DEVELOPMENTAL
BIOLOGY
66,
50-57
(1978)
Axon Initiation
by Ciliary
Neurons
in Culture
FRANK COLLINS’ Department
of Biological
Sciences, Stanford
University,
Stanford,
Received December 29, 1977; accepted March
California
94305
3, 1978
A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 pm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend fdopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum. INTRODUCTION
cells (Helfand et al., 1976). Under these conditions, a high percentage of nerve cells extend neurites which are many hundreds of microns in length and which have the ultrastructural appearance of axons (Wessells et al., 1976). The present work demonstrates the extraordinary speed and synchrony with which ciliary neurons on a polyomithine substratum initiate axons in response to the addition of heart cell-conditioned medium (HCM). Also, time-lapse microcinematography was used to follow the early events of axon formation in this system. Several major changes in cell behavior were observed, whose implications both for the mechanisms of initiation and the mode of action of HCM are discussed. Axon formation in this system probably represents a process of regeneration, since the ciliary neurons used have formed axons in uiuo (Landmesser and Pilar, 1974) which are cut and retract into the cell body during dissociation. It is not yet known whether the events of regenerative initiation described here are identical to those which occur when a ciliary neuron sends out an axon for the first time.
Although neurite elongation has been studied in detail using nerve cell cultures (Bray, 1973; Bunge, 1973; Luduena and Wessells, 1973), the process of neurite initiation (i.e., the emergence of a neurite from the nerve cell body) has not received much study. This neglect is undoubtedly a result of the difficulty of following initiation in typical nerve cell culture systems. What is required for the optimal study of initiation is a culture system in which a high percentage of neurons, free from contact with any other cell, can be induced to begin neurite formation synchronously in a short period of time. A culture system with these properties is described in the present communication. This culture system is based on the observation that dissociated ciliary neurons from chick embryos can be cultured at low cell densities on a polyornithine-coated substratum in medium previously “conditioned” by the growth of embryonic heart ’ Present address, to which reprint requests should of Anatomy, College of Medicine, University of Utah, Salt Lake City, Utah 84132. be sent: Department
50
0012.1606/78/0651-0050$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
FRANK MATERIALS
COLLINS
Axon Initiation
AND METHODS
Single cell suspensions. Ciliary ganglia and hearts were dissected from 8 to g-day White Leghorn chick embryos, as described previously (Helfand et al., 1976). Ganglia or minced heart pieces were dissociated by incubation at 37°C in Ca’+-Me-free Hank’s salt solution for 20 min (ganglia) or 30 min (hearts), followed by incubation at 37°C in 0.1% trypsin (Difco Bactotrypsin) in Ca2+-Me-free Hank’s for 20 min (ganglia) or 30 min (hearts) and then transfer to culture medium (seebelow) and dissociation into single cells by repeated pipetting. The cell suspension was pelleted by centrifugation and resuspended in the appropriate medium. Culture conditions. Cell cultures were kept at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Media were equilibrated to the temperature and pH of the incubator before use. Unconditioned, control medium was modified Ham’s F-12 (Spooner, 1970) with 10% fetal calf serum. Heart cell-conditioned medium (HCM) was prepared from control medium as follows: Eight hearts were dissociated and the cell suspension (ca. 2 x lo7 cells) was plated in 50 ml of control medium into a 150-cm2 tissue culture flask (Corning). After 16 hr the medium overlying the cell monolayer was discarded and replaced with 50 ml of control medium. The first batch of conditioned medium was collected 96 hr later, and another 50 ml of control medium was added to the monolayer for 96 hr before being collected as the second batch of conditioned medium. Each 50-ml batch of conditioned medium was diluted with 25 ml of control medium and filtered through a 0.2pm pore disposable filter (Nalge). Plastic tissue culture dishes (Falcon) were coated with polyornithine or polylysine as follows: Each 35-mm diam dish was incubated with 2 ml of 1 mg of poly-DLornithine HBr/ml (type I-B, Sigma) or 1 mg of poly-DL-lysine HBr/ml (type VII-B, Sigma) in 0.15 M borate buffer, pH 8.3, for
in Vitro
51
18 to 24 hr; then dishes were washed in sterile, distilled water and stored dry for up to 2 weeks at room temperature before use. Nerve cell structures. Dissociated ciliary ganglia were cultured in 35-mm diam polyornithine or polylysine-coated dishes in 2 ml of medium per dish. There were approximately 2500 neurons and 2500 non-neuronal cells per dish (i.e., one fourth to one half of a dissociated ganglion per dish). At this density most cells are widely separated from one another. Neurons were distinguished from non-neuronal cells by the following criteria: Neurons are about 15 pm in diameter and remain rounded and phaserefractile throughout the period of culture. Non-neuronal cells, before spreading, are only about one third the diameter of neurons and have pebbly surfaces, Most nonneuronal cells flattened out onto the substratum during the period of culture and were not observed to extend net&e-like processes. Assays of axon outgrowth. Axon outgrowth was followed by phase contrast microscopy of living cells. To determine the percentage of initiated neurons, 100 neurons were examined per dish for each time point. Any neuron which possessedone or more neurites at least 15 pm (ca. one nerve cell body diameter) in length was scored as being initiated (e.g., Fig. 1F). The titer of a sample of HCM was determined using serial twofold dilutions of the HCM with control medium, which were placed into separate polyornitbine-coated dishes. Dissociated neurons were added and the percentage of initiated neurons was determined 12 hr later. The endpoint was defined as the greatest dilution which still gave greater than 10% initiated neurons. Titers are expressed as the endpoint dilution. The rate of growth of cone movement was determined on neurons cultured on a polyornithine-coated tissue culture plastic dish by following a given field of growth cones with time-lapse microcinematography for 30 min. The distance covered by
