Biomedical subjects

M E Hatten

Publications and source records attributed to M E Hatten.

At least 55 records · Page 3Linked to original sources

PubMed1991

Astrotactin provides a receptor system for CNS neuronal migration.

CNS neuronal migration is a specialized form of cell motility that sets forth the laminar structure of cortical regions of brain. To define the neuronal receptor systems in glial-guided neuronal migration, an in vitro assay was developed for mouse cerebellar granule neurons, which provides simultaneous tracking of hundreds of migrating neurons. Three general classes of receptor systems were analyzed, the neuron-glial adhesion ligand astrotactin, the neural cell adhesion molecules of the IgG superfamily, N-CAM, L1 and TAG-1, and the beta 1 subunit of the integrin family. In the absence of immune activities, migrating cerebellar granule neurons had an average in vitro migration rate of 12 microns h-1, with individual neurons exhibiting migration rates over a range between 0 to 70 microns h-1. The addition of anti-astrotactin antibodies (or Fabs) significantly reduced the mean rate of neuronal migration by sixty-one percent, resulting in eighty percent of the neurons having migration rates below 8 microns h-1. By contrast, blocking antibodies (or Fabs) against L1, N-CAM, TAG-1 or beta 1 integrin, individually or in combination, did not reduce the rate of neuronal migration. By video-enhanced contrast differential interference contrast microscopy the effects of anti-astrotactin antibodies were seen to be rapid. Within fifteen minutes of antibody application, streaming of cytoplasmic organelles into the leading process arrested, the nucleus shifted from a caudal to a central position, and the extension of filopodia and lamellopodia along the leading process ceased. Correlated video and electron microscopy suggested that the mechanism of arrest by antiastrotactin antibodies involved the failure to form new adhesion sites along the leading process and the disorganization of cytoskeletal components. These results suggest astrotactin acts as a neuronal receptor for granule neuron migration along astroglial fibers.

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PubMed1990

Mechanisms of glial-guided neuronal migration in vitro and in vivo.

Our laboratory has developed an in vitro model system in which glial-guided neuronal migration can be observed in real time. Cerebellar granule neurons migrate on astroglial fibers by apposing their cell soma against the glial arm, forming a specialized migration junction, and extending a motile leading process in the direction of migration. In vitro assays indicate that the neuronal antigen astrotactin functions as a neuron-glia ligand, and is likely to play a role in the movement of neurons along glial fibers. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on heterotypic glial processes with a cytology, speed and mode of movement identical to that of neuronal migration on homotypic glial fibers, suggesting that glial fibers provide a permissive pathway for neuronal migration in developing brain. In vivo analyses of developing cerebellum demonstrate a close coordination of afferent axon ingrowth relative to target cell migration. These studies indicate that climbing fibers contact immature Purkinje neurons during the migration and settling of Purkinje cells, implicating a role for afferents in the termination of migration.

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PubMed1990

Riding the glial monorail: a common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain.

In vitro studies from our laboratory indicate that granule neurons, purified from early postnatal mouse cerebellum, migrate on astroglial fibers by forming a 'migration junction' with the glial fiber along the length of the neuronal soma and extending a motile 'leading process' in the direction of migration. Similar dynamics are seen for hippocampal neurons migrating along hippocampal astroglial fibers in vitro. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on astroglial processes with a cytology and neuron-glia relationship identical to that of homotypic neuronal migration in vitro. In all four cases, the migrating neuron presents a stereotyped posture, speed and mode of movement, suggesting that glial fibers provide a generic pathway for neuronal migration in developing brain. Studies on the molecular basis of glial-guided migration suggest that astrotactin, a neuronal antigen that functions as a neuron-glia ligand, is likely to play a crucial role in the locomotion of the neuron along glial fibers. The navigation of neurons from glial fibers into cortical layers, in turn, is likely to involve neuron-neuron adhesion ligands.

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PubMed1990

Antibodies that recognize astrotactin block granule neuron binding to astroglia.

To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-E16 chicks, were radiolabeled with [35S]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, L1, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, L1, N-cadherin, and integrin also did not impair neuron binding to glia.

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PubMed1990

Meander tail reveals a discrete developmental unit in the mouse cerebellum.

