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Biomedical subjects

M Schachner

Publications and source records attributed to M Schachner.

At least 415 records · Page 23Linked to original sources
PubMed1984

Maintenance of immunocytologically identified Purkinje cells from mouse cerebellum in monolayer culture.

Purkinje cells were identified in monolayer cultures obtained from trypsin-dissociated cerebella of embryonic and early postnatal mice by the Purkinje cell-specific monoclonal antibodies PC1, PC2, PC3 and UCHT1. These cells also expressed the neuronal marker L1 antigen but not the glial markers, glial fibrillary acidic protein or 04 antigen. They also expressed tetanus toxin receptors, PC4, M1 and Thy-1 antigens. Survival of Purkinje cells was best: (a) when cerebella were taken from mice not older than one day of age: (b) when cells were seeded at higher plating densities; and (c) cultured in chemically defined medium which facilitates the survival of neurons. No Purkinje cells could be detected in cultures from mice older than 6 days. PC1 antigen expression developed in vitro on the same time scale as in vivo, i.e. it was first detectable at the equivalent of postnatal days 3-4. At this stage cell bodies had a size of 13-14 micron in diameter and few processes. Dendrite-like arborizations, with more than one primary dendrite, extension of usually only one thin and long (0.5-1.6 mm) axon-like process and collaterals directed preferentially towards other Purkinje cells, developed with time in culture until the final form was reached by the equivalent of approximately day 16. Cell body size was 18-19 micron in diameter at this stage. Cell shapes were reminiscent of those described in certain cerebellar mouse mutants and in experimentally produced agranular cerebella. Many ultrastructural features of these cells correlated with those described for the in vivo counterpart. However, there was a lack of spiny branchlets and abnormally long persisting somatic spines. Synaptic contacts of the 'en passant' type could be seen at the Purkinje cell soma. Gray type I synapses were seen on Purkinje cell dendrites and spines.

Animals↗
PubMed1984

Depolarization of cultured oligodendrocytes by glutamate and GABA.

Subpopulations of cultured oligodendrocytes from mouse spinal cord respond with depolarization to glutamate and GABA. Heterogeneity in the oligodendrocyte population was indicated by the observation that some cells respond to both GABA and glutamate, while others respond to only one and some are not responsive to either. Depolarizations are not mediated by an increase of [K+]o released from neurons. The response to GABA was blocked in Na+-free solution and is not accompanied by a change in input resistance. Several other neurotransmitters did not induce changes in membrane potential.

Animals↗
PubMed1984

Synapse formation and synaptic activity in mammalian nerve-muscle co-culture are not inhibited by antibodies to neural cell adhesion molecule L1.

Co-cultures of rat myotubes and spinal cord explants from mouse embryos were maintained in the presence of Fab fragments of polyclonal antibodies to neuronal cell surface antigen L1. Microscopic observation showed that neurite outgrowth was not blocked by anti-L1. By intracellular recording, no effect was observed on the number of myotubes that showed endplate potentials, nor on the efficiency of synaptic contacts. As was demonstrated by indirect immunofluorescence, added Fab fragments remained bound to the neurite surface and were present in the medium for at least two days in culture, after which time antibodies were replaced during the medium change. Taken together, these observations show that L1 antigen is not involved in synapse formation between nerve and muscle.

Animals↗
PubMed1984

Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, but not of monoclonal antibody, both of which are known to react with the surface membrane of these cells.

Animals↗
PubMed1984

The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2.

The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies.

Animals↗
PubMed1984

Glial cells in the pineal gland of mice and rats. A combined immunofluorescence and electron-microscopic study.

Antigenic markers characteristic of astrocytes and their differentiative states (i.e., glial fibrillary acidic protein (GFAP), vimentin, and M1 and C1 antigens) were investigated in the pineal gland of mouse and rat using double immunolabeling techniques. In both species the so-called interstitial cells as characterized by TEM were shown to be astrocytes, since they expressed vimentin, but neither fibronectin (a marker for fibroblasts and endothelial cells) nor the neuron-specific L1 antigen or tetanus toxin receptors. Subpopulations of vimentin-positive pineal astrocytes were also GFAP- and C1- antigen-positive. M1- antigen-positive cells were not detected. It is concluded that a considerable proportion of interstitial cells in the pineal gland of rat and mouse are immature astrocytes which, in contrast to other parts of the central nervous system, persist into adulthood.

