Glycoproteins of cultured explants of fetal cerebellum.
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Alkaloids↗
Biomedical subjects
M M Bird
Publications and source records attributed to M M Bird.
Microtubules and their relationships with other cytoskeletal components at cholinergic tectal synapses in culture.
Cholinergic synapses (identified by selective staining with tannic acid) of explant cultures of avian optic tectum were examined by electron microscopy after exposure of the cultures to a microtubule-stabilising drug, Taxol. The presynaptic components of such synapses commonly contained microtubules but they were never in close association with the presynaptic specialisation. Postsynaptically, microtubules commonly ran into, or close to, the postsynaptic density and/or were linked to it by 3-5 nm filaments, which, along with other filamentous cytoskeletal elements, were a prominent feature of tannic acid-stained cholinergic synapses. This study also provided some evidence that acetylcholine receptors may be present in both presynaptic and postsynaptic membranes at early stages of synapse formation and in the postsynaptic membrane alone at later stages.
Animals↗
Cytoplasmic inclusions within developing mouse retinal photoreceptor cells maintained in culture: possible type A retrovirus particles?
Unusual cytoplasmic inclusions were consistently observed in photoreceptor cells of explants of embryonic mouse retina maintained in culture for 21 days or longer. The inclusions were chiefly spherical (some appeared to be short cylinders) and about 76 nm in diameter. They consisted of a granular/amorphous electron-dense periphery and an electron-lucent core and were usually in clusters, often close to the Golgi apparatus or plasma membrane. They were also commonly present in photoreceptor cell synaptic terminals in the outer plexiform layer. They displayed close relationships with cisterns and tubules of the endoplasmic reticulum and many were located within dilated sacs of reticulum. Narrow, smooth-surfaced tubules appeared to run into or through the cores of some of the inclusion bodies linking them to others. Similar inclusions were not seen in any other cell types in the explants and have not been reported in previous in vivo and in vitro studies of developing or adult mouse retina. The possibility that these inclusions represent Type A retrovirus particles is considered.
Animals↗
An ultrastructural study of embryonic chick retinal neurons in culture.
The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study. At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form. Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated. These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.
Animals↗
Establishment of synaptic connections between explants of embryonic neural tissue in culture: experimental ultrastructural studies.
The development of synaptic interconnections between co-cultured explants of central and peripheral nervous tissue from chick embryos has been investigated by light and electron microscopy. Two sets of co-cultured explants were used: (a) dorsal root ganglion (DRG) and spinal cord and (b) retina and tectum. Both sets of co-cultured explants became linked by bundles of fibres but the most consistent results were obtained with the DRG-spinal cord explants. Thus axons from the DRG extended large distances across the culture substrate to reach and enter mainly the dorsal horn region of the spinal cord explants. In contrast retina-tectum links were less frequently established and were less extensive, possibly because there are fewer cells in retinal explants capable of establishing contacts in tectal explants than there are cells in DRG explants capable of establishing contacts in the spinal cord. In order to distinguish between synapses involving only neuronal elements within an explant and those involving ingrowing fibres, fibre bundles linking adjacent explants were transected and the preparations fixed two to six hours later. Electron microscope study of such cultures revealed degenerating neurites and terminals in the spinal cord explants receiving DRG fibres but none in the corresponding DRG explants. Retinal explants contain numerous synapses of many types but degenerating terminals could not be found within the retinal explants after nerve fibre transections. Degenerating neurites and terminals were found within tectal explants but they were fewer and more difficult to locate than those found within spinal cord explants. The reasons for such differences are discussed.
Animals↗
A note on presynaptic dendrites in explant cultures of embryonic chick tectum.
Explants from embryonic chick tectum maintained in culture for at least two weeks contained presynaptic dendrites, characterised by focal presynaptic specialisations in addition to the usual fine structural features of dendrites. The explants also contained bouton-like structures with both pre- and postsynaptic specialisations, interpreted as the terminal parts and appendages of the presynaptic dendrites. The dendrites and their appendages engaged in various serial synapses including triplet and reciprocal synapses. Although it is known that cells which do not normally have presynaptic dendrites may be induced to develop them under abnormal developmental or pathological conditions, the development of presynaptic dendrites in the present material is more likely to be a manifestation of organotypic development, since the adult avian tectum normally contains cells with presynaptic dendrites.
Animals↗
Regions of putative acetylcholine receptors at synaptic contacts between neurons maintained in culture and subsequently fixed in solutions containing tannic acid.
Spinal cord neurons from 9-day chick embryos were maintained in culture for up to 35 days and then fixed in 4% cacodylate-buffered glutaraldehyde containing 2% tannic acid. After about 15 days in culture a small percentage of the synaptic specializations present were characterized by striking electron-dense striations averaging 15 nm in width, oriented perpendicular to the postsynaptic membrane. These structures increased in frequency with time in culture (to a maximum of about 10% of all synapses in the oldest cultures); they were asymmetrical, protruding approximately 8 nm into the synaptic cleft, and more deeply (approximately 15-18 nm), into the postsynaptic cytoplasm. On the basis of earlier work by Sealock (1980) they are interpreted as concentrations of acetylcholine receptors. Similar membrane differentiations were also seen associated with active-zone areas of a few presynaptic membranes, and the possibility that these represent presynaptic acetylcholine receptors is discussed. Additional observations reported are (1) the presence of striations resembling those seen at the postsynaptic membrane in the membranes of some postsynaptic vesicles, and (2) filamentous links between the striations and cytoskeletal elements of the postsynaptic cell.
