Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1858-1861, August 1971

Biochemical Differentiation in Reaggregating Brain Cell Culture (morphology/ assays/mouse embryo) NICHOLAS W. SEEDS Department of Biophysics and Genetics and Department of Psychiatry, University of Colorado Medical Center, Denver, Colo. 80220 Communicated by Theodore T. Puck, June 11, 1971

ABSTRACT Dissociated cells from embryonic mouse Cell culture brain reassociate in rotation culture to form aggregates. During cell culture the specific activities of acetyl- 25-ml Erlenmeyer flasks containing 3.5 ml of basal Eagle's (EC 2.3.1.6), (EC 3.1.1.7), medium with 0.4% glucose and 10% fetal calf serum were and (EC 4.1.1.15) in the aggre- inoculated with 1-2 X 107 cells. The flasks were gassed with gates increase up to twenty-fold, a phenomenon that at approximates some of the biochemical events in the de- C02-air (5:95) and incubated 370C with constant rotation velopment of the mouse brain. (70 rpm). After 2 days, the cultures were transferred to 50- ml flasks and 5 ml of fresh medium containing 15% fetal calf Elucidation of factors regulating the biochemical and electro- serum was added. Thereafter, every 2 days 5-6 ml of medium physiological development of the central nervous system has was removed and replaced with fresh basal Eagle's medium been primarily limited to in vivo studies; thus, the role of containing 0.4% glucose and 15% fetal calf serum. cell-cell interactions in development has been difficult to de- Clonal cell lines were kept in logarithmic growth on 150-mm termine. The few in vitro studies of nerve cell development have Falcon plastic dishes. Neuroblastoma N-18 was cultured in dealt with tissue explants or monolayers of dispersed cells. Al- Dulbecco's modification of Eagle's medium with 10% fetal though differentiated cells may be maintained as explants or calf serum. Glial cell C-6 was grown in basal Eagle's medium monolayers, cells rarely acquire the characteristics of a dif- with 0.4% glucose, 10% fetal calf serum, and a 4 X concen- ferentiated state under these conditions (1). trate of essential amino acids. Chinese hamster ovary K-1 The technique of reaggregation allows dissociated cells to was maintained in F-12 medium with 10% fetal calf serum. reestablish specific cell-cell contacts, thus producing histo- typic patterns characteristic of the original tissue (2). Ag- Enzyme assays gregate structures that are identical to the in vivo hippocampal architecture have been demonstated by DeLong (3) and Enzymatic assays were always performed on the day after Sidman (4). Therefore, reaggregating cell culture may be the the culture medium was changed. Aggregates were allowed to most favorable for promoting differentiation. settle out and washed twice in 8 ml of Saline 1 with 0.4% In this report, brain cell aggregate cultures that show glucose. Washed aggregates were homogenized in 0.05 M patterns of biochemical differentiation similar to the de- KHP04 (pH 6.8)-0.5% Triton X-100-1 mM EDTA (K+). An veloping mouse brain are described. aliquot of the cell extract that contained Triton was adjusted to 0.5 mM and 2 mM 2- METHODS mercaptoethanol and used for assay of glutamate decarbox- Cell dissociation ylase activity. The aggregates for the phosphogluconate C57B1/6J mice were used in all studies. Pregnant females were pathway assay were washed with 0.24 M sucrose-0.02 M either purchased from the Jackson Laboratory or bred in Tris - HCl(pH 7.3) and homogenized in 0.05 M Tris - HCl- this laboratory. Mice 16-18 days pregnant were the source of 1 mM EDTA. fetuses for cell culture. The whole brains were removed and Choline acetyltransferase was assayed according to the finely diced. The tissue was dissociated in 0.25% trypsin procedure of Schrier and Shuster (5). The 50-jsl reaction mix- (Difco 1:250) in Saline 1 (0.138 M NaCl, 54 mM KC1, 1.1 mM ture had 12.5 mM choline I-, 0.5 mM neostigmine MeSO4, Na2HPO4, 1.1 mM KH2PO4, 0.4% glucose, and 0.01% CaCl2), 1.1 mM acetylCoA, 0.2 M NaCl, and 2.2 X 105 cpm of [1-14C] and incubated at 370C with constant rotation for 20 min. acetylCoA (48.6 Ci/mol). Acetylcholinesterase was assayed The tissue was triturated several times and incubation was by the procedure of Blume et al. (6). The 50-,A reaction mix- continued for an additional 15 min. The suspension was ture had 0.2 M NaCl, 2.5 mM I-, and 105 cpm triturated several times and passed through a nylon screen to [1-'4C]acetylcholine I- (2.4 Ci/mol). Duplicate reaction complete its dissociation into single cells. After the addition of mixtures with and without B.W. 284-C51 [1,5-bis-(4 allyl- 10% fetal calf serum, the cells were collected by centrifuga- dimethylammoniumphenyl) pentan-3-one dibromide], a tion. specific inhibitor of acetylcholinesterase (7), were used to distinguish acetylcholinesterase from . The glutamate decarboxylase assay of Roberts and Simonsen (8) was used. The 50-al reaction mixtures contained Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase 2.5 mM iglutamate (K+), 2.4 X 105 cpm [1-14C]iglutamate (EC 3.1.1.7); ChAT, choline acetyltransferase (EC 2.3.1.6); (20 Ci/mol), 0.5 mM pyridoxal phosphate, and 2 mM 2- Gluase, glutamate decarboxylase (EC 4.1.1.15). mercaptoethanol. Duplicate assays contained 10-1 M amino- 1858 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Brain Cell Culture Differentiation 1859