Animals↗
PubMed1984

Interstitial and parenchymal cells in the pineal gland of the golden hamster. A combined thin-section, freeze-fracture and immunofluorescence study.

A combined thin-section/freeze-fracture study was performed on the superficial pineal gland of the golden hamster, comparing the parenchymal and interstitial cells of this animal with those previously investigated in rats. In contrast to rats, no gap junctions and gap/tight junction combinations could be found between pineal parenchymal cells of the hamster. Furthermore, the interstitial cells of the hamster pineal gland were found to have large flat cytoplasmic processes, which abut over large areas equipped with tight junctions. In thin sections, profiles of interstitial cell processes were seen to surround groups of pinealocytes. Interstitial cells and their sheet-like, tight junction-sealed processes thus appear to delimit lobule-like compartments of the hamster pineal gland. Because the classification of the interstitial cells uncertain, the expression of several markers characteristic of mature and immature astrocytes and astrocyte subpopulations has been investigated by indirect immunohistology. Many of the non-neuronal elements in the pineal gland are vimentin-positive glial cells, subpopulations of which express glial fibrillary acidic protein (GFA) and C1 antigen. The astroglial character of these cells is supported by the lack of expression of markers for neuronal, meningeal and endothelial cells. M1 antigen-positive cells have not been detected.

Animals↗
PubMed1984

Electrical properties of oligodendrocytes in culture.

The electrical properties of immunocytologically identified oligondendrocytes from embryonic mouse spinal cord maintained in culture for 3 to 6 weeks studied by passing current and recording potential changes with two separate intracellular electrodes. The average input resistance was 3.3 M omega and ranged from 0.7 to 16 M omega (n = 35). The input resistance increased by 19% with depolarization and decreased by 9% with hyperpolarization of 25 mV. The membrane time constant determined from the slope of the late exponential tail was 3.45 +/- 2.5 ms SD (n = 15). The specific membrane resistance of three cells was determined by a simplified square pulse analysis combined with measurement of membrane area. Membrane area was estimated from photomicrographs of cells injected with Lucifer Yellow CH and stained with the cell surface-reactive antibody 04 and from electron micrographs. An average specific membrane resistance of 1.3 X 10(3) omega cm2 and specific capacitance of 1.7 mu F/cm2 were calculated. Increasing [K+]o depolarized the cells and decreased the input resistance and the time constant.

Animals↗
PubMed1984

Some immature tetanus toxin-positive cells share antigenic properties with subclasses of glial cells. An immunofluorescence study in the developing nervous system of the mouse using a new monoclonal antibody S1.

Monoclonal antibody S1 reacts in monolayer cultures with the cell surfaces of oligodendrocytes and a subclass of astrocytes derived from early postnatal mouse cerebellum, cerebrum and spinal cord, as well as with some glial cells in mouse retina but not in dorsal root ganglia. At earlier developmental stages S1 antigen is present in addition to oligodendrocytes and astrocytes on some tetanus toxin-positive neurons. S1 antigen is a developmentally early marker, detectable already in freshly trypsinized single cell suspensions from cerebella of 13-day-old embryos. Immunocytolysis of S1 antigen-bearing cells leads to reappearance of S1 positive glial cells but not tetanus toxin receptor-positive neurons. S1 antigen is also expressed in rat, rabbit, chicken and human. When cultured cells are permeabilized with denaturing agents, S1 antibody not only labels cell surfaces of some glial cells and, depending on the developmental stage, some neuronal cells but also intracellular components of all astrocytes, oligodendrocytes, neurons and fibroblasts.

Animals↗
PubMed1984

Specificity of histiotypic organization and synaptogenesis in reaggregating cell cultures of mouse cerebellum.

