared as the last of its cells migrated inward (adult 3-layer configuration). The Purkinje cell population was established by 13 weeks, though the cerebellum was destined subsequently to increase several orders of magnitude in surface area and volume. The increase was achieved in part by cell growth, but mainly by extensive cell proliferation in the external granular layer. At 22 weeks, about 30% of the external granular cells incorporated thymidine-H3 upon a single supravital exposure; the external granular layer attained maximum cell number at some stage after birth. At the 5-layer stage from about 21 to 32 weeks, the interrelationships between various classes of young neurons in the cerebellar cortex became very complex. The Purkinje cells developed ascending branched dendritic processes with growth cones and displayed transient short cytoplasmic processes that extended from the soma in all derections. Basket cell neurons had formed but their axons appeared not to envelop the Purkinje somas as yet. Less mature, smaller cells were beginning to migrate from the external granular layer inward past the Purkinje somas. Their cell bodies in the newly forming granular were separated from the Purkinje cell bodies by a dense tangle of axons in the lamina dissecans. Many of these axons terminated in swellings interpreted tentatively as immature mossy endings, while others passed outward to enclose the cell bodies and proximal dendrites of the Purkinje cells. Some general points emerged from a comparison of cerebellar development in man and animals. The time of cell origin cannot be inferred necessarily from the time of overt differentiation; deep cerebellar neurons and Purkinje neurons arise in the first trimester, but the former cells differentiate much earlier. Purkinje cells acquired characteristic shapes by the middle of gestation, when very few granule cell neurons had yet formed, and thus appear to develop relatively independently of the granule cells. Although the adult cerebellum appears to be organized similarly among mammals, a developmental component, the lamina dissecans, has been illustrated only in man and whale; its appearance may reflect the combination of early Purkinje cell and late granule cell differentiation in species with a prolonged period of development. One of the most intriguing features of the lamina dissecans is that it appears to contain axon terminals at a time prior to the arrival of the postsynaptic cells.

Abstract: The generation cycle of germinative cells (external matrix cells) in the external granular layer of the cerebellar cortex of the 10-to 11-day-old mouse was studied by radioautography following repeated injections of H3-thymidine. The generation time is 19 hr, presynthetic time 8.5 hr, DNA-synthetic time 8 hr, postsynthetic time 2 hr, and mitotic time 0.5 hr. These proliferating cells occupy the outer half of the external granular layer and make up the external matrix layer. Neuroblasts are differentiated from the external matrix cell, migrate out from the layer and accumulate in the inner half of the external granular layer to form the external mantle layer. The transit time of the neuroblasts in the external mantle layer is 28 hr. Thereafter, they migrate farther into the molecular layer and the internal granular layer. By means of long-term cumulative labeling, the rate of daily production of neuroblasts from the external matrix cell is studied in quantitative terms. It becomes clear that the entire population of the inner granule neurons arises postnatally in the external granular layer between 1 and 18 days of age and that 95% of them is produced between postnatal days 4 and 15. Finally, the fate of the cells in the external granular layer at its terminal stage was studied by marking the cells with H3-thymidine during 15–16 days of life and following their subsequent migration and developmental changes up to 21 days of life. Comparison of radioautographs taken before and after the migration disclosed that the external matrix cells give rise to a small number of neuroglia cells. This finding revealed their multipotential nature.

Abstract: We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.

Abstract: A continuum of transitional forms between the cells in the external (transitory) granular layer of the cerebellar cortex and the granule cells in the internal (definitive) granular layer has been identified with the electron microscope in chick embryos 17–20 day old. This confirms that at least a number of the granule neurons is derived from the cells of the external granular layer, a concept based on studies with Golgi and autoradiographic methods. The differentiating granules in the molecular layer have a vertical orientation (vertical bipolar cells) and are shaped as flattened spindles, with the larger horizontal diameter in the direction of the parallel fibres. The cytoplasmic organelles in the vertical bipolar cells show a distinct polarization. As these cells are encountered in deeper positions in the cortex they have an increasing number of ergastoplasmic cisternae, and their mitochondria lose their dense granules and DNA-filaments. Near the internal granular layer these cells become rounded. Their cytoplasmic membrane systems appear more prominent, extending even into the axon hillock, and thin processes (primitive dendrites) are emitted from the cell body. Mossy fibre knobs are found to make synapse-like contacts not only with the dendritic tips of granule neurons, as usual in adult animals, but also with their dendritic trunks and perikarya. The knobs contain tubular profiles of the smooth endoplasmic reticulum, the number of which in many instances seem to be inversely proportional to that of the synaptic vesicles. In the developing glomeruli dendro-dendritic attachment plaques (desmosomes) are absent. Astrocytes and mitotic astroblasts have been observed at all levels of the cerebellar cortex except in the outer portion of the molecular layer and in the external granular layer.