ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Karyosphere of the oocytes of the common frog, Rana temporaria]

Karyospheres of ca. 200 mcm in diameter were isolated from the common frog oocytes of definitive size. An electron microscope study has revealed in the karyosphere fibrillar nucleoli and micronucleoli, modified synaptinemal complexes sometimes connected with chromatin and fibrillar material containing a great number of, mostly atypical, pore complexes resembling those of nuclear membrane and forming "pseudomembranes". An electrophoresis of the isolated karyosphere has revealed 12 distinct protein bands, of 3 which correspond to the protein triplet characteristic of the nuclear matrix and the rest 9 represent high molecular weight components with the molecular weight from 130 to 200,000 D.     

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.     

Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiation and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult 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.     

Cerebellar plasma membrane proteins and their antisera

Plasma membranes have been isolated from neonatal through adult cerebella by a sequence of differential centrifugation, aqueous two-phase polymer fractionation and density gradient centrifugation. The protein composition of cerebellar membranes from various aged mice was compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Increases in the relative amount of membrane proteins with molecular weights (X 10(-3)) of 400, 340, 270, 220, 54, 44, and 9.5 were most pronounced, while a protein of 66,000 Mr disappeared between birth and Day 25. The relationship of these proteins and others to specific cell types in the cerebellum was examined by preparing membrane fractions from isolated granule and Purkinje cells, as well as from the cerebella of neurological mutant mice: reeler, weaver, staggerer, and nervous. In addition, those membrane proteins on the surface of dissociated cerebellar cells were identified by lactoperoxidase-catalyzed iodination, while glycoproteins were identified by galactose oxidase treatment and NaB3H4 reduction. Rabbit antisera were prepared toward those SDS-PAGE membrane proteins which appeared cell specific or developmentally regulated. Sera from these rabbits were used with indirect immunoperoxidase and immunofluorescence to stain frozen sections of mouse cerebellum and dissociated cerebellar cell cultures. In tissue sections antiserum toward the 400,000 Mr protein (p400) and antiserum p14.7 gave strong reactions with Purkinje cells while anti-p130 reacted preferentially with Purkinje cell somas, anti-p220 stained small cells in the internal granule layer and anti-p30 displayed a coarse, grainy staining of the granule and molecular layers, characteristic of a synaptic localization. Only anti-p220 and anti-p130 bound to freshly dissociated cells or cultured cerebellar cells. Large phase-bright cells in the cultures bound antiserum p130. Anti-p220 reacted specifically with a subpopulation of small round viable cells that bound tetanus toxin and decreased in number from 9% at Day 3 to 0.5% of the cells by Day 11, suggestive of granule neurons.     

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7–1.5 µm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5–2.5 µm) have a higher level of activity, contain 4–7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5–3.5 µm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18–22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2–7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.     

Presence of protein I, a phosphoprotein associated with synaptic vesicles, in cerebellar granule cells

Abstract: The cerebellar levels of Protein I, a synapse-specific neuronal phosphoprotein, have been investigated in the cerebellar mouse mutants staggerer ( sg ), weaver ( wv ), nervous ( nr ), and Purkinje cell degeneration ( pcd ). The Protein I concentration was reduced by about 66% in sg and wv mutants, representing a 90% loss of Protein I per cerebellum. A heterozygote effect was observed in the wv mutant. These results indicate that a great majority of Protein I in the normal cerebellum may be present in the granule cells. in nr mutants the cerebellar Protein I concentration was reduced by only 12% in 62-day-old mice, suggesting that Purkinje cells contribute little to cerebellar Protein I. However, a greater reduction was observed in pcd mutants, which may reflect on the nature of the pcd mutation.     

