Neuronal Cells from Chick Embryo Cerebral Hemispheres Cultivated on Polylysine-Coated Surfaces

Plasma membranes isolated from chick embryo neurons after 2 days (division phase) and 5 days (maturation phase) in culture showed no differences in the phospholipid (PL) pattern or in the plasma membrane average fluidity. Significant ganglioside (Ggl) variations were only found in GM1 and GD3 which represent 35% of the total plasma membrane N-acetylneuraminic acid (NeuNac) content (1.8-fold GM1 NeuNac increase, 1.4-fold GD3 NeuNac reduction). Lactoperoxidase-catalyzed radioiodination (¹²⁵I) under saturating conditions indicated that the hydrophobic core of cell surface Ggl was more exposed to the hydrophilic extracellular environment than that of PL. Nevertheless, when interneuronal contacts start to form, the ¹²⁵I-labelling of both PL and Ggl dropped progressively throughout the maturation phase. Further analysis revealed that (1) plasma membrane GD3 was not accessible to the enzymatic labelling, and (2) surface phosphatidylethanolamine (PE) and GT1b became more accessible to the external medium during development, whereas the opposite was found for phosphatidylcholine (PC) and GM1. The overall profile of radioiodinated PL and Ggl was unaltered when poly-L-lysine was substituted for polyethyleneimine as culture substratum, except for an increase in GT1b labelling 24 h after plating. These results suggest the existense of a ''buffering'' mechanism protecting the neuronal plasma membrane during cellular growth from marked changes in fluidity, polar lipid composition and relative localization of polar lipids with regard to the extracellular environment. In addition, our findings suggest that: (1) GD3, which is at maximal levels during the division phase, is not exposed to the extracellular environment; (2) PE, PC, GM1 and GT1b undergo subtle topological reorganization as cellular maturation proceeds, and (3) the culture substratum may modulate the exposure of certain cell surface glycolipid components.

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“…(16). Cells were plated as described (3,17] at an initial density of (1.5-5 x 106cells/ml. Media were renewed every other day.…”

Section: Preparation O F Culture Dishes With Polyaminesmentioning

confidence: 99%

Expression of Plasma Membrane and Cell Surface Phospholipids and Gangliosides of Chick Embryo Neurons Grown in Primary Cultures: Developmental Studies

Lelong

Frechard²,

Crémel³

et al. 1991

Dev Neurosci

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“…The majority of neuronal cultures previously described (4,5) contain serum. Its ill-defined and variable composition and its ability to promote the proliferation of glial cells, which produce neuronal growth factors (6), have prevented investigators from defining the molecular growth requirements of neurons.…”

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confidence: 99%

Changes in insulin and transferrin requirements of pure brain neuronal cultures during embryonic development.

Aizenman¹,

Weichsel²,

Vellis³

1986

Proc. Natl. Acad. Sci. U.S.A.

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A pure neuronal culture grown in a defined serum-free environment has been developed and characterized. Insulin was the only hormone found to enhance the growth of neurons obtained from embryonic chicken brains during the early proliferative stage, a time when many neurons survived without the addition of any growth factors to the culture. With increasing embryonic age, there was an increase in the number of neurons requiring transferrin. By the time neurons reached a postmitotic state in older brains, they were completely dependent on both insulin and transferrin for survival and growth. Because this culture is free of glial cells and serum, it provides an effective basis for investigating molecular mechanisms underlying neuronal development.Current understanding of neuronal development has progressed slowly due to the complexity of the developing brain, in which astrocytes, oligodendrocytes, neurons, and their precursors interact in an ever-changing chemical environment. This complexity has been a major obstacle to defining the stages and characterizing the biochemical changes through which neurons progress during development. Our approach towards unravelling the developmental process has been to isolate, culture, and study a population composed of a single cell type as it proliferates and differentiates. This investigation defines the requirements for neuronal growth in vitro and the changes occurring in these requirements as the neurons develop in vivo.We have studied 6-to 9-embryonic day (ED) chicken brains because they are composed of a nearly pure population of neuronal cells that during this period progress in vivo from a dividing to a postmitotic state (1-3). The majority of neuronal cultures previously described (4, 5) contain serum. Its ill-defined and variable composition and its ability to promote the proliferation of glial cells, which produce neuronal growth factors (6), have prevented investigators from defining the molecular growth requirements of neurons. The only previous attempt to culture chicken brain cells in a defined medium, after an exposure to serum, did not delineate the factors critical for neuronal survival and, in addition, did not characterize the cell types of the culture (7).In this study, we have investigated the essential growth requirements for pure neuronal populations obtained from embryos of various ages. We found these requirements to change with development. The neuronal cultures were characterized by immunocytochemical and biochemical means and were found to be essentially free of glial cells. This eliminated the possibility of the factors acting on, or via, glial cells and increases the effectiveness of this culture system for studying neuronal development. MATERIALS AND METHODSCell Culture Preparation. Neuronal cultures were prepared from 6-, 7-, 8-, or 9-ED brains. After the brains had been dissected from the chicken embryos and the meninges had been stripped off, the tissue was put in 1:1 mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 medium...

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“…The dis sociated cells were cultured in polylysine-coated plastic Petri dishes following the detailed description of Sensenbrenuer and co-workers [24,37]. After incuba tion for various periods of time the cells were washed three times with buffered saline and harvested by using a rubber policeman.…”

Section: Preparation O F Culturesmentioning

confidence: 99%

“…Primary cultures of dissociated nervous tissue are considered to be good models of morphological and biochemical development of brain cells [3,12,15,24,26,37,38,42,45]. Isolated neurons can be grown in primary cul ture where they develop from single-cell sus pension into mature neurons with a well-form ed neuronal network and synaptic connec tions [24,37].…”

Section: Introductionmentioning

confidence: 99%

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Developmental Changes of the GABA and Polyamine Systems in Isolated Neurons in Cell Culture

Seiler¹,

Sarhan²,

Roth-Schechter³

1981

Dev Neurosci

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Dissociated cells of cerebral hemispheres from 8-day-old chick embryos were cultivated for 8 days in polylysine-coated Petri dishes. Changes of DNA, RNA, total proteins, putrescine, spermidine, spermine, free amino acids and enzymes involved in polyamine and GABA metabolism were studied throughout neuronal development in culture. The presence of GABAergic neurons in the cultured cell population was demonstrated. There were time-dependent changes in cellular polyamine concentrations and the activities of the enzymes involved in polyamine metabolism. Since no significant proliferation took place after the first day in culture, the observed changes indicate a role of polyamine metabolism during neuronal differentiation; it was not possible, however, to attribute the observed changes to specific functions. This culture system seems especially useful for the study of biochemical and of functional correlations between GABA and polyamine metabolism and morphological and functional developments of neurons.

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Neuronal Cells from Chick Embryo Cerebral Hemispheres Cultivated on Polylysine-Coated Surfaces

Cited by 130 publications

References 18 publications

Expression of Plasma Membrane and Cell Surface Phospholipids and Gangliosides of Chick Embryo Neurons Grown in Primary Cultures: Developmental Studies

Expression of Plasma Membrane and Cell Surface Phospholipids and Gangliosides of Chick Embryo Neurons Grown in Primary Cultures: Developmental Studies

Changes in insulin and transferrin requirements of pure brain neuronal cultures during embryonic development.

Developmental Changes of the GABA and Polyamine Systems in Isolated Neurons in Cell Culture

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