DEVELOPMENTAL BIOLOGY
52
each growth cone was measured on tracings from the projected film and corrected to the true distance by comparison with the projected image of a micrometer scale filmed at the same magnification. Time-lapse microcinematography of axon initiation. A glass coverslip was sealed with paraffin over a hole cut in the bottom of a tissue culture dish, and the modified dish was coated with polyornithine, as described above. Neurons were seeded onto such dishes in control medium and an individual neuron was filmed both before and after the addition of HCM. During filming the pH was maintained by sealing the lids with Vaseline petroleum jelly and the temperature was kept at 37°C with an air curtain incubator (Sage Instrumerits) . Glass coverslips were washed in concentrated nitric acid, rinsed thoroughly in distilled water, and dried. They were then coated with a barely visible layer of carbon in a vacuum evaporator and heated at 300°C overnight. The coverslips were sealed onto dishes and coated with polyornithine as above. Omission of carbon-coating and baking resulted in poor axon outgrowth. RESULTS
Ciliary dium
Neurons
in Unconditioned
Me-
Dissociated ciliary neurons do not extend axons in unconditioned medium, although they survive and are active in other ways for many hours. When dissociated neurons are plated in unconditioned medium onto a polyornithine-coated glass or tissue culture plastic surface, within 15 min virtually all cells are attached sufficiently to resist being dislodged by swirling. Attached cells are widely separated from one another at the plating densities used. The neurons, which are easily distinguished from the non-neuronal cells (see Methods), begin within 2 hours to extend filopodia around their perimeters (Figs. 1A and 1B). As observed by time-lapse microcinematography,
VOIAJME 65, 1978
these filopodia are in constant motion. For example, Figs. IA and 1B were taken 6 set apart, and during that brief time interval many of the filopodia moved. These filopodia move over the substratum and eventually bend back to the cell body, where they disappear; they give no suggestion of attaching to the substratum. Also, darkened areas form at various points around the cell’s periphery which lift up and move back toward the cell body, where they disappear. The arrow in Fig. 1B indicates one such area which has just formed. Although positive identification of these active, darkened areas awaits high-resolution scanning electron microscopy, they closely resemble the membrane ruffles seen on mature growth cones or the leading edge of fibroblastic cells (Bray and Bunge, 1973), and they will be referred to as membrane ruffles for convenience. Neurons in unconditioned medium continue to form active tilopodia and membrane ruffles without extending axons for 12 to 18 hr and then begin to show signs of deterioration (i.e., vacuolization and gradual loss of refractility).
The Response of Neurons to Heart Conditioned Medium (HCM)
Cell-
When the unconditioned medium on a culture of the type described above is replaced by HCM under the appropriate conditions (see below), the following changes in neuronal behavior are observed: By 20 min many of the filopodia are no longer in constant motion but remain in one position for several minutes; for example, Figs. 1C and 1D were taken 4 min apart, and during this interval many of the filopodia did not move. This lack of motion is considered to result from attachment of filopodia to the substratum (Albrecht-Buehler, 1976). These adherent filopodia are typically of greater average length than those observed in unconditioned medium (compare Fig. 1A with 1C). Occasionally such filopodia are observed to flatten out thinly onto the substratum (not shown). By 30 min after the addition of HCM, the formation of filopodia
Frc ;. 1. A cihary neuron before and after the addition of HCM. Ciliary ganglia were dissociated and medium onto a polyornithine-coated glass coverslip dish for time-lapse microcinematog in uric ,onditioned medium was replaced with HCM and filming was resumed. These photo After 4 hr the unconditioned were tmlarged from the 16-mm film. A and B were taken 6 set apart at 3 hr, 52 min after the neuron was in uric :onditioned medium. C and D were taken 4 min apart at 21 and 25 min after the addition of HCM. of HCM. Filopodia are the finger-like projl taken at 32 min, and F was taken at 56 min after the addition which I measure 0.1 to 0.2 pm in diameter and 5 to 15 pm in length. x1000; bar = 10 pm. 53
plated mdv. graphs plated E was Ections
54
DEVELOPMENTALBIOLOGY
and membrane ruffles begins to be restricted to areas of the periphery where growth cones are forming. As these growth cones (arrows in Fig. 1E) move away from the cell body, the remainder of the cellular perimeter stops exhibiting filopodial movements and membrane ruffles, so that the once active surface of the rounded cell body (the perikaryon) becomes quiescent. By 45 min approximately 50% and by 90 min greater than 80% of all neurons in such cultures bear one or more axons which are at least one nerve cell body diameter (ca. 15 pm) in length; such neurons (e.g., Fig. 1F) are referred to as initiated in quantitative assays of axon outgrowth (Figs. 2-4). Growth cones remain motile for at least 24 hr, the time when cultures were terminated, and axon lengths of many hundreds of microns are achieved. Variables Which of Initiation
