Experimental Sey mouse chimeras reveal the developmental deficiencies of Pax6-null granule cells in the postnatal cerebellum

Pax6 has been implicated in cerebellar granule cell development, however the neonatal lethality of the Sey/Sey mutant has precluded a more detailed study of this late developing neuronal type. In this study we use experimental mouse chimeras made from wildtype and Pax6-null embryos to circumvent early lethality and assess the developmental potential of mutant cells in the construction of the cerebellum. We have identified the granule cell as a direct target of mutant gene action, with glia and Purkinje cells being affected in what is largely a non-cell autonomous manner.Most dramatically, in postnatal day 21 (P21) chimeras, mutant cells are largely absent in the anterior and posterior cerebellum while present in central lobules, but amidst disorganized cerebellar architecture. Analysis of P0/1 and P10 chimeras demonstrates a profound temporally based defect where mutant cells colonize the anterior and posterior EGL but fail to migrate to the IGL. Mutant granule cells in the central lobules can reach the IGL in an abnormal manner, with large streams of cells forming raphes through the molecular layer.These studies provide new insights into the role of Pax6 in postnatal cerebellar development that pinpoint the granule cell as an intrinsic target of the mutant gene and key events in the life of the developing granule cell that depend upon normal Pax6 expression.     

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.     

Cultured granule cells and astrocytes from cerebellum differ in metabolizing sphingosine

Sphingosine metabolism was studied in primary cultures of differentiated cerebellar granule cells and astrocytes. After a 2-h pulse with [C3-(3)H]sphingosine at different doses (0.1-200 nmol/mg of cell protein), both cell types efficiently incorporated the long chain base; the percentage of cellular [(3)H]sphingosine over total label incorporation was extremely low at sphingosine doses of <10 nmol/mg of cell protein and increased at higher doses. Most of the [(3)H]sphingosine taken up underwent metabolic processing by N-acylation, 1-phosphorylation, and degradation (assessed as (3)H(2)O released in the medium). The metabolic processing of exogenous sphingosine was extremely efficient in both cells, granule cells and astrocytes being able to metabolize, respectively, an amount of sphingosine up to 80- and 300-fold the cellular content of this long chain base in 2 h. At the different doses, the prevailing metabolic route of sphingosine was different. At lower doses and in a wide dose range, the major metabolic fate of sphingosine was N-acylation. With increasing doses, there was first increased sphingosine degradation and then increased levels of sphingosine-1-phosphate. The data demonstrate that, in neurons and astrocytes, the metabolic machinery devoted to sphingosine processing is different, astrocytes possessing an overall higher capacity to synthesize the bioactive compounds ceramide and sphingosine-1-phosphate.     

The electro-dynamics of the dendritic space in Purkinje cells of the cerebellum

The functional geometry of the reconstructed dendritic arborization of Purkinje neurons is the object of this work. The combined effects of the local geometry of the dendritic branches and of the membrane mechanisms are computed in passive configuration to obtain the electrotonic structure of the arborization. Steady-currents applied to the soma and expressed as a function of the path distance from the soma form different clusters of profiles in which dendritic branches are similar in voltages and current transfer effectiveness. The locations of the different clusters are mapped on the dendrograms and 3D representations of the arborization. It reveals the presence of different spatial dendritic sectors clearly separated in 3D space that shape the arborization in ordered electrical domains, each with similar passive charge transfer efficiencies. Further simulations are performed in active configuration with a realistic cocktail of conductances to find out whether similar spatial domains found in the passive model also characterize the active dendritic arborization. During tonic activation of excitatory synaptic inputs homogeneously distributed over the whole arborization, the Purkinje cell generates regular oscillatory potentials. The temporal patterns of the electrical oscillations induce similar spatial sectors in the arborization as those observed in the passive electrotonic structure. By taking a video of the dendritic maps of the membrane potentials during a single oscillation, we demonstrate that the functional dendritic field of a Purkinje neuron displays dynamic changes which occur in the spatial distribution of membrane potentials in the course of the oscillation. We conclude that the branching pattern of the arborization explains such continuous reconfiguration and discuss its functional implications.     

Postnatal differentiation of the granule cells in the hippocampus and cerebellum: A histochemical study

Summary Postnatal differentiation of the granule cells in the fascia dentata and the granule cell layer of cerebellum is described in terms of enzymatic activity with NADPH-R, NADH-R, SDH and Ac. Ph. In relation to this the neuropil, where the granule cells have their dendritic ramifications, has been studied. The granule cells in both the regions, after a progression of enzymatic changes, achieve an asymptotic level of enzyme activity around 16 days postnatally.Parts of this paper were presented at the XI. Symposion, Gesellschaft für Histochemie, München 1966.Technical assistance of Miss. W. Komp and Miss. E. Hartwieg is gratefully acknowledged.     

