Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3537-3541, July 1979 Neurobiology

Isolation and culture of motoneurons from embryonic chicken spinal cords (ventral spinal neurons/cell separation/development in vitro/formation of neuromuscular synapses)

SADAHIKO MASUKO*, HIROSHI KUROMIt, AND YUTAKA SHIMADA* *Department of Anatomy and tBrain Research Institute, School of Medicine, Chiba University, Chiba 280, Japan

Communicated by Aron A. Moscona, April 16, 1979

ABSTRACT A method is described for isolating cholinergic a motoneurons from the spinal cord of chicken embryos at stage 17-18 (Hamburger and Hamilton numbering), at the time when it has been shown that motoneurons withdraw from the mitotic cycle but neurons of other types and glia are still mitotic. Fragments of the ventral half of the spinal cord are incubated for 24 hr in the presence of 10 ,uM 1-fl-D-arabinofuranosylcytosine in order to eliminate dividing cells and are subsequently dissociated into a suspension of single cells. The following evidence has been obtained and suggests that these cells are neuronal and appear to be a motoneurons: (i) they are resistant to the lethal effect of arabinofuranosylcytosine, and thus are postmitotic at stage 17-18; (ii) when grown in vitro, they exhibit morphological characteristics similar to those of ventral spinal neurons, which include the ability to be stained with silver, Nissl, methylene blue vital stain at pH 6.5-7.0, and choline acetyltransferase histochemistry; (iii) they have high choline acetyltransferase activity; (iv) they are capable of forming functional synapses with muscle.

Embryonic chicken spinal cords have been a favored source of neural tissue for culture. Recently, particular interest in them has been stimulated by the evidence that neurons dissociated from this tissue will form synapses with skeletal muscle cells in vitro (1-4). Although this culture system possesses the advantages of accessibility for analysis by microelectrodes, scanning electron microscopy, and other methods related to cell surface studies, it is complicated in that the dissociated spinal cord contains, in addition to a motoneurons, a variety of nerve cell types that do not usually make synaptic contacts with muscle in vivo; such cells may make nonspecific contacts with muscle in vitro and thus interfere with analysis of the properties exhibited by motoneurons in associations with muscle. Further, this culture, over a longer period of time, is overwhelmed by proliferating fibroblastic cells, glial cells, or both. In the present study, an attempt was made to isolate and culture a homogeneous population of a motoneurons from embryonic spinal cords. The procedure is based on the earlier observation that many of the cells destined for the ventral horn withdraw from the mitotic cycle in the developing chicken embryo by stage 17-18 of Hamburger and Hamilton (5), whereas other types of neurons as well as glia display an upward trend of mitosis at this state (6-8). Taking advantages of this difference in the period of mitosis, we were able to prepare the cell suspensions enriched in the former cells. By incubating fragments of ventral spinal cords (VSCs) in a medium containing an inhibitor of DNA synthesis (1-3-D-arabinofuranosylcytosine, Ara-C), dividing cells (non-motoneurons and glia) are eliminated. They are subsequently dispersed into a suspension of single cells for culture.

MATERIALS AND METHODS Preparation of Cultures. Spinal cords from chicken embryos at 60-66 hr of incubation [stage 17-18 (5)] were used. By removal of the lateral body folds,visceral tissues and notochord, the ventral surface of the spinal cord was exposed; the connective tissues destined to form the vertebrae and somites are loosely attached to the spinal cord (Fig. la). After a layer of these overlying tissues had been peeled off, the spinal cords could be obtained (Fig. lb). With a razor blade, each side of the cords was cut longitudinally along the mediolateral line into ventral and dorsal halves (Fig. ic). The VSCs, which include the ventral horn, were then cut into small pieces (approximately 0.5 mm3). Fragments from five embryos were suspended in a 3-ml culture medium containing 10 ,iM Ara-C in 25-ml erlenmeyer flasks. The flasks were placed on a gyratory shaker rotating at 70 rpm at 37°C for 24 hr. After this treatment, the fragments were dissociated with trypsin and dispersed into single cells according to our standard procedure (1). The cells obtained were plated at a concentration of 3 X 105 cells in 1 ml of culture medium within collagen-coated 35-mm plastic dishes. In some experiments, nerve and muscle cell cocultures were prepared. Suspensions of embryonic skeletal muscle cells were obtained from breast muscles of 12-day-old chicken embryos by the standard trypsinization procedure. Cell suspensions enriched in myogenic cells, prepared by the differential adhesion procedure (9), were used. They were again plated at a concentration of 3 X 105 cells in 1 ml of culture medium within collagen-coated 35-mm dishes. After muscle cells had been grown for 2-3 days, 2 X 104 nerve cells prepared by the procedure described above were added to each muscle culture dish. The culture medium consisted of Eagle's minimal essential medium with glutamine, 15% horse serum, 5% embryo extract, and penicillin/streptomycin in concentrations of 50 units/ml and 50 ,g/ml, respectively. All cultures were kept at 370C in an atmosphere of 5% CO2 in air at a saturation level of humidity. The culture medium was changed every 2-3 days. Autoradiography. VSC fragments from 60 to 66-hr chicken embryos were cultured in the medium containing 0.5 ,Ci (1 Ci = 3.7 X 1010 becquerels) of [3H]thymidine (The Radiochemical Centre, Amersham, England) for 24 hr with or without 10 lM Ara-C. They were then fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4. After dehydration, they were embedded in glycol methacrylate (10) and cut at 2 ,tm. The serial sections mounted on glass slides were dipped in Sakura NR-M2 emulsion and exposed for 2-4 weeks. The sections were then developed and stained with toluidine blue.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: Ara-C, 1-3-D-arabinofuranosylcytosine; CAT, choline acetyltransferase; VSC, ventral spinal cord; WSC, whole spinal cord. 3537

