Replacement of Nerve-Growth Factor by Ganglionic Non-Neuronal Cells for the Survival In Vitro of Dissociated Ganglionic Neurons (culture neuroglia)

Transcription

1 Proc. Nat. Acad. Sci. USA VoL 69, No. 12, pp , December 1972 Replacement of Nerve-Growth Factor by Ganglionic Non-Neuronal Cells for the Survival In Vitro of Dissociated Ganglionic Neurons (culture neuroglia) PATRICIA BURNHAM, CHARLES RAIBORN, AND SILVIO VARON Department of Biology, University of California, San Diego, La Jolla, Calif Communicated by R. Galambos, August 25, 1972 ABSTRACT Nerve-growth factor is known to cause a considerable increase in the number of neurons putting out processes and surviving in cell cultures of dissociated dorsal-root and sympathetic ganglia from embryonic chicks. Similar effects of nerve-growth factor have now been noted with cultures of dissociated dorsal-root ganglia from newborn mice or rats. In all three sensory ganglionic systems, the effects of the nerve-growth factor on fiber production and neuronal survival could be mimicked, in the absence of the factor, by adequate increase of the nonneuronal cells in the cultures. The results suggest a hypothesis that views the role of the nerve-growth factor as subordinate to that of the non-neuronal cells. The nerve-growth factor (NGF) is generally described as a protein agent, or group of agents, capable of inducing growth and differentiation of sympathetic and embryonic spinal sensory (DRG) neurons (1). The characteristics of NGF proteins obtained from mouse submaxillary gland have been. well investigated in the last five years (2, 3). However, little real progress has been made toward understanding the effects of NGF and the mechanisms by which they are achieved. In fact, many traditional interpretations of past experiments are actually being questioned in the light of more recent ones. Thus, the higher biosynthetic activity seen in ganglia incubated with NGF appears likely to reflect a maintenance rather than a stimulatory effect of the agent (ref. 4, also Burnham and Varon, in preparation), the fiber outgrowth elicited in vitro from explanted ganglia can no longer be viewed as the consequence of an NGF-stimulated RNA synthesis (4), and late embryonic or postnatal DRGs have been recognized as tissues responsive to NGF in spite of their failure to provide a halo response in the classical NGF bioassay (5). A question that has rarely been asked and hardly ever investigated is whether NGF addresses itself to the neurons (as has been implicitly assumed), or to the non-neuronal cells of the target tissue, or to both. The use of dissociated ganglia offers the opportunity to manipulate the relative proportions of neuronal and non-neuronal elements and to analyze the consequences on the behavior of the cultured neurons. Recent investigations of cell cultures obtained from dissociated sympathetic ganglia of embryonic chicks (6) and DRGs of newborn mice (5) demonstrated that most dissociated neurons did not survive in culture, even in the presence of NGF, unless adequately provided with non-neuronal cells. In addition, nonneuronal support was able to replace the support provided by Abbreviations: DRG, dorsal root (spinal sensory) ganglion; NGF, nerve-growth factor NGF to mouse ganglionic neurons (5) for a short period. The present paper extends these findings and proposes a working hypothesis on the relative roles of NGF and non-neuronal cells in neuronal performance in vitro. MATERIALS AND METHODS White-Leghorn 8-day chick embryos, DBA x C57BL6 hybrid newborn mice, and Wistar Albino newborn rats were the source animals. DRGs were collected and dissociated as described for chick (7) and mouse (5) ganglia; the latter technique was also used for rat ganglia. Briefly, ganglia were dissected out into a low-bicarbonate (0.22 g/liter), Ca++, Mg++free saline solution, ph 7.5, incubated 30 min at 370 in 0.25% trypsin (in same saline), washed once in culture medium-(see below), and dispersed in culture medium by multiple aspirations (25, 50, and 25 for mouse, rat, and chick DRGs, respectively) through a 0.5-mm bore Pasteur pipet. Tissue was dispersed into a cell suspension directly in culture medium [Eagle's basal medium with NaHCO3 (final concentration, 2.2 g/liter), glucose (final concentration, 6 g/liter), 200,000 units of penicillin/liter, and 5% of heat-inactivated fetal-calf serum]. Cell suspensions were differentially counted for neurons and non-neuronal cells and diluted with culture medium as desired for seeding into culture. In some cases, 7S NGF (8) was added to the culture medium at final concentrations of Biological Units/ml (9); use of Beta-NGF (2) at equal activity yielded the same results. Non-Neuronal Cultures were prepared by seeding 2-ml aliquots of DRG cell suspensions in NGF-free media into collagen-coated, 35-mm plastic Falcon