Analyses of phenotypic mutations with altered patterns of cellular organization in invertebrate systems have lead to the identification of genes important to histogenesis. Efforts to identify genes defining segments or compartments in mammalian systems and demonstrate a role for such genes in the establishment of the cellular architectonics of the brain have been hampered by the absence of phenotypic mutations that reveal compartments. Evidence is presented that in the cerebellum of the mutant mouse, meander tail, there is an abrupt transition from the normal cytoarchitecture seen in the posterior lobes to a severely disorganized cellular pattern. Thus, in the anterior lobes the Purkinje cells are positioned randomly throughout the cortex, and other cellular layers, including the internal granule cell layer, do not form. In addition, radial Bergmann glial processes are virtually absent. One explanation for the discrete boundary of the affected area in the anterior portion of this mutant cerebellar cortex is that the meander tail gene influences compartmental cellular organization in mammalian brain.

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PubMed1990

Central nervous system neurons migrate on astroglial fibers from heterotypic brain regions in vitro.

In different regions of the developing mammalian brain, neurons follow the processes of radial glial cells over very different trajectories to reach their destinations in specific neuronal layers. To investigate whether the movement of neurons along glial fibers is specified by glia in a given region or whether glia provide a permissive substrate for migration in different brain regions, we purified neurons and astroglial cells from developing cerebellum and hippocampus and analyzed neuronal migration on heterotypic glial fibers with time-lapse, video-enhanced differential interference microscopy in vitro. Granule neurons purified from early postnatal rat cerebellum migrated on astroglial processes of glia purified from late embryonic or early postnatal rat hippocampus with a cytology, neuron-glial relationship, and dynamics of movement that were indistinguishable from those of mouse granule cells migrating on cerebellar astroglial processes in vitro [Edmondson, J. C. & Hatten, M. E. (1987) J. Neurosci. 7, 1928-1934]. In the reciprocal combination, hippocampal neurons migrated on cerebellar glial processes in a manner that was also remarkably similar to migration along homotypic, hippocampal glial fibers [Gasser, U. E. & Hatten, M. E. (1990) J. Neurosci. 10, 1276-1285]. In all cases, migrating neurons had a characteristic appearance, apposing their cell soma against the glial fiber and extending in the direction of migration a motile, leading process that enfolded the glial fiber with short filopodia and lamellipodia. As seen by video microscopy, neurons moved along homotypic and heterotypic glial processes by translocation of the soma and were not "pulled" forward by the leading process. As the neuron moved, the nucleus remained in the posterior portion of the cell and cytoplasmic vesicles moved forward from the soma into the leading process. The dynamics of the movement of neurons along heterotypic glial substrates, including the speed and periodicity of motion, was identical to that of neurons migrating along homotypic glial substrates. These experiments suggest that the mechanism of movement of neurons along glial fibers is conserved in these two brain regions during development.

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PubMed1990

Neuron-glia interactions of rat hippocampal cells in vitro: glial-guided neuronal migration and neuronal regulation of glial differentiation.

To examine neuron-glia interactions of hippocampal cells, including glial-guided neuronal migration, glial organization of neuronal positioning and neuronal regulation of astroglial differentiation, rat hippocampal tissue, harvested between embryonic day 16 (E16) and postnatal day 3 (P3), was dissociated into a single cell suspension and plated in glass coverslip microcultures (Hatten and Liem, 1981; Hatten et al., 1984). Immunostaining the cells with antibodies against the glial filament protein (AbGFP) revealed developmental stage-specific changes in the number and extent of morphological differentiation of hippocampal astroglial cells. At E16-E18, fewer than 5% of the cells were AbGFP-positive; stained cells were immature, bearing very short processes. By E19-E20, the number of stained cells increased to 15% of the total cell population. Three forms of differentiated glial cells predominated, a bipolar form bearing processes 30-50 microns, an elongated form which resembled the radial glia of hippocampus, bearing processes 120 microns in length, and a stellate form with 3 or more processes 30-50 microns in length. At P0-P3, glial morphological differentiation varied with the culture substratum; differentiated forms resembling those seen at E20 occurred on Matrigel, but not on polylysine. Quantitation of the distribution of neurons relative to AbGFP-stained glial processes revealed developmental stage-specific changes in glial organization of neuronal positioning in the cultures. In cultures of E16-E18 hippocampal cells, the neurons did not preferentially associate with astroglial cells. By E19-E20, extensive neuron-glia interactions occurred, with 80-90% of the neurons being located within 5-10 microns of a glial process. In addition to their organization of neuronal positioning, E20 hippocampal astroglial cells supported extensive neuronal migration. Migrating hippocampal neurons displayed a cytology and neuron-glia cell apposition identical to that described for migrating cerebellar granule cells in vitro (Edmondson and Hatten, 1987), closely apposing their cell soma against the hippocampal glial process and moving along the glial arm by extending a thickened, leading process. Migration was seen only along highly elongated glial profiles resembling radial glial seen in vivo. The morphological differentiation of hippocampal glial cells in vitro was dependent on cell-cell interactions with neurons. In the absence of neurons, purified hippocampal astroglia had flat, undifferentiated profiles and proliferated rapidly. The addition of hippocampal neurons rapidly arrested glial growth and induced glial process extension.