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Table 1. Effect of Ara-C on cell number* in the VSC of stage 17 chicken embryos Cells Samples counted per embryo 6 112,000 + 13,000 Before Ara-C treatment After culture with 10 pM Ara-C 44 37,000 + 7,000 for 24 hr After culture without Ara-C 14 347,000 ± 80,000 for 24 hr * Cell number (mean ± SD) was determined after dissociation of the tissues with trypsin.

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FIG. 1. Isolation of a spinal cord from a stage 17 chicken embryo

and removal of its dorsal half. (a) The lateral body folds, visceral tissues and notochord have been removed. The ventral surface of the

spinal cord (SC) is seen between somites (SM). (b) The entire spinal cord has been stripped and is opened along the roof plate. (c) The spinal cord has been hemisected into the ventral (V) and dorsal (D) halves on one side. (a and b X12, bars 1 mm; c X30, bar 0.5 mm.)

labeled except those with picnotic nuclei, and a large number of degenerating cells without any label were found (Fig. 2a). In the absence of Ara-C, labeled as well as unlabeled cells were found (Fig. 2b). The unlabeled cells appeared to be those that had withdrawn from the mitotic cycle before the culture. The effect of 100 ,uM Ara-C was indistinguishable from that of Ara-C at 10 MtM. The decrease in cell number with the of 1 MM Ara-C was never as great as after treatment treatment @ with 10 MM; autoradiographic examination of the VSCs treated at this lower concentration occasionally revealed labeled and seemingly viable cells. Development in Culture and Histology. Of the cells dissociated from the Ara-C-treated VSC fragments, 85-90% routinely excluded trypan blue, a vital dye for viability tests. In the '

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dh the cells ettl rapdl from esuspension, ed culture attached to the substrate, and extended short processes within

the first 24 hr of culture (Fig. Sa). These processes increased in length and number, and also began to branch out during the subsequent days (Fig. 3b). By 5-7 days they had assumed a

Histology. Cell cultures were observed daily with an inverted phase contrast microscope. After 7 days of culture, some cul-

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tures were fixed in 10% formalin in 0.1 M cacodylate buffer at pH 7.4 and impregnated with silver (11), or stained with thio-

nine to demonstrate Nissl substance. Other cultures were vitally stained with methylene blue at pH 6.5-7.0 to demonstrate the cholinergic nerves (12). Still other cultures were subjected to histochemical staining for choline acetyltransferase (CAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) (13). Choline Acetyltransferase. CAT activity was assayed radiochemically by measuring the synthesis of [1-'4C]acetylcholine from [1-14C]acetyl-CoA (The Radiochemical Centre, Amersham, England). Cultures were scraped and homogenized in 0.2 ml of a solution containing 50 mM NaPO4 at pH 7.4, 0.2 M NaCl, 0.5% Triton X-100, and bovine serum albumin at 5 mg/ml. Duplicate assays were performed according to the method of Fonnum (14).

Electrophysiology. Electrophysiological experiments were

performed on the stage of an inverted microscope. Membrane potentials of individual muscle fibers were measured with intracellular glass microelectrodes filled with 3 M KCl (resistance 10-30 MU). Focal extracellular stimulation was achieved through a microelectrode filled with 2 M NaCl.