dishes, allowing 2 hr for non-neuronal attachment, replacing medium and unattached cells with fresh culture medium, and incubating the cultures in a water-saturated, 5% C02-95% air atmosphere. After a few days, no neurons were detectable in these cultures, either because they had been removed before attachment (mouse) or because they had failed to survive without NGF (chick, rat). Neuron-free cell cultures from chick DRGs were used directly (see below) 24 hr after seeding. With mouse (5) or rat DRGs, the neuron-free cell cultures were grown to confluency and harvested by use of trypsin (0.25% in Ca++, Mg++-free saline), appropriate aliquots were seeded again into fresh, collagen-coated dishes. Medium and unattached cells were removed after a 2-hr attachment period, and the attached non-neuronal cultures were used immediately. To examine the influence of additional numbers of nonneurons on the performance of dissociated neurons in culture,

2 Proc. Nat. Acad. Sci. USA 69 (1972) we set up ganglionic cell cultures by seeding 2-ml aliquots of freshly prepared and appropriately diluted DRG cell suspensions, without or with NGF, either directly on collagen-coated Falcon dishes ("unsupplemented" cultures) or on previously attached, homologous non-neuronal cells ("supplemented" cultures). Cultures were incubated in 5% C02-95% air, and media were renewed at 24 hr. For quantitative examination (4, 5), dishes were briefly removed from the incubator and attached cells (total neurons, neurons bearing fibers andwhere possible-non-neuronal cells) were differentially counted by phase contrast microscopy over a representative area (1/25th) of the dish. Triplicate dishes were counted at each time interval, and averaged; replicate counts in the same dish or across replicate dishes varied by no more than ±45% from the average, and only slightly more so among repeat experiments with different DRG cell suspensions and nonneuronal preparations. RESULTS Comparative effects of NGF and non-neuronal supplementation on cultured DRG neurons Neuronal responses to either or both conditions are most effectively investigated in terms of three neuronal parameters: attachment (total number of neurons found attached to the culture floor, regardless of whether they exhibit a process); fiber production (number of neurons found to bear newlygrown fibers, irrespective of their length); and longer-term survival (number of neurons persisting for more than 1 day in culture). Newborn Mouse DRGs. Characteristics of this culture have been reported (5). Fig. 1 illustrates the time courses of neuronal attachment (A) and fiber production (B). The presence of NGF in unsupplemented ganglionic cultures (curves b) increased maximal attachment 5-fold to 70% of the seeded neurons, advanced fiber production by several hours to involve all the neurons attached initially, and supported the survival of this maximal number of fiber-bearing neurons for more than A. ATTACHMENT B. FIBER PRODUCTION ~/I.I, ]Al1So1'"' L La Mouse DRG cultures: effects of NGF and non-neuronal FIG. 1. supplementation. Dissociation yielded 5500 neurons and 6000 non-neuron cells per ganglion. All culture dishes received 25,000 neurons and 30,000 non-neurons: non-neurons achieved a maximal attachment of,000 by 2 hr. Some culture dishes ( ) contained 21,000 previously attached non-neurons (out of 100,000 seeded). In some cultures (X), the medium contained 10 Biological Units of 7S NGF per ml. (a) No NGF, no supplementary non-neurons; (b) NGF only; (c) non-neuronal supplementation only; (d) NGF and non-neuronal supplementation. Replacement of NGF by Non-Neuronal Cells 3557 B. FIBER PRODUCTION FIG. 2. Rat DRG cultures: effects of NGF and non-neuronal supplementation. Dissociation yielded 2,500 neurons and-16,000 non-neuron cells per ganglion. All culture dishes received 25,000 neurons and 160,000 non-neuron cells: non-neurons achieved a maximal attachment of 24,000 by 4 hr. Some culture dishes ( ~) contained 54,000 previously attached non-neurons (out of 100,000 seeded). The concentration of 7S NGF and symbols are the same as in Fig. 1. one day with nearly no losses. Seeding of DRG cells onl previously attached non-neuronal cultures (about 20,000 cells per dish), with or without NGF, advanced by about 1 hr the onset of both the attachment and the fiber production of the neurons. More importantly, the non-neuronal supplementation in the absence of NGF (curves c) provided the cultured neurons with at least two of the NGF-inducible gains, namely a similar 5-fold higher attachment, and an early and full conversion to fiber-bearing neurons, but appeared to fail in a longer-term maintenance role. When NGF was used in addition to non-neuronal supplementation (curves d), neuronal attachment and conversion were further accelerated, longerterm survival was again supported, but