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Biomedical subjects

M E Hatten

Publications and source records attributed to M E Hatten.

At least 73 records · Page 4Linked to original sources
PubMed1984

Two forms of cerebellar glial cells interact differently with neurons in vitro.

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.

Animals↗
PubMed1984

Neuron-astroglial interactions in vitro and their implications for repair of CNS injury.

To study neuron-glial interactions, our laboratory has developed an in vitro model system that, when used with cell type-specific antisera, allows visualization of contacts between cerebellar granule neurons and astroglia. When cells were dissociated from early postnatal mouse cerebellum and plated in microcultures, the neurons aligned along glial filament protein (GFP)-containing astroglial processes. The behavior of the neurons depended on the shape of the particular astroglial cell that they contacted. Neuronal migration commonly occurred along highly elongated astroglial processes of Bergmann-like glia but was inhibited when neurons nestled among the arms of stellate astroglia. To analyze the influence of neurons on the astroglial shapes associated with neuronal migration, cerebellar granule neurons and astroglia were purified and recombined. In the absence of neurons, cerebellar astroglia assumed a flattened shape and proliferated rapidly. In the absence of astroglia, neurite outgrowth was severely impaired. When neurons were recombined with purified astroglia, astroglial proliferation slowed markedly, the shape of the astroglia transformed into complex forms, and neuron-glial interactions were seen. In tissue sections, immature forms of glia were found in the developing cerebellar axon tracts, but no obvious relationship could be discerned between the growing axonal tips and the glia. At P7, a period when the growth of cerebellar axons slows markedly, a transient natural gliosis was seen in the putative white matter. These studies underscore the interdependence of neurons and astroglia during periods of neuron differentiation and neurite outgrowth. In addition, they raise the possibility that the disruption of normal neuronal-astroglial contacts suffered during CNS injury could lead to defects in astroglial form and surface properties that, in turn, might impair axon regrowth.

Animals↗
PubMed1984

Defects in specific associations between astroglia and neurons occur in microcultures of weaver mouse cerebellar cells.

The neurological mutation weaver is characterized by defects in granule cell migration along Bergmann glial processes and by subsequent death and disposition of granule cells. Immunocytochemical localization of antisera raised against purified glial filament protein (AbGF) and transmission electron microscopy were used to visualize specific associations between granule neurons and astroglia in microcultures of cerebellar cells dissociated from normal (+/+), heterozygous (+/wv), and homozygous (wv/wv) B6CBA-w mouse cerebella. In microcultures of cells dissociated from normal B6CBA-Aw-J-wv (+/+) cerebella, staining with AbGF closely resembled results previously reported for cells taken from C57BL/6J (+/+) tissue. Two forms of stained astroglia were seen, one with a larger perikaryon and shorter processes, among which 12 to 20 unstained cells nestled; and another with a smaller cell soma and longer processes, along which a few unstained cells were seen. The first resembled astrocytes of the internal granular layer and white matter, the second Bergmann astroglia. In microcultures of heterozygous animals (+/wv), the number of granule cells was reduced slightly. Many stained astroglia resembled those from +/+ cerebella, but others had thickened processes and enlarged terminal "endfeet." Granule cells associated with both forms of stained astroglia. Cultures from homozygous weaver (wv/wv) cerebella contained very few, if any, granule cells and did not exhibit specific neuronal/glial interactions characteristic of +/+ cells. Both forms of stained wv/wv glial cells had enlarged cell somata giving rise to stunted processes, suggesting that both Bergmann glia and astrocytes are affected by the weaver gene. By both immunocytochemical staining and electron microscopy, accumulation and tangling of glial filaments were seen. Immunocytochemical staining of weaver cerebellar tissue with AbGF also revealed abnormalities in astrocytes of the internal granular layer and white matter in addition to previously reported defects in Bergmann glia. Agglutination with five plant lectins, each with a different carbohydrate-binding specificity, revealed that postnatal weaver cerebellar cells agglutinate with the lectins concanavalin A and the wheat germ agglutinin, suggesting the persistence postnatally of embryonic cell surface elements on postnatal weaver cells.

Animals↗
PubMed1984

Embryonic cerebellar neurons accumulate [3H]-gamma-aminobutyric acid: visualization of developing gamma-aminobutyric acid-utilizing neurons in vitro and in vivo.

