Postnatal Development Of Rat Cerebellum Research Articles

Distribution and expression of Kirre, an IgSF molecule, during postnatal development of rat cerebellum

Immunoglobulin superfamily (IgSF) molecules are actively involved in cell-cell adhesion, neuronal migration, axonal guidance and synapse formation in the nervous system. Kirre, as a member of this family, has been implicated in mammalian neuronal differentiation and development. Although the distribution of rKirre (a rat homologue of Drosophila Kirre) mRNA was previously analyzed in adult rat cerebellum by in situ hybridization, the expression levels of transcript and protein were not well studied. Here, we showed that the expressions of rKirre mRNA and protein significantly increased during postnatal development of rat cerebellum. rKirre mRNA was mainly expressed in the granular layers and Purkinje cell layer in the developing cerebellum, revealing a possible involvement of rKirre in granule cell migration and Purkinje cell development. An essential relationship between rKirre and Purkinje cells was implied by the co-localization of rKirre and NF-200 on the cell bodies of Purkinje cells. These results suggest that rKirre may play a potential role in postnatal developing rat cerebellum.

Isoform-dependent immunolocalization of 14-3-3 proteins in developing rat cerebellum

We investigated the expression of 14-3-3 protein and its 7 isoforms during postnatal development of rat cerebellum with immunoblot and immunohistochemistry with isoform-specific antibodies. The relative amounts of total 14-3-3 protein, probed by an antibody (14-3-3 COM) recognizing a sequence shared among its isoforms, exhibited no significant changes from postnatal day 2 (P2) to P100. 14-3-3 COM-like immunoreactivity (IR), initially in the apical portion of Purkinje cells at P2, extended to Purkinje cell bodies at P14 and to their dendrites (P100) with increasing intensity. Molecular layer (after P7) and cerebellar nucleus neurons (after P14) were also immunolabeled with this antibody. These chronological changes were shared with those obtained with beta, gamma, and eta isoforms. In contrast, epsilon isoform-like IR was initially identified in processes of radial and Bergmann glia at P2 prior to its appearance in the molecular layer at P7 with subsequent intensification also in Purkinje cells after P14. Zeta and tau isoform-like IR was identified in the white matter and/or in oligodendroglial cells. The sigma isoform was the only isoform exhibiting a significant quantitative change with a peak at P14. Immunolocalization of sigma isoform was initially restricted in several cells in Purkinje cell layer at P2 and shifted to nuclei of external and internal granule cells and Purkinje cells after P14, whereas its immunolabeling was markedly weaker at P100. Different immunolocalizations of the 7 isoforms suggest that 14-3-3 protein isoforms individually associate with the neuronal and glial proliferation, differentiation, migration and development during postnatal formation of rat cerebellum.

Transient abundance of presenilin 1 fragments/nicastrin complex associated with synaptogenesis during development in rat cerebellum

Immunolocalization and expression of endogenous nicastrin (NCT) and presenilin 1 (PS1) fragments during postnatal development of rat cerebellum were investigated with fragment-specific antibodies. Immunoblotting for NCT revealed the expected mature and immature species, which gradually declined during development. In contrast, the expression of PS1 N-terminal fragment exhibited a peak at postnatal day 14 (P14) and declined thereafter. This chronological change was similarly observed with PS1 C-terminal fragment. Immunoprecipitation of NCT indicated its physical association with PS1 fragments. Colocalization of these molecules to the endoplasmic reticulum in cerebellar Purkinje cells indicates that they are organized into a complex in developing neurons. In addition, active sites of synaptogenesis, the base of the external granular layer and glomeruli, contained PS1 fragments and smaller amount of NCT. Isolated synaptic fraction contained both PS1 and NCT, suggesting their functional association within synapses. Transient abundance of NCT and PS1 fragments as a complex, when (P14) and where synaptogenesis is active, is consistent with intracellular trafficking of this complex in developing neurons and suggests its role as γ-secretase in synaptogenesis.

Active caspase-3 expression during postnatal development of rat cerebellum is not systematically or consistently associated with apoptosis.

