Developing Rat Cerebellar Cortex Research Articles

Insulin-Like Growth Factor-1 Receptor Is Differentially Distributed in Developing Cerebellar Cortex of Rats Born to Diabetic Mothers.

Insulin-like growth factor-1 (IGF-1) has an important role in development of the central nervous system (CNS). Maternal diabetes is associated with a higher risk of developmental abnormalities in their offspring including motor dysfunction and learning deficits. The present study aimed to investigate the effects of maternal diabetes on the distribution pattern of IGF-1 receptor (IGF-1R) in the developing rat cerebellar cortex. Wistar female rats were maintained diabetic from a week before pregnancy through parturition, and male offspring was killed at P0, P7, and P14. In spite of P0, there was a significant increase in the total cerebellar volume in the pups born to diabetic mothers. In diabetic group, the IGF-1R+ granular cell densities in internal granular (IGL) and molecular (ML) layers were increased at P0. Moreover, the number of positive granular and Purkinje cells in the IGL of diabetic neonates' cerebellum was reduced in comparison with the control group at P7 and P14. There were no differences either in volume or in the number of IGF-1R+ cells in the layers of the cerebellar cortex between the insulin-treated diabetic group and controls. Our data indicate that diabetes in pregnancy strikingly influence the localization of IGF-1R in the developing cerebellar cortex.

TGFβ-Smad2 Signaling Regulates the Cdh1-APC/SnoN Pathway of Axonal Morphogenesis

Axon growth is critical to the establishment of neuronal connectivity. The E3 ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) and its substrate the transcriptional modulator SnoN form a cell-intrinsic pathway that orchestrates axonal morphogenesis in the mammalian brain. How the Cdh1-APC/SnoN pathway is controlled in the nervous system remained unknown. Here, we report that the TGFbeta-regulated signaling protein Smad2 plays a key role in regulating the Cdh1-APC/SnoN pathway in neurons. We find that Smad2 is expressed in primary granule neurons of the developing rat cerebellar cortex. The Smad signaling pathway is basally activated in neurons. Endogenous Smad2 is phosphorylated, localized in the nucleus, and forms a physical complex with endogenous SnoN in granule neurons. Inhibition of Smad signaling by several distinct approaches, including genetic knock-down of Smad2, stimulates axonal growth. Biochemical evidence and genetic epistasis analyses reveal that Smad2 acts upstream of SnoN in a shared pathway with Cdh1-APC in the control of axonal growth. Remarkably, Smad2 knock-down also overrides the ability of adult rat myelin to inhibit axonal growth. Collectively, our findings define a novel function for Smad2 in regulation of the Cdh1-APC/SnoN cell-intrinsic pathway of axonal morphogenesis, and suggest that inhibition of Smad signaling may hold therapeutic potential in stimulating axonal growth after injury in the CNS.

Stabilization of neurites in cerebellar granule cells by transglutaminase activity: identification of midkine and galectin-3 as substrates

The formation of covalent isopeptide cross-links between cell surface protein molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. et al. (1995) Neuroscience 65, 1063–1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebellar cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryonic dorsal rhombic lip, which contain granule neuron precursors. A possible biological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-007. Inhibition of cross-linking activity did not influence the number of neurites formed by granule neurons, but caused the destabilization of neurites during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formation (bifurcation). Inactivation of cross-linking activity prevented the formation of fascicles between neurites only when cells were cultured on a biomatrix surface. Two glial proteins involved in cell–extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possible non-pathological role for transglutaminase in the control of axon collateral branching by developing granule neurons in the cerebellar cortex is discussed.

Metabotropic glutamate receptor 2/3 immunoreactivity in the developing rat cerebellar cortex.

In adult rat cerebellar cortex, the metabotropic glutamate receptors (mGluRs) 2 and 3 (mGluR2/3) are present in somata, dendrites, and terminals of Golgi cells as well as in presumed glial processes (Ohishi et al. [1994], Neuron 13:55-66). In the present study, spatiotemporal changes in immunostaining for mGluR2/3 were examined in postnatal rat cerebellar cortex. mGluR2/3-immunoreactive Golgi cell somata appeared first in the internal granular layer at postnatal day 3 (P3) and were restricted to lobules IX and X; however, by P5, they were present in all lobules. Immunoreactive Golgi cell axons were adult-like, appearing as tortuous fibers with clusters of varicosities. They were observed first in the internal granular layer at P7 and increased in number and complexity with time. It was confirmed that mGluR2/3-immunoreactive Golgi cell axon terminals belong to the synaptic glomerulus by P10. Immunoreactive Golgi cell dendrites extending into the molecular layer became prominent after P15. By that time, the immunostaining pattern was characteristic of Golgi cells, as seen typically in adults. Many intensely immunoreactive radial processes existed at birth (P0). These traversed the molecular and external granular layers, reaching the pial surface in every cerebellar lobule. Because they showed coimmunoreactivity for glial fibrillary acidic protein, they were confirmed to be Bergmann glial fibers. After P9, they began to lose immunoreactivity at the portion corresponding to the molecular layer, while an immunostained granular pattern appeared in that layer. Immunoreactive radial processes, however, remained in the external granular layer, and finally, at P21, they disappeared together along with the external granular layer. Granular staining in the molecular layer reached background levels at this time. These spatiotemporal changes in mGluR2/3 distribution suggested that there may be distinct roles for mGluR2/3 in Golgi cells and Bergmann glial cells during the early postnatal period. mGluR2/3 in Golgi cells might be associated closely with systemic maturation, whereas mGluR2/3 in Bergmann glia might be needed for neuron-glia interactions related to granule cell development.

