DEVELOPMENTAL
Survival
BIOLOGY
50, 541-547
(1976)
and Development in Culture of Dissociated Neurons from Ciliary Ganglia STEPHEN Department
L. HELFAND, o/Biological
GARY A. SMITH, Sciences,
Stanford
Accepted
AND NORMAN
University,
January
A number of characteristics of parasympathetic motor neurons make it advantageous to develop culture conditions that will permit study of such cells free of the organism, and free of contact with undefined cell types. The avian ciliary ganglion extends postganglionic fibers to innervate the intrinsic muscles of the eye (1). Two types of neuron are present: small choroid cells that serve the smooth muscle of vascular choroid tissue; and larger ciliary cells that innervate the striated iris and ciliary muscles (2-4). The latter innervation may be the only case of interaction between the autonomic nervous system and striated muscle (5, 6). Neurons of ciliary ganglia follow a pattern of developmentally triggered cell death (7, 8). Between stages 35 and 38 (9) in chicken embryos, approx 50% of the differentiated neurons die; even more cells become necrotic if the normal target organ, the eye, is absent (8, 10). A unique synapse formation occurs in the ganglion: whereas choroid cells have typical bouton synapses, ciliary cells assume a calyx formation prior to forming boutons (11). The two neuronal types in the ganglia also differ in that ciliary cells are one of the few types in which synapses form directly on a myelinated soma (3, 4). These features, plus the
WESSELLS 94305
ganglia survive in low density A charged substratum, polyornivigorous neurite formation. The is active after dialysis. Neurites charged substratum provided in
fact that parasympathetic neurons tend to have discrete and easily isolated end organs, make this system ideal for the study of development of neuromuscular junctions and of neuronal specificity. Hoosima et al. (12) have succeeded recently in eliciting axonal outgrowth from intact chick embryonic ciliary ganglia and in getting survival “over several days.” No success with dissociated ganglia was reported. In the current paper, a procedure is outlined that permits at least short-term (ca. 7 days) survival of some single neurons from dissociated ganglia. METHODS
Ciliary ganglia were dissected from 8day (stage 33-34) White Leghorn chicken embryos as follows: a severed head was placed in Hank’s balanced salt solution in a petri dish, the bottom of which was covered with paraffin or Sylgard (Dow Corning). Two sterile insect pins were inserted at 45” angles through the neck and the beak-upper jaw region, and into the paraffin to hold the head upright. Starting posteriorly on one side, watchmaker’s forceps or iridectomy scissors were used to cut through the skin and subjacent connective tissue; the cut was run forward, dorsal to the eye, then across the front of the head, and finally, posteriorly above the other eye 541
Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved
K.
California
8, 1976
Parasympathetic neurons from avian embryonic ciliary culture when neurons are free from contact with other cells. thine, and a conditioned medium permit cell survival and heart-conditioned medium must be present continuously and elongate rapidly, branch extensively, and follow patterns of the culture dish. INTRODUCTION
Stanford,
Parasympathetic
542
DEVELOPMENTAL BIOLOGY
(rotation of the dish for the posteriad cut helps). Next, starting anteriorly just above the forward insect pin, the skin and brain are pulled upward and posteriad; using two forceps virtually the whole brain can be removed down to the base of the skull and discarded. The dish is rotated to its original position and a cut is made behind each eye if dense connective tissue is still present there. The scissors or forceps are slipped between the superior rectus muscle and the eyeball, and the muscle is severed. To dissect the right eye, a pair of forceps held in the left hand is pushed downward through the base of the skull with one point on each side of the region where the optic nerve exits the right eye (the optic nerve cannot be seen yet; let experience be the guide). Then, forceps in the right hand are inserted so that the points are just inside the tips of the first pair of forceps and also on each side of the optic nerve. The left forceps holds the head stationary as the right pair is pulled laterally to tear the eye sideways. The optic nerve breaks and tissues medial to the eye rupture. In nearly every case the ciliary ganglion can be seen located directly posterior to the severed optic nerve and resting against the eyeball. Fine nerve trunks lead to the ganglion, which is the only oval-shaped body in the vicinity. The ganglion can easily be teased free and lifted from the eye using forceps. After transfer to a dish containing medium, the nerve trunks are removed with iridectomy knives. With practice, ca. four ganglia can be obtained in 5 min. Control medium consisted of modified F12 (13) containing 10% by volume of fetal calf serum. Ganglia were dissociated using trypsin according to Letourneau (18, 26) except that 20-min incubation periods were used. Hemacytometer counts revealed 1.6-1.8 x lo4 cells per ganglion (neurons plus “glia”). Cells were pelleted after counting and resuspended in the medium to be tested. Three to five x lo4 cells in 3 ml of medium were placed in Falcon 35-mm diameter culture dishes (30-ml cul-