The fine structure of reaggregating cultures of cells from 6- to 7-day-old mouse cerebellum was studied at intervals between 3 and 21 days in vitro (DIV). The resulting aggregates consisted mainly of small neurons (granule, stellate and basket cells), neuroglial cells and their processes. Large neurons were rarely present. By 7 DIV the previously loosely packed components had tightened into a more compact mass. A peripheral plexiform layer had formed which had many fine axons arranged into fascicles of parallel fibers. Deep to this zone was a cellular region containing clusters of neurons interspersed with small areas of neuropil. Axosomatic synapses appeared on neurons which resembled stellate or basket cells but not on granule cells. Axo-dendritic synapses formed in the neuropil of the cellular zone and, less frequently, in the outer plexiform layer. After 3 weeks glial cell processes had increased in volume at the expense of neurons. When cerebellar cells were cultured with cells from pons and medulla, which are normal sources of mossy fiber input, aggregates formed in which synaptic glomeruli were found. They were not seen in aggregates containing cells from retina and olfactory bulb cultured with cerebellum. Our observations suggest: that natural histogenetic mechanisms persist after dissociation and reaggregation of cerebellar cells resulting in a separation of an outer, 'molecular'-like layer from an inner granule cell layer and that neurons retain specificity of their synaptogenic capabilities both with regard to appropriate cell types and the morphological form that the synapses take.

Animals↗
PubMed1984

Specificity of monoclonal antibody N1 for cell surfaces of mouse central nervous system neurons.

Monoclonal antibody N1 reacts by indirect immunofluorescence with the cell surface of tetanus toxin-positive neurons from early postnatal mouse cerebellum. In freshly trypsinized single cell suspensions from early postnatal mouse cerebellum, 5-10% of all viable cells express N1 antigen on their surface. After 3-24 h of maintenance in vitro all N1 antigen-positive cells are tetanus toxin-positive. After culture periods of 3-4 days, most (approximately 90%) tetanus toxin-positive cells express N1 antigen on their surface. When horse serum-supplemented medium (HSSM) is used for cultivation, neurons begin to lose N1 antigen from their surface after about one week in vitro, until after two weeks in vitro, N1 antigen is no longer detectable, although some tetanus toxin-positive neurons can be shown to survive in culture. In defined medium, however, N1 antigen-positive neurons can still be detected after 34 days in vitro, the longest culture period examined so far. Complement-dependent immunocytolysis deletes all N1 antigen-positive and approximately 90% of all tetanus toxin-positive neurons from cultures. The remaining neurons reveal a morphology different from the one of the majority of small neurons, the granule cells. They have slightly larger cell bodies and several branched and unbranched cellular processes. Neonatal cerebellar cells show the same temporal sequence of appearance and disappearance of N1 antigen on most tetanus toxin-positive neurons in HSSM, and a persistence of N1 antigen on neurons in defined medium. N1 antigen becomes first detectable at embryonic day 17, and never becomes detectable in cell cultures derived from cerebella of younger mice. At all stages studied, N1 antigen expression is restricted to tetanus toxin-positive neurons, while it is absent from the cell surfaces of astrocytes, oligodendrocytes and fibroblasts. N1 antigen is also found in cultures derived from early postnatal mouse cerebrum, but is not detected in cultures derived from mouse retina, spinal cord, dorsal root ganglion, and embryonic telencephalon. It is also not detectable in cerebellar cultures from rabbit, rat, chicken and human. When N1 antibody is applied to fixed cultures where intracellular antigens are accessible, all cell types are labeled intracellularly, with astrocytes and fibroblasts revealing a fibrillary, vimentin-like staining pattern.

Animals↗
PubMed1984

Expression of neural cell adhesion molecule L1 during development, in neurological mutants and in the peripheral nervous system.

Neural cell adhesion molecule L1 consists of two glycoprotein bands of 140 and 200 kdaltons at all developmental stages studied (from birth to adulthood in murine cerebellum and cerebral hemispheres) and in the 4 neurological mouse mutants reeler, weaver, staggerer and Purkinje cell degeneration. In histological sections L1 antigen is detectable at birth in the Purkinje cell layer and fiber tracts in the prospective white matter, but not in the external granular layer. From postnatal day 4 onwards L1 antigen additionally appears in the inner part of the external granular layer, the zone of postmitotic premigratory granule cell neurons. The outer part of the external granular layer remains L1 antigen-negative until it disappears at approximately day 12. From then onwards, the antigen remains prominent in the nascent molecular layer and is less detectable in white matter and internal granular layer, the location of the cell bodies of postmigratory granule cells. The four neurological mouse mutants show development of L1 antigen expression analogous to the normal situation, despite an abnormal cellular architecture. In contrast to the central nervous system. Western blots of adult sciatic nerve show a more complex pattern of L1 immunoreactive bands. L1 antigen is detectable on most, if not all Schwann cells in histological sections of sciatic nerve from 17-day-old embryos. At postnatal day 2, only some Schwann cells appear L1 antigen-positive. From then onwards L1 seems most prominently associated with non-myelinating Schwann cells. In monolayer cultures of neonatal dorsal root ganglia the antigen is observed on the surface of neurons and of some Schwann cells. The mutant, trembler, shows a more immature staining pattern for L1 antigen in adult sciatic nerve.