Expression of dna topoisomerase II isoforms and apoptosis in developing rat cerebellar cortex

Expression of dna topoisomerase II isoforms and apoptosis in developing rat cerebellar cortex

1120 Expression of DNA topoisomerase II isoforms and apoptosis in developing rat cerebellar cortex

1120 Expression of DNA topoisomerase II isoforms and apoptosis in developing rat cerebellar cortex

Localization of HPC-1/syntaxin 1 in developing rat cerebellar cortex.

In adult rat cerebellum, HPC-1/syntaxin 1 is detected at high density on the plasma membrane of the non-synaptic region of parallel fibers in addition to the synaptic terminal membranes and the synaptic vesicles (Koh, S., Yamamoto, A., Inoue, A., Inoue, Y., Akagawa, Y., Kawamura, Y., Kawamoto, Y., and Tashiro, Y. (1993). J. Neurocytology 22: 995-1005). To assess the possibility that HPC-1/syntaxin 1 participates in the morphogenesis of the nervous system, we examined changes in the localization of HPC-1/syntaxin 1 during postnatal development of the molecular layer of the rat cerebellum. HPC-1/syntaxin 1 appeared in the granule cells in the outer granule cell layer in 3-days-old rat cerebellum when the formation of synapses and the appearance of a synaptic vesicle protein, synaptophysin, had not yet been observed in the molecular layer. At this stage, the granule cells began to form parallel fibers. Confocal laser microscopy and immuno-electron microscopy showed that HPC-1/syntaxin 1 was localized on the extruding plasma membrane of the granule cells to form parallel fibers. In 8-days-old rats, synapses formed between the parallel fibers and the developing dendrites of Purkinje cells, and the HPC-1 immunoreactivity appeared on the axons of parallel fibers and on the synapses. In 21-days-old rats, the HPC-1/syntaxin is involved in the formation of the molecular layer, especially in the axonal growth of the parallel fibers.

Development of cholinesterase histochemical staining in cerebellar cortex: Transient expression of “nonspecific” cholinesterase in Purkinje cells of the nodulus and uvula

Patterns of “nonspecific” cholinesterase (ChE) and acetylcholinesterase (AChE) activity were studied in developing rat cerebellar cortex by enzyme histochemistry and light and electron microscopy. Three types of ChE histochemical reaction product were observed in cerebellar cortex: (i) ChE is found in capillary endothelium throughout the cerebellum. Capillary ChE staining is present by the time of birth and continues into adulthood. (ii) ChE is found in radial glial fibers and their parent cell bodies, the Golgi epithelial cells. Radial glial fiber staining is most intense during the first 3 weeks of postnatal life. (iii) ChE is found in Purkinje cells of the nodulus and ventral uvula. No ChE staining of Purkinje cells was seen in other parts of the cerebellum. ChE staining of Purkinje cells appears to be transient, first appearing at Postnatal Day 2 (P2), reaching peak intensity at P7–9, and decreasing to adult levels by P16. AChE activity displays a pattern markedly different from ChE, with staining in deep cerebellar nuclei, in putative mossy fiber terminals, and in Golgi neurons of cerebellar cortex. No evidence was found for transient AChE staining in Purkinje cells in any part of the cerebellum. The function of transiently expressed ChE activity in developing Purkinje neurons is unknown, but may be related to reorganization of cerebellar cortical circuitry associated with growth of mossy fiber afferents.