VOLUME 50, 1976
ture flasks were also used successfully). Medium was changed at 48-hr intervals, using great care to avoid dislodgement of cells. (In recent experiments successful single-cell cultures have been prepared using ganglia from 4-g-day embryos.) Heart-conditioned medium (HCM) was prepared by dissociating whole hearts from the 8-day chicken embryos as in (14), and plating at a density of 3.2 x lo7 cells per 50 ml of F12SlO in 150-cm2 Corning tissue culture flasks. After 48 hr, just prior to attainment of confluency, the medium was decanted, centrifuged at 940 g for 10 min at room temperature to remove heart cells (14), and used fresh or stored at 4°C for up to 2 weeks prior to use. In recent experiments, medium left on heart cells for 72 hr has yielded better results on nerve growth (pH adjusted to 7.3 with NaOH). HCM was dialyzed in lo-ml batches using dialysis tubing with 24-A pore diameter, presumably allowing passage of molecules of less than 10,000 daltons. Dialysis was carried out against loo-ml samples of F12SlO at 6°C with constant stirring, and with the F12SlO changed every 24 hr for 6 days. Alternatively, lo-ml aliquots of HCM were concentrated by a factor of 10 using CO, pressure and an Amicon PM10 filter (allows passage of molecules of 10,000 daltons and less). Three-tenths milliliter of the concentrate was added to 2.7 ml of F12SlO containing dissociated cells. Filtrate that passed the Amicon filter (0.3 ml plus 2.7 ml F12SlO) was found to lack ability to support neuronal survival or neurite outgrowth. Nerve growth factor (NGF; Wellcome Reagents, Ltd.) was used at levels ranging from 0.1 to 50 units per ml of HCM or F12SlO. Tests on intact 8-day-chick dorsal root ganglia showed abundant neurite outgrowth at many levels, so ciliary ganglion cells were tested at 5 and 50 units per ml. “Anti-NGF,” which is horse serum containing antibodies to purified NGF (Wellcome Reagents, Ltd.), was used at 20% by
BRIEF NOTES
vol in HCM or F12SlO. Coated culture dishes were prepared as in Letourneau (18, 26) by placing 2 ml of poly-L-ornithine-HBr (PORN; Nutritional Biochemicals product was preferable to that of Sigma; stock 1 mg per ml in 0.15 M borate buffer, pH 8.4) solution in a 35-mm dish for 24 hr at room temperature. Then the dishes were washed four times with sterile triple-distilled water and used or stored dry for up to 2 weeks prior to use. Time lapse cinematography was as in (27). Cells prepared for scanning electron microscopy were cultured on PORN-palladium coated cover slips (as in 261, fixed as in (27), dehydrated through acetones, dried in a critical point apparatus using liquid COz, coated with gold-palladium on a rotating stage in a Denton evaporator, and viewed in a Coates and Welter field emission SEM. RESULTS
AND
DISCUSSION
Of many culture conditions tested, the most satisfactory involve use of a highly charged substratum and of a conditioned medium. Ciliary ganglion cells dissociated and suspended in heart-conditioned medium, and plated on polyornithine (PORN)-coated dishes survive and develop. Thus, several thousand neurons with neurites (Fig. 1; processes at least 30pm long and tipped with active growth cones) are found at 24 or 48 hr of culture after 3 to 5 X lo4 cells (glia plus neurons) are plated. If unconditioned control medium (F12SlO; 13) is used instead, only lo20 neurons initiate neurites during the first 24 hr, and most of those cells die during the ensuing 24-48 hr. Dorsal root ganglion cells prepared by the same techniques and cultured in the same medium (but with NGF) on a PORN substratum survive and develop extensive neuritic trees (as in 14, 18, 27). Therefore, the F12SlO is not simply toxic to all neurons; apparently the parasympathetic cells have special needs met by the conditioning process.
543
If freshly dissociated cells suspended in HCM are plated in plastic tissue culture dishes or flasks, then very few cells survive or initiate neurite formation. Similar cells in HCM plated in plastic dishes containing small pieces of PORN-coated cover slips, survive, and form abundant neurites if they happen to land on the cover slips, but fail to initiate neurites or survive if they happen to attach to the plastic substratum. Thus, both HCM and attachment to the PORN substratum are essential (in recent work by S. Johnson, collagen substratum and HCM permit some neuron survival and neurite formation). The HCM stimulation of survival and neurite formation occurs despite exhaustive dialysis (see Methods) or concentration of the medium using Amicon membranes (see Methods). The possibility that HCM acts by depositing active substances on the culture substratum was investigated by treating PORN-coated dishes for 3 days with HCM, washing (three baths of excess Hanks salt solution), and then