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PubMed1988

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor.

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.

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PubMed1988

Astrotactin: a novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures.

A microculture system for mouse cerebellar cells has been used to identify an immune activity, raised in rabbits against postnatal cerebellar cells, that blocks neuron-glial interactions in vitro. In the presence of blocking antibodies, stable neuron-glial contacts did not form and neuronal induction of glial process outgrowth did not occur. Subsequently, neurons were randomly arranged in the cultures rather than organized along the arms of astroglia. We have named the immune activity that blocks neuron-astroglial interactions anti-astrotactin. Partial purification of the anti-astrotactin blocking antibodies was obtained by cellular absorption with PC12 cells, a clonal cell line which expresses both the N-CAM and NILE (Ng-CAM, L1) glycoproteins. Subsequent absorption with purified cerebellar granule cells, but not with astroglial cells, removed the blocking activity, suggesting that the antigen(s) bound by blocking antibodies are neuronal. Immunoprecipitation of [35S]methionine- or [3H]fucose-radiolabeled Triton extracts of early postnatal cerebellar cells showed that the unabsorbed antiserum recognized a large number of proteins. Among these were bands with apparent molecular masses of N-CAM (180 and 140 kD) and NILE (230 kD). After absorption of the immune serum with PC12 cells, the number of bands recognized by the antiserum was reduced to a prominent band at 100 kD and a diffuse smear of material between 80 and 90 kD. The prominent band at 100 kD was removed by subsequent absorption of the immune serum with granule cells, a step which removed the blocking activity in the cerebellar microculture assay. Further evidence suggests that the astrotactin activity is missing or defective on granule cells from the neurological mutant mouse weaver, an animal that suffers a failure of glial-guided neuronal migration. When anti-astrotactin Fab fragments were pre-absorbed with weaver cerebellar neurons and then tested in the functional assay of neuron-glial interactions, the immune blocking activity was not removed. In contrast, wild-type cerebellar neurons removed the anti-astrotactin blocking activity under the same conditions. Subsequently, when [3H]fucose-radiolabeled Triton extracts of weaver and normal cells were immunoprecipitated with whole or PC12-absorbed anti-astrotactin antiserum, the intensity of the band at 100 kD was reduced by 95% in weaver cells.

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PubMed1988

The extending astroglial process: development of glial cell shape, the growing tip, and interactions with neurons.

To analyze how astroglial cells attain the complex shapes that support neuronal migration and positioning in vitro (Hatten et al., 1984; Hatten 1985), early postnatal mouse cerebellar cells were plated in microcultures, and glial process outgrowth was monitored by high-resolution time-lapse video microscopy combined with immunocytochemical localization of antisera to glial filament protein (GFP), and by electron microscopy. The 2 principal astroglial forms seen in these cultures, stellate and Bergmann-like (Hatten et al., 1984), begin to develop their distinctive shapes by the outgrowth of processes in the first 8 hr after the cells are plated. Glial process extension is most vigorous in this period, resulting predominantly in stellate forms. A second population of glial cells, having fewer, longer processes reminiscent of Bergmann glia in vivo, first appears about 5 hr after plating. During the next 16-24 hr, while the stellate cells only slightly increase their process length, the bipolar cells double their length. The most striking feature of the elongating glial process is its highly motile tip, which rapidly extends microspikes and lamellopodia. Unlike the neuronal growth cone, which is the expanded terminal of a thin neurite shaft, the glial growing tip forms the end of a wide, paddle-like process that is filled with motile mitochondria and masses of glial filaments, and is bordered by an undulating lamella fringed by microspikes. Soon after the emergence of glial processes, cell-cell interactions between the growing glial process tip and granule neurons occur. Within minutes of an initial encounter between the glial process and the neuron, contact relationships that are stable during the observation period form between the cells. Subsequently, many neurons extend a small neurite onto the glial process, and astroglial process extension continues by the movement of the glial growing tip out beyond the neuron. Thus, cerebellar astroglia in vitro develop complex shapes in the same fashion as do neurons: the outgrowth of processes tipped by a motile ending. The growing tips of astroglial processes interact with neurons, resulting in the stable association of neurons and glia.