oxyacetic acid hemihydrochloride and 0.15 M KCl to dis- TABLE 1. Comparison of enzyme specific activities* in brain tinguish the chloride-sensitive, pyridoxal-dependent neuronal aggregate and monolayer culture decarboxylase activity from the glial or mitochondrial activity Phos- that is independent of pyridoxal phosphate and is stimulated Choline Glu- phoglu- by chloride (9). acetyl- Acetylcholine tamate conate Glucose-6-phosphate dehydrogenase and 6-phosphogluco- trans- esterase decarb- path- nate dehydrogenase were assayed together using [1-14C]- Cells ferase (-)t (+ )t oxylase way D-glucose-6-phosphate and measuring the 14CO2 released. Dissociated cells 0.08 2.98 0.44 0.17 2.56 Reactions were performed in a manner analogous to those for Monolayer-Day 11 0.05 2.43 0.43 0.08 1.48 glutamate decarboxylase. The 50-,l reaction mix contained Aggregate-Day 11 0.51 27.50 1.70 0.41 1.46 10 mM MgCl2, 0.1 mM EDTA (K+), 2 mM glucose-6-phos- phate, 1 mM NADP, and 2.4 X 105 cpm [1-14C]D-glucose-6- Brain tissue from embryonic mice was dissociated into in- phosphate (10 Ci/mol). No 14CO2 was released during the dividual cells that were placed in plastic dishes and grown as a reaction if [1-14C]D-glucose was replaced with [6-14C]D- monolayer culture or placed in flasks on a rotary shaker to form glucose. aggregates. After 11 days of culture, the enzymatic activities All assays were incubated at 370C for 10 min. Duplicate of the cells grown under both conditions were compared to the assays were performed at several concentrations of enzyme activities of the original dissociated cells. and values given represent initial rates. The protein con- * All specific activities given as nmoles of product formed per centration of the homogenates was measured by the pro- min per mg protein. i an cedure of Lowry (10). t BW-284-C51, inhibitor of acetylcholinesterase. Radioactive products were suspended in toluene-Perma- fluor (Packard Instrument Co.) or Triton-toluene-Permafluor scintillation fluid and counted at 80% efficiency. All radio- Embryonic mouse brain was used as a source of cells be- isotopes were checked for purity prior to use and purified cause these cells have essentially basal levels of the enzymatic if necessary. activities in question and also form much better aggregates. The dissociated brain cells rapidly reassociate in rotation RESULTS culture to form spherical aggregates of about 500 um in The central nervous system of mice is very poorly developed diameter. Histological examination of an aggregate that had at birth, and the greatest development of many biochemical been in culture for 17 days shows several different sizes of activities related to nerve transmission occurs postnatally. cells, many of which possess -like processes. The large This is demonstrated by the increased specific activity of and small cells often segregate and align themselves into choline acetyltransferase (ChAT) (11) acetylcholinesterase definite patterns (Fig. 2A and B). (AChE) (12), and glutamate decarboxylase (Gluase) (13). The specific activities of ChAT, AChE, and Gluase in These activities in the whole brains of C57Bl/6J mice are the aggregates as a function of time in culture are shown in shown in Fig. 1. The cholinesterase activity resistant to BW- Fig. 3. ChAT increases over 20-fold to an activity 70% 284-C51 has been subtracted from the values shown. This that of the adult mouse brain in vivo. Similarly, a 10-fold resistant activity represents 5-8% of the total acetylcholine increase in AChE activitiy is observed in the cultures. The hydrolytic activity in 18-day-old embryonic brain and 2-5% maximum level is 40% of the adult brain. Furthermore, the of the activity in adult mouse brain. Furthermore, Gluase BW-284-C51-resistant activity decreases from 9% in freshly activity represents both pyridoxal phosphate dependent dissociated cells to less than 3% in 21-day-old cultures. The (neuronal) and independent (glial) activities (9). In 15-day- Gluase activity increases 5-fold and is about 30% of the level old embryonic brain, 20-25% of the Gluase activity is in adult mouse brain. In addition, the pyridoxal phosphate independent of pyridoxal phosphate and stimulated by Cl-, independent and chloride-stimulated activity decreases dur- whereas, in adult mouse brain, 2-5% is the glial form. ing cell culture. The greatest increase in all three activities occurs between The activity of cultures grown as monolayers or aggregates the first and third weeks after birth, with maximal activity is compared in Table 1. The specific activities of all attained by 4-6 weeks postnatally. Likewise, most synapse three tested decreased in the monolayer cultures formation and dendritic branching occurs during this same as compared to the starting cells. In contrast, all three ac- time period (14). tivities increased during the same time interval in the ag-

