D. W. James

An electron-microscopic study of the early outgrowth from chick spinal cord in vitro

Summary1.A simple method is described for the location and ultrathin sectioning of selected areas of the outgrowth from chick spinal cord in vitro.2.What appear by light microscopy to be single outgrowing fibres are commonly (but not invariably) bundles of processes. Some are so small as to be beyond the limits of light microscopical resolution. Over appreciable distances bundles may have no supporting cells associated with them.3.Within the outgrowth, synaptic profiles have been observed, and it is suggested that they are newly formed during culture.

Association of glial cells with the terminal parts of neurite bundles extending from chick spinal cord in vitro

SummaryThe terminal parts of radially directed neurite bundles growing out from chick embryo spinal cord in vitro have been examined by phase and electron-microscopy,A type of ending is described in which the terminal parts of the neurites are associated with a glial cell. The latter sends a single major process proximally towards the explant. Distally it is attached to the substrate, and the neurite ends are related to its dorsal (nonsubstrate) aspect. Appearances suggesting a mechanism of adhesion of neurites to each other and to the gial cell are described.“Growth vesicles” were found in both neurites and glia.It is suggested that movements of terminal glial cells may affect the pattern of outgrowth of their attached neurite bundles.

An electron-microscopic study of the de novo formation of neuromuscular junctions in tissue culture

SummaryTrypsin dissociated somatic muscle from 11-day chick embryos was cultured upon a collagen substrate, and later confronted with 7–9 day chick lumbar spinal cord. Regions of contact between outgrowing nerve and muscle were identified light microscopically and prepared for electron microscopy. Neuromuscular junctions comparable to those found in the developing chick embryo formed de novo between homologous explants. Appearances were also found suggesting that mechanisms may exist to maintain apposition between nerve and muscle in the early stages of development of such junctions.

Synaptic profiles in the outgrowth from chick spinal cord in vitro

SummarySynaptic profiles have been identified in the outgrowth from chick embryo spinal cord maintained in vitro for short periods. Profiles corresponding to types that may be excitatory and inhibitory in the intact central nervous system have been found. Their presence outside expiants, and in occasional relation to glial cells, suggests that neurites themselves may possess a generalised capacity for synapse formation under appropriate circumstances, rather than be limited to specific targets.

The development of synapses in vitro between previously dissociated chick spinal cord neurons.

SummaryThe formation and development of synaptic contacts between dissociated chick spinal cord neurons has been investigated. By the 6th day in vitro “immature” profiles with few vesicles were observed. By 14–18 days “mature” types with numerous vesicles were found, indistinguishable from those of newly hatched chick spinal cord. After this period degeneration occurred, and was especially marked in the post-synaptic element. Such degeneration could be postponed by the addition of small numbers of somatic muscle cells. The Kanaseki and Kadota (1969) technique was applied to the study of coated vesicles at various stages of synaptic development.

Scanning electron microscopic observations of chick embryo fibroblasts in vitro, with particular reference to the movement of cells under others.

SummaryChick embryo fibroblasts in vitro have been studied with the scanning electron microscope. It has been found that when an advancing ruffled membrane reaches and “crosses” another cell, it retains contact with the substrate upon which it is moving. It is concluded that failure of contact inhibition is not explicable in terms of any hypothesis suggesting that advancing ruffled membranes adhere preferentially to the free (i. e. facing the nutrient medium) aspects of cells they encounter, rather than to their substrate.

Outgrowth from chick embryo spinal cord in vitro, studied with the scanning electron microscope

SummaryCultures of chick spinal cord have been grown upon a plastic substrate, using the Maximov flying coverslip technique. After fixation, drying and coating in vacuo with carbon and gold, they have been examined with a Cambridge Instrument Company “Stereoscan” scanning electron microscope. A number of cell types — fibroblasts, oligodendrocytes and astrocytes — have been identified in the outgrowth, and in a single case possibly a neuron. The appearance of these cells as seen with scanning electron microscopy is described. What appear under light microscopic examination to be single processes often possess a multicomponent structure, and are axon bundles rather than single axons. Both bundles and cells are attached to the substrate by discreet finger-like processes. Some relations of glial cells to axon bundles are shown, and appearances suggestive of early myelination are recorded.

The culture of previously dissociated embryonic chick spinal cord cells on feeder layers of liver and kidney, and the development of paraformaldehyde induced fluorescence upon the former

SummarySpinal cord cells from embryonic chicks were cultured upon liver and kidney feeder layers of similar species origin. Successful cultures were obtained with inocula of cord cells containing as few as 15 000 cells ml−1, whereas without feeder layers at least 200 000 ml−1 are ordinarily required. Upon liver, many neurons and processes became intensely fluorescent, a property seldom shared by those grown upon kidney. Many processes upon liver contained large numbers of dense-cored vesicles, significantly larger and more numerous than in those grown upon kidney. We conclude that association with liver feeder layers has the consequence of producing in cord cells fluorescence and ultrastructural characteristics appropriate to catecholamine content, through a mechanism as yet unknown.

The development and ultrastructure of previously dissociated foetal human cerebral cortical cells in vitro.

SummaryCells 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.

An ultrastructural and electrophysiological study of the development of neuro-muscular junctions between previously dissociated foetal rat cells in vitro

SummaryThe development of neuro-muscular junctions between previously dissociated foetal rat spinal cord and somatic muscle has been investigated. The first indications of junction formation, both ultrastructurally and electrophysiologically, were observed after circa 18 days in vitro. The junctions contained numerous vesicles, but no secondary folds were developed even after 6 weeks in culture, and synaptic densities were not well marked. Functional endplates were found, and action potentials, endplate potentials and miniature endplate potentials recorded.