Relocalization of a microtubule-anchoring protein,ninein, from the centrosome to dendrites during differentiation of mouse neurons

Microtubules in typical cells form radial arrays with their plus-ends pointing toward the cell periphery. In contrast, microtubules in dendrites of neurons are free from centrosomes and have a unique arrangement in which about half have a polarity with a minus-end distal orientation. Mechanisms for generation and maintenance of the microtubule arrangement in dendrites are not well understood. Here, we examined dendritic localization of a centrosomal protein, ninein, which has microtubule-anchoring and stabilizing functions. Immunohistochemical analysis of developing mouse cerebral and cerebellar cortices showed that ninein is localized at the centrosome in undifferentiated neural precursors. In contrast, ninein was barely detected in migrating neurons, such as those in the intermediate layer of the cerebral cortex and the internal granular layer of the cerebellar cortex. High expression was observed in thick dendrite-bearing neurons such as pyramidal neurons of the cerebral cortex and Purkinje neurons in the cerebellar cortex. Ninein was not detected at the centrosome of these cells, but was diffusely present in cell soma and dendrites. In cultured cortical neurons, ninein formed granular structures in soma and dendrites, being not associated with γ-tubulin. About 60% of these structures showed resistance to detergent and association with microtubules. Our observations suggest that the minus-ends of microtubules may be anchored and stabilized by centrosomal proteins localized in dendrites.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time-dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T- or Y-shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Find of coccidians of the genus Isospora (Coccidiida) in the Central Asian jackal

During the period of 1981-1982 9 central asiatic jackals were caught in the Chimkent region and "Kent-Bulak" area on the left bank of the Syr-Darja of the Kzyl-Orda region. In two jackals caught in the Kzyl-Orda region one species of coccidians of the genus Isospors was found. Oocysts of I. kzilordiniensis are oval without micropyle, the membrane is monolayer, smooth, greenish, 1.5 mu thick. The size of oocysts is 26.1-31.9 X 17.4-20.3.     

Release of Endogenous Taurine and γ-Aminobutyric Acid from Brain Slices from the Adult and Developing Mouse

The spontaneous and potassium-stimulated release of endogenous taurine and gamma-aminobutyric acid (GABA) from cerebral cortex and cerebellum slices from adult and developing mice was studied in a superfusion system. The spontaneous release of GABA was of the same magnitude in slices from adult and developing mice, but the spontaneous release of taurine was considerably greater in the adults. The potassium-stimulated release of GABA from cerebral cortex slices was about five times greater in adult than in 3-day-old mice, but the potassium-stimulated release of taurine was more than six times greater in 3-day-old than in adult mice. In cerebellar slices from 7-day-old mice, potassium stimulation also evoked a massive release of taurine, whereas the evoked release from slices from adult mice was rather negligible. Also in cerebellar slices the potassium-stimulated release of GABA exhibited the opposite quantitative pattern. The stimulated release of both GABA and taurine was partially calcium dependent. The results suggest that taurine may be an important regulator of excitability in the developing brain.     

The use of Herr four-and-a-half clearing fluid for the rapid microscopic examination of thick sections of normal and neoplastic tissues.

We have demonstrated that Herr's 4 1/2 clearing fluid, developed for use with plant tissues, can be successfully used for the microscopic examination of thick sections of normal and neoplastic mammalian tissues. Rat Novikoff hepatoma, rat liver, and human colon and skin samples were fixed in Bouin's, stained with iron hematoxylin, treated with Herr's 4 1/2 clearing fluid and examined by phase contrast microscopy. Tissue architecture and cytological detail were easily observed by focusing through tissue sections as thick as 70 mu. The method permits rapid microscopic examination of mammalian tissues and enables the investigator to detect readily morphological abnormalities within a tissue.     

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.     