plating freshly dissociated cells suspended in unconditioned F12SlO in the hypothetically “conditioned” dishes. No survival or neurite formation occurred other than that characteristic of the control F12SlO cultures described above. As an additional control, the same HCM used for the 3 days in the attempt to “condition” the dishes was tested for residual activity. Nerve cells suspended in this “used” HCM and plated in fresh PORN-coated dishes survived and differentiated identically to cells plated in fresh HCM. These two results suggest that the HCM effect is not due to deposition of materials on the surface of the culture dish. Therefore, the results are like those of Luduena (14) for spinal ganglion neurons and heart-conditioned medium, but unlike those of Konigsberg and Hauschka (15) who showed that conditioning factor for muscle cell differentiation can be deposited on a culture dish. It has been proposed that low levels or absence of serum from culture media
544
DEVELOPMENTAL BIOLOGY
causes an increase in adhesiveness between neurons and culture substrata and thereby stimulates axon formation (1618). Possible equivalence of HCM to serum-free medium was tested by suspending and plating freshly dissociated cells in the modified F12 (13) used to prepare HCM. Within 14 hr of plating on PORN, most neurons in the F12 were lysed or visibly degenerating, and no neurites formed. Therefore, HCM is not simply F12SlO that has lost serum during the conditioning process. The HCM effect does not appear to involve NGF (see 19). NGF at doses of 5 or 50 units per ml of F12SlO elicits no increase in survival or neurite formation. Similarly, NGF at levels up to 50 units per ml neither enhances nor hinders the HCM effect in an observable manner. Anti-NGF at levels up to 20% (see Methods) in HCM showed no effect on neuronal growth or viability (Fig. lb). This proportion of antiNGF serum inhibited NGF-stimulated outgrowth of neurites from B-day dorsal root ganglia (with NGF at 5 or 50 units per ml; see 19) runs concurrently with the parasympathetic cell cultures that showed no such inhibition. As a further control, horse serum lacking antibodies to NGF had no effect, pro or con, on neurite outgrowth or parasympathetic cell survival when used at levels up to 20% by vol in F12SlO or HCM; aliquots of the same cell suspension used to test anti-NGF activity were employed in these cases. Overall, these results indicate, as expected, that parasympathetic neurons in culture are insensitive to NGF. To test whether HCM is required only
VOLUME 50, 1976
during cell attachment and neurite initiation, or is needed continuously, cells were cultured in HCM or F12SlO. After 24 hr, half of each set of cultures was washed with and transferred to the other medium. Cells switched from HCM to F12SlO were visibly degenerating 24 hr later. Of the cells initially plated in F12SlO and then transferred to HCM, some long axons were observed 24 hr later that were not present at the time of the transfer (Fig. lc). This low level of rescue plus the rapid degeneration in going from HCM to unconditioned medium suggests that the HCM factor(s) must be present continuously. Survival of ciliary ganglion cells in vitro allows comparison of neuronal morphology with that in situ (l-4). Whereas all of the ganglionic neurons in situ are said to be unipolar and not to branch extraoccularly (3, 4), most nerve cells in culture have at least two neurites. It is not uncommon for the cells to possess from three to six neurites emerging from the cell soma (Fig. lf; the PORN may stimulate neurite initiation; see 18). Lengths and diameters of neurites vary. Many cells have thin neurites (less than 2-pm wide) with total lengths of ca. 1000 pm at 24 hr; other cells have thick neurites (diameters greater than 7 km) and lengths of 150-200 pm at 24 hr. Most neurites branch profusely, often in a symmetrical pattern (22,23) and with regular branch angles (23). So-called “glial” cells assume a fibroblastic morphology on the PORN substratum (Figs. lh, i), show frequent ruffling activity, move about the dish, and undergo mitosis (monolayers can be obtained by ca. 3 weeks continuous culture). In time-lapse
FIG. 1. (a) A parasympathetic neuron after ca. 24 hr in HCM and on PORN. Ca. 650x. (b) A neuron after 48-hr culture in the presence of anti-NGF (20%, see text). Ca. 250x. (cl A “rescued” neuron cultured for 24 hr in F12S10, followed by 24 hr in HCM. Ca. 300x (d) A neuron on the 10th day of culture in HCM, on PORN. Ca. 300x (e) Neurons after 48hr culture on a patterned substratum. The light squares are palladium, the dark aisles, PORN. Ca. 120 x (f) A parasympathetic cell after 24 hr, showing the rounded cell body, multiple neurites originating from the soma, and the highly flattened growth cones (a PORN effect, see 26). Ca. 1100x. (g) Two growth cones near the edges of PORN aisles (palladium, as in Fig. le, appears above and below). Ca. 1200x. (h), (i) “Glial” cells in a typical flattened configuration on PORN, and showing abundant ruffles and filopodia at the cell margins. Ca. 2000x, 1500x, respectively.
