Cytotoxic effects of methylnitrosourea on developing brain

The effect of methylnitrosourea (MNU) on cerebellar and cerebral DNA, RNA, protein, lysosomal enzymes (acid DNase, RNase, phosphatase, and beta-glucuronidase), and 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activities was studied in rats from birth through 12 days of age. Subcutaneous injection of MNU in a dose of 0.625 mmol/kg caused a suppression of increase in weights and content of DNA, RNA, and protein of cerebellum, but no changes in those of the cerebrum or in body weight. Ratios of protein and RNA to DNA were substantially elevated by MNU in the cerebellum but not in the cerebrum. Acid DNase and acid RNase activities of MNU-treated rats were significantly elevated beyond the increase of these activities in controls in the cerebellum, but no change in these activities by MNU was observed in the cerebrum. A slight elevation in acid phosphatase activity was observed in the cerebellum but not in the cerebrum after MNU pretreatment. Beta-glucuronidase and 2',3'-CNPase activities were not changed in the cerebellum or in the cerebrum. These results suggest that in the developing brain, especially in the cerebellum at the mitotic stage, MNU caused cell damage and inhibited cell mitosis.     

Impaired Structural and Functional Development of Cerebellum Following Gestational Exposure of Deltamethrin in Rats: Role of Reelin

Reelin is an extracellular matrix molecule that is involved in the normal development of the cerebellar lamination, Bergmann glial fibres alignment, Purkinje cell monolayer arrangement and granule cell migration. In this study, we have examined the effects of maternal exposure of deltamethrin (DLT), a type II pyrethroid insecticide, on the structural and functional development of rat cerebellum during postnatal life. DLT (0.75 mg/kg body weight, intraperitoneally dissolved in dimethylsulphoxide) was administered in timed pregnant rats during two different gestational time periods, i.e. gestational days of 7–10 and 11–14, respectively. In DLT exposed rats, a significant overexpression of reelin was observed in the cells of the external granule cell layer (EGL) and internal granule cell layer along with an ectopic expression of reelin in the EGL as well as in the migrating granule cells just below the EGL, revealing an arrest of granule cell migration in this zone. Mis-orientation and hypertrophy of the Bergmann glial fibres further hampered the journey of the granule cells to their final destination. Possibly reelin overexpression also caused misalignment of the Purkinje cells and inhibited the neurite growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbour. Thus, it is proposed that the DLT exerts its neurotoxic effects possibly via the intracellular accumulation and low release of reelin leading to an impaired granule cell and Purkinje cell migration, inhibition of neurite outgrowth and reduced spine density. Such impaired cerebellar development leads to motor coordination deficits.     

Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

Effects of methylazoxymethanol given at different stages of postnatal life on development of the rat brain. Comparison with those of thyroid deficiency

Newborn rats were treated at different stages of their development with low doses of methylazoxymethanol acetate. The postnatal increase of the DNA content of the cerebrum did not differ from that of controls. In the cerebellum, the DNA content was transitorily reduced, but later, the external granular layer became thicker and DNA deposition increased in comparison with controls; finally, the cerebellar DNA returned to a normal value. Morphological abnormalities of the cerebellum, abnormal orientation of migrating cells, scattering of Purkinje cell bodies within the internal granule cells and specially striking abnormalities of the morphology and orientation of Purkinje cell dendrites were noted in rats treated with MAM from birth to day 3. The effects on the Purkinje cell morphogenesis persisted but were much less marked when MAM was given from 4 to 7 or from 8 to 11 days. Neonatal thyroid deficiency, as MAM-treatment between days 0 and 3, leads to an abnormal position of Purkinje cell bodies within the cerebellar cortex; it also leads to morphological abnormalities of their dendritic arborization which closely resemble those observed after MAM-treatment during the second postnatal week. It also alters the cell formation in the cerebellum. Thyroid deficiency probably exerts its effect on cell formation earlier than previous biochemical studies have shown. On another hand, the morphological abnormalities of Purkinje cell arborizations in the thyroid-deficient animals may be partly due to the perturbations of cell formation which persist later in the cerebellum.     

