Cerebellar External Granule Cell Layer Research Articles

Intact Numbers of Cerebellar Purkinje and Granule Cells in Sudden Infant Death Syndrome

Despite much research during recent decades, the etiology and pathogenesis of sudden infant death syndrome (SIDS) remain unknown. Because of the role of the cerebellum in respiratory and cardiovascular control, it has been proposed that it plays an important role in the pathogenesis of SIDS. To date, 5 postmortem studies on the cerebellum of SIDS cases have yielded conflicting results. Using a rigorous design-based stereologic approach, we investigated postmortem cerebella from 9 SIDS patients who died between 2 and 10 months of age and from 9 age- and sex-matched control children. Neither the volumes of the cerebellar external granule cell layer, molecular layer, internal granule cell layer (including the Purkinje cell layer), and white matter nor the total numbers of Purkinje cells, granule cells in the internal granule cell layer, and the number of granule cells per Purkinje cell showed statistically significant differences between the SIDS cases and the controls. Based on these observations, we conclude that structural alterations in cerebellar development are not involved in the etiology and pathogenesis of SIDS.

Medulloblastoma in Mice Lacking p53 and PARP: All Roads Lead To Gli

Medulloblastomas, embryonal neoplasms arising in the cerebellum, are the most common malignant brain tumors in children. They are composed of primitive cells with the potential to differentiate along neuronal and glial lines. Similar lesions, known as primitive neuroectodermal tumors (PNETs), arise outside the posterior fossa, albeit rarely. Several molecular pathways important in cerebellar development have been implicated in medulloblastoma pathogenesis (reviewed in 1-3 ). A better understanding of the cells and signaling events involved in medulloblastoma tumorigenesis will be critical to the development of targeted therapies for these aggressive neoplasms, as evidenced by the inhibition of medulloblastoma growth by drugs blocking Hedgehog pathway activity. 4 In this issue of The American Journal of Pathology, Tong and colleagues 5 describe a new medulloblastoma model in which tumors arise from the cerebellar external granule cell layer (EGL) of mice lacking p53 and PARP. The up-regulation of the Hedgehog pathway effector Gli in all of their tumors suggests a more general role for Hedgehog signaling than was previously appreciated. In medulloblastomas, some cellular pathways may be more equal than others.

Localization of mRNAs for subfamily of guanine nucleotide-exchange proteins (GEP) for ARFs (ADP-ribosylation factors) in the brain of developing and mature rats under normal and postaxotomy conditions

ADP-ribosylation factors (ARFs) play important roles in vesicular trafficking and cytoskeletal regulation and its activation depends on guanine nucleotide-exchange proteins (GEPs). By way of in situ hybridization histochemistry, the localization of mRNAs for subfamily members of low-molecular-weight ARF-GEPs in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression for msec7-1 was distinct in the ventricular zone while that for msec7-1, -3 and EFA6 in the mantle zone. In early postnatal brain, the expression for msec7-1, -2, -3 and EFA6 was seen widely in various loci of the gray matter with different intensity, and the expression of msec7-1 and -2 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for the four ARF-GEPs decreased more or less in most gray matter and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression of EFA6 mRNA was also evident in the molecular layer of the hippocampus and dentate gyrus. No obvious gene expression for cytohesin-4 and ARF-GEP100 was detected in the brain at any stages of development. The present findings suggest that ARF-GEPs are differentially involved in some processes essential to neuronal differentiation and maturation in association with ARFs.

Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy

ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons. Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy. In the embryonic brain, the expression for ARF-1, -4, -5, -6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3, -4, -5 in the mantle zone. In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4, -5, -6 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression levels of ARF-2 and -4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs. The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation.

Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm.

