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Biomedical subjects

S Varon

Publications and source records attributed to S Varon.

At least 199 records · Page 11Linked to original sources
PubMed1980

Survival, proliferation and morphological specialization of mouse Schwann cells in a serum-free, fully defined medium.

Neonatal mouse dorsal root ganglionic (DRG) cell dissociates were cultured in a synthetic medium with horse serum or the serum-free supplement N1 (insulin, transferrin, progesterone, putrescine, selenium). Serum-supplemented cultures with added nerve growth factor (NGF) yielded neurons, small flat and spindle cells (Schwann) and large flat cells (fibroblastic elements). However, in serum-free, N1-supplemented medium plus exogenous NGF, neurons and Schwann cells predominated, with very few large flat cells. In the N1 medium most Schwann cells assumed a typical spindle shape and were associated with neuritic processes when neurons were present. Upon addition of serum, virtually all of the Schwann cells appeared to abandon physical contact with the neurites and develop a more flattened morphology. In N1 medium without NGF (no neurites), most Schwann cells still assumed a spindle shape and formed characteristic chain-like associations. Autoradiographic techniques, as well as numerical analyses, demonstrated that in N1 medium Schwann cells were able to proliferate when associated with neurites but only slightly so in their absence. These Schwann cells showed a marked increase in proliferation when serum was added regardless of the presence or absence of neurites. The above observations may provide a basis for the preparation of purified Schwann cells, alone or in combination with their neurons.

Animals↗
PubMed1980

Cholinergic neuronotrophic factors: fractionation properties of an extract from selected chick embryonic eye tissues.

An aqueous extract derived from selected intraocular tissues of 15-day chick embryos contains a soluble macromolecular agent which is capable of ensuring the survival of 8-day chick embryonic ciliary ganglionic neurons in monolayer culture. When this ciliary neuronotrophic factor (CNTF) was concentrated using ultrafiltration and subjected to Sephadex G100 and G200 chromatography, activity was detected in most of the eluted fractions. A peak of the most active fractions was eluted in a region corresponding to a molecular weight of 35-40 X 10(3) and contained about 20-30% of the applied protein. CNTF activity bound readily to DE-52 cellulose resin at neutral pH and was eluted with NaCl in a narrow region containing about 20-40% of the applied protein. Gel electrophoretic staining profiles of the active DE52 fraction indicated considerable (but still only partial) simplification in protein composition. While significant CNTF activity losses were incurred in response to each of the above treatments, an active material could be conveniently generated in one working day in milligram amounts having a specific activity of 60,000 trophic units/mg protein. This trophic activity is in the same range as that of the only other known neuronotrophic factor, Nerve Growth Factor.

Animals↗
PubMed1979

Cholinergic neuronotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia.

Two key components of neural development and regeneration, survival of the involved neurons and elongation of neuritic elements, are likely to depend on the availability of an appropriate trophic drive to these neurons. At present, only one trophic factor, Nerve Growth Factor, is known to ensure both survival and neuritic growth for its target neurons. A search for a second such agent, a putative cholinergic neuronotrophic factor (CNTF), has been undertaken using as indicators neuronal survival, neurite outgrowth and choline acetyltransferase (CAT) activity in monolayer cell cultures. Eight-day chick embryo ciliary ganglia yielded two monolayer culture systems which appear to be well suited for a CNTF assay. Ciliary ganglionic dissociates, seeded on a highly adhesive collagen substratum, show no neuronal survival by 24 h if the medium is supplemented only with serum or chick embryo extract. However serum and embryo extract combined support survival of, and extensive neuritic outgrowth from, nearly the theoretical number of ganglionic neurons seeded. Alternatively, ciliary ganglionic neurons can be made to survive and produce a profuse neuritic outgrowth on polyornithine-coated dishes if supplied with medium conditioned over chick embryo heart muscle cultures, as already described by other laboratories. The two trophic sources differ markedly in their effects on the ganglionic neurons when tested on collagen or polyornithine substrata, and in some cases when different serum supplements are used. Neuronal survival, neurite production and, possibly, CAT activity appear to be subject to independent regulation. The culture systems used in this study can be developed into quantitative bioassays for the isolation of the different agents responsible for neuronal survival and neurite promotion, and for the investigation of their activities.

Animals↗
PubMed1979

Sodium dependence of the nerve growth factor--regulated hexose uptake in chick embryo ganglionic cells.

Embryonic dorsal root ganglionic cells, when incubated in vitro in the absence of nerve growth factor (NGF) undergo a general metabolic degeneration which is preceded by certain changes in permeation properties. Previous studies demonstrated that NGF can rapidly modulate permeation properties which regulate the availability to the cell of an important energy source, glucose. Hexose uptake was determined by measuring the ability of the cells to accumulate [3H]labeled 2-deoxy-D-glucose. The work reported here shows that the NGF-dependent portion (about one-third) of the total specific hexose uptake was also dependent on the presence of Na+, with the apparent uptake constant (Kt) for deoxyglucose varying inversely with an external Na+ concentration of 70-140 mM; Vmax was unaffected in this range. Preincubation of ganglionic cells with 10 mM ouabain for 15-60 min, followed by a pulse with [3H]-deoxyglucose, also resulted in 50-95% reduction of the NGF-sensitive uptake. A similar pretreatment of cells with veratridine gave a 25-50% reduction in uptake. The NGF-controlled hexose uptake was also energy dependent, being diminished 50-95% after a 30-90 min preincubation with 2 mM 2,4-dinitrophenol. Uptake activities for other substrates (alpha-aminoisobutyric acid, uridine) which exhibited NGF regulation were likewise Na+-sensitive. These results indicate that availability of major energy substrates to NGF-dependent dorsal root ganglionic neurons is controlled by sodium gradients across their membranes. It is conceivable that NGF provides for maintenance and development of its target neurons by acting on such sodium gradients and, consequently, regulating the intake of essential nutrients.