Development is a dynamic process that includes an intricate balance between an increase in cell mass and an elimination of excess or defective cells. Although caspases have been intimately linked to apoptotic events, there are a few reports suggesting that these cysteine proteases can influence the differentiation and proliferation of cells. Specifically, the active form of caspase-3, which has been classified as an executor of apoptosis, recently has been implicated in a nonapoptotic role in the regulation of the cell cycle, cell proliferation, and cell differentiation. This study investigated the nonapoptotic function and phenotypic expression of active caspase-3-positive cells in the external granule cell layer (EGL) of the postnatal rat cerebellum by using biochemical and immunohistochemical analyses, respectively. Evidence that negates an apoptotic function for the caspase-3-positive EGL cells includes a failure to exhibit chromatin condensation (assessed with TOPRO), phosphatidyl serine externalization (Annexin V labeling), or DNA fragmentation (TUNEL labeling). Proliferative (Ki67-positive) and differentiated (TUJ1-positive) cells within the EGL exhibited a cytosolic expression of caspase-3, whereas terminally differentiated granule cells (NeuN-positive) in the internal granular layer and the migrating granule cells did not express active caspase-3. Thus, this study supports a nonapoptotic role for active caspase-3 in cells residing in the EGL and suggests a possible involvement in EGL proliferation and differentiation.

Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum.

Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.

Quantitative analysis of nitric oxide synthase expressed in developing and differentiating rat cerebellum

In order to investigate quantitatively the differentiation and maturation process of granule cells in the postnatal development of rat cerebellum, nitric oxide synthase (NOS) activities were determined in the micro-dissected developing cerebellar layers, using our microassay method. NOS activities were increased in the molecular and internal granular layers (IGLs) during development and the activity was measurable in the neuroblastic external granular layers (EGLs). Newly devised micro-immunoblot analysis semi-quantitatively showed more amount of endothelial NOS (eNOS) and non-negligible amount of neuronal NOS (nNOS) in microsamples from EGL, compared with other developing and adult cerebellar layers. nNOS mRNA was also detected using RT-PCR (reverse transcriptase-polymerase chain reaction) in the microsamples from this germinal layer. Intraperitoneal administration of N G-nitro- l-arginine inhibited NOS activity in vivo and disturbed the layer formation in developing cerebellum.

Synaptic 5′-nucleotidase is transient and indicative of climbing fiber plasticity during the postnatal development of rat cerebellum

The transient appearance of 5′-nucleotidase, an adenosine-producing ecto-enzyme, was studied during specific stages of postnatal synaptogenesis in the rat cerebellum. For ultrastructural detection of 5′-nucleotidase activity, an enzyme-cytochemical technique was used. Between postnatal days 4 and 6, enzymatic reaction product was present in the synaptic clefts of climbing fibers contacting the perisomatic spines, apical cones and emerging dendrites of Purkinje cells (CF-PC synapses). Labeled parallel fiber synapses were observed on dendritic shafts of cerebellar interneurons. At postnatal days 9 and 12, enzyme-positive parallel fiber terminals were in addition numerous on the spines of peripheral Purkinje branchlets, and gradually disappeared thereafter. Between postnatal days 8 and 15, labeling of perisomatic CF-PC contacts persisted. In contrast, climbing fiber synapses on Purkinje dendrites were only occasionally labeled. Between postnatal days 18 and 21, synaptic reaction product was restricted to mossy fibers. At the same time, association of 5′-nucleotidase with glial profiles was prominent throughout the cerebellar layers. In adult cerebellum (from 24 days onwards) all synapses were devoid of enzymatic activity. Throughout development, basket, stellate and Golgi cell synapses were devoid of enzymatic activity. We conclude that 5′-nucleotidase is present in excitatory cerebellar synapses during part of their generation period. The transient nature of this phenomenon suggests that 5′-nucleotidase may serve as a novel, cytochemical marker for a specific state of synaptic maturation, and in particular for climbing fiber plasticity. A role of 5′-nucleotidase in purinergic neuromodulation and cellular contact formation could be significant in these processes.

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.