maximal numbers of neurons were barely, if at all, increased. Newborn Rat DRGs. In vitro studies of this material have been limited. Rat DRGs-like mouse DRGs, until recently (5)-have not been proven to be susceptible to NGF influence and, in fact, were assumed not to be so at least beyond-an early fetal developmental stage. Dissociation and culture of newborn-rat DRGs are now under investigation, and only some attachment and fiber-production rate curves are presented here (Fig. 2). The maximal amount of neuronal attachment (A) was the same in all cases (80-90% of the neurons seeded) and was reached with about the same time course, except when both NFG and non-neuronal supplementation were used. Considerable differences, however, were noted after maximal attachment had been achieved: while in the absence of both supports (curves a), progressive losses of neurons were evident from the first day onward, longer-term survival could be supported by non-neuronal supplementation (about 50,000 cells per dish) alone (curve c) almost as effectively as by NGF alone (curves b) or by a combination of both (curves d). The influence of the additional non-neuronal cells was clearly evident even in the earlier phase of the culture when fiber production was examined (B). Thus, like those from the mouse, rat DRG neurons were responsive to either NGF or I

3 3558 Cell Biology: Burnham et al. ~~~~~~~I \ ~~~X \% X X--- X 1 x hours in c B. FIBER PRODUCTION Fx d Jx / ii 'I/IX / I_ I FIG. 3. Chick (8-day embryo) DRG cultures: effects of NGF and non-neuronal supplementation. Dissociation yielded 12,000 neurons and 30,000 non-neuron cells per ganglion. All culture dishes received 100,000 neurons and 250,000 non-neurons: the non-neurons achieved a maximal attachment of 50,000 by 2 hr. Some culture dishes ( ) contained 53,000 previously attached non-neurons (out of 250,000 seeded as NGF-free DRG cell suspension). non-neuronal supplementation. This system, however, differed from the previous one in at least two respects, namely, the ability of the ganglionic neurons to attach maximally even without supplementation with non-neuron cells (or NGF) and the competence of the non-neuronal supplement to provide the third NGF-inducible gain-a longer-term survival of neurons. It should also be noted, coincidentally with these differences, that both the unsupplemented ganglionic cultures and the previously attached cultures of non-neuronal cells used here contained more than twice as many non-neuron cells as did the corresponding mouse cultures. 8-Day Embryonic Chick DRGs. Neurons dissociated from DRGs of 7- to -day chick embryos have been known to be dependent on NGF for their survival in culture (10, ). As Fig. 3 shows, the time patterns for neuronal attachment (A) and fiber production (B) differed considerably in this system from those described in the preceding sections. The number of attached neurons reached a maximum of 60% of the seed after about 6 hr of culture; this was the same under all conditions tested, but declined rapidly thereafter, while the number of fiber-bearing neurons was increasing. The levels at which the neuronal losses subsided were the same as those at which conversion to fiber-bearing neurons ceased to increase, and differed considerably with the kind of support made available. NGF without nonneuronal supplementation (curves b) raised this level by about 4-fold over that of the controls (curves a). Non-neuronal supplementation (about 50,000 cells per dish) without NGF had approximately the same effect (curves c). Use of both factors (curves d) raised the level by another 2-fold. In both cases of non-neuronal supplementation (c, d), fiber production started several hours earlier-an advance even greater than that seen with the mouse cells. On the longerterm basis, all levels appeared to hold for 2-3 days and decline only slowly thereafter. Thus, maximal neuronal attachment required no exogenous help in the chick, as in the rat, DRG system. The "intrinsic" non-neuronal component of these ganglionic cultures, however, seemed to provide insufficient support (with or without the help of NGF) for fiber production and persistance of all attached neurons even on the shorterterm basis-a situation closely resembling that already reported for cultures of dissociated sympathetic ganglia from 1 1-day chick embryos (6). Similarly, supplementation with non-neuronal cells provided partial but not full support, at least at the densities applied in these experiments. Graded effects of numbers of supplementary non-neuron cells on mouse DRG cultured neurons Mouse DRG cultures appeared the most suitable, among the three systems tested, for a finer analysis of the effects of nonneuronal supplementation. Fig. 4 illustrates neuronal attachment in a series of experiments where fresh DRG cell suspensions