Animals↗
The effects of taxol on embryonic chick tectum maintained in culture: an electron microscope study.
Tectal explants from chick embryos, established in culture for 2-3 weeks, were exposed to taxol-enriched media for 1-7 days, fixed, and studied by transmission electron microscopy. Taxol treatment resulted in no apparent disruption of the overall integrity of the organization of the explants nor in grossly increased cell death, but caused marked abnormalities of cytoskeletal elements. Intermediate filaments were increased in number in both neuronal and glial cells and very large numbers of microtubules were present, some aligned below the plasma membrane but most as components of large bundles in neuronal cell bodies and processes. Some such microtubules were associated with a network of intermicrotubule substance, consisting of 10-nm filaments running parallel to the microtubules, in hexagonal arrays surrounding individual microtubules, together with a very fine amorphous or filamentous component which was drawn into thread-like structures that linked the larger filaments to one another and formed the sides of the hexagons. Taxol treatment also resulted in the formation of concentric rings of microtubules separated by cylindrical sheets of electron-dense material. These observations extend previous descriptions of the effects of taxol on cytoskeletal elements, add to growing evidence for heterogeneity of microtubules within neurons, and suggest that taxol may be useful in studies of the functions of cytoskeletal elements and of microtubule heterogeneity in neurons.
Alkaloids↗
Neurons and glial cells in long term cultures of previously dissociated newborn mouse cerebral cortex.
In long term cultures of newborn mouse spinal cord neurons, glial cells and macrophage-like cells may frequently be located in their entirety. Many neurons possessed processes which could be traced from their cell bodies to their growth cones, a feature which indicated that the cells remained immature. The identification of neurons with certainty is a major problem when live cells are observed by light microscopy, but their size, nuclear and cytoplasmic content and the arrangement and number of their processes were features which helped to locate them. After many weeks in culture, however, astrocytes also became extremely large and it became increasingly difficult to separate them from neurons unless features within the cells were clearly seen. The shape and size of glial cells, especially astrocytes, may alter rapidly in culture, and a wide range of features was present for each cell type. It was therefore often impossible to categorize them with certainty when viewed live, but the identification of many was subsequently confirmed with the use of electron microscopic sections. Macrophage-like cells were numerous in older cultures where they engulfed the ever increasing number of dead cells. The size of the cell bodies of many macrophages matched those of neurons, but their stumpy processes and largely granular content were generally adequate to identify them. The persistent immaturity of the neurons and their apparent dependence both on environmental conditions and synaptic relations, and the characteristics of glial cells in long term cell cultures are discussed.
Animals↗
Transport media for Campylobacter fetus venerealis.
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Letter↗
Ultrastructural observations on rapid formation of neuro-muscular junctions in vitro.
The development of neuro-muscular junctions (mouse, rat) from the time of first contact between neurons and myotubes in culture and the changes which lead to the formation of functional synaptic contacts have been investigated using light microscopy and ultrastructural techniques. An extensive basal lamina was present when the neuronal cell population was added to the developing myotubes in culture. The nerve cells were initially strongly attracted to each other and nerve cell aggregates formed rapidly. It was only when nerve fibers began to grow out of these aggregates to contact developing myotubes that changes within the cytoplasm of the two adjacent cells were observed. These developments included accumulations of filaments, membrane densities, mitochondria and large clear vesicles within both cells in the region of contact. In addition, collections of glycogen granules and an extensive membrane reticular complex were found within myotubes, and an extensive granular material filled many of the nerve processes. The basal lamina within the intercellular space appeared more electron-dense that elsewhere and was traversed by strands linking the two cell membranes. These features all appeared to be stages of the initial formation of neuro-muscular junctions. It was only after these events had occurred that presynaptic vesicles gradually appeared within the future nerve terminal. The results of this paper therefore support the view that synaptic transmissions at developing mammalian neuro-muscular junctions is not necessarily dependent on the presence of presynaptic vesicles.
Animals↗
The morphology of synaptic profiles in explants of foetal and neonatal mouse cerebral cortex maintained in a magnesium-enriched environment.
The present study examines the ultrastructure of synaptic profiles developed in explant cultures of immature mouse cerebral cortex, maintained for prolonged periods in a magnesium-rich environment. The ethanolic phosphotungstic acid method was employed in addition to conventional preparation procedures so that paramembranous densities could be clearly observed. Although presynaptic terminals were frequently packed to capacity with vesicles in cultures maintained in magnesium-rich media, there was always a proportion which contained loosely collected vesicles. Few other changes in synaptic morphology were apparent and the paramembranous densities were unaffected. The reasons for the effectiveness of the transmission block, the absence of any change in the morphology of paramembranous densities, and the excessive crowding of presynaptic vesicles are considered and discussed.
