DEVELOPMENT&
58, 106-113
BIOLOGY
Quantitative
(1977)
in Vitro Studies on the Nerve Growth Requirement of Neurons II. Sensory
Factor
(NGF)
Neurons
LLOYD A. GREENE Department Retardation
of Neuropathology, Research Center,
Received
Harvard Children’s
September
Medical School, and Department of Neuroscience, Hospital Medical Center, 300 Longwood Avenue, Massachusetts 02115
16,1976;
accepted
in revised
form
March
Mental Boston,
2,1977
Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (9 The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of processbearing cells derived from S-day (E8) embryonic DRGs is 0.5 nglml (-2 x 10-l’ M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few processbearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from El7 and El8 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in Go. These results are discussed with respect to the role of NGF in development of sensory neurons. INTRODUCTION
DRG sensory neurons appear to resemble those which it has on sympathetic neurons. One major difference between the two neuronal types, however, is that, while sympathetic neurons appear to be effected by NGF throughout life, sensory DRG neurons appear to be effected by the factor only during a limited period of their development (14, 16, 17, 27). As previously pointed out (ll), dissociated cell culture is a useful system in which to study the effects of NGF on neurons. Besides lending itself to quantitative studies, the advantages of this approach are that it is possible to culture neurons from ganglia of widely varying embryonic ages and that it is possible, by means of suitable techniques (211, to remove from the cultures virtually all cell types other than neurons. In the present report, we have employed cell cultures of dissociated chick embryo
Among the several known targets for nerve growth factor protein (NGF) are embryonic dorsal root ganglionic (DRG) sensory neurons. In uiuo, the medio-dorsal neurons of responsive DRGs undergo an increase in size and extent of outgrowth of neurites when exposed to elevated levels of NGF (16, 18). In vitro, NGF stimulates neurite outgrowth from responsive explanted ganglia (3, 7, 16, 20) and enhances the survival in such explants of mediodorsal neurons (25,261. Also, when responsive embryonic DRGs are dissociated, the neurons do not survive unless the medium is supplemented with NGF (8,151. Sensory ganglia which can respond to NGF morphologically also show metabolic responses to the factor (5,161 and have been shown to possess specific high-affinity NGF receptors (9, 13, 14). In many respects, the effects of NGF on 106 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
LLOYD
A.
GREENE
Sensory
DRGs to study (a) the amount of NGF required by responsive sensory neurons for their survival, (b) the loss during development by DRG neurons of their survival requirement for NGF, and (c) the regulation in. vitro of this loss. MATERIALS
AND
METHODS
Cell cultures and media. Dorsal root ganglia were dissected from chick embryos of 8 to 18 days of incubation and were trypsinized, dissociated, plated, and maintained according to the methods described previously for sympathetic ganglia (11). Cells were seeded at a density of 75,000150,000 neurons per 35mm dish and with a plating efficiency ranging from approximately 20-40%. Media were also as previously indicated (11) except that, as noted, in some cases, media were supplemented with chick embryo extract at a final proportion of 2%. Removal of NGF from established cultures was carried out by washing the plates three times and then feeding them with NGF-free medium. In certain experiments, ganglionic dissociates were preplated in uncoated plastic tissue culture dishes in order to remove non-neuronal cells which preferentially attached to the plastic substrate (21). After 2 hr of preplating, the culture medium (containing unattached cells which were for the most part neuronal in morphology) was quantitatively transferred to collagen coated dishes. Further reduction in the number of non-neuronal cells was achieved by treating the cultures with the mitotic inhibitor 0.6 fl cytosine arabinoside (Sigma) between Days 1 and 4 in uitro. This concentration of drug was empirically found to suppress cell growth without causing detectable damage to the neuronal population. Differential cell counts revealed that, after 2 weeks in vitro, cultures treated in this way contained ratios of neurons to non-neurons of between 1O:l and 3:l. CeZZ counts. Cell counts were carried out as previously described for cultures of dis-
Neuron
NGF
Requirement
107
sociated sympathetic ganglia (11) and are expressed (unless otherwise stated) as relative to the number of process-bearing cells present 24 hr after plating in cultures which contained NGF. Process-bearing cells were considered to be those which extended fibers to a distance at least twice the diameter of their cell bodies. Such cells also displayed other morphological features of neurons including rounded phasebright cell bodies for the first several days in culture and, at times thereafter, prominent nuclei and nucleoli as well as extensive fiber outgrowth. In other studies (221, cells with these morphological characteristics have been shown to be electrically excitable. In cultures derived from embryos incubated longer than 14 days, cells were also observed for the first several days after plating which were round and phasebright but which did not extend processes. By our criteria, such cells were not included in neuronal counts. RESULTS
NGF Requirement of Responsive Neurons 24 hr after Plating
DRG
Figure 1 shows the relative number of process-bearing cells (neurons) observed in cell cultures of dissociated g-day embryonic (EB) DRGs 24 hr after plating in the presence of various concentrations of NGF. In the absence of added NGF, few processbearing cells were observed, and the cultures contained floating debris of what appeared to be degenerated neurons. Significant enhancement in neuronal survival was obtained in the presence of as little as 0.05 rig/ml of NGF; maximal numbers of neurons were observed at a minimum NGF concentration of 0.5 rig/ml. In agreement with previous findings for cultured sympathetic (10, 11) and sensory (4) neurons, the number of process-bearing cells present in the cultures progressively decreased at NGF concentrations greater than 5 rig/ml when the medium contained 10% fetal bovine serum. However, neu-
108
DEVELOPMENTAL
BIOLOGY
VOLUME
58, 1977
accompanied by the loss of the NGF requirement for survival, DRGs from embryos incubated from 8 to 18 days were dissociated and cultured with or without the presence of NGF (50 rig/ml). Figures 2 and 3 summarize the results of these experiments in which cell counts were made at times varying from 1 to 10 days after plating. In cultures derived from E8 (see
8 ++---+-3 ?-+..-. ,,a ““““” FIG. 1. Relative number of process-bearing chick embryo DRG cells observed in culture 24 hr after plating in the presence of various concentrations of NGF and in various media. Media varied as indicated with respect to their contents of horse serum (HS), fetal bovine serum (FBS1, and chick embryo extract (EE). Data are expressed relative to the number of process-bearing cells (21,904) observed in cultures plated in medium containing 5% HS, 2% EE, and 0.5 nglml of NGF. Values are based on the averages of cell counts on duplicate cultures.