545
546
DEVELOPMENTALBIOLOGY
movies and repeated still-photographs of selected fields, the “glial” cells have never been seen to extend long neuritelike processes or to assume morphologies that would lead them to be mistaken for neurons. The multipolarity and branching of ciliary ganglion cells in culture is similar to that of cells from sympathetic and dorsal root ganglia (14, 18, 22, 24, 25). Extrinsic factors such as adhesiveness of the substratum are thought to influence the number of axons, their branching, their rates of elongation, and their directions of growth (18, 26). Letourneau (26) found that sensory axons can be directed along experimentally chosen pathways, according to the pattern of the more adhesive substratum available in a culture dish. The parasympathetic neurons of ciliary ganglia behave similarly. Growth cones showing intense ruffling and microspike activity (confirmed by cinematography) tend to move along the more adhesive substratum (PORN) and to avoid the less adhesive one (palladium) in shadowed dishes (Figs. le, g). Thus, motor as well as sensory neurons can be guided by routes of materials with differing adhesivities. It is not known whether both types of ciliary ganglion neurons survive under these culture conditons. The only morphological difference in situ at 8 days of incubation in chick embryos is that ciliary cells are larger than choroid cells (l-4, 7). Cell somas of surviving neurons with neurites in vitro vary between 10 and 30 pm in diameter. Scanning electron microscopy shows such somas to be rounded so that differing degrees of flattening do not account for such size differences. Nuclei of many neurons are placed eccentrically in the somas, just as is observed in uiuo (1, 3, 7). This arrangement is seen both in newly attached cells that have not yet initiated axon formation in vitro, and later during stages of neurite elongation. Though it is not clear which types of ganglionic neurons survive in culture, it is
VOLUME 50, 1976
of interest to define a typical survival curve. At 24 hr under standard conditions, approx lo-15% of the neurons reported to be present in intact ganglia (6500 * 500; 7, 8, 28) survive and possess neurites longer than 30 Frn (cells from ca. 2.3 ganglia are present in each dish; therefore, about 1.5 x lo4 neurons should have been plated per dish). There is a marked drop in neurons per dish, from several thousand to several hundred, between Days 4 and 6 in uitro, and a steady deterioration to only a few healthy looking neurons by lo-12 days (Fig. Id). The majority of cells surviving at this time is found near the periphery of the dishes and appears to be totally isolated from contact with other living cells. At 812 days most surviving neurons are multipolar (two to six neurites) with short (ca. 400 pm), thick (ca. 10 pm) neurites and active growth cones. In recent experiments in which 3-day HCM (see Methods) was used, a much larger number of cells survived 9 days. It is not known whether the deterioration and death seen in vitro will be alleviated by further improvements in conditioned media, or whether such things as the lack of end-organ interactions accounts for neuronal morbidity. Nevertheless, it is worth emphasizing that some individual neurons from 8-day embryos survive for up to 10 days in vitro, apparently free of contact with all other cells. This total time is well beyond the point where 92% of ciliary ganglion neurons die when the eye is missing in uiuo (8). Landmesser and Pilar (7, 8) have shown that the periphery is not required for the cytological and functional differentiation of ciliary ganglion cells, but that after differentiating the neurons appear to require proper peripheral connections to survive. On the other hand, Coughlin (29) found that parasympathetic neurons of submandibular ganglia will only extend axons in vitro if the prospective end organ, the submandibular epithelium, is nearby. For some other nerve types, contact with or culture in the presence of non-neuronal
BRIEF NOTES
cell types affects important parameters of neuronal differentiated phenotype (30-32). It will be of interest, therefore, to determine whether the conditioning effect in medium (which is also seen for neuroblastoma; 31) is: (i) merely a culture-related phenomenon; (ii) reflects the requirement for a parasympathetic “growth factor” (see 29); or (iii) is equivalent to factors normally derived from end organs. Special gratitude is expressed to Dr. Willem F. Stevens of the Netherlands for stimulating our interest in ciliary ganglia and for instructing us in dissection procedures. Thanks also go to our colleagues Robert Nuttall, Richard Roth, Rae Nishi, and Stanley Johnson. Ms. Nishi performed the experiments using PORN cover slips in uncoated dishes. Supported by Grant No. H.D. 04708 from NIH. Messrs. Helfand and Smith are currently firstyear students at Albert Einstein and the U.S.C. Schools of Medicine, respectively. REFERENCES 1. CARPENTER, F. (1906). Bull. Mus. Comp. 2001. Harvard 48, 139-243. 2. SETO, H. (1931). J. Orient. Med. 15, 123-135. 3. HESS, A. J. (1965). J. Cell Biol. 25, l-19. 4. MARWITT, R., PILAR, G., and WEAKLY, J. (1971) Brain Res. 25, 317-334. 5. HESS, A. J. (1966). Anat. Rec. 154, 356-357. 6. PILAR, G., and VAUGHN, P. (1971). J. Physiol. 219, 253-266. 7. LANDMESSER, L., and PILAR, G. (1974). J. Physiol. 241, 715-736. 8. LANDMESSER, L., and PILAR, G. (1974). J. Physiol. 241, 737-749. 9. HAMBURGER, V., and HAMILTON, H. (1951). J. Morph. 88, 49-92. 10. COWAN, W., and WENGER, E. (1968). J. Exp.