Affect
the Time- Course
The rapid initiation of axons in response to HCM is only achieved after a period of cell “recovery” following dissociation of the ganglion. When neurons are plated in HCM immediately after dissociation, there is a lag time of about 2 hr before axons begin to appear (Fig. 2, solid line). The much more rapid response to HCM described above is achieved by culturing neurons in unconditioned medium for at least 3 hr after dissociation before adding HCM (Fig. 2, dashed line). This result suggests either that some time must elapse for cell repair after dissociation, or that some time is required for neurons to prepare to reextend axons, or both. Neurons undergo a similar “recovery” in plastic petri or plastic tissue culture dishes, to which they do not attach. Neurons incubated in such dishes (in unconditioned medium or HCM) for several hours after dissociation may then be removed, resuspended in HCM, and plated onto a polyornithine-coated tissue culture plastic dish. Such neurons initiate axons much more rapidly than do neurons plated under these conditions immediately after
VOLUME 65,1978
dissociation. Therefore, “recovery” can occur in unconditioned medium or HCM and does not require attachment to a substratum. Only certain batches of HCM promote the most rapid initiation, the speed of initiation being directly related to the HCM titer (Fig. 3). Typically, the second batch of HCM produced by a monolayer of heart cells is much more effective than the first (Fig. 3). Such differences in the time course of initiation are apparently due to differences in the concentration of “active factors” between various batches of HCM. A similar effect can be produced by diluting a high-titer batch of HCM with increasing amounts of unconditioned medium, which causes an increasing delay in the onset of initiation (Fig. 4). The data of Fig. 4 show a clear effect of HCM concentration on the time of onset of initiation. These data require additional comment. It is clear from the preceding results that HCM is required for the formation of growth cones by the nerve cell body. It is reasonable, therefore, that the delayed initiation in dilute or low titer HCM is due to the delayed formation of growth cones. The assay for an “initiated” SO
r
‘0
1
JI
,
2
,
3
,
4
,
5
; 6
TIME
AFTER
PLATING
,
7
, 8
(hr)
FIG. 2. A comparison between the kinetics of initiation in freshly dissociated vs “recovered” neurons. Freshly dissociated ciliary ganglia were plated at 0 hr onto polyornithine-coated tissue culture plastic dishes, either in HCM (solid line) or in unconditioned medium, which was replaced with HCM at 6 hr (dashed line).
FRANK
TIME AFTER
ADDITION
OF HCM
COLLINS
Axon Initiation
(hr)
FIG. 3. The time course of axon initiation in different batches of HCM. Dissociated ciliary ganglia were plated onto polyornithine-coated tissue culture plastic dishes in unconditioned medium. After 4 hr (0 hr on the abscissa) unconditioned medium was replaced by aliquots of different batches of HCM (lines A-D) or by fresh unconditioned medium (line E). C and A are the first and second batches of HCM produced by one heart cell monolayer. D and B are the first and second batches from another monolayer. Titers: A = 1:8; B = 1:8; C = 1:2; and D = 1:l. Each point represents the average and range for duplicate cultures.
neuron, however, requires the growth cone to move at least 15 pm away from the cell body. Therefore, it is possible that some of the delay occurring in the events shown in Figs. 3 and 4 is because of a reduction in the rate of growth cone movement in dilute or low-titer HCM. To test this possibility, direct measurement of the rate of growth cone movement in two different dilutions of HCM has been made. Although there seemsto be a tendency for growth cones to move faster in more concentrated HCM (61 +- 15 pm/hr vs 39 + 16 pm/hr for a twofold change in the HCM concentration), the results so far observed are not highly statistically significant because of the great variability in the rate of growth cone movement within a single culture. DISCUSSION
These results demonstrate the potential usefulness of the HCM-polyornithine nerve cell culture system for studying the events of axon formation. Conditions are described
in Vitro
55
in which a population of ciliary neurons, each of which is attached to the substratum and free of contact with other cells, can be induced to begin axon formation within 20 min by adding HCM. Within 90 min, greater than 80% of the neurons bear one or more axons which are at least 15 pm in length. Under these conditions each neuron initiates an average of 3 to 4 axon-like processes.In the embryo, ciliary neurons apparently have only one axon (Landmesser and Pilar, 1974). Thus, the extracellular environment can modify the number of axons possessedby ciliary neurons. To my knowledge, this work is the first description of changes in cell surface behavior during the initiation of neurite outgrowth. In this system, initiation consists of an apparent redistribution of the capacity of parts of the neuronal surface to generate movement (i.e., filopodial extension and membrane ruffling). Initially the capacity to generate such movements is distributed randomly and rather uniformly around the cell periphery. Initiation leads to the for-
RELATIVE
CONCENTRATION
OF HCM
FIG. 4. The effect of diluting HCM with unconditioned medium on the time course of initiation. Dissociated ciliary ganglia were plated onto polyornithine-coated tissue culture plastic dishes in unconditioned medium. After 4 hr the unconditioned medium was replaced either with undiluted HCM (relative concentration = 1) or with serial twofold dilutions of HCM with unconditioned medium (relative concentrations of 0.5 to 0.125). The time course of initiation was followed at hourly intervals. The time to reach the half-maximal percentage of initiated neurons (41 2 6%) is plotted against the relative concentration of HCM.