Animals↗
The development and ultrastructure of previously dissociated embryonic chick corpus striatum cultured on feeder layers of liver cells.
Embryonic chick corpus striatum neurons were dissociated and maintained on liver feeder layers in culture. Although some large dark-cored vesicles were present in many nerve processes and presynaptic boutons they were substantially less numerous than chick spinal cord neurons grown under identical conditions. Paraformaldehyde-induced fluorescence, although observed in a few culture batches in aggregates of corpus striatum neurons, was otherwise absent and no decisive evidence was obtained to suggest that fluorescent corpus striatum neurons were commonly developed on liver feeder layers in culture. Microtubules filled most cell bodies and nerve processes, and extended well into synaptic boutons often approaching the active zones. They were much more abundant in cultures of corpus striatum than in comparable spinal cord preparations and formed the principal organelle of many nerve fibres. These differences between chick spinal cord and corpus striatum neurons are both interesting and difficult to interpret. It is possible that fewer appropriate cholinergic neurons are available for transformation into adrenergic neurons within the corpus striatum, and that excessive numbers of dark-cored vesicles indicate only a greatly increased rate of acetylcholine production and storage.
Acetylcholine↗
Scientists teaching gross anatomy.
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Anatomy↗
Presynaptic and postsynaptic organelles of synapses formed in cultures of previously dissociated mouse spinal cord.
This study describes some of the ultrastructural features of presynaptic and postsynaptic organelles at synapses developed in cultures of previously dissociated mouse spinal cord cells. Particular attention was paid to the agranular reticulum which is well developed at many presynaptic and postsynaptic sites, either in the form of simple tubules or cisternae, or more complex networks and often closely associated with mitochondria. In addition, the disposition of microtubules at and close to synaptic specializations is described. These and other features of synaptic zones, such as granular vesicles in presynaptic sites, are discussed in relation to cultures developed on feeder layers and synapses in vivo, and in relations to possible degenerative and regenerative events in the cell cultures.
Animals↗
Microsurgical transection of small nerve fibre bundles in vitro. Effects on axons, growth cones and glial cells.
Fibres growing from neurons of explanted dorsal root ganglia from 10 day chick embryos were transected and subsequently observed by light and electron microscopy after periods of a few to fifty minutes. Changes immediately proximal and distal to the cut together with alterations further away from the site of injury on both sides of the cut were recorded. Observations were also made on the growth cones of damaged axons and on changes in associated glial cells. Reactive and degenerative changes including the rotation, retraction and swelling of cutaxons occurred rapidly. Electron microscopy revealed tracts of filamentous material close to the sealed-off ends of axons, swollen organelles such as mitochondria, and lamellar bodies of varying dimensions. Proximal to the injury and closer to the explant, damaged and degenerating axons mingled with normal processes. Many contained only a fine granular material, others clumps of organelles, particularly mitochondria. Distal to the cut, microspikes were lost from some growth cones. The dense granular material filling microspikes and growth cones remained unchanged. Clumps of large clear vesicles, lamellar bodies and swollen degenerating mitochondria were present, not only within growth cones, but also in all parts of the axon distal to the cut. Glial cells associated with transected axons soon developed an electron dense cytoplasm containing swollen organelles. Large numbers of vesicles filled with a particulate substance were also found. The possible significance of the changes observed after transection are considered and discussed.
Animals↗
The development and ultrastructure of previously dissociated foetal human cerebral cortical cells in vitro.
Cells from foetal human cerebral cortex were mechanically dissociated and subsequently maintained in vitro for periods ranging between three and twenty-eight days. The ultrastructure of these cells at different stages of their development in culture was extensively examined. Nuclear and cytoplasmic features were extremely variable and a wide range of cell types was evidently represented. Of the three principal cell types found i.e. neurons, neuroglia and mesenchymal cells, only a minority of cells was classified with confidence, particularly during the first two weeks in culture. Extensive intercellular junctions of the adhaerens variety, common after 14 days in vitro were present at an earlier stage of development than synaptic profiles. First indications of synapse formation were observed after 21 days in vitro and after 24 days presynaptic sites filled with synaptic vesicles and with well defined presynaptic and postsynaptic thickenings were found. The significance of some of the features observed are both considered and discussed.
Cerebral Cortex↗
Microtubule fascicles in the stem processes of cultured sensory ganglion cells.
Microtubule fascicles, resembling those characterizing the initial segment of multipolar neurons, have been observed by electron microscopy within and close to the origin of the stem process of some unipolar ganglion cells in explant cultures of embryonic chick dorsal root ganglia. Each fascile comprised 2-6 closely spaced parallel microtubules linked by electron dense cross-bridges. Since similar observations have been made on stem processes in vivo, the possibility that linked microtubules occur commonly in this site is considered. The observations are discussed in relation to a possible correlation between the presence of microtubule fascicles and the initiation of action potentials.
Action Potentials↗