Expression of proenkephalin mRNA in developing cerebellar cortex of the rat: expression levels coincide with maturational gradients in Purkinje cells

The cellular localization of proenkephalin (PE) mRNA expression was systematically examined in midsagittal (vermal) sections of the developing rat cerebellar cortex by in situ hybridization. PE mRNA was initially detected in Golgi cells of postnatal day 7 (PND 7) rats and in each group thereafter. Moreover, PND 7 rats also displayed an intense layer of PE mRNA hybridization signal over the Purkinje mell layer. By PND 14,mdistinct cellular labeling was observed in a subpopulation of Purkinje cells in all lobules of the vermis except lobule III. At PND 7 and 14, the area and level of intensity of Purkinje cell associated PE mRNA hybridization signal followed a gradient that was most intense caudally but then decreased rostrally. At PND 21, the proportion of labeled Purkinje cells and the intensity of PE hybridization signal was evenly dispersed between the anterior and posterior lobules of the cerebellar vermis. PE hybridization signal was not detected in the developing neural cells of the external granular layer or the interneurons of the molecular layer in the vermis. These results indicate that the ontogeny of PE mRNA expression in Purkinje cells is developmentally regulated since levels of expression closely follow the chronological order of settling and maturation of these neurons. Based on prior evidence that endogenous opioids inhibit the growth of Purkinje cell dendrites and dendritic spines, PE expression is likely to be important for Purkinje cell maturation.

Extralysosomal localization of acid paranitrophenylphosphatase activity in the developing rat cerebellar cortex.

Using para-nitrophenyl phosphate as substrate, extralysosomal localization of acid phosphatase activity was investigated in the developing and adult rat cerebellar cortex, particularly in the Purkinje (P) cells. Light microscopic observations revealed that acid para-nitrophenylphosphatase (acid NPPase) activity appeared increasingly in the P cell and molecular layers throughout their postnatal development. Under an electron microscope, the acid NPPase activity was found in the cisterns of nuclear envelope and endoplasmic reticulum of the P cells, in addition to the lysosomal activity. In the synapses on the P cells, the acid NPPase activity was predominantly localized in the presynaptic membrane of such parallel fibers. The activity of nonlysosomal acid NPPase was inhibited by fluoride (0.1mM) and CuSO4 (1.0mM), but not by L(+)-tartrate (10mM). The present results are discussed in a possible relationship to an activity of phosphotyrosine protein phosphatase in the developing rat cerebellar cortex.

Effects of Hypothyroidism on the Detyrosylation of α-Tubulin in the Parallel Fibers of the Developing Rat Cerebellar Cortex

The immunocytochemical staining of tyrosylated alpha-tubulin using the specific monoclonal antibody YL 1/2 decreases dramatically in the parallel fibers of the rat cerebellum during development. Detyrosylation starts at 14 days in the deeper part of the molecular layer and then spreads over the entire molecular layer within a week. Thyroid deficiency induces a marked delay in alpha-tubulin detyrosylation in the parallel fibers. Detyrosylation begins only at the age of 28 days in hypothyroid animals. Immunobinding assays of detyrosylated and tyrosylated alpha-tubulin concentrations in the cerebellar supernatant of developing rats confirm that thyroid deficiency increases the tyrosylated alpha-tubulin content at 21 and 35 days. Short-term thyroxine treatment (daily from day 8, 10 or 12, to day 14) do not induce the normal pattern of tubulin detyrosylation starting at 14 days, although they do induce parallel fiber lengthening. Daily treatment from birth is required to produce the alpha-tubulin modification found in normal rats. We propose that the microtubule detyrosylation which occurs during axonal maturation is not a prerequisite for axon growth and synaptic differentiation, but may rather be implicated in the stabilization of microtubules in the mature axons.

Regulatory expression of cellular src protein in developing rat cerebellar cortex and olfactory bulb

Regulatory expression of cellular src protein in developing rat cerebellar cortex and olfactory bulb

Autoradiographic localization of ornithine decar☐ylase in cerebellar cortex of the developing rat with [ 3H]α-difluoromethylornithine

Ornithine decar☐ylase was autoradiographically localized in the developing rat cerebellar cortex after intracisternal injection of [ 3H]α-difluoromethylornithine, a specific, irreversible inhibitor of the enzyme. At nine days of age, when cerebellar ornithine decar☐ylase activity is maximal, auto-radiographic grains were distributed over all layers of the cerebellar cortex and throughout the brain stem. Within cerebellar folia, the highest grain density was associated with the molecular layer, whereas the internal and external granule cell layers were less densely labeled. Enhancement of ornithine decar☐ylase activity by intracisternally-administered isoproterenol correspondingly increased the autoradiographic grain density over each layer. Thus much of the polyamine biosynthetic capability needed to support neuronal and/or glial differentiation appears to be associated with the developing cell processes. The combination of [ 3H]α-difluoromethylornithine autoradiography with localized injection techniques provides a potentially powerful tool for the study of the involvement of polyamine biosynthesis in brain development.