The developmental control genes of the Pax family are essential for brain development. Several Pax genes are also involved in chromosomal translocations causing malignancies in humans, and Pax5 expression is deregulated in medulloblastomas. We have investigated whether Pax5 can induce tumors in the developing mouse brain. Primary mouse embryonic neuroectodermal cells were retrovirally transduced with mouse Pax5 and transplanted into the brain of syngeneic host mice. No tumors developed in 36 transplants after one year, and there were no alterations in the differentiation pattern of the neural transplants. We then generated transgenic mice expressing human Pax5 under control of the Engrailed-2 promoter, which is expressed in the cerebellar external granule cell layer and in medulloblastomas. Sustained expression was achieved in the cerebellum of transgenic animals throughout lifetime. Expression levels were similar to those observed in human medulloblastomas. Again, cerebellar morphogenesis was undisturbed, and no tumors arose. These results strongly argue against a dominant transforming activity of PAX5 in NEC and in cerebellar granule cell precursors of mice, and underline the restricted tissue-specificity of PAX5 related oncogenesis.

Localization of mRNAs for Novel, Atypical As Well As Conventional Protein Kinase C (PKC) Isoforms in the Brain of Developing and Mature Rats

Using in situ hybridization histochemistry, the localization of mRNAs for 10 isoforms of protein kinase C (PKC) in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression was positive only for PKCepsilon, mu, lambda, and zeta with the former three more evident: The expression for PKCmu and lambda in the ventricular germinal zone and that for PKCepsilon, zeta, and lambda in the mantle zone. In the postnatal brain, the expression for PKCdelta, eta, and theta was detected differentially in a few circumscribed loci such as the thalamus, the habenula, the septum, and the cerebellar granule cells, whereas that for the other isoforms was seen widely in various loci of the gray matter with different intensity. The expression in the cerebellar external granule cell layer was positive only for PKCbeta (betaI and betaII), mu, and lambda with that for PKCbeta confined to its inner zone. There is a general tendency for all PKC isoforms that the expression levels reach at peaks in early postnatal brain and decreases more or less in adult specimens. This is the first report on the spatio-temporal heterogeneity in the gene expression for the whole members of PKC family in the brain throughout development, especially at embryonic days.

Localization of mRNA for fatty acid transport protein in developing and mature brain of rats.

Gene expression for rat fatty acid transport protein (FATP) and fatty acid translocase (FAT) were examined by Northern and in situ hybridization analysis. In Northern blot analysis of developing brain, FATP mRNA was detected weakly throughout all developing stages without any changes in the expression level, while no gene expression for FAT mRNA was detected at any stages. By in situ hybridization histochemistry, intense expression was seen in the ventricular germinal zone on pre- and perinatal stages, whereas distinct expression was observed in the cerebellar Purkinje and granule cell during postpostnatal development. No expression was detected in the cerebellar external granule cell layer. Because of the high expression of FATP mRNA in the embryonic ventricular zone and the postnatal cerebellar cortical neurons in parallel to the gene expression for fatty acid binding protein (FABP) which we have recently reported, co-operated involvement of FATP and FABP in the active uptake of long-chain fatty acids is plausible in these cells.

Spatio-temporally differential expression of genes for three members of fatty acid binding proteins in developing and mature rat brains

The chronological changes in the gene expression for three species of cytosolic fatty acid-binding proteins (FABPs) in the rat brain were examined by Northern and in situ hybridization analyses. The expression for heart(H)-FABP became evident after birth, with a gradual increase and confined to the gray matter, suggesting that the expression of H-FABP mRNA is neuron-specific in postnatal brain. The expression for brain(B)-FABP was very intense in the ventricular germinal zone, without expression in the cerebellar external granule cell layer, suggesting the dominant expression in the cells of glial lineage. B-FABP mRNA was transiently expressed in perinatal gray as well as white matter and the expression in glial cells persists only in the olfactory nerve fiber layer at the adult stage. On the other hand, the expression for skin type(S)-FABP was evident in the both ventricular germinal zone and cerebellar external granule cell layer, suggesting the expression in cells of neuronal lineage. The expression for S-FABP was evident in the prenatal gray matter and S-FABP mRNA was expressed in glial cells at early postnatal stage, whereafter the expression decreased to, but remained at weak levels in the adult brain. Discrete functions of the three FABPs were suggested in neurons and glia differentially at various developmental stages.