ronal survival was unaltered at NGF concentrations up to 5000 rig/ml when the cultured medium contained 2% embryo extract in addition to 10% fetal bovine serum. Similar results pertained in the presence of 5% horse serum with or without embryo extract.
“““““.
FIG. 2. Relative survival as a function of time in culture of process-bearing cells derived from DRGs at various embryonic stages plated with (darkened circles) or without (blank circles) NGF (50 nglml) in the medium. For each embryonic age, data are given as relative to the number of process-bearing cells observed 24 hr after plating in the presence of NGF (27,615; 19,731; 33,973; 13,170; 14,394; and 19,952 at ages Ell, E13, E14, E16, E17, and E18, respectively). Each value represents the mean of counts on three sister cultures; vertical lines represent one standard deviation from the mean.
Effect of NGF on the Survival of ProcessBearing Cells in Cultures Derived from DRGs of Different Embryonic Stages Preliminary experiments indicated that, among the various media employed above, optimal long-term maintenance of neurons occurred in medium containing 5% horse serum and 2% embryo extract. This medium, therefore, was used in all subsequent experiments. Previous reports have indicated that explanted DRGs from chick embryos older than Days E13-El6 do not respond to NGF by extending processes (14, 16, 271. In order to test whether this disappearance of NGF-induced process outgrowth was also
ow
5 DAYS
a
IN cuLT”RE
FIG. 3. Relative proportion of process-bearing cells derived from dissociated DRGs of various embryonic ages which survive in culture in the absence of NGF. Data are from Fig. 2, except the values for day ES which are from Fig. 1. Ordinate denotes the ratio (x loo), at (A) each time point or at (B) 24 hr after plating, of the number of process-bearing cells observed in cultures maintained without NGF to that in cultures maintained with NGF.
LLOYD
A.
GREENE
Sensory
Figs. 1 and 4 for cell counts) and El1 ganglia, very few neurons survived in medium not supplemented with NGF. In cultures derived from ganglia of Day El3 and older, however, the number of process-bearing cells which survived in the absence of NGF increased as a function of the age of the ganglia. By ages El7 and EM, the numbers of process-bearing cells observed in cultures with and without NGF were nearly identical. It has been previously shown that nonneuronal DRG cells, when present in suffcient numbers, may support the survival of otherwise NGF-dependent sensory neurons (6, 231, probably via the secretion of a molecule which is identical or similar to NGF (24). In order to test whether the survival of neurons without exogenously added NGF in the present experiments was due to support by non-neuronal cells, El7 and El8 ganglia were dissociated, preplated, and then subsequently treated
FIG. 4. Effect of withdrawal of NGF at various times after plating on the survival of process-bearing DRG cells. Sister cultures were prepared from ES DRGs and were plated either in the presence (50 rig/ml) or absence of NGF. At the times indicated by arrows, the medium of pairs of cultures which had been previously maintained in the presence of NGF was changed to one which was not supplemented with the factor. Cell counts for plates containing NGF are given as darkened circles and solid lines, whereas those for plates without NGF are given as blank circles and dashed lines. Data are averages of counts on duplicate cultures and are expressed relative to the average number of process-bearing cells (37,976) observed in NGF-containing dishes 24 hr after plating. Cultures were fed daily.