人体小脑神经元发育的定量观察

目的:研究人体小脑神经元的发育过程。方法:应用体视学方法,对18例不同时期人体小脑组织Golgi染色后进行观察,观测小脑皮质分层出现的时间,观测并计算神经元的数密度、体密度和表面积密度。结果:6月龄时,小脑皮质出现较明显的分子层、蒲肯野细胞层和颗粒层;星形细胞、篮状细胞、蒲肯野细胞、颗粒细胞和高尔基细胞的的数密度随月龄/年龄的增长而减少,体密度和表面积密度随月龄/年龄的增长而增加,但这些减小和增大是不等速的,6-8月龄变化最明显。结论:人体小脑神经元的发育呈现快慢交替、不均速发展,6～8月是小脑神经元发育的重要时期。     

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.     

Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice.

The content of glutamate, GABA, aspartate, glycine and alanine was determined in the cerebellum, brain stem and cerebrum of three different mutant mice which have been named ‘staggerer’, ‘weaver’ and ‘nervous’ on the basis of neurological symptoms. In the ‘staggerer’ and ‘weaver’ mutants there is an almost complete absence of granule cells in the cerebellar cortex while in the ‘nervous’ mutant there is a loss of Purkinje cells (and to a lesser extent a loss of granule cells) in the cerebellar cortex. In the cerebellum of the ‘weaver’ mutant, the content of glutamate was signficantly lower (P < 0.025) than control values (8.77 ± 0.76 vs 12.0 ± 1.3 μmol/g tissue wet wt) and the contents of GABA and glycine were significantly greater than normal levels. In the cerebellum of the ‘staggerer’ mutant, the content of glutamate was significantly lower (6.62 ± 0.70 μmol/g) and the contents of glycine and alanine significantly higher than control values. In the cerebrum and brain stem regions of the staggerer mutant, weaver mutant and the normals the contents of the five amino acids were the same. The contents of glycine and alanine in the cerebellum, GARA and glycine in the brain stem and GABA and alanine in the cerebrum of the nervous mutants were higher than control values. The data are discussed in terms of a possible role for glutamate functioning as an excitatory transmitter when released from the cerebellar granule cells.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea pig, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

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.     

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.     

Light and electron microscopic study of the mitochondria in human embryonic nervous tissue cells in vitro

After a 5-min heating of isolated Tradescantia leaves at 49 degrees C the granular component in the nucleoli of parenchyma cells disappears and the nucleoli grow more compact and exclusively fibrillar in structure. Simultaneously, the ability of chloroplasts of these cells for phototaxis is completely inhibited. In leaves placed in a moist chamber at room temperature 48 h after heating a normal ultrastructure of nucleoli and the ability of chloroplasts to respond by phototaxis to change in illuminations are restored almost synchronously.     

Neurochemical and histological analysis of motor dysfunction observed in rats with methylnitrosourea-induced experimental cerebellar hypoplasia

Histological and neurochemical changes related to motor dysfunction observed in rats after neonatal treatment with nitrosoureas were examined. Neonatal rats received subcutaneous injections of methylnitrosourea (MNU: 0.125 mmol/kg, s.c.) or ethylnitrosourea (ENU: 0.25 mmol/kg, s.c.) daily at 4,5,6 and 7 days post partum, a period of cerebellar granule cell, stellate cell and basket cell formation. At 14 days and 45 days after birth, MNU-treated rats displayed a lowering in motor coordination skills measured by tests of retainment ability on a rod of 26 mm diameter, chinningclimbing ability on parallel rods or retainment ability on a rotating rod. Histological examination at 14 days after birth showed a cerebellar hypoplasia with reduced cellularity of the internal granule cell layer and a disperse disposition of Purkinje cells in the granule cell layer. Cerebellar growth and cerebellar content and concentration of DNA were remarkably reduced in the MNU-treated rat. The degree of the reduction in cerebellar content of glutamic acid paralleled the degree of the cerebellar hypoplasia at 14 and 45 days after birth. In contrast, the concentrations of gamma-aminobutyric acid, acetylcholine, 5-hydroxytryptamine and norepinephrine were significantly increased by MNU treatment. ENU treatment control did not exert any significant changes in the neurotransmitters and motor coordination. These results suggest that the motor dysfunctions observed in MNU treated rats are induced by unbalanced output activities from Purkinje cells to motor neurons.