Chick Embryonic Neurons Research Articles

Ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24- ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha- ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24- ras and c-Ha- ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.

Induction of glutamine synthetase in rat astrocytes by co-cultivation with embryonic chick neurons.

Co-cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co-cultivation and the number of neurons present in the co-culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.

Sympathetic neurons grown in culture generate ATP by glycolysis: correlation between ATP content and [ 3H]norepinephrine uptake and storage

The purpose of the present study was twofold: one, to establish the metabolic pathway responsible for the synthesis of ATP of chick embryonic sympathetic neurons maintained in culture and supplemented with nerve growth factor; two, to establish the relationship between ATP content and the uptake and storage of [3H]norepinephrine. We show that ATP content of 12-day-old cultured neurons was not significantly reduced by uncouplers (dinitrophenol and dicumarol) and other inhibitors (cyanide, azide, etc.) of oxidative metabolism, but was reduced by iodoacetate, meta-arsenite or glucose deprivation. Uptake and storage of [3H]norepinephrine in cultured neurons remained virtually unimpaired when ATP levels were reduced over 85% by iodoacetate. These observations suggest that the major route of ATP production is via anaerobic glycolysis, and only a small fraction of the total ATP store is used for uptake and storage of the sympathetic transmitter.

Interchangeability of nerve growth factor and high potassium in the long-term survival of chick sympathetic neurons in serum-free culture medium

The results of the present experiments demonstrate that embryonic sympathetic neurons initially selected in culture by nerve growth factor could be subsequently supported by raising potassium concentration of the medium to 35 mM. Alternatively, neurons initially selected by 35 mM potassium could be supported in a normal culture medium but supplemented with nerve growth factor. It is concluded that nerve growth factor and high concentrations of potassium act on the same subpopulation of chick embryonic sympathetic neurons to promote their long-term survival in a serum-free culture medium.

Sciatin: immunocytochemical localization of a myotrophic protein in chicken neural tissues.

A mytotrophic protein (sciatin) purified from chicken sciatic nerves has "trophic" or "maintenance" effects on cultured muscle. We have elicited a specific antiserum against purified sciatin in rabbits. Using this antiserum, we investigated the distribution of sciatin in embryonic and adult chicken tissues by an unlabeled peroxidase-an-tiperoxidase method at the light microscopic level. The antiserum stained adult chicken neural tissues in situ and cultured embryonic chick neurons. Staining was intense in the cell bodies of spinal cord neurons and the axoplasm of sciatic nerves. These was reaction product seen in the outer margins of myelin sheaths that corresponded to the Schwann cell cytoplasm. Cerebral cortical neurons were weakly stained by the antiserum. No staining was apparent in oligodendrocytes or astrocytes. Nonneural tissues, such as skeletal, smooth and cardiac muscle, kidney, and liver, were also unstained by the antiserum. Cultured spinal cord neurons, cerebral cortical neurons, and sensory neurons were stained immunocytochemically by the antiserum. There was no reaction product seen in the glial cells that are usually present in neuronal cultures or cultured cells from liver, kidney, skeletal muscle, smooth muscle, and cardiac muscle. Our results thus show that the myotrophic protein is localized in neuronal perikarya and their processes in vivo as well as in vitro.

Effects of Anticonvulsant Drugs on Chick Embryonic Neurons and Glia in Cell Culture

The responses of neurons and glial cells to diphenylhydantoin (DPH), 5-(p- hydroxyphenyl)-5-phenylhydantoin (HPPH) and phenobarbital were studied in cell cultures of dissociated chic

The development of axonal transport of proteins and glycoproteins in the optic pathway of chick embryos

Axonal transport in chick embryonic retinal neurons was studied after intraocular injection of [ 3H]proline or [ 3H]fucose. Rapid transport to the contralateral optic tectum could be demonstrated from the 10th day of incubation, and the rate of rapid transport increased with age. As compared to proline-labelled proteins, a relatively larger proportion of retinal fucose-labelled glycoproteins was transported to the tectum throughout the development, with the maximal difference at 18 days. Sodium dodecyl sulphate (SDS) gel electrophoresis of leucine- or fucose-labelled tectal proteins revealed that a large number of relatively high mol. wt. polypeptides reached the optic tectum by a rapid phase of axonal transport.

Axonal agranular reticulum and synaptic vesicles in cultured embryonic chick sympathetic neurons.

Cultured chick embryonic sympathetic neurons contain an extensive axonal network of sacs and tubules of agranular reticulum. The reticulum is also seen branching into networks in axon terminals and varicosities. The axonal reticulum and perikaryal endoplasmic reticulum resemble one another in their content of cytochemically demonstrable enzyme activities (G6Pase and IDPase) and in their characteristic membrane thicknesses (narrower than plasma membrane or some Golgi membranes). From the reticulum, both along the axon and at terminals, there appear to form dense-cored vesicles ranging in size from 400 to 1,000 A in diameter. These vesicles behave pharmacologically and cytochemically like the classes of large and small catecholamine storage vesicles found in several adrenergic systems; for example, they can accumulate exogenous 5-hydroxydopamine. In addition, dense-cored vesicles at the larger (1,000 A) end of the size spectrum appear to arise within perikaryal membrane systems associated with the Golgi apparatus; this is true also of very large (800-3,500 A) dense-cored vesicles found in some perikarya.