Meningeal cells stimulate and direct the migration of cerebellar external granule cells in vitro

The external granular layer is a secondary proliferative zone that arises from the caudolateral margin of the cerebellar ventricular zone and then spreads beneath the pial surface, eventually covering the entire cerebellar anlage. Here, both a part of the Bergmann glia and granule cells are generated. Selective destruction of the leptomeningeal cell layer during development in vivo disrupts the subpial extension of the external granular layer and the laminar deposition of its descendant cells. The mechanisms by which meningeal fibroblasts exert their controlling influence on cortical development have remained unclear but could involve diffusible factors and/or interactions mediated by direct cellular contacts. In order to test these assumptions, we have co-cultivated cerebellar slice explants with meningeal cells with and without interposition of a microfilter barrier. In this setup, meningeal cells by a diffusible factor stimulated the emigration of immature neurons exclusively from the external granular layer. This effect could also be elicited by fibroblasts from other tissues but not by nonfibroblastic cells such as, e.g., astroglia. In the Boyden chamber assay, the migration of undifferentiated neurons isolated from the external granular layer was chemotactically oriented towards the source of meningeal cell-conditioned media. In comparison, neurons from the internal granular layer did not respond to this stimulus. The attraction of immature neurons towards the pial surface could (1) represent a mechanism for the establishment of (subpial) secondary proliferative zones and (2) hypothetically also play a role in the outward-directed migration of postmitotic cells, e.g., in the isocortical anlage.     

Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.     

Activation of glutamate receptors inhibits Na/K-ATPase of cerebellum granule cells

Na/K-ATPase prepared from cerebellum granule cells of 10-12-day-old mice is inhibited by glutamate and its agonists, NMDA (ligand for ionotropic receptors) and ACPD (ligand for metabotropic receptors). The inhibition is specific and prevented by subsequent antagonists (MK-801 for ionotropic NMDA-receptors and MCPG for metabotropic receptors). The inhibiting effect of NMDA is significantly reversed by cysteine and that of ACPD by chelerythrine or indolyl maleimide. It is concluded that ionotropic receptors inhibit Na/K-ATPase because of intracellular production of reactive oxygen species, and metabotropic receptors mediate their effect via protein kinase C.     

Postnatal development of the granule cell in the kitten cerebellum

Newborn to 9-week-old kittens were anesthetized and then sacrificed by inserting a scalpel into the thoracic cavity and severing the descending aorta. The cerebella were removed and processed according to Golgi-Cox and rapid Golgi modifications. The results indicate that the dendritic processes of the granule cells undergo a marked postnatal maturation even though there is little postmigratory change in the size and shape of the cell body. A club-shaped swelling of the distal portions of the dendrites is observed by the end of the first week; this "claw" becomes multilobated by three weeks and displays elaborate digitiform projections by nine weeks. The sequence of postnatal development in the kitten appears to follow a time course similar to that observed in other species. A possible correlation between the postnatal development of the granule cell and the postnatal development of the spincocerebellar system is discussed.     

Murine numb regulates granule cell maturation in the cerebellum

Notch is a key regulator of vertebrate neurogenesis and the cytoplasmic adaptor protein Numb is a modulator of the Notch signaling pathway. To address the role of murine Numb in development of the central nervous system, we used a conditional gene ablation approach. We show that Numb is involved in the maturation of cerebellar granule cells. Although the specification of neural cell fates in the cerebellum is not affected in the absence of Numb, the transition from a mitotic progenitor to a mature granule cell is aberrant and migration of postmitotic granule cells to the internal granule cell layer is delayed. In some animals, this results in a complete agenesis of granule cells and a strong ataxia. We confirmed these findings in vitro and found that Numb-deficient cerebellar progenitor cells show a marked delay in granule cell maturation. Our results suggest that Numb plays a role in the transition of a mitotic progenitor to a fully differentiated granule cell in the cerebellum. In addition, the maturation of Purkinje cells is also delayed in Numb-deficient mice.     

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.     

Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4'-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.     

Early development of the granule cell in the cerebellum of the chick embryo

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.     

A transient outward current dependent on external calcium in rat cerebellar granule cells

Summary The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to anI _A-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca²⁺, but significantly inhibited by 3mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED₅₀=0.7mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca²⁺, Mn²⁺ and Mg²⁺. In 2.8mM Mn²⁺, the transient potassium conductance was comparable to that in 2.8mM Ca²⁺, while in 2.8mM Mg²⁺ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca²⁺ was present, Mg²⁺ up to 5mM had no effect. The transient current increased with increasing concentrations of external Ca²⁺, [Ca²⁺] _o , and the maximum conductancevs. [Ca²⁺] _o curve could be approximated by a one-site model. In addition, the current recorded with 5.5mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca²⁺ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.     

Protective effect of (+/-) isoborneol against 6-OHDA-induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine (DA) may play an important role in the pathogenesis of Parkinson's disease (PD). (+/-) Isoborneol is a monoterpenoid alcohol present in the essential oils of numerous medicinal plants and is a known antioxidant. In this study, we investigated the neuroprotective effect of isoborneol against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun N-terminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA. Our results indicate that the protective function of isoborneol is dependent upon its antioxidant potential and strongly suggest that isoborneol may be an effective treatment for neurodegenerative diseases associated with oxidative stress.