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.     

Histological and ultrastructural features in the early stage of Purkinje cell degeneration in the cerebellar calcification (CC) rat.

The cerebellar calcification (CC) rat is a new neurodegenerative mutant with severe Purkinje cell loss and symmetrical calcifications in the cerebellar cortex manifesting ataxia: lack of coordination in body movements. In the present study, histopathological features were examined in the Purkinje cell degeneration in postnatal homozygous suckling rats without clinical signs, which were genotyped by microsatellite markers. In addition, the calcified Purkinje cells were investigated ultrastructurally and elemental analysis was performed on the deposits. Body weight of the homozygous (cc/cc) rats was already slightly lower compared with the heterozygotes (cc/+) in the neonatal stage. The degeneration of the Purkinje cells in the cc/cc rats was recognized obviously in lobules VI, VII, VIII and IX from 14 days after birth, a few days before the appearance of the ataxic behavior. The Purkinje cells in the region along the fissure between the VIII and IX lobule areas were intensely positive for periodic acid-Schiff reaction specific to glycoconjugates, and in this region, calcium depositions were weakly positive for von Kossa's stain. Electron microscopy also revealed that the calcified Purkinje cells possessed numerous electron-dense bodies containing inclusions with cystic structures such as vesicles, mitochondria and lysosomes, and these bodies were mainly composed of calcium and phosphorous. These findings suggest abnormal storage of glycoconjugates might be a trigger of Purkinje cell degeneration and serves as a matrix for accumulation of calcium phosphate in the cerebellum of CC rats.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Cellular Distribution of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Staggerer Mutant

The distribution of cerebellar gangliosides was studied in staggerer (sg/sg) mutant mice, where the majority of granule cells die after completing their migration across the molecular layer. In addition, the external granule cell layer in sg/sg mice persists longer than in normal mice. Moreover, in the sg/sg cerebellum, Purkinje cells are significantly reduced in number, and almost none have tertiary branchlet spines. The loss of Purkinje cells and granule cells in sg/sg mice is accompanied by an early-onset reactive gliosis that continues through adulthood. By correlating changes in ganglioside composition with the well-documented histological events of cerebellar development in normal and sg/sg mice, we obtained strong evidence for a nonrandom cellular distribution of gangliosides. The sharpest reduction in the GD1a content of sg/sg cerebellum occurred after 15 days of age, coincident with granule cell loss. GT1a, on the other hand, was significantly reduced from 15 through 150 days in the sg/sg mice. GD3 is a major ganglioside of the undifferentiated granule cell, but it becomes rapidly displaced by the more complex gangliosides with the onset of granule cell maturation. In the sg/sg mice, GD3 persisted at abnormally high levels from 15 to 28 days and then accumulated through adulthood. These findings, and those from other cerebellar mouse mutants, suggest that GD1a is enriched in granule cells and that GT1a is enriched in Purkinje cells. Our findings also suggest that GT1a is more concentrated in branchlet spines than in other regions of the Purkinje cell membrane. GT1b appears to be enriched in both granule cells and Purkinje cells, whereas GM1 appears to be enriched in myelin. Furthermore, the apparent persistence of the embryonic ganglioside GD3 in sg/sg mice results from an early-onset reactive gliosis, together with a partial retardation in granule cell maturation. The accumulation of GD3 beyond 28 days reflects the continued accretion of GD3 in reactive glia.     

An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies]

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

Chromatin matrix activity in Purkinje cells and granular cells of the rat cerebellar cortex during postnatal differentiation

Moore's method used for the examination of chromatin template activity in Purkinje and granule cells of 7, 14, 30 days and 3 months old rate cerebellar cortex has shown the age-dependent changes during differentiation period. The histograms for Purkinje cells have demonstrated that all neurons were distributed into 3 groups of activity according to their nuclear labelling. The cell percentage in each group varied during ontogenesis.     

Mapping the development of cerebellar Purkinje cells in zebrafish

The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1174–1188, 2015     

Granule cell raphes in the developing mouse cerebellum

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.     