Distribution of GAP-43 mRNA in the immature and adult cerebellum: a role for GAP-43 in cerebellar development and neuroplasticity

Expression of GAP-43 mRNA in the rat cerebellum and inferior olivary nucleus was examined at birth, during postnatal development and in the adult by both Northern and in situ hybridization. Northern blot analysis revealed that cerebellar GAP-43 mRNA expression increases from birth to postnatal day (PD) 7 and then declines to a lower level in the adult. At birth, in situ hybridization experiments showed intense labeling of GAP-43 mRNA in the premigratory, but not the germinal, zone of the cerebellar external granule cell layer. Localization of GAP-43 within the premigratory zone, a layer containing post-mitotic granule cells, indicates that granule cells begin expressing GAP-43 mRNA after final mitosis and during axonal outgrowth of the parallel fibers. The deep cerebellar nuclei and the inferior olive were also intensely labeled at birth. GAP-43 mRNA was localized in granule cells during their migration through the molecular layer of the developing cerebellum and after their arrival in the internal granule cell layer. By PD 21, the pattern of GAP-43 expression was similar to that observed in the adult; GAP-43 mRNA was localized to the internal granule layer and the inferior olive with minimal to no hybridization in the deep cerebellar nuclei and none in the molecular layer. Purkinje cells were devoid of GAP-43 mRNA throughout the postnatal and adult periods. In light of our observations, we propose that GAP-43 is a critical factor in granule cell differentiation/migration, as well as in parallel and climbing fiber axonal outgrowth and synaptogenesis during development. Localization of GAP-43 mRNA within granule and inferior olivary cells of adult animals indicates that GAP-43 protein observed in the molecular layer is transported from these cells to their terminals in the molecular layer suggesting that GAP-43 is also an intrinsic presynaptic determinant in cerebellar neuroplasticity.

Cerebellar dysplasias in humans: development and possible relationship to glial and primitive neuroectodermal tumors of the cerebellar vermis.

Cerebellar dysplasias are commonly found in the white matter and nodulus of the vermis in newborns and are particularly prominent in infants with trisomy 13-15 and trisomy 18 syndromes. Little is known of the developmental biology of these structures. We have studied the development of cerebellar dysplasias in human fetuses ranging from 15 to 32 weeks gestational age and from 11 days to 15 months postnatal. The expression of developmentally regulated neuronal and glial polypeptides was investigated by immunohistochemistry using a panel of extensively characterized monoclonal antibodies. Dysplasias were first observed at 15 weeks gestation as irregularly distributed, parenchymal or perivascular clusters of primitive cells in the inferior vermis. These cell clusters resembled primitive neuroepithelial cells or cells of the cerebellar external granule cell layer and they persisted into postnatal life. They retained the capacity to undergo cell division and were weakly reactive for the low affinity nerve growth factor receptor but were negative for all other neuronal or glial proteins at all gestational ages. At about 20 weeks gestation, cerebellar dysplasias become more complex with the appearance of ganglion cells which matured histologically and phenotypically in parallel with normal dentate neurons and Purkinje cells. These dysplasias often contained a prominent glial component which was identified by immunostaining for glial fibrillary acidic protein. Our findings suggest that midline cerebellar dysplasias are normal variants of development. Whether the mitotically active cells comprising these dysplasias are targets for neoplastic transformation into cerebellar primitive neuroectodermal tumors or other types of childhood tumors such as pilocytic astrocytomas or atypical teratoid/rhabdoid tumors remains speculative.

Postnatal taurine deficiency in the kitten results in a persistence of the cerebellar external granule cell layer: correction by taurine feeding.

Dietary deprivation of taurine in pregnant cats from approximately 1 week prior to giving birth is sufficient to reduce substantially the taurine concentration in feline milk but does not result in any abnormalities in kittens at birth. Kittens nursing on this low taurine milk have a lower growth rate than normal, have lower tissue taurine concentrations, and 8 weeks after birth have a persistence of cells in the cerebellar external granule cell layer. Mitotic figures are present also, indicating that cell division is occurring still, an event which normally is completed 3-4 weeks after birth. Daily oral supplementation with 40 mumoles taurine increases the growth rate almost to the level of normally nurtured kittens and results in normal tissue taurine concentrations and apparently normal migration of cells in the cerebellum. These findings indicate that nutritional taurine supplied in the milk is involved in the normal ontogeny of the cerebellum and that a taurine deficiency at this stage of development results in a maturational delay.