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Effect of Ara-C Treatment. The total cell numbers in the VSC fragments before Ara-C treatment, in those after culture for 24 hr with 10MgM Ara-C, and in those cultured for 24 hr in the absence of Ara-C were determined (Table 1). With the Ara-C treatment, the number of cells in the VSC decreased by increased the number number increased the treatment treatment the third, whereas whereas without without the one third, 3-fold over the 24-hr culture period. Autoradiographic examination revealed that in the presence of Ara-C no cells were

medium conof VSCs cultured in(a)the FIG. 2. Autoradiographs and absence (b) hr in the presence taining VSC24has no labeled viable cells, but has many of Ara-C.[3H]thymidine Ara-C-treated for degenerating cells (arrows). Nontreated VSC has unlabeled (motor) and labeled (nonmotor) cells. (X700, bars 10 Aim.)

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Proc. Natl. Acad. Sci. USA 76 (1979)

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FIG. 3. Phase contrast micrographs of dissociated cells from Ara-C-treated VSC after 1 (a), 3 (b), and 6 days (c) in culture. (a x200; b and

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large multipolar shape with cable-like extensions, and resembled morphologically the ventral spinal neurons (Fig. Sc). These cells with processes could be stained with silver (Fig. 4a), and their cell bodies could be stained with Nissl stain (Fig. 4b). Further, 78.2% of the cells (n = 220) were vitally stained with methylene blue at pH 6.5-7.0 (Fig. 4c), and 88.8% of the cells (n = 214) were stained with a histochemical demonstration for CAT (Fig. 4d). Thus, these cells are neuronal and most of them appear to be cholinergic. In the early phases of culture, fibroblast-like cells were completely absent or at least very few in number, but in long-term cultures some nonneuronal cells generally aggregate or adhere to large multipolar cells. CAT Activity. Dissociated cells from Ara-C-treated VSCs, nontreated VSCs, and nontreated whole spinal cords (WSCs) from stage 17 embryos were inoculated at the same concentration (3 X 105 cells per plate) and, after 1 week, the CAT activities were compared. These CAT activities were assayed because they are a measure of acetylcholine synthesis in spinal cord and muscle cultures (15). The specific activity of CAT was not measured because in the latter two cultures most of the protein is located in the proliferating fibroblastic and/or glia cells. Thus, the CAT activity of each culture was used as an index. The CAT activity in cultures of Ara-C-treated VSCs was from 1.5 to 4 times higher than in nontreated VSCs, and from 4 to 5 times higher than in nontreated WSCs (Fig. 5). Because neurons in these three cultures extended axonal or dendritic processes to almost the same degree, the differences in the CAT activities do not appear to be due to any difference in neuronal cell maturation. This result indicates that the cell cultures of . N-

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VSCs treated with Ara-C must have been substantially enriched in cholinergic neurons. In parallel with the cultures mentioned above, WSCs from 4-day-old embryos (stage 24) without previous treatment with Ara-C were dissociated according to the procedure of Berg and Fischbach (15) and cultured at the same number of cells per plate as described above. The CAT activity of this culture after 1 week was about one-fourth of that of the cells from Ara-Ctreated VSCs (Fig. 5). Nerve-Muscle Synapses. Neuromuscular junction formation was examined in myotubes cocultured for 4-5 days with separated spinal neurons from Ara-C-treated VSCs. Spontaneously depolarizing changes in potentials were detected from the myotubes that were contacted by the processes of nearby neurons (Fig. 6a). These changes were small (range 0.2-3.0 mV) and occurred in a random manner at a low rate (1-30/min)

(Fig. 6b). Furthermore, stimulation of-nerve processes through an extracellular electrode could repeatedly evoke postsynaptic potentials in a nearby myotube contacted by the neuron. Both the spontaneous and evoked potentials were completely inhibited by addition of d-tubocurarine, but were reversed after washout of the drug (Fig.6c and d). Thus, these spontaneously depolarizing potential changes are similar to the miniature end-plate potentials and the evoked postsynaptic potentials are equally similar to the end-plate potentials in muscle cells cultured with spinal neurons, as reported in previous investigations (3, 16, 17). These results indicate that the nerve cells prepared by our present procedure possess the ability to form functional cholinergic synapses with muscle cells.

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FIG. 4. Cells, released from the Ara-C-treated VSC, cultured for 7 days. Stained with silver (a), Nissl (b), methylene blue at pH 6.5-7.0 (c), and histochemical demonstration for CAT (d). (X600, bars 10 ,m.)