56
DEVELOPMENTAL
BIOLOGY
mation of localized growth cones, in which the surface movements are more concentrated spatially and perhaps more coordinated, and to the cessation of surface movements elsewhere. It is possible that these changes occur either (1) because an agent essential for surface movements becomes localized in the growth cones or (2) because surface movements in regions other than the growth cones are actively suppressed. The cessation of activity at the surface of the perikaryon may be reversible; however, under present conditions, no new axons emerge from the perikaryon once surface movements cease (30-40 min after the addition of HCM). The start of locomotion in fibroblastic cells is also accompanied by the restriction of an originally uniform surface activity to a broad leading edge (Vasiliev and Gelfand, 1973). The mechanism of localization of surface movements is unknown, although changes in the cell cortex seem to be important. Actively moving regions of the cell surface in fibroblasts (and neurons) are typically associated with a latticework of microfilaments in the underlying cell cortex (Wessells et aZ., 1973). At the quiescent surfaces of locomotory fibroblasts this latticework is replaced by microfilament bundles (Wessells et al., 1973). There is also evidence, from the effect of colcemid on fibroblast locomotion, that microtubules are important for maintaining the leading edge (Gail and Boone, 1971). Since microfilament bundles and microtubule arrays near the cell surface have not been observed in nerve cell bodies (Luduena and Wessells, 1973), it will be of interest to determine what changes in the cell cortex and cytoskeleton accompany the restriction of surface motility to growth cones. How does HCM induce axon initiation? The following observations would seem to be especially relevant to this question: (1) Axon initiation occurs within 20 to 30 min of the addition of HCM and would perhaps occur even sooner in more concentrated preparations of HCM than are presently
VOLUME
65,1978
available (Fig. 4); (2) The first visible response of neurons to HCM is the replacement of nonadherent filopodia with longer, apparently adherent, filopodia which occasionally flatten onto the substratum; and (3) I have recently observed that the action of HCM in the present culture system seems to depend on its deposition of material onto the polyornithine-coated culture substratum (Collins, manuscript in preparation; this manuscript has been submitted to Proc. Natl. Acad. Sci. USA). Together these results have suggested the working hypothesis that HCM deposits material onto the polyornithine substratum, which provides appropriate sites for fdopodiai adhesion, and (possibly in combination with other effects) leads to the process of axon outgrowth. I would like to thank Dr. Norman Wessells and Randal Johnston for their comments on the manuscript and Belen Palmer for her excellent technical assistance. This work was supported by a National Institutes of Health postdoctoral fellowship to the author and by NIH Grant No. HD 04708 to Dr. Wessells. REFERENCES ALBRECHT-BUEHLER, G. (1976). Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function? J. Cell Biol. 69,275-286. BRAY, D. (1973). Branching patterns of individual sympathetic neurons in culture. J. Cell Biol. 56, 702-712. BRAY, D., and BUNGE, M. B. (1973). The growth cone in neurite extension. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 195-209. Elsevier, New York. BUNGE, M. B. (1973). Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56, 713-735. GAIL, M. H., and BOONE, 6. W. (1971). Effect of colcemid on fibroblast motility. Exp. Cell Res. 65, 221-227. HELFAND, S. L., SMITH, G. A., and WESSELLS, N. K. (1976). Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia. Develop. Biol. 50, 541-547. LANDMESSER, L., and PILAR, G. (1974). Synapse formation during embryogenesis of ganglion cells lacking a periphery. J. Physiol. 241, 715-736. LUDUENA, M. A., and WESSELLS, N. K. (1973). Cell locomotion, nerve elongation, and microfilaments.
FRANK
COLLINS
Develop. Biol. 30, 427-440.
Axon Initiation
in Vitro
57
The expression of differentiathyroid in cell culture. J. Cell
WESSELLS, N. K., NUTTALL, R. P., WRENN, J. T., and JOHNSON, S. (1976). Differential labeling of the cell surface of single ciliary ganglion neurons in vitro.
VASILIEV, J. M. and GELFAND, I. M. (1973). Interactions of normal and neoplastic fibroblasts with the substratum. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 311-329. Elsevier, New York.
WESSELIS, N. K., SPOONER, B. S., and LUDUENA, M. A. (1973). Surface movements, microfilaments, and cell locomotion. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 53-76. Elsevier, New York.
SPOONER, B. S. (1970). tion by chick embryo
Physiol. 75.33-48.
Proc. Nat. Acad. Sci. USA 73,4100-4104.