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PubMed1988

Mouse cerebellar granule neurons arrest the proliferation of human and rodent astrocytoma cells in vitro.

To understand the control of glial tumor cell proliferation, we have examined the effects of neurons on a number of human and rodent glioma lines. These included C6, G26-24, U-251, HTB-16, and A-172 cells of astroglial lineage and G26-20 of bipotential astrocytic and oligodendrocytic lineage. Rapid, specific binding of granule neurons to the human A-172, HTB-16, and U-251 and mouse G26-24 cell lines occurred, after which 3H-thymidine incorporation by these astrocytoma cells dropped 2-5-fold within 12 hr. The number of glial cells remained constant for 5-7 d when the glia were cocultured with granule neurons. Thereafter many neurons detached from the glial cells and glial proliferation commenced again. No effects on glial cell number were seen when PC12 cells were substituted for cerebellar granule neurons. To test the mechanism of neuronal control of glioma cell growth, we added granule neurons or PC12 cells that had been fixed lightly with paraformaldehyde, a plasma membrane fraction of purified granule cells, PC12 cells or astrocytoma cells, or medium conditioned by either granule cells or a mixed population of cerebellar neurons and astroglia. The proliferation of responsive glioma cell lines ceased in the presence of either fixed granule neurons or plasma membranes purified from granule neurons. The addition of fixed PC12 cells or plasma membranes purified from PC12 cells, 3T3 cells, or astrocytoma cells had no effect on glial cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1988

Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro.

In developing mammalian brain, many neurons migrate to their final position by moving in direct apposition to radially oriented glial cells. Glial-guided migration can be visualized in microcultures of mouse cerebellar cells by the combined use of cellular antigen markers and high resolution time-lapse video microscopy (Hatten et al., 1984; Edmondson and Hatten, 1987). Such studies have demonstrated the behavior of migrating cells and revealed a motile leading process on the migrating neuron that resembles an axonal growth cone and grows along extended glial fibers. To study the fine structural details of the migrating neuron and its neuron-glial apposition, we identified and monitored neurons in microcultures with video microscopy and examined the cytology and cellular contacts of the same cells with transmission electron microscopy. The cytology of the soma and leading process of migrating cells closely matches that described for granule cells in intact brain (Rakic, 1971). Newly observed structures include the presence of longitudinally oriented microtubules extending from a basal body in the soma into the leading process, and microfilament-rich filopodia arising from the soma and leading process. The most striking feature of actively migrating neurons is a specialized junction between the neuronal cell soma and apposing glial fibers. At this junction, here termed "interstitial density," the extracellular space is dilated to 20 nm and filamentous material in the intracellular cleft either spans the cleft or runs parallel to the cell membranes. Some interstitial fibrils are contiguous with, or are transmembranous extensions of, submembranous cytoskeletal elements that attach to microtubules. Interstitial junctions were not found between neurons that did not translocate in the observation period before fixation. Instead, stationary cells formed desmosomes (puncta and macula adhaerentia) at appositions with glial processes.

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PubMed1987

Neuronal inhibition of astroglial cell proliferation is membrane mediated.

Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fluorescence microscopy or with [35S]methionine-labeled cells indicated that the kinetics of the binding of the neurons to astroglial cells are rapid, occurring within 10 min of the addition of the neurons to the growing glia. 6 h after neuronal attachment, astroglial DNA synthesis decreases, as shown by a two- to fivefold decrease in [3H]thymidine incorporation, and glial growth ceases. No effects on astroglial cell growth were seen after adding medium conditioned by purified cerebellar neurons cultured in the absence of astroglia, by astroglia cultured in the absence of neurons, or by a mixed population of cerebellar cells. This result was unchanged when any of these media were concentrated up to 50-fold, or when neurons and astroglia were cultured in separate chambers with confluent medium. Two groups of experiments suggest that membrane-membrane interactions between granule neurons and astroglia control astroglial cell growth. First, neurons fixed with dilute amounts of paraformaldehyde (0.5%) bound to the astroglia with the same kinetics as did living cells, inhibited DNA synthesis, and arrested glial growth within hours. Second, a cell membrane preparation of highly purified granule neurons also bound rapidly to the glia, decreased [3H]thymidine incorporation two- to fivefold and inhibited astroglial cell growth. The rate of the decrease in glial growth depended on the concentration of the granule neural membrane preparation added. A similar membrane preparation from purified cerebellar astroglial cells, PC12 cells, 3T3 mouse fibroblasts, or PTK rat epithelial cells did not decrease astroglial cell growth rates. Living neurons were the only preparation that both inhibited glial DNA synthesis and induced the astroglial cells to transform from the flat, epithelial shapes they have when they are cultured without neurons to highly differentiated forms that resemble Bergmann glia or astrocytes seen in vivo. These results suggest that membrane-membrane interactions between neurons and astroglia inhibit astroglial proliferation in vitro, and raise the possibility that membrane elements involved in glial growth regulation include neuron-glial interaction molecules.

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PubMed1987

Glial-guided granule neuron migration in vitro: a high-resolution time-lapse video microscopic study.

To study neuronal migration, migrating granule neurons in microcultures prepared from early postnatal cerebellum have been analyzed with time-lapse, video-enhanced differential interference contrast microscopy. The morphology of migrating neurons resembles the elongated forms of migrating neurons described both in vivo and in vitro (Rakic, 1971; Hatten et al., 1984). The neuron closely apposes its soma along the glial fiber and extends a thickened leading process in the direction of migration. This leading tip is highly motile, with several filopodial extensions. Intracellular vesicular structures extend from the nucleus into the leading process of migrating neurons in vitro. Quantitation of the motions of migrating neurons revealed a saltatory pattern of advance along the glial fiber. Periods of cell soma movement at the rate of 56 +/- 26 micron/hr along the glial fiber are punctuated by periods during which the cell soma slows to a complete stop. The overall rate of migration is 33 +/- 20 micron/hr. The growing tip of the leading process rapidly extends and retracts, resulting in a net advance along the glial fiber. However, the periods of the extension and retraction of the leading process growing tip are not synchronized with the motions of the cell soma.

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PubMed1986

Weaver mouse cerebellar granule neurons fail to migrate on wild-type astroglial processes in vitro.

To study the regulation of glial-guided neuronal migration, we have analyzed the behavior of cerebellar granule neurons purified from the homozygous weaver (wv/wv) B6CBA-w mouse, an autosomal recessive genetic mutation that suffers a failure of granule cell migration along Bergmann glial processes (Rakic and Sidman, 1973a, b; Rezai and Yoon, 1972), on the processes of astroglia purified from homozygous normal B6CBA-Aw-J-wv (+/+) mouse cerebella. When co-cultured with normal astroglia, weaver granule neurons failed to form neuron-glia contacts characteristic of migrating neurons and impaired normal astroglial morphological differentiation. Normal astroglial cells co-cultured with weaver granule cells had enlarged cell somata with stunted processes and enlarged endfeet compared to normal astroglia co-cultured with normal granule cells. In contrast, normal neurons associated with weaver astroglia, forming tight appositions seen for migrating neurons in vivo, and enhanced weaver astroglial morphological differentiation. Weaver astroglia co-cultured with normal granule cells contained a more normal complement of glial filaments and had a smaller perikaryon with longer, more tapered processes than their counterparts co-cultured with weaver neurons. These results suggest, in agreement with the study of Goldowitz and Mullen (1982) on heterozygous mutant chimeras, that the granule neuron is a primary site of action of the weaver gene, and further support our previous findings that neuron-glia interactions regulate astroglial morphological differentiation (Hatten, 1985).

