Int. J. Devl. Neuroscience, Vol. 1, No. 6, pp. 403-409, 1983.
0736-5748/83 $3.00+0.00 Pergamon Press Ltd. (~) 1983 ISDN
Printed in Great Britain.
SURVIVAL OF CHICK EMBRYO SYMPATHETIC NEURONS IN CELL CULTURE JOHN LEAH* a n d CHEV KIDSON Queensland Institute of Medical Research and Biochemistry Department, Queensland University, Brisbane, Australia
(Accepted 16 August 1983)
Abstract--Changes in neuronal numbers during the development of the chick embryo paravertebral sympatheticnervous systemhave been examined usingcell culture techniques. Early sympatheticganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequentlythere is a large decrease in the number of neurons and an increasein the populationof non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-dayor older embryos than those from earlier stages of development. Key words: Cell death, Sympatheticneurons, Chick embryo.
Extensive cell death during normal development occurs in many neural systems in a variety of species, and has been the concern of several reviews .6,7,22,30 The peak period of degeneration of the developing neurons coincides with their establishing peripheral connections, and it has been proposed that an interaction with appropriate target cells to acquire trophic agents is a primary determinant of neuronal survival. 14'15'29 H e r e we use the cell culture method to detail changes in neuronal numbers during in vivo ontogenesis of the chick sympathetic nervous system, to determine the relation between the period of neuronal death and timing of innervation, and to examine the efficacy of Nerve Growth Factor (NGF) 3 in preventing this ontogenic neuronal death in vitro.
MATERIALS AND METHODS Paravertebral sympathetic chains were dissected from thirty 51/2- to 21-day chick embryos (aged according to H a m b u r g e r & Hamilton), 16 freed to adherent connective tissue, and the total number of ganglia obtained counted. In early embryos where individual ganglia are not entirely clear and many neurons are in the connectives between ganglia, 2° the number of ganglia was defined by the number of vertebra. These were incubated at 37°C for 30 min in 5 ml calcium- and magnesium-free phosphate buffered saline (PBS) containing 0.1% trypsin (Worthington) and 0.01% DNase (Miles). One hundred ~1 ice-cold foetal calf serum (FCS) was added to halt proteolysis and the ganglia dissociated into single cells by gentle trituration through a fine-bored pasteur pipette. Cell suspensions were then centrifuged at 200 g for 5 min. The cell pellet was resuspended in growth medium [Minimal Essential Medium, (MEM) ( G I B C O ) plus 4% FCS] and 2 × 105 to 106 cells added to collagen or polylysine (Sigma type I) coated 33 mm tissue culture plates (Kayline) containing 2 ml growth medium; or 105 cells placed in the 15 mm wells of a 24-well culture dish (Costar) containing 1 ml growth medium. The use of polylysine and collagen enhanced cell attachment and prevented their aggregation into clusters, so that individual neurons and neurites, and nonneuronal cells remained discernible. Growth medium also contained 2-5 ng/ml N G F 14 and was renewed each 2-3 days. N G F was obtained from Collaborative Research and Wellcome, or prepared from mouse submaxillary glands by the method of Varon et al. 35 To determine in vitro survival, neurons were initially counted at × 200 magnification with phase contrast optics, 12 h after plating and at the end of each of six subsequent 24 h periods. Twenty or more fields were counted per culture dish and at least 4 cultures were used in each survival experiment. Neurite outgrowth was quantified by examining the cultures 12-18 h after plating at × 200 magnification and counting the number of individual neurites and their branches per field. * Present address: Department of Anatomy, Universityof Queensland, St. Lucia, Queensland 4067, Australia. 403
404
John Leah and Chev Kidson RESULTS
Immediately after dissociation of the ganglia, cell numbers were counted in a hemacytometer under phase contrast optics. Cells from 5- to 9-day embryo ganglia then appeared uniformly small but after 12-24 h in culture'more than 98% had rounded, phase bright cell bodies, often with extended neurites, and were electrically excitable. 24 Few fibroblasts or other non-neuronal cells, characterized by flat, phase dark, polygonal shapes, were evident; although after a further 1-2 days in culture these began replication and increased in number. Thus for practical purposes all cells in sympathetic chains obtained from these early embryos were counted as neurons. After dissociation, the ganglia from older embryos were seen to contain both neurons and other cell types; the neurons being distinguished by their larger (approximately 18 I~m diameter), highly refractile cell bodies which usually retained one or more axonal stumps. This criterion for recognizing and counting neurons from the older ganglia was verified by again determining the percent neurons in the population after 12-24 h in culture. At this time the fibroblasts and other non-neuronal elements had attached to the culture dish and assumed their characteristic flat grey appearance but had not begun significant replication, while the neurons remained spherical, extended long neurites and were electrically excitable and sensitive to iontophoretically applied acetylcholine. ~ Figure 1 shows the number of neurons dissociated per ganglion as a function of embryo age. During days 5-9 this number increases approximately three-fold. The neuronal density then peaks, and approximately 70% of the neurons are lost during the next 3-4 days. The remaining population then decreases by 50% during the 7 days until hatching.