Amino Acids↗
PubMed1979

Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons.

Chick ciliary ganglionic neurons require an interaction with their peripheral targets for survival during a critical period of their embryonic development in vivo. It has recently been shown that survival of these neurons in dissociated cell cultures is supported by extract from whole chick embryo. In this study, an assay system based on microwell cultures of ciliary ganglionic neurons was used to demonstrate that a very rich source of trophic factor for them is the intraocular target tissues they innervate. Out of 8000 trophic units present in a 12-day embryo, 2500 were contained in the eye. A subdissection of the eye showed its activity to be localized in a fraction containing the ciliary body and choroid coat, with a specific activity almost 20-fold higher than that of the whole embryo. This selective intraocular distribution at a time when survival or death of ciliary ganglionic neurons is decided in vivo suggests that this soluble factor may be involved in the normal development of the ciliary ganglion.

Animals↗
PubMed1979

Nerve growth factor action on 2-deoxy-D-glucose transport in dorsal root ganglionic dissociates from chick embryo.

Dorsal root ganglionic cells, when incubated in vitro in the absence of nerve growth factor (NGF), undergo a general metabolic degeneration which is preceded by loss of certain permeation properties. To determine in which ways an absence of NGF can also affect the capacity of these cells to take up an important energy source, namely glucose, experiments were carried out in which cells were incubated with or without NGF for varying times, and then presented with the factor and tested for the ability to take up 3H-labeled 2-deoxy-D-glucose. As with exogenous uridine, hexose transport in DRG cells was reduced by NGF deprivation and restored by delayed NGF administration (up to 6 h). Both the initial rate and equilibrium level were affected in an NGF dose-dependent fashion. Calculation of apparent Kt and V max in NGF-deprived and NGF-supported cells showed about two-fold differences between NGF-controlled and NGF-independent hexose transports, suggesting corresponding differences between NGF-dependent and other ganglionic cells. Restoration of hexose transport by delayed NGF administration took place within minutes of presentation of the factor. The delay before onset of restoration and the speed with which restoration was achieved have been found also to be dependent on the NGF concentration, suggesting that they reflect equilibration kinetics between NGF and its binding sites rather then the development of the response within cells. Thus, NGF can rapidly modulate permeation properties which regulate the availability of major energy substrates for the cell. This effect of NGF is discussed in the content of current views on the mode of action of the factor.

Animals↗
PubMed1979

The culture of chick embryo dorsal root ganglionic cells on polylysine-coated plastic.

Polylysine-coated culture surfaces are strongly adhesive for neural cells, restrict locomotion on nonneuronal elements, but do not inhibit neurite elongation. In the present study, culture dishes were pre-treated with poly-D-lysine (PDL) at various concentrations, seed with dissociates from 8-day chick embryo dorsal root ganglia, and incubated under conditions that normally support both neuronal survival and nonneuronal proliferation. Pretreatment with low (0.1 mg/ml) PDL concentrations had no effect on neuronal survival and neuritic growth, but entirely prevented an increase in ganglionic nonneurons, yielding a numericallly stable culture greatly enriched in neurons. Higher PDL concentrations caused increasing losses in both cell classes. The 50% levels of cell loss were achieved at about the same PDL dose, but earlier for neurons that nonneurons and still with no impairment of neuritic growth from the surviving neurons. A procedure was developed to compare acid-soluble and acid-precipitable accumulation of radioactivity under 1-hr pulses of [3H]uridine, which was applicable even to poorly attached cells. The cytotoxic effects of higher PDL pretreatments was revealed as early as 6 hr after seeding by 2- to 4-fold lower radioaccumulation. The data are discussed in terms of possible regulations of cell permeability and metabolism by adhesive interactions between cells and their substratum, or other cells.

Animals↗
PubMed1979

Development, reactivity and GFA immunofluorescence of astroglia-containing monolayer cultures from rat cerebrum.

This report describes detailed protocols for the dissociation, seeding and growth in vitro of monolayer cultures derived from neonatal rat cerebrum. Primary cultures derived by using different seeding densities and in vitro ages were examined qualitatively and quantitatively for morphological composition in terms of two major cell classes (flat cells and process-bearing cells) and for the presence within these classes of glial fibrillary acidic protein (GFA) as detected by immunofluorescence histochemistry. Also examined was the reaction of the cells to serum withdrawal plus the administration of dibutyryl cyclic AMP in terms of the conversion of flat cells into process-bearing cells. Conditions are defined for the generation of in vitro cell populations, more than 90% of which are GFA-containing flat cells which can all be experimentally converted into cells with processes. These well-defined culture preparations will serve as useful models for future studies of astroglial behaviour.

Animals↗
PubMed1979

A procedure for purifying neuron-like cells in cultures from central nervous tissue with a defined medium.

A serum-free medium (N1) containing the supplements insulin, transferrin, progesterone, putrescine and selenium was used to culture cells from a variety of embryonic chick central nervous system tissues, namely, optic lobe, neural retina, spinal cord and telencephalon. The N1 medium supported the survival of fiber-bearing cells (features typical of cultured neurons) as well as or better than horse serum, while permitting no, or almost no flat cells. Survival and growth of chick flat cells required fetal calf, but not horse serum. Cultivation of newborn mouse telencephalon under these conditions yielded similar results, except that either fetal calf or horse serum supported flat cells.

Animals↗