were seeded, in the absence of NGF, over homologous previously attached non-neuronal cultures obtained by previously seeding 0-300,000 non-neuron cells per dish: for comparison, aliquots of DRG cell suspensions were also cultured in the presence of NGF (broken lines) with no or about 70,000 (200,000 seed) of previously attached non-neuron cells in the dishes. A progressive increase of the non-neuronal supplementation up to 8800 cells per dish (30,000 seed) gradually raised the rates and maximal levels of neuronal attachment to about those induced by NGF alone (A). Further increases (B) in the number of supplementary nonneuron cells advanced the onset of attachment but enhanced only minimally the attachment rates and maximal levels. Previously attached, non-neuronal cultures of nearly 70,000 cells per dish provided the neurons with a support that could not be further improved by additional non-neuron cells or by the combined use of NGF, suggesting that a ceiling of neuronal performance intrinsic to the present system had been reached. Furthermore, the previously noted inadequacy of non-neuronal supplementation to support longer-term neuronal survival without NGF (e.g., Fig. 1, curves c) also was attached neurons / dish 15,000,000 A Proc. Nat. Acad. Sci. USA 69 (1972) I I NGF I A * >70.0 * 66.0 'K\ 42.0 ii 22.0 I 12.5 ~~~~~~~~~~I 1, 0 4 NGF 6 8I FIG. 4. Mouse DRG cultures: graded neuronal attachment with various numbers of supplementary non-neuron cells. Ganglionic cell suspensions were seeded, as described in Fig. 1, without NGF, into culture dishes containing previously attached mouse DRG non-neuron cells in the numbers (X 10-3) indicated for each curve and derived from non-neuronal seeds between 0 and 30,000 (A) or 50,000 and 300,000 (B). For comparison 7S NGF (10 Biological Units per ml) was added to some cultures ---) containing no or 66,000 previously attached non-neurons.

4 Proc. Nat. Acad. Sci. USA 69 (1972) found to decrease with increasing numbers of supplementary non-neuron cells, and to be entirely overcome when 70,000 or more supplementary non-neurons per dish were provided. The dose-dependent relationship between neuronal performance and non-neuronal supplementation was fully confirmed by an analysis of the appearance of fiber-bearing neurons (Fig. 5). DISCUSSION The study of dissociated sympathetic ganglia (6) had revealed that neurons deprived of their non-neuronal partners failed to survive in culture despite the presence of NGF. Conversely, neuronal-fiber production and survival were progressively increased beyond the levels achieved in an unfractionated ganglionic culture if increasing numbers of non-neuron cells were supplied to the sympathetic neurons. A dependence of cultured neurons on adequate availability of non-neuron cells was again observed with dissociated DRGs from newborn mouse that were found to respond to NGF with a 5-fold increase in neuronal attachment, fiber production, and survival (5). Mouse DRGs, however, added another dimension to the finding, namely that non-neuronal supplementation of a ganglionic culture provided the cultured neurons with apparently the same advantages as an NGF-containing medium, though only for a brief period. The data presented here on the use of various amounts of non-neuronal supplementation demonstrate that the longer-term inadequacy (see Results) was only a matter of insufficient supplementation and that appropriate numbers of supplementary non-neurons did fully substitute for the presence of NGF. Finally, two other NGFresponsive systems-drgs from 8-day chick embryos and newborn rats---have now been shown to (i) respond also to non-neuronal supplementation, and (ii) exhibit the same, or nearly the same, neuronal performance when supported by either NGF or non-neuron cells. It is likely that an examination of different amounts of non-neuronal supplementation will confirm with these systems, as it did with the mouse DRGs, that NGF support could be fully replaced by sufficient nonneuronal support. The similarity of these two effects does not, however, necessarily imply identity of roles. NGF alone failed to support longer-term survival of neurons deprived of all or most of their non-neuronal partners in mouse DRGs (5) and chickembryo sympathetic ganglia (6) while absence of NGF did not prevent longer-term survival supported by an adequate non-neuronal supplement. Similarly, the effects of NGF alone on fiber production could not be increased with 100-fold excesses of the agent (data not presented), yet the neuronal gains from NGF could be further expanded by the concomitant use of non-neuronal supplementation in all four ganglionic systems examined. The possibility cannot be at present excluded that NGF and non-neuronal supports may eventually prove to be two independent phenomena. However, the available data (Fig. 