Interactions between cerebellar Purkinje cells and their associated astrocytes

Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic spines, but the Purkinje cells had astrocytic sheaths, their somata were not hyperinnervated, the somatic spines had disappeared, complex spike discharges predominated, and membrane input resistance was like that of Purkinje cells in untreated control cultures. When cerebellar cultures without granule cells and glia were transplanted with granule cells and/or glia from another source, a series of changes occurred that included stripping of excess Purkinje cell axosomatic synapses by astrocytic processes, reduction of heterotypical axospinous synapses in the presence of astrocytes, disappearance of Purkinje cell somatic spines with astrocytic ensheathment, and proliferation of Purkinje cell dendritic spines after the introduction of astrocytes. Dendritic spine proliferation was followed by formation of homotypical axospinous synapses when granule cells were present or persistence as unattached spines in the absence of granule cells. The results of these studies indicate that astrocytes regulate the numbers of Purkinje cell axosomatic and axospinous synapses, induce Purkinje cell dendritic spine proliferation, and promote the structural and functional maturation of Purkinje cells.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Structure of the fetal sheep brain in experimental growth retardation

A quantitative morphometric study of brain development has been made in growth-retarded fetal sheep. Intrauterine growth retardation was induced by removal of endometrial caruncles in the ewe prior to conception thereby reducing the size of the placenta in a subsequent pregnancy. Total brain and cerebellar weights were reduced by 21% (P less than 0.002) and the cerebrum by 20% (P less than 0.05) in the growth-retarded fetuses at 139 +/- 1 day (term = 146 days) compared with age matched control fetuses. Measurements of mean neuronal diameters were made on Purkinje cells, cerebellar granule cells, cortical cells in the motor and visual areas and hippocampal pyramidal cells; none were significantly different from control values. In growth-retarded fetuses compared with controls, there was a significant reduction in the thickness of the motor and visual cortices and the numerical density of neurones was significantly higher in these areas. In the cerebellar vermis, the number of Purkinje cells per unit surface area of Purkinje cell layer was higher, the numerical density of granule cells was significantly higher concomitant with a reduction in the area of the inner granular layer, and the area of the molecular layer was also reduced. In the hippocampal formation, the numerical density of pyramidal neurones was higher and the width of the stratum moleculare (dentate gyrus) was reduced. Migration of pyramidal neurones from the germinal layer to stratum pyramidale was not affected. These findings indicate that intrauterine growth retardation does not markedly affect cell size or neuronal migration (in the hippocampus) but does cause a significant reduction in the growth of the neuropil in the cerebellum, motor and visual cortices and the hippocampal formation.     

DEVELOPMENTAL FEATURES OF CEREBELLAR HYPOPLASIA AND BRAIN BILIRUBIN LEVELS IN A MUTANT (GUNN) RAT WITH HEREDITARY HYPERBILIRUBINAEMIA 1

Abstract— The marked cerebellar hypoplasia found in the homozygous (jj) Gunn rat with hereditary unconjugated hyperbilirubinaemia may provide an explanation of bilirubin neurotoxicity in vivo. In the jj Gunn rat. Purkinje cells were nearly selectively affected in the cerebellar cortex, and the cerebellar weight showed no increase after 10 days of age. The development-dependency of the cerebellar lesion was supported by the observation that the cerebellar lobuli which developed earlier were less affected. Brain bilirubin in the developing jj Gunn rat was determined by a spectrophotometric method, and was found to be extremely low (1–3 μg). The level of brain bilirubin decreased after birth, and showed little correlation with the level of bilirubin free of albumin which correlated clearly with total serum bilirubin level even in the neonate. These findings suggest that there is an affinity of brain tissue for bilirubin associated with the blood-brain barrier to bilirubin. No significant difference was found between the levels of bilirubin in the cerebellum and those of other brain regions in jj Gunn rat. These results seem to imply that the development-dependency of cerebellar hypoplasia in the jj rat may be due to the characteristic nature of rat cerebellar development, i.e. the postnatal neurogenesis. and not to changes in brain bilirubin levels. In the jj Gunn rat. cerebellar cell proliferation appears to be in some way affected by bilirubin during cerebellar development.     