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Fig. 1. Changes in the number of neurons per ganglion during development of the chick paravertebral sympathetic nervous system.
The pattern of neuronal loss seen during development in vivo (Fig. 1) appeared to be maintained when the neurons were grown in cell culture in the presence of NGF. Thus 80% of neurons dissociated from 8- to 9-day embryos died within 4-5 days in vitro, whereas neurons from older embryos which had survived the critical period in vivo continued to survive well in culture (Fig. 2). As reported previously, ~4 the slow degeneration in these older neurons could be halted more completely by daily addition of N G F (Fig. 3). The loss of the younger neurons could not be prevented by this procedure. Their failure to survive for extended culture periods did not appear to be attributable to inadequate NGF, in the sense that the N G F used was both sufficient and required for the in vitro maintenance of neurons from 13-day or older embryos (Figs 3 and 4). The N G F used also supported neurons cultured from 19-day embryos over long periods in culture, even in the complete absence of non-neuronal cells. 10 The N G F used in the present experiments was also effective in eliciting neurite outgrowth from the dissociated SG neurons (Fig. 5); although neurons from 13-day embryos were more responsive to the N G F than those from 8- to 9-day embryos.
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Fig. 3. Percent survival of neurons from 14-day embryos as a function of the time in culture. N G F was added daily until day 4 after which it was washed, without replacement from some cultures and replaced daily in others. All cultures received cytosine arabinoside on days 2 and 3 to inhibit the overgrowth of background non-neuronal cells.
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Fig. 4. Percent survival, after 5 days in culture, of sympathetic neurons dissociated from 9- and 16-day embryo ganglia. N G F was added once at the time of plating.
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DISCUSSION Changes in cell division rates and neuronal numbers during the development of a number of neural centers have previously been quantified using histological techniques, where neurons are identified by RNA content 37 or Nissl substance in stained cells. 8"22'3°However, at a very early stage of development in vivo a distinction between neuronal and non-neuronal species is not always possible. 17 In the present study neurons in the population of cells dissociated from embryonic ganglia could readily be distinguished, in vitro, by their spherical phase bright cell bodies, and the presence of long branching neurites and electrical excitability at the time of counting, 12-24 h in culture. Epithelial, Schwann and other non-neuronal elements assumed a flat, polygonal phase dark form. The rarity of these background cells in culture derived from early sensory and sympathetic ganglia has been noted previously 5"24"34 and in chick sympathetic and rat superior cervical ganglia their numbers increase during subsequent in vivo development.15"18 The present studies indicate that early chick sympathetic ganglia contain predominantly neurons and that, in contrast to non-neuronal cells, their numbers decrease during development of the ganglia. The results of Varon & Raiborn,34 obtained using a procedure similar to that described here, suggest that the number of neurons increases maximally during days 9-12, without later decrement. In the present experiments more neurons could be dissociated from the ganglia than obtained by these authors, especially with ganglia of embryonic days 7-10. We have found that only minimal numbers of these early neurons are damaged if low trypsin concentrations (0.1%) and gentle trituration are used. Additionally, the use of DNase ensures that cells do not clump, and are not lost due to failure to centrifuge down in the presence of chromatin release from a few lysed cells. A possibility for the high number of neurons, and the rarity of non-neuronal cells observed in the early ganglia in culture in this and other studies, is that removal of the immature cells from the in vivo environment, or perturbation of some cell control functions following exposure to trypsin, results in a form of pluripotentiality. On transfer to the culture environment cells destined to become non-neuronal species might then acquire neuronal phenotypes, giving an overestimate of the neuronal density in early ganglia. However, the pattern for the in vivo genesis of the chick sympathetic ganglia described here is similar to that of the chick trochlear 8 and mesencephalic nucleus, 32 and ciliary ganglia23 in the time of peak neuronal numbers (days 8-9) and the duration and ~xtent of primary neuronal loss (days 9-14, 60-80%). The pattern is also consistent with the result 9f Yates, 37 which indicates that cells of this sympathetic chain high in cytoplasmic RNA content ipresumative neurons) cease replication by embryonic day 8. The results suggest that neurons are lost from the developing chick sympathetic ganglia in two 9hases; a short critical phase during which many neurons degenerate, and a secondary slower phase vhere the loss is less extensive. A similar biphasic cell loss also appears to occur in the chick
Chick embryo sympathetic neurons in cell culture
407