BIOLOGY
66,
50-57
(1978)
Axon Initiation
by Ciliary
Neurons
in Culture
FRANK COLLINS’ Department
of Biological
Sciences, Stanford
University,
Stanford,
Received December 29, 1977; accepted March
California
94305
3, 1978
A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 pm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend fdopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum. INTRODUCTION
cells (Helfand et al., 1976). Under these conditions, a high percentage of nerve cells extend neurites which are many hundreds of microns in length and which have the ultrastructural appearance of axons (Wessells et al., 1976). The present work demonstrates the extraordinary speed and synchrony with which ciliary neurons on a polyomithine substratum initiate axons in response to the addition of heart cell-conditioned medium (HCM). Also, time-lapse microcinematography was used to follow the early events of axon formation in this system. Several major changes in cell behavior were observed, whose implications both for the mechanisms of initiation and the mode of action of HCM are discussed. Axon formation in this system probably represents a process of regeneration, since the ciliary neurons used have formed axons in uiuo (Landmesser and Pilar, 1974) which are cut and retract into the cell body during dissociation. It is not yet known whether the events of regenerative initiation described here are identical to those which occur when a ciliary neuron sends out an axon for the first time.
Although neurite elongation has been studied in detail using nerve cell cultures (Bray, 1973; Bunge, 1973; Luduena and Wessells, 1973), the process of neurite initiation (i.e., the emergence of a neurite from the nerve cell body) has not received much study. This neglect is undoubtedly a result of the difficulty of following initiation in typical nerve cell culture systems. What is required for the optimal study of initiation is a culture system in which a high percentage of neurons, free from contact with any other cell, can be induced to begin neurite formation synchronously in a short period of time. A culture system with these properties is described in the present communication. This culture system is based on the observation that dissociated ciliary neurons from chick embryos can be cultured at low cell densities on a polyornithine-coated substratum in medium previously “conditioned” by the growth of embryonic heart ’ Present address, to which reprint requests should of Anatomy, College of Medicine, University of Utah, Salt Lake City, Utah 84132. be sent: Department
50
0012.1606/78/0651-0050$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
FRANK MATERIALS
COLLINS
Axon Initiation
AND METHODS
Single cell suspensions. Ciliary ganglia and hearts were dissected from 8 to g-day White Leghorn chick embryos, as described previously (Helfand et al., 1976). Ganglia or minced heart pieces were dissociated by incubation at 37°C in Ca’+-Me-free Hank’s salt solution for 20 min (ganglia) or 30 min (hearts), followed by incubation at 37°C in 0.1% trypsin (Difco Bactotrypsin) in Ca2+-Me-free Hank’s for 20 min (ganglia) or 30 min (hearts) and then transfer to culture medium (seebelow) and dissociation into single cells by repeated pipetting. The cell suspension was pelleted by centrifugation and resuspended in the appropriate medium. Culture conditions. Cell cultures were kept at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Media were equilibrated to the temperature and pH of the incubator before use. Unconditioned, control medium was modified Ham’s F-12 (Spooner, 1970) with 10% fetal calf serum. Heart cell-conditioned medium (HCM) was prepared from control medium as follows: Eight hearts were dissociated and the cell suspension (ca. 2 x lo7 cells) was plated in 50 ml of control medium into a 150-cm2 tissue culture flask (Corning). After 16 hr the medium overlying the cell monolayer was discarded and replaced with 50 ml of control medium. The first batch of conditioned medium was collected 96 hr later, and another 50 ml of control medium was added to the monolayer for 96 hr before being collected as the second batch of conditioned medium. Each 50-ml batch of conditioned medium was diluted with 25 ml of control medium and filtered through a 0.2pm pore disposable filter (Nalge). Plastic tissue culture dishes (Falcon) were coated with polyornithine or polylysine as follows: Each 35-mm diam dish was incubated with 2 ml of 1 mg of poly-DLornithine HBr/ml (type I-B, Sigma) or 1 mg of poly-DL-lysine HBr/ml (type VII-B, Sigma) in 0.15 M borate buffer, pH 8.3, for
in Vitro
51
18 to 24 hr; then dishes were washed in sterile, distilled water and stored dry for up to 2 weeks at room temperature before use. Nerve cell structures. Dissociated ciliary ganglia were cultured in 35-mm diam polyornithine or polylysine-coated dishes in 2 ml of medium per dish. There were approximately 2500 neurons and 2500 non-neuronal cells per dish (i.e., one fourth to one half of a dissociated ganglion per dish). At this density most cells are widely separated from one another. Neurons were distinguished from non-neuronal cells by the following criteria: Neurons are about 15 pm in diameter and remain rounded and phaserefractile throughout the period of culture. Non-neuronal cells, before spreading, are only about one third the diameter of neurons and have pebbly surfaces, Most nonneuronal cells flattened out onto the substratum during the period of culture and were not observed to extend net&e-like processes. Assays of axon outgrowth. Axon outgrowth was followed by phase contrast microscopy of living cells. To determine the percentage of initiated neurons, 100 neurons were examined per dish for each time point. Any neuron which possessedone or more neurites at least 15 pm (ca. one nerve cell body diameter) in length was scored as being initiated (e.g., Fig. 1F). The titer of a sample of HCM was determined using serial twofold dilutions of the HCM with control medium, which were placed into separate polyornitbine-coated dishes. Dissociated neurons were added and the percentage of initiated neurons was determined 12 hr later. The endpoint was defined as the greatest dilution which still gave greater than 10% initiated neurons. Titers are expressed as the endpoint dilution. The rate of growth of cone movement was determined on neurons cultured on a polyornithine-coated tissue culture plastic dish by following a given field of growth cones with time-lapse microcinematography for 30 min. The distance covered by