Neuron
NGF
Requirement
109
with cytosine arabinoside. As a result of this treatment, the number of non-neuronal cells in such cultures was greatly reduced; by 10 days after plating the density of such cells was only about 3000ldish (i.e., about one-tenth that of the neuronal population). Neuronal cell counts of such cultures revealed that the number of process-bearing cells was the same both with and without added NGF. Thus, the ability of sensory neurons to survive in cultures without NGF in the present experiments appears to derive from a property of the neurons themselves rather than from the presence of a non-neuronal cell population. Another feature of the experiments summarized in Fig. 2 which is worth noting is that, in cultures derived from El4 to El8 ganglia, the number of process-bearing cells appeared to increase over time in culture. Since increases were also observed in the presence of the mitotic inhibitor cytosine arabinoside, it is unlikely that such effects were due to cell division. A likely source of the increase was a population of cells present in cultures derived from ganglia of ages El4 and older which did not extend processes at times soon after plating, but which possessed the morphological appearance of nerve cell bodies (i.e., round and phase-bright). However, since it was generally not possible to identify positively such cells as neurons (i.e., to distinguish them from dividing or detaching non-neuronal cells), accurate counts of their numbers were not attempted. Observations of the cultures did reveal, nevertheless, that the number of such cells was substantially reduced by 5 to 6 days after plating, presumably because they had either failed to survive, had commenced to extend processes, or were in fact non-neuronal. Loss of the NGF Requirement for Survival by Sensory Neurons in Vitro The increasing ability of DRG neurons from embryos older than day El1 of incubation to survive in dissociated cell culture
110
DEVELOPMENTAL
BIOLOGY
in the absence of exogenous NGF suggests that loss of NGF dependence is part of the normal development of such neurons in. situ. In order to confirm this and to test whether and under what conditions such a transition might also take place in. uitro, sister cultures were prepared from E8 DRGs and were deprived of NGF at various times after plating. The results of this experiment are summarized in Fig. 4. Few process-bearing cells survived by 24 hr after plating in the absence of NGF; by 6 days after plating, such cultures contained no neurons. When NGF was withdrawn from cultures at 24 hr after plating, loss of process-bearing cells occurred within the next 24 hr and continued over the next 4 days. After 5 days following removal of NGF, however, little further loss of process-bearing cells took place. Similarly, when NGF was removed from cultures 3 and 5 days after plating, loss of processbearing cells (relative to controls) occurred for the next several days and then ceased. The number of neurons which continued to survive in the absence of NGF increased as a function of the age of the culture at the time when NGF was removed. Finally, when NGF was withdrawn from cultures which had been previously cultured in its presence for 7 days, survival of processbearing cells relative to controls was no longer affected. The above findings suggest that the loss of NGF-dependence by DRG neurons can take place in vitro as well as in uiuo. However, as previously indicated, an alternate interpretation of such results is that nonneuronal cells, which continue to increase in density with time in vitro, serve to replace the requirement for exogenous NGF. Yet, another possibility is that the neurons themselves secrete sufficient levels of NGF to sustain themselves. In order to test these alternatives, several different types of experiments were carried out. First, medium was collected from cultures which had been maintained for 7 days with (50 rig/ml) and for another 10 days without
VOLUME
58, 1977
NGF. This medium was found to be incapable of supporting the survival of newly plated dissociated DRG neurons unless supplemented with NGF. Second, dissociated E8 DRGs were preplated and cultured in the presence of cytosine arabinoside in order to decrease the number of non-neuronal cells. When NGF was withdrawn from such cultures after 7 days of maintenance in its presence, no loss of process-bearing cells (relative to that occurring in sister cultures from which NGF was not removed) was observed over the next 7 days. In a third experiment, sister cultures were maintained for 1 week with NGF (50 rig/ml) and then for an additional 2 weeks without the factor. Equal numbers of cells from freshly dissociated ES DRGs were then added to each culture, either with or without the presence of exogenous NGF (50 rig/ml). Because of their obvious difference in size, refractility, and extent of process outgrowth, the newly plated neurons were easily distinguishable from those which had been previously maintained in culture for 3 weeks. As shown in Fig. 5, the newly plated neurons did not survive in such cultures for more than a few days unless NGF was added. These experiments strongly suggest that survival of sensory neurons in our cultures in the absence of exogenous NGF is not due to the presence of nonneuronal cells or to the production of an NGF-like material by either non-neuronal cells or neurons. DISCUSSION
Quantitative NGF Requirement sponsive Sensory Neurons
by Re-
In agreement with previous studies (2, 7,8,15), neurons from dissociated E8 chick sensory ganglia were found to be totally dependent on NGF for survival in vitro. The minimal level of this requirement (0.5 rig/ml; 2 x 10-l’ M) is equal to that estimated for cultured chick sympathetic neurons (10, 11). Such findings suggest that NGF may affect survival of sensory and
LLOYD
A.
GREENE
OAYS AFTER SECOND 0 I
RATING 4 I
Sensory
8
Neuron
NGF
Requirement
111
with embryo extract, resembles findings made with sympathetic neurons (11). The mechanism(s) behind this phenomenon is not clear, but the present data are consistent with the previous suggestion (3, 11) that humoral factors may play an important role in the response of neurons to NGF. NGF Requirement by Sensory Neurons from Different Embryonic Stages
0
L+---=L
13
21 DAYS
1 29
AFTER FIRST PLATING
FIG. 5. Failure of established cultures of E8 DRGs to support the survival of process-bearing cells from freshly dissociated ganglia. Cultures of E8 DRGs were maintained for 1 week in the presence of NGF (50 rig/ml) and then 2 weeks in its absence. On the twenty-first day after initial plating, these established cultures were seeded with equal aliquots of freshly dissociated E8 DRG cells either with (50 rig/ml) or without NGF. Differential cell counts were made (based on distinctive morphological properties) of the number of well-established (circles) and newly plated (triangles) process-bearing cells in cultures which either received (darkened symbols) or did not receive (blank symbols) NGF. Data are expressed as relative to the average number of process-bearing cells observed in cultures 13 days after the first plating (19,512) and are the averages of counts made on duplicate cultures of each type.