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2001. 168, 105-124. 11. DELORENZO, A. J. (1960). J. Cell Biol. 7,31-36. 12. HOOISMA, J., SLAAF, D. W., MEETER, E., and STEVENS, W. F. (1975). BF& Res. 85,79-85. 13. SPOONER. B. S. (19701.5. Cell Phrsiol. 75,33-48. 14. LUDIJER~, M. A. (1973). Develop. Biol. 33, 268284. 15. HAUSCHKA, S., and KONIGSBERG, I. (1966). Proc. Nat. Acad. Sci. USA 55, 119-126. 16. SCHUBERT, D., HUMPHREYS, S., DEVITRY, F., and JACOB, F. (1971). Develop. Biol. 25, 514546. 17. LUDUERA, M. A. (1973). Develop. Biol. 33, 470476. 18. LETOURNEAU, P. (1975). Develop. Biol. 44,77-91. 19. LEVI-M• NTALCINI, R. (1964). Science 143, 105110. 20. LEVI-M• NTALCINI, R., and ANGELETTI, P. (1966). Pharm. Revs. 18, 619-628. 21. PILAR, G., and VAUGHN, P. C. (1969). Comp. Biochem. Physiol. 29, 73-87. 22. BRAY, D. (1973). J. Cell Biol. 56, 702-712. 23. STRASSMAN, R., and WESSELL~, N. K. (1973). Tissue and Cell 5, 401-412. 24. BUNGE, M. B. (1973). J. Cell Biol. 56.713-735. 25. YAMADA, K., SPOONER, B., and WESSELLS, N. K. (1971). J. Cell Biol. 49, 614-635. 26. LETOURNEAU, P. C. (1975). Develop. Biol. 44, 92101. 27. LUDUEAA, M. A., and WESSELL~, N. K. (1973). Develop. Biol. 30, 427-440. 28. TERZUOLO, C. (1951). Z. Zellforsch. 36, 255-267. 29. COUGHLIN, M. D. (1975). Develop. Biol. 43, 123158. 30. PATTERSON, P. H., and CHUN, L. Y. (1974). Proc. Nat. Acad. Sci. USA 71, 3607-3610. 31. MONARD, D., SOLOMON, F., RENTSCH, M., and GYSIN, R. (1973). Proc. Nat. Acad. Sci. USA 70, 1894-1897. 32. GILLER, E. L., SCHRIER, B. K., SHAINBERG, A., FISK, H. R., and NELSON, P. G. (1973). Science 182, 588-589.
Survival
BIOLOGY
50, 541-547
(1976)
and Development in Culture of Dissociated Neurons from Ciliary Ganglia STEPHEN Department
L. HELFAND, o/Biological
GARY A. SMITH, Sciences,
Stanford
Accepted
AND NORMAN
University,
January
A number of characteristics of parasympathetic motor neurons make it advantageous to develop culture conditions that will permit study of such cells free of the organism, and free of contact with undefined cell types. The avian ciliary ganglion extends postganglionic fibers to innervate the intrinsic muscles of the eye (1). Two types of neuron are present: small choroid cells that serve the smooth muscle of vascular choroid tissue; and larger ciliary cells that innervate the striated iris and ciliary muscles (2-4). The latter innervation may be the only case of interaction between the autonomic nervous system and striated muscle (5, 6). Neurons of ciliary ganglia follow a pattern of developmentally triggered cell death (7, 8). Between stages 35 and 38 (9) in chicken embryos, approx 50% of the differentiated neurons die; even more cells become necrotic if the normal target organ, the eye, is absent (8, 10). A unique synapse formation occurs in the ganglion: whereas choroid cells have typical bouton synapses, ciliary cells assume a calyx formation prior to forming boutons (11). The two neuronal types in the ganglia also differ in that ciliary cells are one of the few types in which synapses form directly on a myelinated soma (3, 4). These features, plus the
WESSELLS 94305
ganglia survive in low density A charged substratum, polyornivigorous neurite formation. The is active after dialysis. Neurites charged substratum provided in
fact that parasympathetic neurons tend to have discrete and easily isolated end organs, make this system ideal for the study of development of neuromuscular junctions and of neuronal specificity. Hoosima et al. (12) have succeeded recently in eliciting axonal outgrowth from intact chick embryonic ciliary ganglia and in getting survival “over several days.” No success with dissociated ganglia was reported. In the current paper, a procedure is outlined that permits at least short-term (ca. 7 days) survival of some single neurons from dissociated ganglia. METHODS
Ciliary ganglia were dissected from 8day (stage 33-34) White Leghorn chicken embryos as follows: a severed head was placed in Hank’s balanced salt solution in a petri dish, the bottom of which was covered with paraffin or Sylgard (Dow Corning). Two sterile insect pins were inserted at 45” angles through the neck and the beak-upper jaw region, and into the paraffin to hold the head upright. Starting posteriorly on one side, watchmaker’s forceps or iridectomy scissors were used to cut through the skin and subjacent connective tissue; the cut was run forward, dorsal to the eye, then across the front of the head, and finally, posteriorly above the other eye 541
Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved
K.