trochlear nucleus 8 and in the motor nuclei of embryonic X e n o p u s laevis spinal c o r d . 21 Such a pattern could reflect separate causative events or the presence of two populations of neurons having different determinants of survival, such as found in the chick sensory ganglia. 1 Importantly in this context, recent histological studies by Lukenbill-Edds & van Horn 26 have revealed the existence of three types of cells containing different transmitter storage organelles which appear in overlapping stages during ganglionic development. Small intensely fluorescent (SIF) cells are present in early (days 7-8) ganglia as are granule containing cells (days 10-12), but neither of these can be detected in late embryos. It would be of considerable interest to determine if these cell species are those lost during development. During postnatal development of the rat superior cervical ganglion there is a slow loss of only 30% of the neurons. 19 This is similar to the secondary phase of cell death reported here, and it remains to be determined whether an initial, more precipitous neuronal loss occurs in the rat SCG prior to birth. In many systems ontogenic neuronal loss occurs about the time of formation of neuron-target cell junctions. 6,29 Sympathetic nerve fibres have reached the chick embryo heart (and possibly also visceral smooth muscle) before the end of the first week, 33 but these show no fluorescence before day 16, and cardioacceleratory responses to sympathetic nerve stimulation cannot be detected until hatching. 27 Thus the peak loss of neurons in the chick sympathetic system may not temporally be correlated with the functional innervation of target cells. Giacobini ~3has reported a sudden peak in the catecholamine content of embryonic chick sympathetic neurons at about day 9. The specific catecholamine level decreases during the next 2-3 days and increases again about day 18. Thus the initial, transient increase in catecholamine content of the sympathetic neurons does not coincide with their formation of synapses on target cells, about the time of establishment of some functional ganglionic synapses 13 but before the majority of these are established,2° and just prior to the period of neuronal death. It may thus result from a preganglionic trophic signal17'31 or reflect some initial interaction with target cells that the neurons require to make to circumvent degeneration. Our results for the N G F requirement for in vitro survival of neurons from 12-day or older embryos are similar to those obtained by G r e e n e 14 for l l - d a y and older chick paravertebral sympathetic neurons, and by Chun et al. 4 for neonatal rat and mouse superior cervical ganglion neurons. The failure of neurons from 9-day chick embryos to survive extended periods in dissociated cell culture, in the presence of NGF, is in contrast to earlier descriptions of the apparent viability of these neurons in aggregates or explant cultures.25 It is possible that when these early neurons are cultured in an abnormal dissociated state they are less able to utilize N G F , or are more susceptible to inadequacies of the in vitro environment. However, a number of studies do indicate that the neuronotrophic actions of N G F may not extend of the entire period of genesis of the sympathetic system. Our result for N G F induced neurite outgrowth (Fig. 5) is consistent with the report of Partlow & Larrabee 28 which shows that neurite outgrowth from explants of 8-day chick embryo sympathetic ganglia is only minimally enhanced by concentrations of N G F which induce dense neurite halos about explants from 14-day embryos. Moreover, Black & Coughlin 2 report that 14-day embryonic mouse superior cervical ganglia do not require N G F for in vitro neurite extension or development or normal tyrosine hydroxylase levels, whereas ganglia cultured from 18-day embryos show a marked dependence on N G F for continued in vitro development of these characters. In the context of survival, it has been found ~ that N G F supports the in vitro growth for a subpopulation of sensory neurons taken from 8- to 13-day chick embryos, whereas neurons from embryos 16 days or older are insensitive to mouse NGF. Additionally, Crain et al. 9 found that sensory neurons cultured from human fetuses of < 8 weeks are N G F dependent but those from 10- to 12-week fetuses are relatively insensitive to NGF. With sympathetic neurons, N G F is ineffective in maintaining neurons cultured from 8-day embryos but supports some of the neurons cultured from 12-day embryos. ~2 However, in contrast to our experiments and those of Greene, 14 N G F was found to support only about 40% of day 12 neurons, whereas N G F plus heart cell conditioned medium (HCM) increased their survival to 88%. This discrepancy in N G F action on 12-day and older neurons might possibly be explained by different culture conditions, in our experiments and those of Greene 14 N G F was added daily. Edgar et al.'s 12 interpretation of their results with chick embryo sympathetic neurons (and with sensory neurons) 1 is similar to ours: it is proposed that there are two subpopulations of these neurons which respond differentially to N G F at different developmental stages. Further experiments are required to clarify the quantitative differences. DN I:6-D
408
J o h n L e a h a n d C h e v Kidson
Additionally, mouse NGF may be adequate to maintain sympathetic neurons dissociated from 12-day or older chick embryos, but inappropriate for protecting these neurons during the earlier, critical period where they may specifically require chick NGF. Amounts of antiserum to mouse N G F ( r a i s e d in r a b b i t s ) w h i c h p r o d u c e a 7 0 % d e c r e a s e in n e o n a t a l m o u s e s u p e r i o r c e r v i c a l g a n g l i o n t y r o s i n e h y d r o x y l a s e h a v e n o d e t e c t a b l e e f f e c t s o n t h e l e v e l s o f t h i s e n z y m e in c h i c k e m b r y o sympathetic neurons (Hill & Hendry, unpublished data). Thus mouse and chick NGF are i m m u n o l o g i c a l l y d i s t i n c t , as a r e m o u s e a n d h u m a n N G F . 3~ W e c o n c l u d e t h a t m o u s e N G F is a c t i v e in p r e v e n t i n g d e g e n e r a t i o n o n l y d u r i n g t h e s e c o n d a r y phase of neuronal death which occurs during ontogenesis of the chick embryo sympathetic nervous system.