DEVELOPMENTAL BIOLOGY
52
each growth cone was measured on tracings from the projected film and corrected to the true distance by comparison with the projected image of a micrometer scale filmed at the same magnification. Time-lapse microcinematography of axon initiation. A glass coverslip was sealed with paraffin over a hole cut in the bottom of a tissue culture dish, and the modified dish was coated with polyornithine, as described above. Neurons were seeded onto such dishes in control medium and an individual neuron was filmed both before and after the addition of HCM. During filming the pH was maintained by sealing the lids with Vaseline petroleum jelly and the temperature was kept at 37°C with an air curtain incubator (Sage Instrumerits) . Glass coverslips were washed in concentrated nitric acid, rinsed thoroughly in distilled water, and dried. They were then coated with a barely visible layer of carbon in a vacuum evaporator and heated at 300°C overnight. The coverslips were sealed onto dishes and coated with polyornithine as above. Omission of carbon-coating and baking resulted in poor axon outgrowth. RESULTS
Ciliary dium
Neurons
in Unconditioned
Me-
Dissociated ciliary neurons do not extend axons in unconditioned medium, although they survive and are active in other ways for many hours. When dissociated neurons are plated in unconditioned medium onto a polyornithine-coated glass or tissue culture plastic surface, within 15 min virtually all cells are attached sufficiently to resist being dislodged by swirling. Attached cells are widely separated from one another at the plating densities used. The neurons, which are easily distinguished from the non-neuronal cells (see Methods), begin within 2 hours to extend filopodia around their perimeters (Figs. 1A and 1B). As observed by time-lapse microcinematography,
VOIAJME 65, 1978
these filopodia are in constant motion. For example, Figs. IA and 1B were taken 6 set apart, and during that brief time interval many of the filopodia moved. These filopodia move over the substratum and eventually bend back to the cell body, where they disappear; they give no suggestion of attaching to the substratum. Also, darkened areas form at various points around the cell’s periphery which lift up and move back toward the cell body, where they disappear. The arrow in Fig. 1B indicates one such area which has just formed. Although positive identification of these active, darkened areas awaits high-resolution scanning electron microscopy, they closely resemble the membrane ruffles seen on mature growth cones or the leading edge of fibroblastic cells (Bray and Bunge, 1973), and they will be referred to as membrane ruffles for convenience. Neurons in unconditioned medium continue to form active tilopodia and membrane ruffles without extending axons for 12 to 18 hr and then begin to show signs of deterioration (i.e., vacuolization and gradual loss of refractility).
The Response of Neurons to Heart Conditioned Medium (HCM)
Cell-
When the unconditioned medium on a culture of the type described above is replaced by HCM under the appropriate conditions (see below), the following changes in neuronal behavior are observed: By 20 min many of the filopodia are no longer in constant motion but remain in one position for several minutes; for example, Figs. 1C and 1D were taken 4 min apart, and during this interval many of the filopodia did not move. This lack of motion is considered to result from attachment of filopodia to the substratum (Albrecht-Buehler, 1976). These adherent filopodia are typically of greater average length than those observed in unconditioned medium (compare Fig. 1A with 1C). Occasionally such filopodia are observed to flatten out thinly onto the substratum (not shown). By 30 min after the addition of HCM, the formation of filopodia
Frc ;. 1. A cihary neuron before and after the addition of HCM. Ciliary ganglia were dissociated and medium onto a polyornithine-coated glass coverslip dish for time-lapse microcinematog in uric ,onditioned medium was replaced with HCM and filming was resumed. These photo After 4 hr the unconditioned were tmlarged from the 16-mm film. A and B were taken 6 set apart at 3 hr, 52 min after the neuron was in uric :onditioned medium. C and D were taken 4 min apart at 21 and 25 min after the addition of HCM. of HCM. Filopodia are the finger-like projl taken at 32 min, and F was taken at 56 min after the addition which I measure 0.1 to 0.2 pm in diameter and 5 to 15 pm in length. x1000; bar = 10 pm. 53
plated mdv. graphs plated E was Ections
54
DEVELOPMENTALBIOLOGY
and membrane ruffles begins to be restricted to areas of the periphery where growth cones are forming. As these growth cones (arrows in Fig. 1E) move away from the cell body, the remainder of the cellular perimeter stops exhibiting filopodial movements and membrane ruffles, so that the once active surface of the rounded cell body (the perikaryon) becomes quiescent. By 45 min approximately 50% and by 90 min greater than 80% of all neurons in such cultures bear one or more axons which are at least one nerve cell body diameter (ca. 15 pm) in length; such neurons (e.g., Fig. 1F) are referred to as initiated in quantitative assays of axon outgrowth (Figs. 2-4). Growth cones remain motile for at least 24 hr, the time when cultures were terminated, and axon lengths of many hundreds of microns are achieved. Variables Which of Initiation