sympathetic ganglionic neurons by means of a similar primary mechanism. A likely possibility in this regard is a specific membrane receptor for NGF. As have sympathetic ganglia (1, 91, chick embryo DRGs have been shown (9, 13, 14) to possess saturable high-affinity (K, = 2-4 x lo-lo M) NGF receptors. Thus, for both types of ganglia, neuronal survival may require occupation of only lo-20% of NGF receptor sites at any given time. The finding that supramaximal levels of NGF adversely affected survival of sensory neurons in the presence of 10% fetal bovine serum, but not in the presence of horse serum or fetal bovine serum supplemented
There is evidence that dorsal root sensory neurons respond to NGF only during a limited period of their development. For example (a) treatment of neonates with NGF (16) or with antibody to NGF (17) does not bring about morphological changes in sensory ganglia as it does in sympathetic ganglia, and (b) DRGs explanted from chick embryos older than 1316 days of incubation do not show NGFstimulated fiber outgrowth (14, 16, 27). One drawback of the latter type of experiments, however, is that the lack of fiber outgrowth could be due to other factors such as physical barriers imposed by the ganglionic capsule rather than to insensitivity to NGF. Furthermore, it is entirely possible that sensory neurons may lose the ability to respond to NGF morphologically, but still require it for their survival. In contrast to previous work which has emphasized the loss of morphological responsiveness to NGF, the present studies have emphasized the loss of the NGF requirement for survival. An increasingly greater proportion of chick DRG neurons were found to survive in vitro in the absence of NGF over the age range between E8 and El3 The most likely interpretation of such findings is that, during development, NGF-requiring neurons became converted to those which do not require the factor. This is strongly supported by our experiments with ES ganglia in which such a conversion took place in vitro. The time course for loss of the NGF requirement (that is, for conversion from NGFdependent to NGF-independent) in vivo
112
DEVELOPMENTAL
BIOLOGY
would thus be reflected in the relative number of process-bearing cells which survive in vitro in NGF-free medium (as shown in Fig. 3B). The past and present results suggest that the losses of both the response to and the requirement for NGF take place at comparable ages of development. It has recently been demonstrated that the number of saturable high-affinity NGF receptors in chick DRGs undergoes a large drop by day El6 and beyond (14). The temporal correspondence of this drop with the loss of response to and requirement for NGF suggests that both effects may be mediated via a common primary site, namely, the NGF receptor. Neuronal
Populations
Requirement
58, 1977
NGF-dependency in vitro was such that the cells had a total conceptual age of 1416 days, an age similar to that at which the neurons in ouo also appeared to be losing their NGF requirement. Such findings suggest that chick sensory neurons are “programmed” (at least by day E8) to lose their requirement for NGF, and that this program may be carried out in vitro in a temporally conserved manner and without the presence of efferent and afferent connections or of non-neuronal cells. We gratefully acknowledge the expert cal assistance of Tania Sarris and Nancy Supported in part by research grants from tional Foundation-March of Dimes, the (N11557), and the Sloan Foundation.
techniGottier. the NaUSPHS
REFERENCES
Dorsal root ganglia have been shown to contain two distinct neuronal populations, which, on the basis of their positions, have been referred to as ventral-lateral (VL) and medio-dorsal (MD) (12, 19). It has been reported that only the MD neurons respond to NGF either in uiuo (16) or in vitro (16, 17). Since the VL neurons differentiate early in development and are relatively mature by morphologic and cytologic criteria by day E8 (12, 191, it is somewhat surprising that we did not observe appreciable numbers of such neurons in NGF-free dissociated cell cultures derived from E&El1 ganglia. The absence of such neurons implies either that VL neurons do not survive under the present conditions of dissociation and culture, or that such neurons do in fact require NGF for their survival in uitro. Loss of NGF
VOLUME
in Vitro
The present experiments demonstrated that neurons derived from day E8 DRG lost their requirement for exogenously supplied NGF as a function of time in culture, and that non-neuronal cells did not play a necessary role in this phenomenon. Interestingly, the time course of the loss of
1. BANERJEE, S. P., SNYDER, S. H., CUATRECASAS, P., and GREENE, L. A. (1973). Nerve growth factor receptor binding in sympathetic ganglia. Proc. Nut. Acad. Sci. USA 70,2519-2523. 2. BANTHORPE, D. V., PEARCE, F. L., and VERNON, C. A. (1974). Effects of nerve growth factor from the venom of Viperu russelli on dispersed sensory ganglion cells from the embryonic chick. J. Embryol. Exp. Morphol. 31, 151-167. 3. BLOOD, C. A. (1972). Some quantitative effects of nerve growth factor on dorsal root ganglia of chick embryos in culture. J. Anat. 112, 315328. 4. BURNHAM, P. A. (1972). Effects of mouse nerve growth factor on intact and dissociated dorsal root ganglia from chick embryos and neonatal mice. Thesis, University of California, San Diego, Calif. 160 pp. 5. BURNHAM, P. A., and VARON, S. (1974). Biosynthetic activities of dorsal root ganglia in uitro and the influence of nerve growth factor. Neurobiology 4, 57-70. 6. BURNHAM, P., RAIBORN, C., and VARON, S. (1972). Replacement of NGF by ganglionic non-neuronal cells for survival in vitro of dissociated ganglionic neurons. Proc. Nut. Acad. Sci. USA 69, 3556-3560. 7. CHARLWOOD, K. A., GRIFFITH, M. J., LAMONT, M. D., VERNON, C. A., and WILCOCK, J. C. (1974). Effects of nerve growth factor from the venom of Vipera russelli on sensory and sympathetic ganglia from the embryonic chick in culture. J. Embryol. Exp. Morphol. 32, 239259. 8. COHEN, A. I., NICOL, E. C., and RICHTER, W.