REFERENCES 1. Barde Y.-A., Edgar D. & Thoenen H. (1980) Sensory neurones in culture: changing requirements for survival factors during embryonic development. Proc. natn. Acad. Sci., U.S.A. 77, 1199-1203. 2. Black I. V. & Coughlin M. D. (1977) Ontogeny of an embryonic mouse sympathetic ganglion in vivo and in vitro. In Maturation and Neurotransmission (eds Vernadakis A., Giacobini E. & Filogamo G.), pp. 65-75. Karger, Basel. 3. Bradshaw R. A. (1978) Nerve growth factor. Ann. Rev. Biochem. 47, 191-216. 4. Chun L. L. Y. & Patterson P. H. (1977) Role of nerve growth factor in the development of rat sympathetic neurones in vitro. 1. Survival, growth and differentiation of catecholamine production. J. Cell Biol. 75,694-704. 5. Cohen A. [., Nicol E. C. & Richter W. (1964) Nerve growth factor requirement for development of dissociated embryonic sensory and sympathetic ganglia in culture. Proc. Soc. exp. biol. Med. 116, 784-789. 6. Cowan W. M. (1973) Neuronal death as a regulative mechanism in the control of cell number in the nervous system. In Development and Aging in the Nervous System (ed. Rockstein M.), pp. 19-41. Academic Press, New York. 7. Cowan W. M. (1978) Aspects of neural development. In International Review o f Physiology, Vol. 17 (ed. Porter R.), pp. 149-191. University Park Press, Baltimore. 8. Cowan W. M. & Wegner E. (1967) Cell loss in the trochlear nucleus of the chick during normal development and after radical extirpation of the optic vesicle. J. exp. Zool. 164, 267-280. 9. Crain S. M., Peterson E. R., Leibman M. & Schulman H. (1980) Dependence on nerve growth factor of early human fetal dorsal root ganglion neurones in organotypic cultures. Exp. Neurol. 67, 205-214. 10. Dvorak D.. Gipps E. & Kidson C. (1978) Isolation of specific neurones by affinity methods. Nature, Lond. 271,564566. l 1. Dvorak D., Gipps E., Leah J. & Kidson C. (1978) Development of receptors for c~-bungarotoxin m chick embryo sympathetic ganglion neurones in vitro. Life Sci. 22,407-414. 12. Edgar D., Barde Y.-A. & Thoenen H. (1981 ) Subpopulations of cultured chick sympathetic neurones differ in their requirements for survival factors. Nature, Lond. 289, 294-295. 13. GiacobiniE. (1978) Regulation of neurotransmitter biosynthesis during development in the peripheral nervous system. In Maturation ofNeurotransmission (eds Vernadakis A., Gaigobini E. & Filogamo G. ), pp. 40-64. Karger, New York. 14. Greene L. A. (1977) Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. 1. Sympathetic neurons. Devl. Biol. 58, 96-105. 15. Hamburger V. (1975) Cell death in the development of the lateral motor column of the chick embryo. J. comp. Neurol. 160, 535-546. 16. Hamburger V. & Hamilton H. L. (1951) A series of normal stages in the development of chick embryos. J. Morphol. 88, 49-92. 17. Hendry I. A. (1976) Effects of axotomy on the trans-synaptic regulation of enzyme activity in adult rat superior cervical ganglia. Brain Res. 107, 105-116. 18. Hendry I. A. (1977) Cell division in the developing sympathetic nervous system. J. Neurocytol. 6, 299-309. 19. Hendry I. A. & Campbell J. (1976) Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment. J. Neurocytol. 5, 351-360. 20. Hruschak K. A., Fredrich V. L. Jr. & Giacobini E. (1982) Synaptogenesis in chick paravertebral sympathetic ganglia: A morphometric analysis. Devl. Brain Res. 4, 229-240. 21. Hughes A. (1961) Cell degeneration in the laval ventral horn of Xenopus laevis (Daudin). J. Embryol. exp. Morphol. 9, 26%284. 22. Jacobson M. (1978) Developmental Neurobiology, pp. 281-292. Plenum Press, New York. 23. Landmesser L. & Pilar G. (1974) Synaptic transmission and cell death during normal ganglionic development. J. Physiol. Lond. Z41,737-749. 