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PubMed1985

Neuronal regulation of astroglial morphology and proliferation in vitro.

To analyze the interdependence of neurons and astroglia during central nervous system development, a rapid method for purifying early postnatal cerebellar neurons and astroglia, and recombining them in vitro, has been developed. The influence of neurons on astroglial shape and proliferation has been evaluated with an in vitro model system previously used to describe the role of cerebellar astroglia in neuronal migration and positioning (Hatten, M. E., and R. K. H. Liem, 1981, J. Cell Biol., 90:622-630; and Hatten, M. E., R. K. H. Liem, and C. A. Mason, 1984, J. Cell Biol., 98:193-204. Cerebellar tissue harvested from C57Bl/6J mouse cerebellum on the third or fourth day postnatal was dissociated into a single cell suspension with trypsin, and enriched glial and neuronal fractions were separated with a step gradient of Percoll. Highly purified astroglial and neuronal fractions resulted from subsequently preplanting the cells on a polylysine-coated culture surface. In the absence of neurons, astroglia, identified by staining with antisera raised against purified glial filament protein, assumed a flattened shape and proliferated rapidly. In the absence of astroglia, cerebellar neurons, identified by staining with antisera raised against the nerve growth factor-inducible large external (NILE) glycoprotein and by electron microscopy, formed cellular reaggregates, had markedly impaired neurite outgrowth, and survived poorly. When purified neurons and isolated astroglia were recombined, astroglial proliferation slowed markedly and the flattened shape expressed in the absence of neurons transformed into highly elongated profiles that resembled embryonic forms of cerebellar astroglia. After longer periods (48-72 h) in the presence of neurons, astroglia had "Bergmann-like" or "astrocyte-like" shapes and neurons commonly associated with them. These results suggest that neurons influence the differentiation of astroglia.

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PubMed1984

Embryonic cerebellar astroglia in vitro.

Three types of astroglia appear during cerebellar development--radial glia and Bergmann glia, which are thought to facilitate neuronal migration, and astrocytes, which are thought to compartmentalize mature granule neurons. Cells resembling Bergmann glia and astrocytes have been described in cultures of cerebellar cells harvested from early postnatal cerebellum. In this study, we have used cell-type specific antisera to visualize embryonic forms of cerebellar astroglia and their interaction with embryonic neurons in vitro. When cells were dissociated from mouse cerebellum on the thirteenth embryonic day (E13), 3 forms of cells were stained with antisera raised against purified glial filament protein ( AbGF ), all of which had more elongated processes and less complex shapes than astroglia from postnatal day 7. The vast majority of embryonic cerebellar neurons did not contact these immature forms of astroglia.

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PubMed1984

Gliotoxic effects of alpha-aminoadipic acid on monolayer cultures of dissociated postnatal mouse cerebellum.

The cytotoxic effects of DL-, D- and L-alpha-aminoadipic acid, a six-carbon homologue of glutamate, were investigated in cell cultures of dissociated postnatal mouse cerebellum. Treatment with alpha-aminoadipic acid resulted in rapid nuclear and cytoplasmic swelling and, after longer periods of exposure, karyopyknosis of astrocytes, identified by indirect immunofluorescence labelling with anti-human glial fibrillary acidic protein antiserum. The number of astrocytes with pyknotic nuclei depended on the concentration of alpha-aminoadipic acid as well as on the duration of drug action. The presence of 0.21 mM DL-alpha-aminoadipic acid or 0.10 mM L-alpha-aminoadipic acid for 40 h caused karyopyknosis in 50% of the astrocytes. In contrast, D-alpha-aminoadipic acid, had little gliotoxic activity. None of the cytotoxic effects of DL-alpha-aminoadipic acid or L-alpha-aminoadipic acid observed for astrocytes were seen for the neurons present in the cultures when the drug was added after 4 days in vitro. Neurotoxic effects were evident, however, when alpha-aminoadipic acid was included in the culture medium at plating. These results indicate that alpha-adminoadpic acid can be used to substantially reduce the number of astroglia in cerebellar cultures and that dissociated cell cultures will provide a useful model with which to study the mechanisms of alpha-aminoadipic acid induced glial toxicity.

2-Aminoadipic Acid↗