California
8, 1976
Parasympathetic neurons from avian embryonic ciliary culture when neurons are free from contact with other cells. thine, and a conditioned medium permit cell survival and heart-conditioned medium must be present continuously and elongate rapidly, branch extensively, and follow patterns of the culture dish. INTRODUCTION
Stanford,
Parasympathetic
542
DEVELOPMENTAL BIOLOGY
(rotation of the dish for the posteriad cut helps). Next, starting anteriorly just above the forward insect pin, the skin and brain are pulled upward and posteriad; using two forceps virtually the whole brain can be removed down to the base of the skull and discarded. The dish is rotated to its original position and a cut is made behind each eye if dense connective tissue is still present there. The scissors or forceps are slipped between the superior rectus muscle and the eyeball, and the muscle is severed. To dissect the right eye, a pair of forceps held in the left hand is pushed downward through the base of the skull with one point on each side of the region where the optic nerve exits the right eye (the optic nerve cannot be seen yet; let experience be the guide). Then, forceps in the right hand are inserted so that the points are just inside the tips of the first pair of forceps and also on each side of the optic nerve. The left forceps holds the head stationary as the right pair is pulled laterally to tear the eye sideways. The optic nerve breaks and tissues medial to the eye rupture. In nearly every case the ciliary ganglion can be seen located directly posterior to the severed optic nerve and resting against the eyeball. Fine nerve trunks lead to the ganglion, which is the only oval-shaped body in the vicinity. The ganglion can easily be teased free and lifted from the eye using forceps. After transfer to a dish containing medium, the nerve trunks are removed with iridectomy knives. With practice, ca. four ganglia can be obtained in 5 min. Control medium consisted of modified F12 (13) containing 10% by volume of fetal calf serum. Ganglia were dissociated using trypsin according to Letourneau (18, 26) except that 20-min incubation periods were used. Hemacytometer counts revealed 1.6-1.8 x lo4 cells per ganglion (neurons plus “glia”). Cells were pelleted after counting and resuspended in the medium to be tested. Three to five x lo4 cells in 3 ml of medium were placed in Falcon 35-mm diameter culture dishes (30-ml cul-
VOLUME 50, 1976
ture flasks were also used successfully). Medium was changed at 48-hr intervals, using great care to avoid dislodgement of cells. (In recent experiments successful single-cell cultures have been prepared using ganglia from 4-g-day embryos.) Heart-conditioned medium (HCM) was prepared by dissociating whole hearts from the 8-day chicken embryos as in (14), and plating at a density of 3.2 x lo7 cells per 50 ml of F12SlO in 150-cm2 Corning tissue culture flasks. After 48 hr, just prior to attainment of confluency, the medium was decanted, centrifuged at 940 g for 10 min at room temperature to remove heart cells (14), and used fresh or stored at 4°C for up to 2 weeks prior to use. In recent experiments, medium left on heart cells for 72 hr has yielded better results on nerve growth (pH adjusted to 7.3 with NaOH). HCM was dialyzed in lo-ml batches using dialysis tubing with 24-A pore diameter, presumably allowing passage of molecules of less than 10,000 daltons. Dialysis was carried out against loo-ml samples of F12SlO at 6°C with constant stirring, and with the F12SlO changed every 24 hr for 6 days. Alternatively, lo-ml aliquots of HCM were concentrated by a factor of 10 using CO, pressure and an Amicon PM10 filter (allows passage of molecules of 10,000 daltons and less). Three-tenths milliliter of the concentrate was added to 2.7 ml of F12SlO containing dissociated cells. Filtrate that passed the Amicon filter (0.3 ml plus 2.7 ml F12SlO) was found to lack ability to support neuronal survival or neurite outgrowth. Nerve growth factor (NGF; Wellcome Reagents, Ltd.) was used at levels ranging from 0.1 to 50 units per ml of HCM or F12SlO. Tests on intact 8-day-chick dorsal root ganglia showed abundant neurite outgrowth at many levels, so ciliary ganglion cells were tested at 5 and 50 units per ml. “Anti-NGF,” which is horse serum containing antibodies to purified NGF (Wellcome Reagents, Ltd.), was used at 20% by
BRIEF NOTES
vol in HCM or F12SlO. Coated culture dishes were prepared as in Letourneau (18, 26) by placing 2 ml of poly-L-ornithine-HBr (PORN; Nutritional Biochemicals product was preferable to that of Sigma; stock 1 mg per ml in 0.15 M borate buffer, pH 8.4) solution in a 35-mm dish for 24 hr at room temperature. Then the dishes were washed four times with sterile triple-distilled water and used or stored dry for up to 2 weeks prior to use. Time lapse cinematography was as in (27). Cells prepared for scanning electron microscopy were cultured on PORN-palladium coated cover slips (as in 261, fixed as in (27), dehydrated through acetones, dried in a critical point apparatus using liquid COz, coated with gold-palladium on a rotating stage in a Denton evaporator, and viewed in a Coates and Welter field emission SEM. RESULTS
AND
DISCUSSION
Of many culture conditions tested, the most satisfactory involve use of a highly charged substratum and of a conditioned medium. Ciliary ganglion cells dissociated and suspended in heart-conditioned medium, and plated on polyornithine (PORN)-coated dishes survive and develop. Thus, several thousand neurons with neurites (Fig. 1; processes at least 30pm long and tipped with active growth cones) are found at 24 or 48 hr of culture after 3 to 5 X lo4 cells (glia plus neurons) are plated. If unconditioned control medium (F12SlO; 13) is used instead, only lo20 neurons initiate neurites during the first 24 hr, and most of those cells die during the ensuing 24-48 hr. Dorsal root ganglion cells prepared by the same techniques and cultured in the same medium (but with NGF) on a PORN substratum survive and develop extensive neuritic trees (as in 14, 18, 27). Therefore, the F12SlO is not simply toxic to all neurons; apparently the parasympathetic cells have special needs met by the conditioning process.