24. Leah J. D. & Kidson C. (1978) Differentiation of sympathetic neurones in cell culture. Proc. A ust. Physiol. Pharmac. Soc. 10, 55 pp. 25. Levi-Montalcini R. & Angeletti P. U. (1963) Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Devl Biol. 7,653-659. 26. Luckenbill-Edds L. & van Horn C. (1980) Development of chick paravertebral sympathetic ganglia. I. Fine structure and correlative histofluorescence of catecholaminergic cells. J. comp. Neurol. 191, 65-76. 27. Pappano A. J. & Loffelholz K. (1974) Ontogenesis of adrenergic and cholinergic neuro-effector transmission in chick embryo heart. J. Pharmac. exp. Ther. 190, 467-478. 28. Partlow L. M. & Larrabee M. G. (1971) Effects of a nerve-growth factor, embryo age and metabolic inhibitors on growth of fibres and on synthesis of ribonucleic acid and protein in embryonic sympathetic ganglia. J. Neurochem. 18, 2101-2118.
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29. Prestige M. C. (1967)Thec•ntr•••f•e••numberinthe•umbarventra•h•rnsduringthedeve••pment•fXen•puslaevis tadpoles. J. Embryol. exp. Morphol. 18, 359-387. 30. Prestige M. C. (1974) Differentiation, degeneration, and the role of the periphery: quantitative considerations. In The Neurosciences Second Study Program (eds Schmitt F. O. & Woden F. G.), pp. 73-82. Rockefeller University Press, New York. 31. Purves D. & Lichtman J. W. (1978) Formation and maintenance of synaptic connections in autonomic ganglia. Physiol. Rev. 58, 821-862. 32. Rogers L. A. & Cowan W. M. (1973) The development of the mescephalic nucleus of the trigeminal nerve in the chick. J. comp. Neurol. 147,291-320. 33. Romanoff A. L. (1960) The Avian Embryo. Macmillan, New York. 34. Varon S. & Raiborn C. (1972) Dissociation, fractionation and culture of chick embryo sympathetic ganglionic cells. J. Neurocytol. 1,211-221. 35. Varon S., Nomura J. & Shooter E. M. (1967) The isolation of the mouse nerve growth factor protein in a high molecular weight form. Biochemistry 6, 2202-2209. 36. Walker P., Weichsel M. E. & Fischer D. A. (1980) Human nerve growth factor: lack of immunocrossreactivity with mouse nerve growth factor. Life Sci. 26, 295-300. 37. Yates R. D. (1961) A study of cell division in chick embryonic ganglia. J. exp. Zool. 147, 167-176.
0736-5748/83 $3.00+0.00 Pergamon Press Ltd. (~) 1983 ISDN
Printed in Great Britain.
SURVIVAL OF CHICK EMBRYO SYMPATHETIC NEURONS IN CELL CULTURE JOHN LEAH* a n d CHEV KIDSON Queensland Institute of Medical Research and Biochemistry Department, Queensland University, Brisbane, Australia
(Accepted 16 August 1983)
Abstract--Changes in neuronal numbers during the development of the chick embryo paravertebral sympatheticnervous systemhave been examined usingcell culture techniques. Early sympatheticganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequentlythere is a large decrease in the number of neurons and an increasein the populationof non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-dayor older embryos than those from earlier stages of development. Key words: Cell death, Sympatheticneurons, Chick embryo.
Extensive cell death during normal development occurs in many neural systems in a variety of species, and has been the concern of several reviews .6,7,22,30 The peak period of degeneration of the developing neurons coincides with their establishing peripheral connections, and it has been proposed that an interaction with appropriate target cells to acquire trophic agents is a primary determinant of neuronal survival. 14'15'29 H e r e we use the cell culture method to detail changes in neuronal numbers during in vivo ontogenesis of the chick sympathetic nervous system, to determine the relation between the period of neuronal death and timing of innervation, and to examine the efficacy of Nerve Growth Factor (NGF) 3 in preventing this ontogenic neuronal death in vitro.