TABLE 2. Enzyme specific activities* of brain cell aggregate and three clonal cell lines

Choline Phospho- acetyl- Acetylcholine esterase Glutamate decarboxylase gluconate Cells transferase (-)* (-)t (+)t pathway Brain aggregate 1.05 28.5 0.80 0.50 0.06 1.72 Neuroblastoma (N18) 0.02 3.08 0.14 0.06 0.15 2.48 Glialcell (C-6) 0.03 1.31 0.13 0.01 0.03 1.22 Chinese Hamster Ovary (K-1) 0.02 0.13 0.13 ND ND 1.64

The enzymatic activity of brain cell aggregates that had been in culture for 21 days is compared to the activities of clonal cell lines in logarithmic growth. The Gluase specific activities in the Chinese hamster ovary K-1 cells were not detectable (less than 0.005). * See Table 1. t ± . Downloaded by guest on October 2, 2021 1860 Biochemistry: Seeds Proc. Nat. Acad. Sci. USA 68 (1971)

BIRTH 20804 BIRTHbBIRTH 20 0 D

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0 0 0 BIRTH 20 40 0BIRTH 20 40 0BIRTH 20 40 AGE(DAYS) AGE(DAYS) AGE (DAYS) FIG. 1. In vivo development of choline acetyltransferase (a), acetyicholinesterase (b), and glutamate decarboxylase (c) in the whole brains of C57Bl/6J mice. gregates. However, Schrier and coworkers* (personal com- phosphogluconate dehydrogenase (EC 1.1.1.43) (16), and munication) have found ChAT activity to increase in mono- isocitrate dehydrogenase (EC 1.1.1.42) (17). The activity of layer cultures of brain cells. glucose-6-phosphate dehydrogenase and 6-phosphogluconate Since all three enzymatic activities tested in this paper in- dehydrogenase are followed in a single assay that is des- crease during brain development, the behavior of an enzyme ignated as the phosphogluconate pathway. The phospho- that decreases postnatally would be of interest. Although the gluconate pathway activity in C57B1/6J mouse brain has enzyme activity may not decrease in culture, an increase in been found to decrease 3-fold from the 18-day-old embryo to activity would be difficult to explain in a differentiating sys- the 3-week-old mouse (unpublished results). The phos- tem. Several enzymes in rat brain related to the production of phogluconate pathway activity decreases during both ag- NADPH decrease markedly after birth of the rat. These are gregate and monolayer culture (Table 1). glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (15), 6- The availability of two clonal cell lines of neural tissue * B. Schrier, J. Farber, R. Rosenberg, E. Thompson, S. Wilson, origin affords the opportunity to compare them to the brain and M. Nirenberg. Laboratory of Biochemical Genetics, NIH, cell aggregates. Neuroblastoma N-18 is a clone derived from Bethesda, Md. the mouse sympathetic nerve tumor C1300; its properties A