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(1964). Nerve growth factor requirement for development of dissociated embryonic sensory and sympathetic ganglia in culture. Proc. Sot. Exp. Biol. Med. 116, 784-789. FRAZIER, W. A., BOYD, L. F., and BRADBHAW, R. A. (1974). Properties of specific binding of lz51nerve growth factor to responsive peripheral nerves. J. Biol. Chem. 249, 5513-5519. GREENE, L. A. (1974). A dissociated cell culture bioassay for nerve growth factor. Neurobiology 4, 286-292. GREENE, L. A. (1977). Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. I. Sympathetic neurons. Deuelop. Biol. 58, 96-105. HAMBURGER, V., and LEVI-M• NTALCINI, R. (1949). Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. 2001. 111, 457-502. HERRUP, K., and SHOOTER, E. M. (1973). Properties of the nerve growth factor receptor of avian dorsal root ganglia. Proc. Nut. Acad. Sci. USA 70, 3884-3888. HERRUP, K., and SHOOTER, E. M. (1975). Properties of the nerve growth factor receptor in development. J. Cell Biol. 67, 118-125. LEVI-M• NTALCINI, R., and ANCELETTI, P. U. (1963). Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in uitro. Deuelop. Biol. 7, 653-659. LEVI-M• NTALCINI, R., and ANGELETTI, P. U. (1968). The nerve growth factor. Physiol. Reu. 48, 534-569. LEVI-M• NTALCINI, R., and BOOKER, B. (1960). Destruction of the sympathetic ganglia in mammals by antiserum to the nerve-growth promoting factor. Proc. Nat. Acad. Sci. USA 42, 384-391. LEVI-M• NTALCINI, R., and COHEN, S. (1960). In
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uiuo effects of a nerve growth agent isolated from snake venom. Acad. Sci. USA 42, 695-699. LEVI-M• NTALCINI, R., and LEVI, G. (1943). Recherches quantitatives sur la marche du processus de differentiation des neurones dan les ganglions spinaux de l’embryon de poulet. Arch. Biol. (Liege) 54, 183-206. LEVI-M• NTALCINI, R., MEYER, H., and HAMBURGER, V. (1954). In vitro experiments on the effects of mouse sarcomas 130 and 37 on the spinal and sympathetic ganglia of the chick embryo. Cancer Res. 14, 49-57. VARON, S., and RAIBORN, C. W., JR. (1969). Dissociation, fractionation and culture of embryonic brain cells. Bruin Res. 12, 180-199. VARON, S., and RAIBORN, C. (1971). Excitability and conduction in neurons of dissociated ganglionic cell cultures. Bruin Res. 30, 83-98. VARON, S., RAIBORN, C., and BURNHAM, P. A. (1974). Selective potency of homologous ganglionic non-neuronal cells for the support of dissociated ganglionic neurons in culture. Neurobiology 4, 231-252. VARON, S., RAIBORN, C., and BURNHAM, P. A. (1974). Implication of a nerve growth factorlike antigen in the support derived by ganglionic neurons from their homologous glia in dissociated cultures. Neurobiology 4, 317-327. WEIS, P. (1970). The in vitro effect of the nerve growth factor in chick embryo spinal ganglia - a light microscopic evaluation. J. Embryol. Exp. Morphol. 24, 381-392. WEIS, P. (1971). The in vitro effect of the nerve growth factor on chick embryo spinal ganglia: An electron microscopic evaluation. J. Comp. Neurol. 141, 117-132. WINICK, M., and GREENBERG, R. E. (1965). Chemical control of sensory ganglia during a critical period of development. Nature (London) 205, 180-181.
58, 106-113
BIOLOGY
Quantitative
(1977)
in Vitro Studies on the Nerve Growth Requirement of Neurons II. Sensory
Factor
(NGF)
Neurons
LLOYD A. GREENE Department Retardation
of Neuropathology, Research Center,
Received
Harvard Children’s
September
Medical School, and Department of Neuroscience, Hospital Medical Center, 300 Longwood Avenue, Massachusetts 02115
16,1976;
accepted
in revised
form
March
Mental Boston,
2,1977
Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (9 The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of processbearing cells derived from S-day (E8) embryonic DRGs is 0.5 nglml (-2 x 10-l’ M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few processbearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from El7 and El8 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in Go. These results are discussed with respect to the role of NGF in development of sensory neurons. INTRODUCTION
DRG sensory neurons appear to resemble those which it has on sympathetic neurons. One major difference between the two neuronal types, however, is that, while sympathetic neurons appear to be effected by NGF throughout life, sensory DRG neurons appear to be effected by the factor only during a limited period of their development (14, 16, 17, 27). As previously pointed out (ll), dissociated cell culture is a useful system in which to study the effects of NGF on neurons. Besides lending itself to quantitative studies, the advantages of this approach are that it is possible to culture neurons from ganglia of widely varying embryonic ages and that it is possible, by means of suitable techniques (211, to remove from the cultures virtually all cell types other than neurons. In the present report, we have employed cell cultures of dissociated chick embryo
Among the several known targets for nerve growth factor protein (NGF) are embryonic dorsal root ganglionic (DRG) sensory neurons. In uiuo, the medio-dorsal neurons of responsive DRGs undergo an increase in size and extent of outgrowth of neurites when exposed to elevated levels of NGF (16, 18). In vitro, NGF stimulates neurite outgrowth from responsive explanted ganglia (3, 7, 16, 20) and enhances the survival in such explants of mediodorsal neurons (25,261. Also, when responsive embryonic DRGs are dissociated, the neurons do not survive unless the medium is supplemented with NGF (8,151. Sensory ganglia which can respond to NGF morphologically also show metabolic responses to the factor (5,161 and have been shown to possess specific high-affinity NGF receptors (9, 13, 14). In many respects, the effects of NGF on 106 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
LLOYD
A.