Affect
the Time- Course
The rapid initiation of axons in response to HCM is only achieved after a period of cell “recovery” following dissociation of the ganglion. When neurons are plated in HCM immediately after dissociation, there is a lag time of about 2 hr before axons begin to appear (Fig. 2, solid line). The much more rapid response to HCM described above is achieved by culturing neurons in unconditioned medium for at least 3 hr after dissociation before adding HCM (Fig. 2, dashed line). This result suggests either that some time must elapse for cell repair after dissociation, or that some time is required for neurons to prepare to reextend axons, or both. Neurons undergo a similar “recovery” in plastic petri or plastic tissue culture dishes, to which they do not attach. Neurons incubated in such dishes (in unconditioned medium or HCM) for several hours after dissociation may then be removed, resuspended in HCM, and plated onto a polyornithine-coated tissue culture plastic dish. Such neurons initiate axons much more rapidly than do neurons plated under these conditions immediately after
VOLUME 65,1978
dissociation. Therefore, “recovery” can occur in unconditioned medium or HCM and does not require attachment to a substratum. Only certain batches of HCM promote the most rapid initiation, the speed of initiation being directly related to the HCM titer (Fig. 3). Typically, the second batch of HCM produced by a monolayer of heart cells is much more effective than the first (Fig. 3). Such differences in the time course of initiation are apparently due to differences in the concentration of “active factors” between various batches of HCM. A similar effect can be produced by diluting a high-titer batch of HCM with increasing amounts of unconditioned medium, which causes an increasing delay in the onset of initiation (Fig. 4). The data of Fig. 4 show a clear effect of HCM concentration on the time of onset of initiation. These data require additional comment. It is clear from the preceding results that HCM is required for the formation of growth cones by the nerve cell body. It is reasonable, therefore, that the delayed initiation in dilute or low titer HCM is due to the delayed formation of growth cones. The assay for an “initiated” SO
r
‘0
1
JI
,
2
,
3
,
4
,
5
; 6
TIME
AFTER
PLATING
,
7
, 8
(hr)
FIG. 2. A comparison between the kinetics of initiation in freshly dissociated vs “recovered” neurons. Freshly dissociated ciliary ganglia were plated at 0 hr onto polyornithine-coated tissue culture plastic dishes, either in HCM (solid line) or in unconditioned medium, which was replaced with HCM at 6 hr (dashed line).
FRANK
TIME AFTER
ADDITION
OF HCM
COLLINS
Axon Initiation
(hr)
FIG. 3. The time course of axon initiation in different batches of HCM. Dissociated ciliary ganglia were plated onto polyornithine-coated tissue culture plastic dishes in unconditioned medium. After 4 hr (0 hr on the abscissa) unconditioned medium was replaced by aliquots of different batches of HCM (lines A-D) or by fresh unconditioned medium (line E). C and A are the first and second batches of HCM produced by one heart cell monolayer. D and B are the first and second batches from another monolayer. Titers: A = 1:8; B = 1:8; C = 1:2; and D = 1:l. Each point represents the average and range for duplicate cultures.
neuron, however, requires the growth cone to move at least 15 pm away from the cell body. Therefore, it is possible that some of the delay occurring in the events shown in Figs. 3 and 4 is because of a reduction in the rate of growth cone movement in dilute or low-titer HCM. To test this possibility, direct measurement of the rate of growth cone movement in two different dilutions of HCM has been made. Although there seemsto be a tendency for growth cones to move faster in more concentrated HCM (61 +- 15 pm/hr vs 39 + 16 pm/hr for a twofold change in the HCM concentration), the results so far observed are not highly statistically significant because of the great variability in the rate of growth cone movement within a single culture. DISCUSSION
These results demonstrate the potential usefulness of the HCM-polyornithine nerve cell culture system for studying the events of axon formation. Conditions are described
in Vitro
55
in which a population of ciliary neurons, each of which is attached to the substratum and free of contact with other cells, can be induced to begin axon formation within 20 min by adding HCM. Within 90 min, greater than 80% of the neurons bear one or more axons which are at least 15 pm in length. Under these conditions each neuron initiates an average of 3 to 4 axon-like processes.In the embryo, ciliary neurons apparently have only one axon (Landmesser and Pilar, 1974). Thus, the extracellular environment can modify the number of axons possessedby ciliary neurons. To my knowledge, this work is the first description of changes in cell surface behavior during the initiation of neurite outgrowth. In this system, initiation consists of an apparent redistribution of the capacity of parts of the neuronal surface to generate movement (i.e., filopodial extension and membrane ruffling). Initially the capacity to generate such movements is distributed randomly and rather uniformly around the cell periphery. Initiation leads to the for-
RELATIVE
CONCENTRATION
OF HCM
FIG. 4. The effect of diluting HCM with unconditioned medium on the time course of initiation. Dissociated ciliary ganglia were plated onto polyornithine-coated tissue culture plastic dishes in unconditioned medium. After 4 hr the unconditioned medium was replaced either with undiluted HCM (relative concentration = 1) or with serial twofold dilutions of HCM with unconditioned medium (relative concentrations of 0.5 to 0.125). The time course of initiation was followed at hourly intervals. The time to reach the half-maximal percentage of initiated neurons (41 2 6%) is plotted against the relative concentration of HCM.