MATERIALS AND METHODS Paravertebral sympathetic chains were dissected from thirty 51/2- to 21-day chick embryos (aged according to H a m b u r g e r & Hamilton), 16 freed to adherent connective tissue, and the total number of ganglia obtained counted. In early embryos where individual ganglia are not entirely clear and many neurons are in the connectives between ganglia, 2° the number of ganglia was defined by the number of vertebra. These were incubated at 37°C for 30 min in 5 ml calcium- and magnesium-free phosphate buffered saline (PBS) containing 0.1% trypsin (Worthington) and 0.01% DNase (Miles). One hundred ~1 ice-cold foetal calf serum (FCS) was added to halt proteolysis and the ganglia dissociated into single cells by gentle trituration through a fine-bored pasteur pipette. Cell suspensions were then centrifuged at 200 g for 5 min. The cell pellet was resuspended in growth medium [Minimal Essential Medium, (MEM) ( G I B C O ) plus 4% FCS] and 2 × 105 to 106 cells added to collagen or polylysine (Sigma type I) coated 33 mm tissue culture plates (Kayline) containing 2 ml growth medium; or 105 cells placed in the 15 mm wells of a 24-well culture dish (Costar) containing 1 ml growth medium. The use of polylysine and collagen enhanced cell attachment and prevented their aggregation into clusters, so that individual neurons and neurites, and nonneuronal cells remained discernible. Growth medium also contained 2-5 ng/ml N G F 14 and was renewed each 2-3 days. N G F was obtained from Collaborative Research and Wellcome, or prepared from mouse submaxillary glands by the method of Varon et al. 35 To determine in vitro survival, neurons were initially counted at × 200 magnification with phase contrast optics, 12 h after plating and at the end of each of six subsequent 24 h periods. Twenty or more fields were counted per culture dish and at least 4 cultures were used in each survival experiment. Neurite outgrowth was quantified by examining the cultures 12-18 h after plating at × 200 magnification and counting the number of individual neurites and their branches per field. * Present address: Department of Anatomy, Universityof Queensland, St. Lucia, Queensland 4067, Australia. 403
404
John Leah and Chev Kidson RESULTS
Immediately after dissociation of the ganglia, cell numbers were counted in a hemacytometer under phase contrast optics. Cells from 5- to 9-day embryo ganglia then appeared uniformly small but after 12-24 h in culture'more than 98% had rounded, phase bright cell bodies, often with extended neurites, and were electrically excitable. 24 Few fibroblasts or other non-neuronal cells, characterized by flat, phase dark, polygonal shapes, were evident; although after a further 1-2 days in culture these began replication and increased in number. Thus for practical purposes all cells in sympathetic chains obtained from these early embryos were counted as neurons. After dissociation, the ganglia from older embryos were seen to contain both neurons and other cell types; the neurons being distinguished by their larger (approximately 18 I~m diameter), highly refractile cell bodies which usually retained one or more axonal stumps. This criterion for recognizing and counting neurons from the older ganglia was verified by again determining the percent neurons in the population after 12-24 h in culture. At this time the fibroblasts and other non-neuronal elements had attached to the culture dish and assumed their characteristic flat grey appearance but had not begun significant replication, while the neurons remained spherical, extended long neurites and were electrically excitable and sensitive to iontophoretically applied acetylcholine. ~ Figure 1 shows the number of neurons dissociated per ganglion as a function of embryo age. During days 5-9 this number increases approximately three-fold. The neuronal density then peaks, and approximately 70% of the neurons are lost during the next 3-4 days. The remaining population then decreases by 50% during the 7 days until hatching.
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Fig. 1. Changes in the number of neurons per ganglion during development of the chick paravertebral sympathetic nervous system.
The pattern of neuronal loss seen during development in vivo (Fig. 1) appeared to be maintained when the neurons were grown in cell culture in the presence of NGF. Thus 80% of neurons dissociated from 8- to 9-day embryos died within 4-5 days in vitro, whereas neurons from older embryos which had survived the critical period in vivo continued to survive well in culture (Fig. 2). As reported previously, ~4 the slow degeneration in these older neurons could be halted more completely by daily addition of N G F (Fig. 3). The loss of the younger neurons could not be prevented by this procedure. Their failure to survive for extended culture periods did not appear to be attributable to inadequate NGF, in the sense that the N G F used was both sufficient and required for the in vitro maintenance of neurons from 13-day or older embryos (Figs 3 and 4). The N G F used also supported neurons cultured from 19-day embryos over long periods in culture, even in the complete absence of non-neuronal cells. 10 The N G F used in the present experiments was also effective in eliciting neurite outgrowth from the dissociated SG neurons (Fig. 5); although neurons from 13-day embryos were more responsive to the N G F than those from 8- to 9-day embryos.