5 for the clearest overview) warrant the submission of the following working hypothesis: (a) Fiber production and survival in culture (and possibly overall attachment, as shown in the mouse system) of most, if not all, the neurons dissociated from ganglionic tissue depend on the amount of support provided to them by the available non-neuronal population-the amount of such support reflects presumably the potency, as well as the number, of the non-neuron cells. (b) NGF is able to compensate for an insufficient non-neuronal support, but only partially: if this Replacement of NGF by Non-Neuronal Cells FIG. 5. Mouse DRG cultures: graded neuronal fiber production with various non-neuronal supplementation. Experimental details as in Fig. 4. support is totally lacking, NGF will fail to service adequately the neurons and, conversely, NGF will no longer be required with a full non-neuronal support. (c) A similar neuronal dependence on the non-neuronal ganglionic cells also occurs in the undissociated ganglia and NGF is needed, and thus is able to elicit demonstrable responses, only when artificial conditions in vitro or developmental situations int -vivo make the nonneuronal support inadequate. This last point, although not essential for the validity of the first two, is particularly attractive in that it offers plausible interpretations of many recent data (for examples, Burnham and Varon, in preparation; also refs. 4 and 5) not readily inserted into the traditional picture (1) of the NGF phenomenon. Perhaps the most exciting feature of this working hypothesis is that it encourages novel, and potentially testable, speculation on the ways by which NGF operates. Thus, NGF might act on the neurons as a replacement or supplement for NGF-like substances produced by the non-neuron cells. NGF might also affect the ganglionic neurons in such a way that they become more susceptible to the non-neuronal support, and hence, become capable of performing even with an otherwise insufficient support. Lastly, the possibility is raised that NGF has as its direct target cells the non-neuronal, rather than the neuronal, elements of the ganglia and operates by boosting or sustaining their neuron-supportive abilities. Other major questions raised by these investigations of dissociated neural systems concern the nature and the specificity of the supportive role played by the ganglionic nonneurons with respect to neuronal performance in culture. Preliminary experiments, in which mouse DRG cell suspensions were seeded on cultures of fibroblasts from chick embryos or newborn mouse, rather than ganglionic non-neuron cells, have indicated that neuronal attachment can be affected by such non-neural cells, at least temporarily, but no conspicuous gain in fiber production or longer-term survival could be observed. The possibility of a physical interaction, with nonneuron cells providing a better attachment or longer-lasting anchoring surface than bare collagen, cannot at present be ruled out. An alternate possibility, however, is that of a nonneuronal production of a chemical agent or agents, delivered to the neurons through direct contact or through the culture

5 3560 Cell Biology: Burnham et al. medium. Further work remains necessary to determine which cell types within the ganglionic non-neuronal populations are responsible for the supportive action, and to what extent the supportive ability is specific to cells of ganglionic, or at least of neural-tissue origin. Nevertheless, it is tempting to view this role as an expression of glial-neuronal interaction, and to regard the experimental approaches described here as a new way to attack the problem of what roles neuroglia play with respect to the development, maintenance, or function of neurons. This work was supported by American Cancer Society Grant VC-12A and USPHS Grant NS from the National Institute of Neurological Disease and Stroke. P.B. was supported by a USPHS Training Grant GM from the National Institute of General Medical Science. Proc. Nat. Acad. Sci. USA 69 (1972) 1. Levi-Montalcini, R. & Angeletti, P. U. (1968) Physiol. Rev. 48, Greene, L. A., Varon, S., Piltch, A. & Shooter, E. M. (1971) Neurobiology 1, Angeletti, R. A. H. & Bradshaw, R. A. (1971) Proc. Nat. Acad. Sci. USA 68, Partlow, L. M. & Larrabee, M. D. (1971) J. Neurochem. 18, Varon, S., Raiborn, C. & Tyszka, E. (1972) Brain Res., in press. 6. Varon, S. & Raiborn, C. (1972) J. Neurocytol., in press. 7. Varon, S. & Raiborn, C. (1972) Neurobiology, in press. 8. Varon, S., Nomura, J. & Shooter, E. M. (1967) Biochemistry 6, Varon, S., Nomura, J., Prez-Polo, J. R., & Shooter, E. M. (1972) in Methods and Techniques of Neurosciences, ed. Fried, R. (M. Dekker Inc., New York), in press. 10. Levi-Montalcini, R. & Angeletti, P. U. (1963) Develop. Biol. 7, Varon, S. & Raiborn, C. (1971) Brain Res. 30,