Expression of smg p25A, a ras p21-like small GTP-binding protein, during postnatal development of rat cerebellum

Changes in expression and localization of smg p25A, a ras p21-like small GTP-binding protein, in developing rat brain were analyzed in comparison with those of synaptophysin, a well-known synaptic vesicle-specific protein. The smg 25A mRNA was detected in whole brain of rat fetus at 14 days of gestation and its level was increased along with the age and reached the maximum level at postnatal day (P) 20. In postnatal cerebellum, the smg25A mRNA level was also increased age-dependently and the maximum level was observed at P30. Immunoblot analysis with an anti- smg p25A monoclonal antibody (MAb SG-11-7) and an anti-synaptophysin monoclonal antibody (SY 38) showed that expression of both smg p25A and synaptophysin was increased age-dependently in postnatal rat cerebellum. By immunofluorescent cytochemical study with the anti- smg p25A antibody, bright fluorescence was observed in the molecular layer of cerebellum and it was increased in accordance with the cerebellar development. In early postnatal cerebellum, the perikarya of Purkinje cells and the white matter were brightly stained with the antibody, but the fluorescence of these portions was faint in adult cerebellum. The anti-synaptophysin monoclonal antibody also stained the molecular layer of cerebellum but the perikarya of Purkinje cells and the white matter had only a weak immunoreactivity with the antibody irrespective of the age. These results indicate that smg p25A is predominantly present in the nerve terminals and that its amount is increased along with the development of postnatal rat cerebellum. Our results also suggest that smg p25A and synaptophysin have the different kinetics of synthesis, transport, and/or turnover in developing rat cerebellum.

Impaired development of rat cerebellum induced by neonatal injection of the glycoprotein synthesis inhibitor, tunicamycin.

The effects of the protein glycosylation inhibitor tunicamycin on the postnatal development of the rat cerebellum were examined in vivo. Tunicamycin (0.2 micrograms) was injected intracranially into 1-day-old rats. Inhibition of glycosylation of the macromolecules in the cerebellum by tunicamycin treatment was suggested by a reduced incorporation of [3H]glucosamine into the trichloroacetic acid (TCA)-insoluble fraction. The tunicamycin treatment did not affect gain in body weight significantly. However the cerebellar weight was significantly reduced by 30-40% compared with that of the controls. Development of GABAergic and cholinergic innervations in the hypoplastic cerebellum was examined by measuring the activities of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT). The specific activity and the total activity of GAD were significantly reduced in the tunicamycin-treated cerebellum. In contrast the specific activity of ChAT was significantly increased, whereas the total activity of ChAT per cerebellum was identical with that of the controls. These results suggest that the intracranial injection of tunicamycin affects the postnatal development of rat cerebellum, such as GABAergic and cholinergic innervations.

Postnatal development of rat cerebellum: Glycosaminoglycan changes related to variation in water content, cell formation and organ growth

Modifications of glycosaminoglycans were studied during rat cerebellum ontogenesis. Levels of both hyaluronic acid and sulphated glycosaminoglycans increase during development but not at the same rate as cerebellar size or proteins. As in the rest of the brain, cerebellar hyaluronic acid concentration decreases during development but not, as expected, in parallel with the loss of tissue water content. In contrast to forebrain, the concentration of cerebellar sulphated glycosaminoglycans decreases at a slow rate. The correlation, suggested by several authors, between glycosaminoglycans concentration and tissue hydratation, cell migration and myelination were not confirmed in developing rat cerebellum.

Muscarinic receptor during postnatal development of rat cerebellum: an index of cholinergic synapse formation?

Quantitative and qualitative modifications of the specific binding sites for [3H]quinuclidinyl benzylate (QNB), a muscarinic antagonist, were studied during rat cerebellar postnatal development. Specific binding sites for QNB (QNB-sbs), regardless of whether they correspond to muscarinic acetylcholine receptors, are present with the highest density in the archicerebellar cortex, but the total amount per region is about the same in the archi -, paleo-, and neocerebellar cortex regions. Large amounts of QNB-sbs are also present in a cerebellar fraction including central white matter and deep cerebellar nuclei. QNB-sbs are low but present at birth and then accumulate during ontogenic development according to a curve which duplicates, with a delay of a few days, the curve of DNA accumulation. Dissection studies indicated that this curve does not depend on the preferential localization of QNB-sbs in a specific cerebellar region nor on the particular development of this region. The similarity of the QNB-sbs and the DNA developmental curves might indicate that the QNB-sbs are present on granule cells; however, a comparative analysis of the data in the literature suggests that a great many QNB-sbs are located on the Purkinje cell dendrites in the molecular layer, where all or some of them might correspond to the extrajunctional muscarinic acetylcholine receptor detected there by electrophysiology. It would appear that only a small percentage of cerebellar QNB-sbs corresponds to the cholinergic synapses present in cerebellar cortex; hence, the question of muscarinic receptors in the cerebellum should be re-examined.

