Biomedical subjects
Publications and source records attributed to I Selak.
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Microcultures of dissociated neurons from various central and peripheral neural tissues were used for quantitative analysis and characterization of trophic agents directed to these test neurons. Media conditioned by a variety of central and peripheral glial as well as muscle cell cultures contain trophic activities for central neurons which are distinct from the more traditional protein factors directed to peripheral neurons, by at least two features: (1) they reside with low molecular weight (Mr less than 1000) agents which are resistant to heat, extremes of pH, and various proteolytic and peptidolytic enzymes; and (2) they are necessary for the short-term survival of a variety of rodent and avian central neurons, but they fail by themselves to support survival of peripheral neurons under the same culture conditions.
We have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na+, K+-pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na+ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13-E18), NGF becomes less and less required for that control as the neurons gain a self-sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.
Explants of embryonic day 8 (E8) chicken dorsal root ganglia (DRG) have been cultured with medium containing serum or the serum-free supplement N1 on one of three substrata: collagen, polyornithine (PORN), or PORN exposed to a polyornithine-binding neurite-promoting factor (PNPF-PORN). Replicate cultures were maintained with or without nerve growth factor (NGF). NGF elicited its classical neuritic outgrowth on all three substrata in serum-containing or serum-free medium. In the absence of NGF, however, a gradation of increasing neurite growth was seen with: PNPF-PORN greater than PORN greater than collagen. This response occurred in both media. In addition, the neuritic halo in each instance was markedly more developed in the absence of serum, especially on PNPF-PORN. Nonneuronal behaviors reflected both serum and substratum influences: thus, nonneuronal outgrowth consisted mainly of flat cells with serum and collagen, was nonexistent with serum and PORN or PNPF-PORN, and involved mostly Schwann-like scattered cells in the absence of serum on any one substratum. The serum-dependent behaviors of ganglionic neurites were examined further with explants from chicken E11 sympathetic ganglia. A single substratum was used (PORN), without exogenous trophic factor. Neurite outgrowth was depressed by the presence of fetal calf serum, thus supporting the generality of this phenomenon. Lastly, PC12 cells, a clonal line of rat pheochromocytoma, will grow neurites in the presence of NGF after 48 hr in serum-free, but not serum-containing media. Addition of serum to serum-free cultures at this time results in the rapid and complete retraction of neurites.
Cultures of PC12 pheochromocytoma cells were established on a polyornithine substratum in medium supplemented with the chemically defined N1 mixture in the presence or absence of Nerve Growth Factor (NGF). Normal cell proliferation in the absence of NGF was equally competent when fetal calf serum (FCS) was replaced with N1-supplemented medium. The differentiation of PC12 cells, which occurs upon NGF treatment, ultimately results in cell death without the addition of 0.1% FCS to the N1-supplemented medium. The combination of N1, 0.1% FCS, and NGF permits the PC12 cells to develop a neuritic outgrowth much earlier than when higher (1-10%) FCS levels are used. Neurite retraction is caused in a dose-dependent manner by a delayed presentation of FCS. Within 2 days of serum presentation, however, neurites regrow to achieve that percentage of neurite-bearing cells which is seen without a serum challenge. Moreover, the retraction response becomes less pronounced with time over the 8-day culture period for any given serum concentration. Among the N1 ingredients, only insulin and transferrin are needed by PC12 cells for survival whether in the dividing state or not. Neurite growth was not dependent on any of the N1 components.
Neurons dissociated from chick sympathetic ganglia of different embryonic ages can be cultured equally well in serum containing or serum-free (N1) medium on collagen polyornithine, and polyornithine pretreated with neurite-promoting factor (PNPF), in the presence of nerve growth factor (NGF). Quantitative analysis of nerve cell survival is best achieved in serum-free conditions and on PNPF-pretreated polyornithine. Under such conditions, 11-day embryonic (E11) sympathetic neurons showed a specific requirement for insulin, selenium and transferrin. Similar analyses at E8 and E16 revealed that the requirement for transferrin is developmentally regulated since it is not expressed by E8 nerve cells until after 3 days in vitro. These data strongly suggest that, besides NGF, other specific molecules are involved in the control of neuronal survival and that specific requirements may be displayed at different embryonic stages.
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We have previously demonstrated that both peripheral and central neurons from 8 day embryonic chick and newborn mouse can be maintained in a serum-free medium using the N1 supplement consisting of insulin, transferrin, putrescine, progesterone, and selenite. In the present studies we show that dorsal root ganglionic (DRG) neurons from embryonic chick (E7-E15) and neonatal mouse can be cultured in a serum-free environment with only the addition of insulin and transferrin, plus Nerve Growth Factor (NGF). Chick DRG from E10-E15 contain a population of neurons sensitive to a chick embryo eye-derived ganglionic neuronotrophic factor (GNTF), which is distinct from the neuronal subset dependent upon NGF. The GNTF-dependent chick neurons can also be maintained in culture with insulin and transferrin supplements. Neonatal mouse DRG neurons, whether supported by NGF or eye-derived GNTF, likewise survive in serum-free medium with only insulin and transferrin. Limited numbers of neurons survive for the first 24 hours in a serum-free medium lacking insulin or transferrin, but failed to display neurite outgrowth even in the presence of added trophic factor.
We have recently shown that intact and dissociated 8-day embryonic (E8) chick dorsal root ganglia (DRG) lose the ability to regulate their intracellular Na+, K+ levels when deprived of nerve growth factor (NGF) for 6 h; recovery occurs within minutes of NGF presentation. These ganglionic neurons are believed to depend on NGF for survival and neurite production over a defined period of embryonic life--between about E6 and E15 in the chick. Using intact DRG from E6-E16 chick embryos we determined developmental changes in: (i) 22Na+ accumulation in the presence and absence of NGF, or in the presence of ouabain; and (ii) intra- and extracellular fluid spaces. Sodium accumulation, in the presence of NGF, increases from E6 to E10. It parallels the total fluid space under ouabain but then decreases conspicuously between E10 and E16, despite little change in the latter. NGF thus prevents Na+ accumulation during the early period, and becomes increasingly irrelevant for this behavior in later (after E10) development. These data are interpreted as indicating that: (i) NGF is required for ionic control by DRG neurons up to E10; and (ii) indigenous behaviors for the control of ion pump mechanism(s) are progressively acquired by these cells from E10 to E16, in parallel with the decreasing ionic relevance of NGF. These findings are consistent with the view that the ionic responses to NGF correlate closely with the survival and neurite-promoting effects of this factor.
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