Role of reactive oxygen species and NADPH-oxidase in the development of rat cerebellum

Experimental evidence suggests that reactive oxygen species (ROS) could participate in the regulation of some physiological conditions. In the nervous system, ROS have been suggested to act as signaling molecules involved in several developmental processes including cell differentiation, proliferation and programmed of cell death. Although ROS can be generated by several sources, it has been suggested that NADPH oxidase (NOX) could be critical in the production of ROS acting as a signal in some of these events. It has been reported that ROS production by NOX enzymes participate in neuronal maturation and differentiation during brain development. In the present study, we found that during rat cerebellar development there was a differential ROS generation at different ages and areas of the cerebellum. We also found a differential expression of NOX homologues during rat cerebellar development. When we treated developing rats with an antioxidant or with apocynin, an inhibitor of NOX, we found a marked decrease of the ROS levels in all the cerebellar layers at all the ages tested. Both treatments also induced a significant change in the cerebellar foliation as well as an alteration in motor behavior. These results suggest that both ROS and NOX have a critical role during cerebellar development.     

The thiamine pyrophosphatase technique as an indicator of the morphology of the Golgi apparatus in the neurons

Summary Detailed studies have been made on the morphology of the TPPase-positive material in various components of the cerebellum. The various types of nerve fibers in various layers of the cerebellum and the glomeruli are free of any TPPase-positive Golgi material. The stellate and basket cells have a thick plate-like Golgi complex. The Purkinje cells showed either a Golgi network or discrete masses of TPPase-positive material of various sizes and shapes. The Bergman glial cells showed a delicate Golgi network. The Golgi type II cells showed a thick or thin TPPase-positive network. The granule cells showed only discrete vesicles, granules, and comma-shaped masses found at any one pole of each cell. The cells of the deep cerebellar nuclei also showed a network. The results of this study are compared with similar studies made on the spinal cord, olfactory bulb, Ammon's horn, fascia dentata, cerebral cortex, and ganglion cells.     

Sulphur nutrition changes the sources of S in vegetative tissues of wheat during generative growth

During generative growth, developing wheat grains require nitrogen and sulphur to synthesize storage proteins. The hypothesis that the S required for grain growth can be derived from vegetative tissues was examined by growing plants in nutrient culture containing either 50 M S (low-S) or 200 M S (high-S) and terminating the nutrient supply at various times during generative growth. After terminating the nutrient supply, high-S plants redistributed soluble S to developing grains from pools in roots and leaves, whereas low-S plants remobilized insoluble S (protein-S) from the leaves to the grains. A model for the cycling of S within mature leaves during generative growth is presented.     

The architecture of the musculo-fibrous apparatus in the human orbit.

Combining the thick serial (60 mu) histological section technique with the 0.5 cm cleared section technique, a definite structural organization in orbital connective tissue and adipose tissue components lying in between, is revealed. Additionally, an interindividual uniformity and bilateral symmetry in the human orbital connective tissue structures was found. Finally, using the reconstruction technique it became clear that each eye muscle has its own connective tissue system in the orbit and that its architecture highly differs dependent on, which orbital area is approached. It is conceivable that in future the spatial architecture of the musculo-fibrous apparatus in the human orbit might prove to be of importance for clinical diagnosis and surgical approach.     

Electron-microscopic immunocytochemistry of 5-hydroxytryptamine in the ascidian endostyle

Summary Corticotropin releasing factor (CRF)-immunoreactive (IR) perikarya, visualized by the indirect immunoperoxidase method in colchicine-pretreated cats, were localized in many discrete regions of the medulla oblongata. They were found mainly in the dorsal aspect and midline of the medulla oblongata, and more rostrally in the ventrolateral portion. Our results also demonstrated CRF-IR neurons in the rostrocaudal extent of the inferior olive, probably projecting to the cerebellar cortex via thick axons visualized along the lateral edge of the medulla. CRF-IR olivary cells were also found in the pontine cat from which the forebrain was removed, but neither in hypophysectomized nor adrenalectomized cats.     