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Fig. 3. Percent survival of neurons from 14-day embryos as a function of the time in culture. N G F was added daily until day 4 after which it was washed, without replacement from some cultures and replaced daily in others. All cultures received cytosine arabinoside on days 2 and 3 to inhibit the overgrowth of background non-neuronal cells.
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Fig. 4. Percent survival, after 5 days in culture, of sympathetic neurons dissociated from 9- and 16-day embryo ganglia. N G F was added once at the time of plating.
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Fig. 5. Effect of N G F in inducing neurite outgrowth from dissociated sympathetic neurons from 8- and 13-day embryos.
DISCUSSION Changes in cell division rates and neuronal numbers during the development of a number of neural centers have previously been quantified using histological techniques, where neurons are identified by RNA content 37 or Nissl substance in stained cells. 8"22'3°However, at a very early stage of development in vivo a distinction between neuronal and non-neuronal species is not always possible. 17 In the present study neurons in the population of cells dissociated from embryonic ganglia could readily be distinguished, in vitro, by their spherical phase bright cell bodies, and the presence of long branching neurites and electrical excitability at the time of counting, 12-24 h in culture. Epithelial, Schwann and other non-neuronal elements assumed a flat, polygonal phase dark form. The rarity of these background cells in culture derived from early sensory and sympathetic ganglia has been noted previously 5"24"34 and in chick sympathetic and rat superior cervical ganglia their numbers increase during subsequent in vivo development.15"18 The present studies indicate that early chick sympathetic ganglia contain predominantly neurons and that, in contrast to non-neuronal cells, their numbers decrease during development of the ganglia. The results of Varon & Raiborn,34 obtained using a procedure similar to that described here, suggest that the number of neurons increases maximally during days 9-12, without later decrement. In the present experiments more neurons could be dissociated from the ganglia than obtained by these authors, especially with ganglia of embryonic days 7-10. We have found that only minimal numbers of these early neurons are damaged if low trypsin concentrations (0.1%) and gentle trituration are used. Additionally, the use of DNase ensures that cells do not clump, and are not lost due to failure to centrifuge down in the presence of chromatin release from a few lysed cells. A possibility for the high number of neurons, and the rarity of non-neuronal cells observed in the early ganglia in culture in this and other studies, is that removal of the immature cells from the in vivo environment, or perturbation of some cell control functions following exposure to trypsin, results in a form of pluripotentiality. On transfer to the culture environment cells destined to become non-neuronal species might then acquire neuronal phenotypes, giving an overestimate of the neuronal density in early ganglia. However, the pattern for the in vivo genesis of the chick sympathetic ganglia described here is similar to that of the chick trochlear 8 and mesencephalic nucleus, 32 and ciliary ganglia23 in the time of peak neuronal numbers (days 8-9) and the duration and ~xtent of primary neuronal loss (days 9-14, 60-80%). The pattern is also consistent with the result 9f Yates, 37 which indicates that cells of this sympathetic chain high in cytoplasmic RNA content ipresumative neurons) cease replication by embryonic day 8. The results suggest that neurons are lost from the developing chick sympathetic ganglia in two 9hases; a short critical phase during which many neurons degenerate, and a secondary slower phase vhere the loss is less extensive. A similar biphasic cell loss also appears to occur in the chick
Chick embryo sympathetic neurons in cell culture
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trochlear nucleus 8 and in the motor nuclei of embryonic X e n o p u s laevis spinal c o r d . 21 Such a pattern could reflect separate causative events or the presence of two populations of neurons having different determinants of survival, such as found in the chick sensory ganglia. 1 Importantly in this context, recent histological studies by Lukenbill-Edds & van Horn 26 have revealed the existence of three types of cells containing different transmitter storage organelles which appear in overlapping stages during ganglionic development. Small intensely fluorescent (SIF) cells are present in early (days 7-8) ganglia as are granule containing cells (days 10-12), but neither of these can be detected in late embryos. It would be of considerable interest to determine if these cell species are those lost during development. During postnatal development of the rat superior cervical ganglion there is a slow loss of only 30% of the neurons. 19 This is similar to the secondary phase of cell death reported here, and it remains to be determined whether an initial, more precipitous neuronal loss occurs in the rat SCG prior to birth. In many systems ontogenic neuronal loss occurs about the time of formation of neuron-target cell junctions. 