Neurobiology: Masuko et al.

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2 3 4 Experiment FIG. 5. CAT activity in cell cultures of Ara-C-treated VSC (empty bars), nontreated VSC (hatched bars), and nontreated WSC (solid bars) from stage 17-18 embryos, and in those of WSC from stage 24 embryos (stippled bars), assayed ,1 week after plating. Each experiment represents a separate dissociation and culture. Each column represents the mean of duplicate assays. Vertical lines indicate ranges.

DISCUSSION It has been reported that the generation time of the neuroepithelial cells in 32-hr chicken embryos is about 8 hr (18) and that in 6-day embryos about 16.hir (19). Thus, the incubation time used in the present work (24 hr) for 60- to 66-hr spinal cord

fragments with Ara-C may be sufficient for the proliferating cells in them to go through their cell cycle and reach the DNA synthetic period. The virtual absence of labeled viable cells in the fragments of Aia-C-treated VSCs according to [3H]thymidine autoradiography indicates that the proliferating cells were either "killed'; or prevented,.from entering the DNA synthetic period. A great reduction in cell number and the presence of many degenerating cells in Ara-C-treated VSC fragments suggests that the cells, which were still capable of division at the time of the cord extirpation, were likely to have been "killed." As for long-term cultures, a few nonneuronal cells were always found. It is known that some nonneuronal cells in vitro have a generation titme longer than 24 hr (20). Thus, although the mitotic neuronal precursors as well as most of the mitotic nonneuronal cells have been practically eliminated by the Ara-C treatment, it is possible that a small number of some kind(s) of nonneuronal cells escaped the present procedure. In the embryonic spinal cord, many somatic motoneurons undergo their final mitosis by stage 17 (7, 8). Our previous autoradiographic analysis of embryonic spinal cords that had beeh treated with [3H]thymidine at stage 17-18 showed that about 70% of the postmitotic cells are concentrated in the motor column of the VSC (21). In the VSC, interneurons and ventral commissure cells have been "born" by this stage, but there are significantly fewer of these cells than motoneurons (7, 8). Visceral preganglionic neurons have a common origin with somatic motoneurons and both are said to be produced at about the same time (22), but our previous study of embryonic cords that had been treated with [3H]thymidine at stage 17-18 revealed that these preganglionic neurons are labeled and, thus, are still mitotic at this stage (21). In the dorsal portion of the spinal cord, postmitotic neurons are present at stage 17 (7, 8). These neurons account for about 20% of the cells unlabeled with [3H]thymidine in the WSC (21). Because the dorsal half of the WSC was effectively removed in the present experiment, more than 85% of the postmitotic cells in VSCs can be regarded as neuroblasts destined to become motoneurons. a

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FIG. 6. (a) Interference contrast micrograph of isolated neuron and muscle cocultures. Neuronal processes are seen in contact with the myotube (M). (X400, bar 50 ,gm.) (b) Spontaneously depolarizing potential changes were recorded from the myotube in a with an intracellular microelectrode. Arrow indicates the recording point. (c and d) Effect of d-tubocurarine (50 ,ug/ml) on miniature end-plate potentials (c) and (0.10,ig/ml) on end-plate potentials (d), recorded in two different myotubes. A, Control; B, 30 min after addition of the drug; C, 30 min after removal of the drug. (Bars 1 mV and 0.2 sec for b and c; 5 mV and 10 msec for d.)

There can be little doubt that the cells dissociated from Ara-C-treated VSCs can be classified as neuronal elements. The growth in vitro of new processes having the morphological characteristics of axons or dendrites, the general appearance of those cells compared with known neuronal tissue, and the ability to react with silver and Nissl stains, all meet the gross morphology criteria for a nerve cell. Further, 80H-90% of the cells have been demonstrated to possess the ability to be stained vitally with methylene blue at the pH value at which only cholinergic neurons are stained, and the cells are stainable with a histochemical demonstration for CAT. Although these two histological methods are not completely specific for cholinergic neurons (12, 13), the biochemical result that these cultures exhibit high CAT activities gives quantitative evidence of a great deal of enrichment of cholinergic neurons. The observation that these cells possess the ability to form functional synapses with muscle supports the notion that they include a functionally distinct-e.g., cholinergic-class of neurons. Thus, although the actual definition of motoneurons is hard to specify with precision, especially when cells are no longer seen in their normal surroundings, these results and the fact that the cultured cells are survivors in Ara-C-treated VSCs that had been removed from stage 17-18 chicken embryos suggest strongly that a relatively pure population of motoneurons has been separated by our procedure. Recently Berg and Fischbach (15) attempted to enrich spinal cord cultures with motoneurons by two methods. In the first method, large cells were separated from dissociated 7-day embryonic chicken WSCs by velocity sedimentation, assuming a