543
If freshly dissociated cells suspended in HCM are plated in plastic tissue culture dishes or flasks, then very few cells survive or initiate neurite formation. Similar cells in HCM plated in plastic dishes containing small pieces of PORN-coated cover slips, survive, and form abundant neurites if they happen to land on the cover slips, but fail to initiate neurites or survive if they happen to attach to the plastic substratum. Thus, both HCM and attachment to the PORN substratum are essential (in recent work by S. Johnson, collagen substratum and HCM permit some neuron survival and neurite formation). The HCM stimulation of survival and neurite formation occurs despite exhaustive dialysis (see Methods) or concentration of the medium using Amicon membranes (see Methods). The possibility that HCM acts by depositing active substances on the culture substratum was investigated by treating PORN-coated dishes for 3 days with HCM, washing (three baths of excess Hanks salt solution), and then plating freshly dissociated cells suspended in unconditioned F12SlO in the hypothetically “conditioned” dishes. No survival or neurite formation occurred other than that characteristic of the control F12SlO cultures described above. As an additional control, the same HCM used for the 3 days in the attempt to “condition” the dishes was tested for residual activity. Nerve cells suspended in this “used” HCM and plated in fresh PORN-coated dishes survived and differentiated identically to cells plated in fresh HCM. These two results suggest that the HCM effect is not due to deposition of materials on the surface of the culture dish. Therefore, the results are like those of Luduena (14) for spinal ganglion neurons and heart-conditioned medium, but unlike those of Konigsberg and Hauschka (15) who showed that conditioning factor for muscle cell differentiation can be deposited on a culture dish. It has been proposed that low levels or absence of serum from culture media
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causes an increase in adhesiveness between neurons and culture substrata and thereby stimulates axon formation (1618). Possible equivalence of HCM to serum-free medium was tested by suspending and plating freshly dissociated cells in the modified F12 (13) used to prepare HCM. Within 14 hr of plating on PORN, most neurons in the F12 were lysed or visibly degenerating, and no neurites formed. Therefore, HCM is not simply F12SlO that has lost serum during the conditioning process. The HCM effect does not appear to involve NGF (see 19). NGF at doses of 5 or 50 units per ml of F12SlO elicits no increase in survival or neurite formation. Similarly, NGF at levels up to 50 units per ml neither enhances nor hinders the HCM effect in an observable manner. Anti-NGF at levels up to 20% (see Methods) in HCM showed no effect on neuronal growth or viability (Fig. lb). This proportion of antiNGF serum inhibited NGF-stimulated outgrowth of neurites from B-day dorsal root ganglia (with NGF at 5 or 50 units per ml; see 19) runs concurrently with the parasympathetic cell cultures that showed no such inhibition. As a further control, horse serum lacking antibodies to NGF had no effect, pro or con, on neurite outgrowth or parasympathetic cell survival when used at levels up to 20% by vol in F12SlO or HCM; aliquots of the same cell suspension used to test anti-NGF activity were employed in these cases. Overall, these results indicate, as expected, that parasympathetic neurons in culture are insensitive to NGF. To test whether HCM is required only
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during cell attachment and neurite initiation, or is needed continuously, cells were cultured in HCM or F12SlO. After 24 hr, half of each set of cultures was washed with and transferred to the other medium. Cells switched from HCM to F12SlO were visibly degenerating 24 hr later. Of the cells initially plated in F12SlO and then transferred to HCM, some long axons were observed 24 hr later that were not present at the time of the transfer (Fig. lc). This low level of rescue plus the rapid degeneration in going from HCM to unconditioned medium suggests that the HCM factor(s) must be present continuously. Survival of ciliary ganglion cells in vitro allows comparison of neuronal morphology with that in situ (l-4). Whereas all of the ganglionic neurons in situ are said to be unipolar and not to branch extraoccularly (3, 4), most nerve cells in culture have at least two neurites. It is not uncommon for the cells to possess from three to six neurites emerging from the cell soma (Fig. lf; the PORN may stimulate neurite initiation; see 18). Lengths and diameters of neurites vary. Many cells have thin neurites (less than 2-pm wide) with total lengths of ca. 1000 pm at 24 hr; other cells have thick neurites (diameters greater than 7 km) and lengths of 150-200 pm at 24 hr. Most neurites branch profusely, often in a symmetrical pattern (22,23) and with regular branch angles (23). So-called “glial” cells assume a fibroblastic morphology on the PORN substratum (Figs. lh, i), show frequent ruffling activity, move about the dish, and undergo mitosis (monolayers can be obtained by ca. 3 weeks continuous culture). In time-lapse
FIG. 1. (a) A parasympathetic neuron after ca. 24 hr in HCM and on PORN. Ca. 650x. (b) A neuron after 48-hr culture in the presence of anti-NGF (20%, see text). Ca. 250x. (cl A “rescued” neuron cultured for 24 hr in F12S10, followed by 24 hr in HCM. Ca. 300x (d) A neuron on the 10th day of culture in HCM, on PORN. Ca. 300x (e) Neurons after 48hr culture on a patterned substratum. The light squares are palladium, the dark aisles, PORN. Ca. 120 x (f) A parasympathetic cell after 24 hr, showing the rounded cell body, multiple neurites originating from the soma, and the highly flattened growth cones (a PORN effect, see 26). Ca. 1100x. (g) Two growth cones near the edges of PORN aisles (palladium, as in Fig. le, appears above and below). Ca. 1200x. (h), (i) “Glial” cells in a typical flattened configuration on PORN, and showing abundant ruffles and filopodia at the cell margins. Ca. 2000x, 1500x, respectively.