56
DEVELOPMENTAL
BIOLOGY
mation of localized growth cones, in which the surface movements are more concentrated spatially and perhaps more coordinated, and to the cessation of surface movements elsewhere. It is possible that these changes occur either (1) because an agent essential for surface movements becomes localized in the growth cones or (2) because surface movements in regions other than the growth cones are actively suppressed. The cessation of activity at the surface of the perikaryon may be reversible; however, under present conditions, no new axons emerge from the perikaryon once surface movements cease (30-40 min after the addition of HCM). The start of locomotion in fibroblastic cells is also accompanied by the restriction of an originally uniform surface activity to a broad leading edge (Vasiliev and Gelfand, 1973). The mechanism of localization of surface movements is unknown, although changes in the cell cortex seem to be important. Actively moving regions of the cell surface in fibroblasts (and neurons) are typically associated with a latticework of microfilaments in the underlying cell cortex (Wessells et aZ., 1973). At the quiescent surfaces of locomotory fibroblasts this latticework is replaced by microfilament bundles (Wessells et al., 1973). There is also evidence, from the effect of colcemid on fibroblast locomotion, that microtubules are important for maintaining the leading edge (Gail and Boone, 1971). Since microfilament bundles and microtubule arrays near the cell surface have not been observed in nerve cell bodies (Luduena and Wessells, 1973), it will be of interest to determine what changes in the cell cortex and cytoskeleton accompany the restriction of surface motility to growth cones. How does HCM induce axon initiation? The following observations would seem to be especially relevant to this question: (1) Axon initiation occurs within 20 to 30 min of the addition of HCM and would perhaps occur even sooner in more concentrated preparations of HCM than are presently
VOLUME
65,1978
available (Fig. 4); (2) The first visible response of neurons to HCM is the replacement of nonadherent filopodia with longer, apparently adherent, filopodia which occasionally flatten onto the substratum; and (3) I have recently observed that the action of HCM in the present culture system seems to depend on its deposition of material onto the polyornithine-coated culture substratum (Collins, manuscript in preparation; this manuscript has been submitted to Proc. Natl. Acad. Sci. USA). Together these results have suggested the working hypothesis that HCM deposits material onto the polyornithine substratum, which provides appropriate sites for fdopodiai adhesion, and (possibly in combination with other effects) leads to the process of axon outgrowth. I would like to thank Dr. Norman Wessells and Randal Johnston for their comments on the manuscript and Belen Palmer for her excellent technical assistance. This work was supported by a National Institutes of Health postdoctoral fellowship to the author and by NIH Grant No. HD 04708 to Dr. Wessells. REFERENCES ALBRECHT-BUEHLER, G. (1976). Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function? J. Cell Biol. 69,275-286. BRAY, D. (1973). Branching patterns of individual sympathetic neurons in culture. J. Cell Biol. 56, 702-712. BRAY, D., and BUNGE, M. B. (1973). The growth cone in neurite extension. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 195-209. Elsevier, New York. BUNGE, M. B. (1973). Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56, 713-735. GAIL, M. H., and BOONE, 6. W. (1971). Effect of colcemid on fibroblast motility. Exp. Cell Res. 65, 221-227. HELFAND, S. L., SMITH, G. A., and WESSELLS, N. K. (1976). Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia. Develop. Biol. 50, 541-547. LANDMESSER, L., and PILAR, G. (1974). Synapse formation during embryogenesis of ganglion cells lacking a periphery. J. Physiol. 241, 715-736. LUDUENA, M. A., and WESSELLS, N. K. (1973). Cell locomotion, nerve elongation, and microfilaments.
FRANK
COLLINS
Develop. Biol. 30, 427-440.
Axon Initiation
in Vitro
57
The expression of differentiathyroid in cell culture. J. Cell
WESSELLS, N. K., NUTTALL, R. P., WRENN, J. T., and JOHNSON, S. (1976). Differential labeling of the cell surface of single ciliary ganglion neurons in vitro.
VASILIEV, J. M. and GELFAND, I. M. (1973). Interactions of normal and neoplastic fibroblasts with the substratum. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 311-329. Elsevier, New York.
WESSELIS, N. K., SPOONER, B. S., and LUDUENA, M. A. (1973). Surface movements, microfilaments, and cell locomotion. In “Locomotion of Tissue Cells” Ciba Foundation Symposium 14, pp. 53-76. Elsevier, New York.
SPOONER, B. S. (1970). tion by chick embryo
Physiol. 75.33-48.
Proc. Nat. Acad. Sci. USA 73,4100-4104.