GREENE
Sensory
DRGs to study (a) the amount of NGF required by responsive sensory neurons for their survival, (b) the loss during development by DRG neurons of their survival requirement for NGF, and (c) the regulation in. vitro of this loss. MATERIALS
AND
METHODS
Cell cultures and media. Dorsal root ganglia were dissected from chick embryos of 8 to 18 days of incubation and were trypsinized, dissociated, plated, and maintained according to the methods described previously for sympathetic ganglia (11). Cells were seeded at a density of 75,000150,000 neurons per 35mm dish and with a plating efficiency ranging from approximately 20-40%. Media were also as previously indicated (11) except that, as noted, in some cases, media were supplemented with chick embryo extract at a final proportion of 2%. Removal of NGF from established cultures was carried out by washing the plates three times and then feeding them with NGF-free medium. In certain experiments, ganglionic dissociates were preplated in uncoated plastic tissue culture dishes in order to remove non-neuronal cells which preferentially attached to the plastic substrate (21). After 2 hr of preplating, the culture medium (containing unattached cells which were for the most part neuronal in morphology) was quantitatively transferred to collagen coated dishes. Further reduction in the number of non-neuronal cells was achieved by treating the cultures with the mitotic inhibitor 0.6 fl cytosine arabinoside (Sigma) between Days 1 and 4 in uitro. This concentration of drug was empirically found to suppress cell growth without causing detectable damage to the neuronal population. Differential cell counts revealed that, after 2 weeks in vitro, cultures treated in this way contained ratios of neurons to non-neurons of between 1O:l and 3:l. CeZZ counts. Cell counts were carried out as previously described for cultures of dis-
Neuron
NGF
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sociated sympathetic ganglia (11) and are expressed (unless otherwise stated) as relative to the number of process-bearing cells present 24 hr after plating in cultures which contained NGF. Process-bearing cells were considered to be those which extended fibers to a distance at least twice the diameter of their cell bodies. Such cells also displayed other morphological features of neurons including rounded phasebright cell bodies for the first several days in culture and, at times thereafter, prominent nuclei and nucleoli as well as extensive fiber outgrowth. In other studies (221, cells with these morphological characteristics have been shown to be electrically excitable. In cultures derived from embryos incubated longer than 14 days, cells were also observed for the first several days after plating which were round and phasebright but which did not extend processes. By our criteria, such cells were not included in neuronal counts. RESULTS
NGF Requirement of Responsive Neurons 24 hr after Plating
DRG
Figure 1 shows the relative number of process-bearing cells (neurons) observed in cell cultures of dissociated g-day embryonic (EB) DRGs 24 hr after plating in the presence of various concentrations of NGF. In the absence of added NGF, few processbearing cells were observed, and the cultures contained floating debris of what appeared to be degenerated neurons. Significant enhancement in neuronal survival was obtained in the presence of as little as 0.05 rig/ml of NGF; maximal numbers of neurons were observed at a minimum NGF concentration of 0.5 rig/ml. In agreement with previous findings for cultured sympathetic (10, 11) and sensory (4) neurons, the number of process-bearing cells present in the cultures progressively decreased at NGF concentrations greater than 5 rig/ml when the medium contained 10% fetal bovine serum. However, neu-
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accompanied by the loss of the NGF requirement for survival, DRGs from embryos incubated from 8 to 18 days were dissociated and cultured with or without the presence of NGF (50 rig/ml). Figures 2 and 3 summarize the results of these experiments in which cell counts were made at times varying from 1 to 10 days after plating. In cultures derived from E8 (see
8 ++---+-3 ?-+..-. ,,a ““““” FIG. 1. Relative number of process-bearing chick embryo DRG cells observed in culture 24 hr after plating in the presence of various concentrations of NGF and in various media. Media varied as indicated with respect to their contents of horse serum (HS), fetal bovine serum (FBS1, and chick embryo extract (EE). Data are expressed relative to the number of process-bearing cells (21,904) observed in cultures plated in medium containing 5% HS, 2% EE, and 0.5 nglml of NGF. Values are based on the averages of cell counts on duplicate cultures.
ronal survival was unaltered at NGF concentrations up to 5000 rig/ml when the cultured medium contained 2% embryo extract in addition to 10% fetal bovine serum. Similar results pertained in the presence of 5% horse serum with or without embryo extract.
“““““.
FIG. 2. Relative survival as a function of time in culture of process-bearing cells derived from DRGs at various embryonic stages plated with (darkened circles) or without (blank circles) NGF (50 nglml) in the medium. For each embryonic age, data are given as relative to the number of process-bearing cells observed 24 hr after plating in the presence of NGF (27,615; 19,731; 33,973; 13,170; 14,394; and 19,952 at ages Ell, E13, E14, E16, E17, and E18, respectively). Each value represents the mean of counts on three sister cultures; vertical lines represent one standard deviation from the mean.
Effect of NGF on the Survival of ProcessBearing Cells in Cultures Derived from DRGs of Different Embryonic Stages Preliminary experiments indicated that, among the various media employed above, optimal long-term maintenance of neurons occurred in medium containing 5% horse serum and 2% embryo extract. This medium, therefore, was used in all subsequent experiments. Previous reports have indicated that explanted DRGs from chick embryos older than Days E13-El6 do not respond to NGF by extending processes (14, 16, 271. In order to test whether this disappearance of NGF-induced process outgrowth was also
ow
5 DAYS
a
IN cuLT”RE
FIG. 3. Relative proportion of process-bearing cells derived from dissociated DRGs of various embryonic ages which survive in culture in the absence of NGF. Data are from Fig. 2, except the values for day ES which are from Fig. 1. Ordinate denotes the ratio (x loo), at (A) each time point or at (B) 24 hr after plating, of the number of process-bearing cells observed in cultures maintained without NGF to that in cultures maintained with NGF.