gamma-Aminobutyric acid (GABA) is the proposed neurotransmitter for four types of cerebellar neurons-Purkinje, Golgi, basket, and stellate neurons. With this investigation we have begun studies to establish when these neurons acquire their neurotransmitter "identification." Autoradiographic studies of both cultured embryonic (embryonic day 13) cerebellar cells and of intact embryonic cerebellum (embryonic day 13) were conducted with tritiated GABA. Two to 5% of the embryonic cerebellar cells accumulated [3H]GABA in vitro. By morphological and immunocytochemical criteria, labeled cells were large neurons with either a thick, apical process, a multipolar shape, or were bipolar with longer processes. The identification of cells which accumulated [3H]GABA as neuronal precursors was supported by the differential sensitivity to drugs that preferentially inhibit accumulation of [3H]GABA by neurons and glia. The results of the in vitro experiments were confirmed and extended with in vivo experiments. When intact cerebellar tissue was removed at embryonic day 13, stripped of meninges and choroid plexus, exposed to low concentrations of [3H]GABA, and processed for light microscopic autoradiography, heavily labeled cells were seen in the middle of the cerebellar anlage. Labeled cells were not seen in the ventricular zone of proliferating neuroblasts lining the fourth ventricle or in the external granular layer emerging at the lateral aspect of the pial surface. The accumulation of [3H]GABA by these cells also showed the pharmacological characteristics of uptake by neurons. This study shows that among migrating, immature forms of the larger neurons of the embryonic cerebellum, there is a select group which accumulates [3H]GABA and other classes of cells which do not. These results indicate very early acquisition of transmitter expression by cerebellar neurons, far in advance of their final positioning and establishment of synapses.

Animals↗
PubMed1982

Binding of developing mouse cerebellar cells to fibronectin: a possible mechanism for the formation of the external granular layer.

The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible.

Aging↗
PubMed1981

Cell assembly patterns of embryonic mouse cerebellar cells on carbohydrate-derivatized polylysine culture substrata.

Four carbohydrate derivatives of poly-D-lysine have been synthesized and assayed as substrates for the tissue culture of embryonic mouse cerebellar cells. On poly-beta-(D-glucopyranosyl)-poly-D-lysine and on poly-beta-(N-acetyl-D-glucosaminyl)-poly-D-lysine, dissociated cerebellar cells formed a monolayer. On poly-beta-(D-galactopyranosyl)-poly-D-lysine, cellular aggregates were formed and cables of processes were extended between the aggregates. On poly-beta-(L-fucosyl)-poly-D-lysine, cerebellar cells failed to attach and died within 24 h. On poly-(N-acetyl)-poly-D-lysine, cell attachment was identical to that on poly-D-lysine. At low concentrations of underivatized poly-D-lysine (0.5-2.0 microgram/ml) dissociated embryonic cerebellar cells formed cellular aggregates, whereas at higher concentrations of poly-D-lysine monolayering was extensive.

Animals↗
PubMed1981

Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro.

Indirect immunocytochemical staining with antisera raised against purified glial filament protein and a neurofilament polypeptide was used to study cell interactions between astrocytes and neurons dissociated from embryonic and early postnatal cerebellum. Staining with antibodies raised against purified glial filament protein revealed that greater than 99% of all processes present in cerebellar cultures during the 1st wk in vitro were glial in origin. After 1 wk in culture, unstained processes that were presumably neuronal were observed. Stained astroglial processes formed a dense network that served as a template for cerebellar neurons, identified by indirect immunocytochemical localization of tetanus toxin. More than 90% of neurons from postnatal days 1 or 7 were positioned within one cell diameter of a glial process. In contrast, less than 40% of the neurons dissociated from early embryonic cerebellum were located adjacent to a glial process. Staining with antibodies raised against purified glial filament protein also revealed differences in astroglial morphology that were under developmental regulation. Astroglial cells from embryonic cerebellum were fewer in number and had thick, unbranched processes. Those from postnatal day 1 were more slender, branched, and stellate. Those from postnatal day 7 were highly branched and stellate. Some veil-like astroglial processes were also observed in cells from postnatal animals. These morphological changes were also observed when cells from embryonic day 13 were maintained for a week in vitro. No specific staining of embryonic or postnatal cerebellum cells was observed with antibodies raised against purified neurofilament polypeptides.

Animals↗
PubMed1981

Developmental stage-specific changes in lectin binding to mouse cerebellar cells in vitro.

Eleven fluorescein isothiocyanate-conjugated (FITC) lectins, each with distinct carbohydrate-binding properties, were used to assess cell surface glycoconjugates of embryonic and early postnatal cerebellar cells in vitro. Fluorescence staining of embryonic day 13 (E13) cerebellar cells with FITC Ricinus communis agglutinin diminished markedly between 24 and 72 hr in vitro. No staining of postnatal day 0 (P0) or postnatal day 7 (P7) cells was observed with FITC Ricinus communis agglutinin. A similar, but less pronounced decrease in FITC concanavalin A, FITC Lens culinaris, and FITC wheat germ agglutinin was observed between embryonic day 13 and birth. No specific staining of E13, P0, or P7 cultures was observed with FITC peanut agglutinin, FITC Dolichos bifloris agglutinin, FITC soybean agglutinin, FITC Wistaria floribundis agglutinin, FITC Phaseolus vulgaris agglutinin, FITC Limulus polyphemus agglutinin, or FITC Ulex europaeusI agglutinin. Similar results were obtained with 125I-lectin binding assays. Ricinus communis 125-I-agglutinin binding decreased dramatically between embryonic day 13 and birth. Less pronounced decreases were observed in 125I-concanavalin A and wheat germ 125I-agglutinin binding. Very low levels of soybean 125I-agglutinin or Ulex europaeusI 125I-agglutinin were bound by either embryonic or early postnatal cerebellar cells in vitro.

Aging↗
PubMed1979

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.

Agglutination↗
PubMed1978

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.

Agglutination↗