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Masuko et al.

that cholinergic motoneurons are the largest cells present at this stage. It has been reported, however, that large cells in chicken embryo spinal cords are not necessarily only motoneurons; early "born" non-motoneurons are quite as large as motoneurons (23). Their second method was to simply culture dissociated 4-day WSCs. This approach was based upon the observation, as in the present study, that motoneurons are among the first cells to withdraw from the mitotic cycle in the developing embryo (6, 22). But by day 4, in addition to the motoneurons, a large number of non-motoneurons will be produced as well (6, 7). Thus, the possibility of the presence of non-motoneurons in these two cultures cannot be ruled out. In fact, our result showed that the CAT activity level of cell cultures of 4-day WSCs is not as high as that of Ara-C-treated VSCs. In conclusion, Ara-C treatment of VSC fragments from stage 17-18 chicken embryos and their subsequent dissociation provide a useful step in attempts to isolate cholinergic a motoneurons that are capable of forming functional synapses with skeletal muscle. This procedure should assist in facilitating studies of the development of a motoneurons in isolation or in various cocultured combinations. It is especially anticipated that the coculture of these neurons with skeletal muscle cells will be useful in addressing such important neurobiologic questions as the analysis of neuromuscular junction formation and the neurotrophic effects on muscle growth and metabolism. We thank Drs. Y. Hagihara and M. Kano for their kind advice and assistance. This work was supported by grants from the Muscular Dystrophy Association, the Japanese Ministry of Education, the Japanese Ministry of Health and Welfare, and the Yamada Science

Foundation.

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1. Shimada, Y., Fischman, D. A. & Moscona, A. A. (1969) Proc. Natl. Acad. Sci. USA 62,715-721. 2. Fischbach, G. D. (1970) Science 169, 1331-1333. 3. Fischbach, G. D. (1972) Dev. Biol. 28, 407-429. 4. Shimada, Y. & Fischman, D. A. (1973) Dev. Biol. 31, 200-225. 5. Hamburger, V. & Hamilton, H. (1951) J. Morphol. 88, 49-92. 6. Hamburger, V. (1948) J. Comp. Neurol. 88, 221-283. 7. Kanemitsu, A. (1972) Proc. Jpn. Acad. 48, 758-763. 8. Hollyday, M. & Hamburger, V. (1977) Brain Res. 132, 197208. 9. Yaffe, D. (1968) Proc. Natl. Acad. Sci. USA 61, 477-483. 10. Bennett, H. S., Wyrick, A. D., Lee, S. W. & McNeil, J. H. (1976) Stain Technol. 51, 71-97. 11. Sevier, A. C. & Munger, B. L. (1965) J. Neuropathol. Exp. Neurol. 24, 130-135. 12. Richardson, K. C. (1969) Anat. Rec. 164,359-378. 13. Burt, A. M. & Silver, A. (1973) Brain Res. 62, 509-516. 14. Fonnum, F. (1975) J. Neurochem. 24, 407-409. 15. Berg, D. K. & Fischbach, G. D. (1978) J. Cell Biol. 77,83-98. 16. Kano, M. & Shimada, Y. (1971) J. Cell. Physiol. 78,233-242. 17. Obata, K. (1977) Brain Res. 119, 141-153. 18. Langman, J., Guerrant, R. L. & Freeman, B. G. (1966) J. Comp. Neurol. 127,399-412. 19. Fujita, S. (1962) Exp. Cell Res. 28,52-60. 20. Karon, M. & Shirakawa, S. (1969) Cancer Res. 29, 687-696. 21. Masuko, S. & Shimada, Y. (1979) in Integrative Control Functions of the Brain, eds. Ito, K., Tsukahara, N., Kubota, K. & Yagi, K. (Kodansha Scientific, Tokyo/Elsevier, Amsterdam), Vol. 2, in press. 22. Levi-Montalcini, R. (1950) J. Morphol. 86,253-283. 23. Kanemitsu, A. (1971) Proc. Jpn. Acad. 47,432-437.

Isolation and culture of motoneurons from embryonic chicken spinal cords.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3537-3541, July 1979 Neurobiology Isolation and culture of motoneurons from embryonic chicken spinal c...
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