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Figs. 1 and 4 for cell counts) and El1 ganglia, very few neurons survived in medium not supplemented with NGF. In cultures derived from ganglia of Day El3 and older, however, the number of process-bearing cells which survived in the absence of NGF increased as a function of the age of the ganglia. By ages El7 and EM, the numbers of process-bearing cells observed in cultures with and without NGF were nearly identical. It has been previously shown that nonneuronal DRG cells, when present in suffcient numbers, may support the survival of otherwise NGF-dependent sensory neurons (6, 231, probably via the secretion of a molecule which is identical or similar to NGF (24). In order to test whether the survival of neurons without exogenously added NGF in the present experiments was due to support by non-neuronal cells, El7 and El8 ganglia were dissociated, preplated, and then subsequently treated
FIG. 4. Effect of withdrawal of NGF at various times after plating on the survival of process-bearing DRG cells. Sister cultures were prepared from ES DRGs and were plated either in the presence (50 rig/ml) or absence of NGF. At the times indicated by arrows, the medium of pairs of cultures which had been previously maintained in the presence of NGF was changed to one which was not supplemented with the factor. Cell counts for plates containing NGF are given as darkened circles and solid lines, whereas those for plates without NGF are given as blank circles and dashed lines. Data are averages of counts on duplicate cultures and are expressed relative to the average number of process-bearing cells (37,976) observed in NGF-containing dishes 24 hr after plating. Cultures were fed daily.
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with cytosine arabinoside. As a result of this treatment, the number of non-neuronal cells in such cultures was greatly reduced; by 10 days after plating the density of such cells was only about 3000ldish (i.e., about one-tenth that of the neuronal population). Neuronal cell counts of such cultures revealed that the number of process-bearing cells was the same both with and without added NGF. Thus, the ability of sensory neurons to survive in cultures without NGF in the present experiments appears to derive from a property of the neurons themselves rather than from the presence of a non-neuronal cell population. Another feature of the experiments summarized in Fig. 2 which is worth noting is that, in cultures derived from El4 to El8 ganglia, the number of process-bearing cells appeared to increase over time in culture. Since increases were also observed in the presence of the mitotic inhibitor cytosine arabinoside, it is unlikely that such effects were due to cell division. A likely source of the increase was a population of cells present in cultures derived from ganglia of ages El4 and older which did not extend processes at times soon after plating, but which possessed the morphological appearance of nerve cell bodies (i.e., round and phase-bright). However, since it was generally not possible to identify positively such cells as neurons (i.e., to distinguish them from dividing or detaching non-neuronal cells), accurate counts of their numbers were not attempted. Observations of the cultures did reveal, nevertheless, that the number of such cells was substantially reduced by 5 to 6 days after plating, presumably because they had either failed to survive, had commenced to extend processes, or were in fact non-neuronal. Loss of the NGF Requirement for Survival by Sensory Neurons in Vitro The increasing ability of DRG neurons from embryos older than day El1 of incubation to survive in dissociated cell culture
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in the absence of exogenous NGF suggests that loss of NGF dependence is part of the normal development of such neurons in. situ. In order to confirm this and to test whether and under what conditions such a transition might also take place in. uitro, sister cultures were prepared from E8 DRGs and were deprived of NGF at various times after plating. The results of this experiment are summarized in Fig. 4. Few process-bearing cells survived by 24 hr after plating in the absence of NGF; by 6 days after plating, such cultures contained no neurons. When NGF was withdrawn from cultures at 24 hr after plating, loss of process-bearing cells occurred within the next 24 hr and continued over the next 4 days. After 5 days following removal of NGF, however, little further loss of process-bearing cells took place. Similarly, when NGF was removed from cultures 3 and 5 days after plating, loss of processbearing cells (relative to controls) occurred for the next several days and then ceased. The number of neurons which continued to survive in the absence of NGF increased as a function of the age of the culture at the time when NGF was removed. Finally, when NGF was withdrawn from cultures which had been previously cultured in its presence for 7 days, survival of processbearing cells relative to controls was no longer affected. The above findings suggest that the loss of NGF-dependence by DRG neurons can take place in vitro as well as in uiuo. However, as previously indicated, an alternate interpretation of such results is that nonneuronal cells, which continue to increase in density with time in vitro, serve to replace the requirement for exogenous NGF. Yet, another possibility is that the neurons themselves secrete sufficient levels of NGF to sustain themselves. In order to test these alternatives, several different types of experiments were carried out. First, medium was collected from cultures which had been maintained for 7 days with (50 rig/ml) and for another 10 days without
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NGF. This medium was found to be incapable of supporting the survival of newly plated dissociated DRG neurons unless supplemented with NGF. Second, dissociated E8 DRGs were preplated and cultured in the presence of cytosine arabinoside in order to decrease the number of non-neuronal cells. When NGF was withdrawn from such cultures after 7 days of maintenance in its presence, no loss of process-bearing cells (relative to that occurring in sister cultures from which NGF was not removed) was observed over the next 7 days. In a third experiment, sister cultures were maintained for 1 week with NGF (50 rig/ml) and then for an additional 2 weeks without the factor. Equal numbers of cells from freshly dissociated ES DRGs were then added to each culture, either with or without the presence of exogenous NGF (50 rig/ml). Because of their obvious difference in size, refractility, and extent of process outgrowth, the newly plated neurons were easily distinguishable from those which had been previously maintained in culture for 3 weeks. As shown in Fig. 5, the newly plated neurons did not survive in such cultures for more than a few days unless NGF was added. These experiments strongly suggest that survival of sensory neurons in our cultures in the absence of exogenous NGF is not due to the presence of nonneuronal cells or to the production of an NGF-like material by either non-neuronal cells or neurons. DISCUSSION
Quantitative NGF Requirement sponsive Sensory Neurons
by Re-
In agreement with previous studies (2, 7,8,15), neurons from dissociated E8 chick sensory ganglia were found to be totally dependent on NGF for survival in vitro. The minimal level of this requirement (0.5 rig/ml; 2 x 10-l’ M) is equal to that estimated for cultured chick sympathetic neurons (10, 11). Such findings suggest that NGF may affect survival of sensory and
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8
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with embryo extract, resembles findings made with sympathetic neurons (11). The mechanism(s) behind this phenomenon is not clear, but the present data are consistent with the previous suggestion (3, 11) that humoral factors may play an important role in the response of neurons to NGF. NGF Requirement by Sensory Neurons from Different Embryonic Stages
0
L+---=L
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21 DAYS
1 29
AFTER FIRST PLATING
FIG. 5. Failure of established cultures of E8 DRGs to support the survival of process-bearing cells from freshly dissociated ganglia. Cultures of E8 DRGs were maintained for 1 week in the presence of NGF (50 rig/ml) and then 2 weeks in its absence. On the twenty-first day after initial plating, these established cultures were seeded with equal aliquots of freshly dissociated E8 DRG cells either with (50 rig/ml) or without NGF. Differential cell counts were made (based on distinctive morphological properties) of the number of well-established (circles) and newly plated (triangles) process-bearing cells in cultures which either received (darkened symbols) or did not receive (blank symbols) NGF. Data are expressed as relative to the average number of process-bearing cells observed in cultures 13 days after the first plating (19,512) and are the averages of counts made on duplicate cultures of each type.