Changes in TRPC channel expression during postnatal development of cerebellar neurons

In the brain, classical (canonical) transient receptor potential (TRPC) channels are thought to be involved in different aspects of neuronal development. We investigated the developmental expression profile of TRPC channels in rat cerebellum during the first 6 weeks after birth. TRPC3 expression is significantly up-regulated whereas TRPC4 and TRPC6 expression are significantly down-regulated over this period of time. TRPC3 expression is mainly found on Purkinje cells and their dendrites, suggesting that the increase in TRPC3 expression reflects development of the dendritic tree of Purkinje cells. TRPC4 expression was restricted to granule and their precursor cells. TRPC6 expression is found on Purkinje cell bodies, on mature granule cells in the internal granule cell layer (but not their precursors) and interneurons in the molecular layer. The decrease in TRPC4 expression suggests that it is required for proper granule cell development whereas the decrease in TRPC6 expression is presumably correlated with interneuron development. Moreover, we demonstrate the presence of functional TRPC channels on Purkinje cell dendrites that are activated following stimulation of metabotropic glutamate receptors. Our results reveal cell-specific expression patterns for different TRPC proteins and suggest that developmental changes in TRPC protein expression may be required for proper postnatal cerebellar development.     

Development of longitudinal patterns in the cerebellum of the chicken (Gallus domesticus): a cytoarchitectural study on the genesis of cerebellar modules.

The cytoarchitecture of the cerebellum has been studied in chicken embryos from day 3-20 using serial sections stained with cresylviolet, haematoxylin-eosin and toluidine blue. Three periods have been distinguished in cerebellar development on a basis of cytoarchitectonic characteristics. Of these periods the middle one, which lasts from the 8th to the 15th day, is marked by two subsequent transient longitudinal cytoarchitectonic patterns in the cortical anlage. The first pattern, which exists between days 8 and 11, consists of 4 longitudinal Purkinje cell clusters (of the first order) at either side of the midline. The second pattern, which is most distinct and complete during embryonic days 12-14, is caused by specific localizations of otherwise few, early inwardly migrating granule cells from the external cerebellar matrix (so-called granule raphes), which pass through the layer of Purkinje cell clusters of the first order and thus subdivide these latter into smaller entities: Purkinje cell clusters of the second order. The number of these latter (6 or 7 and 11 or 12 in the anterior and posterior lobes, respectively) correspond to the number of parasagittal modules, which can be discerned on a basis of the organization of fiber connections of the adult cerebellar cortex. Thanks to this similarity various hypotheses can be formulated concerning the significance of the transient cytoarchitectonic patterns in the primitive cortex for the genesis of the modular organization of the cerebellum.     

Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures

Purkinje cells are vulnerable to a number of physical, chemical, and genetic insults during development and maturity. Normal development of these cells depends on the cell-cell interactions between granule and astroglial cell populations. Apoptotic death in Purkinje neurons had been shown to be associated with cell cycle activation, and new DNA synthesis is associated with Purkinje cell death in staggerer and lurcher mutant mice. Here using an in vitro organotypic slice culture model from 9 (P9) and 4 days (P4) old postnatal rats we show that the cyclin dependent kinase (cdk) inhibitors (roscovitine, olomoucine, and flavopiridol) protect the Purkinje cells from cell death. The results are more pronounced in the cerebellar sections from P4 rats. Analysis of Purkinje neurons in sections from P4 rats after 1 week of culturing showed that while there were very limited calbindin positive neurons in the untreated sections the cdk inhibitor treated sections had a notably higher number. Although treatment with cdk inhibitors inhibited Purkinje cell loss significantly, the morphology of these neurons was abnormal, with stunted dendrites and axons. Since the retinoblastoma protein (Rb) is the major pocket protein involved in determining the differentiated state of neurons we examined the effect of over-expressing Rb in the organotypic cultures. Rb overexpression significantly inhibited the Purkinje cell death and these neurons maintained their normal morphology. Thus our studies show that the cell death in Purkinje neurons observed in organotypic cultures is cell cycle dependent and the optimal survival requires Rb.