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movies and repeated still-photographs of selected fields, the “glial” cells have never been seen to extend long neuritelike processes or to assume morphologies that would lead them to be mistaken for neurons. The multipolarity and branching of ciliary ganglion cells in culture is similar to that of cells from sympathetic and dorsal root ganglia (14, 18, 22, 24, 25). Extrinsic factors such as adhesiveness of the substratum are thought to influence the number of axons, their branching, their rates of elongation, and their directions of growth (18, 26). Letourneau (26) found that sensory axons can be directed along experimentally chosen pathways, according to the pattern of the more adhesive substratum available in a culture dish. The parasympathetic neurons of ciliary ganglia behave similarly. Growth cones showing intense ruffling and microspike activity (confirmed by cinematography) tend to move along the more adhesive substratum (PORN) and to avoid the less adhesive one (palladium) in shadowed dishes (Figs. le, g). Thus, motor as well as sensory neurons can be guided by routes of materials with differing adhesivities. It is not known whether both types of ciliary ganglion neurons survive under these culture conditons. The only morphological difference in situ at 8 days of incubation in chick embryos is that ciliary cells are larger than choroid cells (l-4, 7). Cell somas of surviving neurons with neurites in vitro vary between 10 and 30 pm in diameter. Scanning electron microscopy shows such somas to be rounded so that differing degrees of flattening do not account for such size differences. Nuclei of many neurons are placed eccentrically in the somas, just as is observed in uiuo (1, 3, 7). This arrangement is seen both in newly attached cells that have not yet initiated axon formation in vitro, and later during stages of neurite elongation. Though it is not clear which types of ganglionic neurons survive in culture, it is
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of interest to define a typical survival curve. At 24 hr under standard conditions, approx lo-15% of the neurons reported to be present in intact ganglia (6500 * 500; 7, 8, 28) survive and possess neurites longer than 30 Frn (cells from ca. 2.3 ganglia are present in each dish; therefore, about 1.5 x lo4 neurons should have been plated per dish). There is a marked drop in neurons per dish, from several thousand to several hundred, between Days 4 and 6 in uitro, and a steady deterioration to only a few healthy looking neurons by lo-12 days (Fig. Id). The majority of cells surviving at this time is found near the periphery of the dishes and appears to be totally isolated from contact with other living cells. At 812 days most surviving neurons are multipolar (two to six neurites) with short (ca. 400 pm), thick (ca. 10 pm) neurites and active growth cones. In recent experiments in which 3-day HCM (see Methods) was used, a much larger number of cells survived 9 days. It is not known whether the deterioration and death seen in vitro will be alleviated by further improvements in conditioned media, or whether such things as the lack of end-organ interactions accounts for neuronal morbidity. Nevertheless, it is worth emphasizing that some individual neurons from 8-day embryos survive for up to 10 days in vitro, apparently free of contact with all other cells. This total time is well beyond the point where 92% of ciliary ganglion neurons die when the eye is missing in uiuo (8). Landmesser and Pilar (7, 8) have shown that the periphery is not required for the cytological and functional differentiation of ciliary ganglion cells, but that after differentiating the neurons appear to require proper peripheral connections to survive. On the other hand, Coughlin (29) found that parasympathetic neurons of submandibular ganglia will only extend axons in vitro if the prospective end organ, the submandibular epithelium, is nearby. For some other nerve types, contact with or culture in the presence of non-neuronal
BRIEF NOTES
cell types affects important parameters of neuronal differentiated phenotype (30-32). It will be of interest, therefore, to determine whether the conditioning effect in medium (which is also seen for neuroblastoma; 31) is: (i) merely a culture-related phenomenon; (ii) reflects the requirement for a parasympathetic “growth factor” (see 29); or (iii) is equivalent to factors normally derived from end organs. Special gratitude is expressed to Dr. Willem F. Stevens of the Netherlands for stimulating our interest in ciliary ganglia and for instructing us in dissection procedures. Thanks also go to our colleagues Robert Nuttall, Richard Roth, Rae Nishi, and Stanley Johnson. Ms. Nishi performed the experiments using PORN cover slips in uncoated dishes. Supported by Grant No. H.D. 04708 from NIH. Messrs. Helfand and Smith are currently firstyear students at Albert Einstein and the U.S.C. Schools of Medicine, respectively. REFERENCES 1. CARPENTER, F. (1906). Bull. Mus. Comp. 2001. Harvard 48, 139-243. 2. SETO, H. (1931). J. Orient. Med. 15, 123-135. 3. HESS, A. J. (1965). J. Cell Biol. 25, l-19. 4. MARWITT, R., PILAR, G., and WEAKLY, J. (1971) Brain Res. 25, 317-334. 5. HESS, A. J. (1966). Anat. Rec. 154, 356-357. 6. PILAR, G., and VAUGHN, P. (1971). J. Physiol. 219, 253-266. 7. LANDMESSER, L., and PILAR, G. (1974). J. Physiol. 241, 715-736. 8. LANDMESSER, L., and PILAR, G. (1974). J. Physiol. 241, 737-749. 9. HAMBURGER, V., and HAMILTON, H. (1951). J. Morph. 88, 49-92. 10. COWAN, W., and WENGER, E. (1968). J. Exp.
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