sympathetic ganglionic neurons by means of a similar primary mechanism. A likely possibility in this regard is a specific membrane receptor for NGF. As have sympathetic ganglia (1, 91, chick embryo DRGs have been shown (9, 13, 14) to possess saturable high-affinity (K, = 2-4 x lo-lo M) NGF receptors. Thus, for both types of ganglia, neuronal survival may require occupation of only lo-20% of NGF receptor sites at any given time. The finding that supramaximal levels of NGF adversely affected survival of sensory neurons in the presence of 10% fetal bovine serum, but not in the presence of horse serum or fetal bovine serum supplemented
There is evidence that dorsal root sensory neurons respond to NGF only during a limited period of their development. For example (a) treatment of neonates with NGF (16) or with antibody to NGF (17) does not bring about morphological changes in sensory ganglia as it does in sympathetic ganglia, and (b) DRGs explanted from chick embryos older than 1316 days of incubation do not show NGFstimulated fiber outgrowth (14, 16, 27). One drawback of the latter type of experiments, however, is that the lack of fiber outgrowth could be due to other factors such as physical barriers imposed by the ganglionic capsule rather than to insensitivity to NGF. Furthermore, it is entirely possible that sensory neurons may lose the ability to respond to NGF morphologically, but still require it for their survival. In contrast to previous work which has emphasized the loss of morphological responsiveness to NGF, the present studies have emphasized the loss of the NGF requirement for survival. An increasingly greater proportion of chick DRG neurons were found to survive in vitro in the absence of NGF over the age range between E8 and El3 The most likely interpretation of such findings is that, during development, NGF-requiring neurons became converted to those which do not require the factor. This is strongly supported by our experiments with ES ganglia in which such a conversion took place in vitro. The time course for loss of the NGF requirement (that is, for conversion from NGFdependent to NGF-independent) in vivo
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would thus be reflected in the relative number of process-bearing cells which survive in vitro in NGF-free medium (as shown in Fig. 3B). The past and present results suggest that the losses of both the response to and the requirement for NGF take place at comparable ages of development. It has recently been demonstrated that the number of saturable high-affinity NGF receptors in chick DRGs undergoes a large drop by day El6 and beyond (14). The temporal correspondence of this drop with the loss of response to and requirement for NGF suggests that both effects may be mediated via a common primary site, namely, the NGF receptor. Neuronal
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NGF-dependency in vitro was such that the cells had a total conceptual age of 1416 days, an age similar to that at which the neurons in ouo also appeared to be losing their NGF requirement. Such findings suggest that chick sensory neurons are “programmed” (at least by day E8) to lose their requirement for NGF, and that this program may be carried out in vitro in a temporally conserved manner and without the presence of efferent and afferent connections or of non-neuronal cells. We gratefully acknowledge the expert cal assistance of Tania Sarris and Nancy Supported in part by research grants from tional Foundation-March of Dimes, the (N11557), and the Sloan Foundation.
techniGottier. the NaUSPHS
REFERENCES
Dorsal root ganglia have been shown to contain two distinct neuronal populations, which, on the basis of their positions, have been referred to as ventral-lateral (VL) and medio-dorsal (MD) (12, 19). It has been reported that only the MD neurons respond to NGF either in uiuo (16) or in vitro (16, 17). Since the VL neurons differentiate early in development and are relatively mature by morphologic and cytologic criteria by day E8 (12, 191, it is somewhat surprising that we did not observe appreciable numbers of such neurons in NGF-free dissociated cell cultures derived from E&El1 ganglia. The absence of such neurons implies either that VL neurons do not survive under the present conditions of dissociation and culture, or that such neurons do in fact require NGF for their survival in uitro. Loss of NGF
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The present experiments demonstrated that neurons derived from day E8 DRG lost their requirement for exogenously supplied NGF as a function of time in culture, and that non-neuronal cells did not play a necessary role in this phenomenon. Interestingly, the time course of the loss of
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