Age Factors↗
PubMed1984

Expression of glial antigens C1 and M1 in the peripheral nervous system during development and regeneration.

The expression of C1 and M1 antigens was studied by indirect immunofluorescence methods in histological sections of peripheral nerves and ganglia of C57BL/6J mice during development and regeneration. In sciatic nerves of adult mice, C1 but not M1 antigen is found in vimentin- and glial fibrillary acidic protein (GFAP)-positive Schwann cells. A similar distribution is also seen in trigeminal nerve, dorsal root and superior cervical ganglia, and olfactory nerve. In all cases vimentin-positive structures outnumber GFAP- or C1 antigen-positive ones. At birth, C1 antigen and vimentin are expressed in sciatic nerves, but GFAP is not yet detectable. M1 antigen cannot be detected in Schwann cells. In monolayer cultures of neonatal mouse dorsal root ganglia, C1 antigen is expressed in a fibrillary staining pattern in some, but not all morphologically identified Schwann cells. In vitro, M1 antigen is not detectable in Schwann cells. After lesioning sciatic nerves of adult mice by cut or crush, detectable levels of C1 antigen rise after 4-6 days: The number of immunofluorescently labeled structures and their relative intensities are drastically augmented, first distally more so than proximally, over control values from non-lesioned, i.e. contralateral nerves. A similar augmentation is also observed for vimentin and GFAP. M1 antigen expression does not reach detectable levels in Schwann cells under these conditions. The increased detectability of C1 antigen persists up to 150 days after lesioning, the longest time period tested.

Animals↗
PubMed1984

gamma-Aminobutyric acid directly depolarizes cultured oligodendrocytes.

gamma-Aminobutyric acid (GABA) depolarizes in a dose-dependent manner approximately one-third of all immunologically identified oligodendrocytes in cultures of mouse spinal cord. Measurements of [K+]o indicate that the response to GABA is not due to K+ released from active neurons. The depolarization is not accompanied by a change in cell input resistance. Replacement of sodium in the bathing solution abolishes the entire response, whereas ouabain only inhibits the repolarization phase. Current clamp experiments with two separate intracellular electrodes show that the depolarization increases at more positive potentials while the repolarization increases at more negative potentials. Bicuculline and picrotoxin but not nipecotic acid reduce the GABA effect. Pentobarbital and chlordiazepoxid also reduce the GABA-induced depolarization. Muscimol produces a depolarization similar to that of GABA. Heterogeneity in the oligodendrocyte population is indicated by the observation that some cells respond to both GABA and glutamate, while others respond only to one and some are not responsive to either.

Animals↗
PubMed1983

Antibody L1 ejected from a micropipette identifies neurons without altering electrical activity.

Antibody L1 which reacts specifically with the cell surface of central nervous system neurons was pressure ejected from a micropipette into the vicinity of a cultured neuron, or applied to the bathing fluid during intracellular recording of activity. No alterations in membrane potential, shape of action potential, firing rates and postsynaptic activities were observed. Binding of antibody was observed by indirect immunofluorescence after injection of Lucifer Yellow. Bath application of antibody resulted in a uniform neuronal staining over the entire culture, whereas pressure ejected antibodies were limited to neuronal structures within about 200 micron of the cell recorded from. Live, L1 antigen-positive neurons could be identified by indirect immunofluorescence prior to recording.

Animals↗
PubMed1983

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro.

The binding of several plant lectins, Concanavalin A (Con A), Lens culinaris A (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA 120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope. In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro- and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.

Agglutination↗