The metamorphosis of the developing cerebellar microcircuit

The cerebellar cortical circuit with its organized and repetitive structure provides an excellent model system for studying how brain circuits are formed during development. The emergence of the mature brain requires that appropriate synaptic connections are formed and refined, which in the rodent cerebellum occurs primarily during the first three postnatal weeks. Developing circuits typically differ substantially from their mature counterparts, which suggests that development may not simply involve synaptic refinement, but rather involves restructuring of key synaptic components and network connections, in a manner reminiscent of metamorphosis. Here, we discuss recent evidence that, taken together, suggests that transient features of developing cerebellar synapses may act to coordinate network activity, and thereby shape the development of the cerebellar microcircuit.     

Effect of nutritional insufficiency on cell proliferation in the matrix zones of the cerebellar anlage in mice

The work has been performed on 62 CBA mice. In the ventricular zone and in the external granular layer of the cerebellar anlage of embryos (13-17 days of the intrauterine development) mitotic index, labelled nuclei index, part of labelled mitoses have been counted. Parameters of the mitotic cycle of the matrix cells have been calculated by means of the graphic method. The proliferative pool value has been calculated. At malnutrition the cerebellar anlage structure retards in its maturation from the norm. For the matrix zones of the cerebellar anlage, higher indices of the proliferative activity are specific. At the same time, duration of the mitotic cycle of the matrix cells increases by 15-17%. It is possible, that retardation of histogenesis of the mouse cerebellar anlage, when developing under conditions of alimentary insufficiency depends on decreased rate of cell proliferation, as a result of prolonged mitotic cycle of the matrix cells.     

Membranocystic lesion in the brain in cerebrotendinous xanthomatosis. Histochemical and ultrastructural study with evidence of its ceroid nature

A case is described of cerebrotendinous xanthomatosis with purely neurological manifestations. Cholestanol deposition in both affected and unaffected brain regions was markedly increased, reaching 18.5-20.8% of the sterol fraction. The unilateral lesions localized in the basal ganglia and cerebellar white matter featured perivascular accumulation of foam cells containing apolar lipid and ceroid. Necrosis with lipid-rich debris was a frequent finding often accompanied by prominent collagen deposition. Within these lesions there were numerous refractile thick membranes which, according to lipid histochemical techniques, could be qualified as ceroid-type lipopigment. It is suggested that the ceroid membranes arise extracellularly directly from the lipid-rich debris. Ultrastructurally, they were composed of convolutes of highly organized trilaminar membranes about 15 nm thick similar to those seen in intracellular ceroid granules. The membranes were embedded in an amorphous substance of low or medium density and were identical in their general appearance, stainability and fine structure to the membranocystic lesion in Nasu-Hakola disease and to the extracellular ceroid in atherosclerotic plaques.     

Polarity of actin filaments at the initial stage of myofibril assembly in myogenic cells in vitro

The polarity of thin filaments in relation to thick filaments in developing muscle cells in vitro was investigated. The majority of thin filaments exhibited the right polarity and spatial position similar to that seen in mature myofibrils. It appears that the interaction between thick and thin filaments exists in the initial phases of myofibrillogenesis. Cortical microfilaments are found to have their polarities arranged randomly.     

pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.     

Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope

Razin, Shmuel (University of Connecticut, Storrs), and Benjamin J. Cosenza. Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope. J. Bacteriol. 91:858-869. 1966-Growth of 11 Mycoplasma strains in liquid media was followed by phase-contrast microscopy. A similar pattern of development was common to all strains. Branching filaments, 0.3 to 0.4 mu thick, characterized the early logarithmic phase of growth. The length of the filaments varied according to the strain tested and the growth medium. Addition of oleic acid to the medium induced the formation of very long filaments by M. laidlawii strain B. Upon aging, the filaments were found to break up into chains of coccoid elements. These chains further fragmented to yield shorter chains and single coccoid elements, which characterized the stationary and decline phases of growth. The size of the coccoid elements increased from 0.3 to 0.4 mu, when formed in the filaments, to 0.6 to 0.8 mu after being released from the chains. Further increase in the size of the cells took place at the decline phase of growth, leading to the formation of very large cells reaching a diameter of 10 to 20 mu. However, these large cells had the appearance of empty vesicles and were apparently nonviable as indicated by viable-count experiments.