6,29 Sympathetic nerve fibres have reached the chick embryo heart (and possibly also visceral smooth muscle) before the end of the first week, 33 but these show no fluorescence before day 16, and cardioacceleratory responses to sympathetic nerve stimulation cannot be detected until hatching. 27 Thus the peak loss of neurons in the chick sympathetic system may not temporally be correlated with the functional innervation of target cells. Giacobini ~3has reported a sudden peak in the catecholamine content of embryonic chick sympathetic neurons at about day 9. The specific catecholamine level decreases during the next 2-3 days and increases again about day 18. Thus the initial, transient increase in catecholamine content of the sympathetic neurons does not coincide with their formation of synapses on target cells, about the time of establishment of some functional ganglionic synapses 13 but before the majority of these are established,2° and just prior to the period of neuronal death. It may thus result from a preganglionic trophic signal17'31 or reflect some initial interaction with target cells that the neurons require to make to circumvent degeneration. Our results for the N G F requirement for in vitro survival of neurons from 12-day or older embryos are similar to those obtained by G r e e n e 14 for l l - d a y and older chick paravertebral sympathetic neurons, and by Chun et al. 4 for neonatal rat and mouse superior cervical ganglion neurons. The failure of neurons from 9-day chick embryos to survive extended periods in dissociated cell culture, in the presence of NGF, is in contrast to earlier descriptions of the apparent viability of these neurons in aggregates or explant cultures.25 It is possible that when these early neurons are cultured in an abnormal dissociated state they are less able to utilize N G F , or are more susceptible to inadequacies of the in vitro environment. However, a number of studies do indicate that the neuronotrophic actions of N G F may not extend of the entire period of genesis of the sympathetic system. Our result for N G F induced neurite outgrowth (Fig. 5) is consistent with the report of Partlow & Larrabee 28 which shows that neurite outgrowth from explants of 8-day chick embryo sympathetic ganglia is only minimally enhanced by concentrations of N G F which induce dense neurite halos about explants from 14-day embryos. Moreover, Black & Coughlin 2 report that 14-day embryonic mouse superior cervical ganglia do not require N G F for in vitro neurite extension or development or normal tyrosine hydroxylase levels, whereas ganglia cultured from 18-day embryos show a marked dependence on N G F for continued in vitro development of these characters. In the context of survival, it has been found ~ that N G F supports the in vitro growth for a subpopulation of sensory neurons taken from 8- to 13-day chick embryos, whereas neurons from embryos 16 days or older are insensitive to mouse NGF. Additionally, Crain et al. 9 found that sensory neurons cultured from human fetuses of < 8 weeks are N G F dependent but those from 10- to 12-week fetuses are relatively insensitive to NGF. With sympathetic neurons, N G F is ineffective in maintaining neurons cultured from 8-day embryos but supports some of the neurons cultured from 12-day embryos. ~2 However, in contrast to our experiments and those of Greene, 14 N G F was found to support only about 40% of day 12 neurons, whereas N G F plus heart cell conditioned medium (HCM) increased their survival to 88%. This discrepancy in N G F action on 12-day and older neurons might possibly be explained by different culture conditions, in our experiments and those of Greene 14 N G F was added daily. Edgar et al.'s 12 interpretation of their results with chick embryo sympathetic neurons (and with sensory neurons) 1 is similar to ours: it is proposed that there are two subpopulations of these neurons which respond differentially to N G F at different developmental stages. Further experiments are required to clarify the quantitative differences. DN I:6-D
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J o h n L e a h a n d C h e v Kidson
Additionally, mouse NGF may be adequate to maintain sympathetic neurons dissociated from 12-day or older chick embryos, but inappropriate for protecting these neurons during the earlier, critical period where they may specifically require chick NGF. Amounts of antiserum to mouse N G F ( r a i s e d in r a b b i t s ) w h i c h p r o d u c e a 7 0 % d e c r e a s e in n e o n a t a l m o u s e s u p e r i o r c e r v i c a l g a n g l i o n t y r o s i n e h y d r o x y l a s e h a v e n o d e t e c t a b l e e f f e c t s o n t h e l e v e l s o f t h i s e n z y m e in c h i c k e m b r y o sympathetic neurons (Hill & Hendry, unpublished data). Thus mouse and chick NGF are i m m u n o l o g i c a l l y d i s t i n c t , as a r e m o u s e a n d h u m a n N G F . 3~ W e c o n c l u d e t h a t m o u s e N G F is a c t i v e in p r e v e n t i n g d e g e n e r a t i o n o n l y d u r i n g t h e s e c o n d a r y phase of neuronal death which occurs during ontogenesis of the chick embryo sympathetic nervous system.
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