A biochemical and immunohistological study of S100 protein in developing rat cerebellum.

Specific immune sera, one directed against S100 protein and one against glial fibrillary acidic protein (both proteins are specific astrocytic markers in rat cerebellum), were used in immunohistological methods to follow the formation and maturation of astrocytes during the postnatal development of rat cerebellum. Throughout development, S100 protein is detected only in astrocytes. Immunofluorescent Golgi epithelial cells and other astrocytes are detected in the cerebellum of newborn rat, their number increases rapidly between birth and the 10th postnatal day and then more slowly until the end of the 3rd week. In contrast, the increase in S100 protein level, measured in cerebellar homogenates by radioimmunoassay, was small until the 10th postnatal day. The greater accumulation subsequently found corresponds to an increase in the content of each astrocyte during the process of maturation.

Localization of hexokinase in neural tissue: immunofluorescence studies on the developing cerebellum and retina of the rat.

Antiserum against purified rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been used in a study of the distribution of hexokinase during the postnatal development of rat cerebellum and retina. The cells of the external germinal layer of the cerebellum exhibit little or no fluorescence. The Purkinje cells exhibit a transient increase in hexokinase levels between 2 and 8 days postnatally, followed by a precipitous decrease (18--12 days) to the relatively low levels found in the mature Purkinje cell. Development of the intensely fluorescent cerebellar glomeruli in the granule cell layer is readily followed during the 3rd and 4th weeks postnatally. With respect to postnatal changes in hexokinase distribution of the retina, perhaps most notable is the observation that even the cytoplasmic protrusions which represent the precursors of the photoreceptor segments are richly endowed with hexokinase. Biochemical differentiation of the photoreceptor segments into hexokinase-rich inner segments and hexokinase-poor outer segments is readily observed long before the growth of the photoreceptor segments has been completed.

Carbonic anhydrase and oligodendroglia in developing rat cerebellum: A biochemical and immunohistological study

In the cerebellum, the isoenzyme II of carbonic anhydrase (CAII) appears to be a specific oligodendrocyte marker. Formation and maturation of oligodendrocytes during the postnatal development of rat cerebellum were followed by using specific immune serum directed against CAII for immunohistofluorescence and radioimmunoassay. Few oligodendrocytes are present in the cerebellum of the newborn rat. Their number increases rapidly between the fourth and the tenth days after birth and then more slowly until the end of the third week. Sequential changes in oligodendrocyte morphology have been observed. Determinations of CAII in cerebellar homogenate by radioimmunoassay show that the CAII level is low from birth to the end of the second week, the increase in the number of oligodendrocytes being accompanied by a small increase in the CAII level. Subsequent cell maturation is accompanied by significant accumulation of CAII.

High affinity glutamate binding during postnatal development of rat cerebellum

High affinity glutamate binding during postnatal development of rat cerebellum

Postnatal development of rat cerebellum: Massive and transient accumulation of concanavalin a binding glycoproteins in parallel fiber axolemma

Modifications of protein-bound sugars during postnatal development of rat cerebellum were studied. Glycoprotein-bound mannose accumulates, in the particulate fractions, at an earlier age than the bulk of glycoprotein sugar. This corresponds to a transient and massive accumulation of glycoproteins which bind to Concanavalin A (Con A). These glycoproteins were localized by using fluorescent Con A and the horseradish peroxidase-Con A method. Cerebellar white matter and the molecular layer bind massive amounts of Con A. The binding in the molecular layer is transient. It follows the same time course as the Con A-binding glycoproteins of particulate fractions, and it is largely confined to the axolemma of parallel fibers. Only growing or newly formed parallel fibers bind Con A. The disappearance of the binding is simultaneous with the maturation of parallel fibers and their synapse formation. These phenomena can be related to fiber growth and maturation and, also, to synapse formation. The possibility of a specific role of Con A-binding glycoproteins is discussed.

Changes in sugar composition of membrane-bound glycoproteins during postnatal development of rat cerebellum.

Changes in sugar composition of membrane-bound glycoproteins during postnatal development of rat cerebellum.

Changes of ganglioside pattern during postnatal development of rat cerebellum.

Changes of ganglioside pattern during postnatal development of rat cerebellum.