David Edgar

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture☆

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.

Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons.

Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.

Modulation of NGF-induced survival of chick sympathetic neurons by contact with a conditioned medium factor bound to the culture substrate.

Abstract Pre-treatment of polyornithine culture substrates with heart cell-conditioned medium increased up to 10-fold the number of chick embryo sympathetic neurons which survive in response to nerve growth factor. The modified substrate alone had no survival activity, but had a permissive effect allowing essentially all neurons of the developmental stages examined to survive in response to lower NGF concentrations.

Both nerve growth factor and high K+ concentrations support the survival of chick embryo sympathetic neurons: Evidence for a common mechanism of action

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K+ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K+, and survival was not increased when both NGF and K+ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K+ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K+ and NGF for the survival of cultured embryonic neurons.

Nerve Growth Factors and Molecules of the Extracellular Matrix in Neuronal Development

SUMMARY The survival of developing neurons is epigenetically regulated by trophic factors. Only one such protein, the nerve growth factor (NGF) has been shown to act in vivo, where it supports the survival of neural-crest-derived sensory and sympathetic neurons. Recently, however, other proteins have been isolated and shown to support the survival of cultured neurons. Furthermore, in addition to the effects of soluble trophic factors, proteins of the extracellular matrix are also able to modulate neuronal survival. Analysis of the basal lamina protein, laminin, shows that when used as a culture substrate it stimulates neurite outgrowth and potentiates neuronal survival via a site associated with its heparin binding domain. On proteolytic cleavage of laminin, however, a cryptic site is unmasked that can also promote neuronal survival and neurite growth. The properties of this cryptic site indicate that it may be similar to that of the laminin-like molecule synthesized by Schwann cells, which although recognized by anti-laminin antibodies is not inhibited by them.

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.

Substrate requirement and media supplements necessary for the long-term survival of chick sympathetic and sensory neurons cultured without serum

Abstract Neurons dissociated from embryonic chick sympathetic and dorsal root (sensory) ganglia were separated from the majority of the ganglionic non-neuronal cells by a preplating technique and then cultured without serum on a polyornithine substrate. The culture medium was F14, supplemented with 5 μg/ml transferrin, 5 μg/ml insulin and 10 ng/ml nerve growth factor (NGF). Under these conditions the neurons rapidly adhered to the substrate, thus obviating the need to initiate the culture in the presence of serum. Quantitative studies showed that the numbers of neurons surviving in this serum-free medium were the same as those surviving under optimal conditions with NGF and serum. Additional supplements to the culture medium—such as progesterone, putrescine, selenium, triiodothyronine, or hydroxycortisone—did not enhance neuronal survival further. The long-term survival of the neurons under the present conditions was, however, improved over that in the presence of serum. Additionally, the few non-neuronal cells remaining in the cultures after preplating were found to proliferate extensively during long culture periods.

Biological and immunological properties of the nerve growth factor from bovine seminal plasma : comparison with the properties of mouse nerve growth factor

Abstract The biological activities of Nerve Growth Factor (NGF) purified from bovine seminal plasma have been compared with those of NGF from mouse submandibular glands in a variety of systems: maintenance of survival in vitro and stimulation of nerve fibre outgrowth from sensory, sympathetic and parasympathetic neurons of the embryonic chick; maintenance of survival in vitro, stimulation of nerve fibre outgrowth and specific induction of tyrosine hydroxylase in neonatal rat sympathetic neurons; stimulation of nerve fibre outgrowth and cellular hypertrophy and specific induction of choline acetyltransferase in pheochromocytoma PC12 cells; induction of tyrosine hydroxylase in bovine adrenal medullary cells; and stimulation of nerve fibre outgrowth from expiants of goldfish retinae. In all cases, the two NGFs had the same effects qualitatively and quantitatively, and with identical dose-dependencies. The results indicate that the wide range of biological effects and target cells delineated in detail for mouse NGF can justifiably be attributed to other NGF proteins, and that they are not exclusively restricted to the mouse NGF molecule. Furthermore, as bovine NGF is free of the renin contaminants so difficult to remove from mouse NGF, the above biological activities can truly be assigned to the NGF molecule. Immunologically, however, mouse and bovine NGFs differ substantially. This is demonstrated by the relatively poor ability of antisera against bovine NGF to inhibit the activity of mouse NGF in vitro, and by the incomplete nature of the immunosympathectomy caused in rats by treatment with antisera to bovine NGF, in contrast to the extensive immunosympathectomy caused in these animals by the administration of comparable quantities of antisera against mouse NGF. Clearly, the biochemical features of the NGF molecules responsible for their biological effectiveness and for their predominant antigenic properties are different.

Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: Correlation with survival factor requirements in culture

The presence of nerve growth factor receptors and the imipramine-sensitive uptake of catecholamines in sympathetic neurons of chick embryos were investigated by autoradiography. Neurons were dissociated from paravertebral ganglia of different embryonic ages and receptors and catecholamine uptake were then determined both at the beginning of culture and after 2 days in culture, at which time the number of surviving neurons is determined by the survival factors present. It was found that while essentially all the neurons specifically bound 125I-NGF both after dissociation and at the end of the culture period, only 60% of the neurons take up [3H]norepinephrine after dissociation, and this proportion remained constant through the culture period under conditions where all the neurons survived. All of the neurons maintained by NGF in culture (35% of the total) displayed this uptake, and in contrast, only one-quarter of those maintained by heart cell-conditioned medium alone (60% of the total) took up catecholamines. The uptake was shown to be neither induced by NGF nor suppressed by heart cell-conditioned medium. These results support the hypothesis that chick sympathetic ganglia contain discrete subpopulations of neurons which may be selected in culture by virtue of their different requirements for survival factors.