Focal axonal swellings in rat cerebellar Purkinje cells during normal development

Focal axonal swellings are characteristic of a wide range of neuropathies. Three neuron-specific monoclonal antibodies have been used to identify focal axonal swellings in the normal developing rat cerebellar cortex. Between 7 and 15 days postnatal, swelling are a common feature of the granular layer and white matter tracts. Using a Purkinje cell-specific antibody, the majority of swellings were shown to occur in Purkinje cell axons. Focal axonal swellings therefore seem to be a normal adjunct of Purkinje cell maturation.

Developmental microvascular architecture of the rat cerebellar cortex.

External and internal microvascular architectures of the developing rat cerebellar cortex, from embryonic day 18 to postnatal day 14 and in adults, were studied using a cerebrovascular casting method for scanning electron-microscopic observation. The external vascularization of the developing cerebellum showed the most significant alteration in vascular morphology at the stage of intensive proliferation of matrix cells in the external granular layer (EGL) from birth to postnatal day 4. It consisted of multiple luminal protrusion of the vessels, septum formation in the lumina, and small, ring-like anastomoses. Moreover, at the end of this stage, these structures of the vessels disappeared and the subarachnoid space was filled with newly-formed microvascular networks. Thereafter, architectural change of the developing pial vessels was mainly accomplished by elongation of each contorted vessel of the network. Concerning internal vascularization, a few vessels connected with the pial vessels were observed in the cerebellar plate forming a loose, simple network in the deeper neural parenchyma before the stage of foliation began. During the period of thickening of the EGL, however, there was no alteration in vascularity of the parenchyma other than the architectural changes proportionate to the newly-formed folia. It was during the synaptogenetic stage in the internal granular layer that the earliest intraneural vascular plexuses were formed. The vascular network in the molecular layer was formed after disappearance of the EGL. These findings suggest that vascular proliferation correlated with EGL-formation pertains to the pial vessels, and not the intraneural ones, which develop after neuronal cell migration in the developing cerebellum has taken place.

Mitochondria in the postnatally developing rat cerebellar cortex: A morphological and biochemical study

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.

Lack of evidence of synaptic contacts by climbing fibre collaterals to basket and stellate cells in developing rat cerebellar cortex

Lack of evidence of synaptic contacts by climbing fibre collaterals to basket and stellate cells in developing rat cerebellar cortex

The effects of early hypo- and hyperthyroidism on the development of rat cerebellar cortex. III. Kinetics of cell proliferation in the external granular layer

The effects of early hypo- and hyperthyroidism on the rates of cell acquisition and proliferation have been studied in the external granular layer (EGL) of the developing rat cerebellar cortex at 10 days of age using quantitative autoradiographic methods. Both altered thyroid reduce the rate of cell acquisition in the EGL, but appear to do so for different reasons. Hyperthyroidism shortens the average length of the cell cycle by decreasing the duration of the pre-DNA synthetic phase (G 1), indicating that excess thyroxine may exert a direct effect on the EGL. This action involves the early onset of neuronal differentiation (cessation of proliferation) 46 which presumably leads to the observed decrease in the rate of cell acquisition (increased doubling time). Such differentiating cells do not, however, leave the proliferative zone or the EGL prematurely, resulting in a reduced labeling index, mitotic index, and growth fraction as non-dividing cells dilute the proliferating cell population. Hypothyroidism, on the other hand, leads to no significant change in the length of the cell cycle or in the mitotic index, but causes a decreased labeling index and growth fraction, as well as a reduced rate of cell acquisition (increased doubling time). No significant change in the amount of cell death in the EGL could be found to explain this apparent discrepancy between the rate of cell proliferation (cell cycle length) and cell acquisition. The answer to this puzzle appears to lie in the mitotic index, which is not affected to the same extent as the labeling index, although it is also slighly reduced. If cells were to remain longer in mitosis, this could result in a decreased labeling index and growth fraction but nearly normal mitotic index and cell cycle length (as measured using the % labeled mitoses method), since those cells dropping out of the cycling population would be counted as mitoses. This hypothesis is supported by the build-up of labeled mitoses with time after [ 3H]thymidine injection as more and more labeled cells reach the border between mitosis and interphase (M/G 1) and fail to pass it, as well as the increased time required for the labeled cell population to double. Such mitotic cells appear to move from the proliferative zone of the EGL (decreased mitotic index) to the subproliferative zone where they accumulate, primarily in anaphase-telophase (increased index of anaphase-telophase mitotic figures), but eventually resume proliferation, as evidenced by a significantly increased labeling index in this zone. These results indicate that the rate of cell proliferation may actually be reduced in hypothyroidism corresponding to the decreased rate of cell acquisition, but this effect is masked by the prolongation of mitosis when analyzed by the method of % labeled mitoses. Possible mechanisms underlying the effects of hypo- and hyperthyroidism on cell acquisition and proliferation in the EGL are discussed. These results indicate the importance of proper levels of thyroid hormone during postnatal development for normal cell acquisition and proliferation in the rat cerebellar cortex.