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~~~b *~~~44 FIG. 2 A and B. Sections through an aggregate after 17 days of culture. Bar in A and B represents 100 Mm and 30 um, respectively. Sec- tions were triple stained with hematoxylin, eosin Y, and azure II. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Brain Cell Culture Differentiation 1861

C r- c 0.60

E E 0.45- .eE .E a 0.30 E 0 0

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0' 4 12 20 4 12 2C 4 12 20 DAYS IN CULTURE DAYS IN CULTURE DAYS IN CULTURE FIG. 3. In vitro development of choline acetyltransferase (a), acetylcholinesterase (b), and glutamate decarboxylase (c) in brain cell aggregates. The activities of the undissociated brain tissue are 0.18, 11, and 0.13, respectively, In c, *-represents neuronal decarboxylase activity as assayed in the presence of pyridoxal phosphate. 0-represents the glial or mitochondrial decarboxylase activity, which is assayed in the presence of aminooxyacetic acid and Cl-.

have been described (18, 6). The properties of glial cell C-6 should offer a relatively simple system for determining the have been described by Benda and coworkers (19). In addi- influence of various hormones and drugs on developmental tion an epithelial cell clone, Chinese hamster ovary K-1 (20), processes. Furthermore, and more importantly, the dis- is included for comparison (Table 2). The brain cell aggre- sociated cells can be fractionated into various cell types prior gates are many-fold more active than logarithmic cultures to reaggregation, thus offering a system for study of the role of the three clones; however, stationary cultures of neuro- of cell-cell interaction in biochemical differentiation of the blastoma N-18 show increased AChE activity (6). Only in the nervous system. phosphoglucornate pathway activity do the clones resemble the aggregate, thus suggesting that the increased enzymatic I acknowledge Dr. S. Wilson's suggestions concerning the Gluase assay, and the expert histological and photographical assistance activities in the brain cells are a true expression of genetic of Miss M. Hertel, Mr. D. Peakman, Mr. G. Barela, and Mr. P. potential that is masked or absent in the clonal cell lines. Wuthier. This investigation has been supported in part by a grant from The Jane Coffin Child Memorial Fund for Medical Research DISCUSSION and the National Institutes of Health (NS-09818). This paper is As shown in the studies presented here, the culturing of brain number 471 from the Eleanor Roosevelt Institute for Cancer cells as aggregates appears to have a distinct advantage over Research and the Department of Biophysics and Genetics, University of Colorado Medical Center. the more conventional technique of culturing in plastic dishes (Table 1). Aggregation allows the reformation of preexisting 1. Moscona, A., in Cells and Tissues in Culture, ed. B. M. cell-cell contacts, which may be necessary for biochemical Willmer (Academic Press, New York, 1965), p. 483. differentiation. do not attach well to the plastic 2. Moscona, A., in Growth in Living Systems, ed. M. X. Zarrow dishes; therefore, many are discarded at the first medium (Basic Books, New York, 1961), p. 197. change. Cellular division is restricted in the aggregates, thus 3. DeLong, G. R., Develop. Biol., 22, 563 (1970). 4. Sidman, R. L., in The Neurosciences Second Study Program, In possibly favoring differentiation. contrast, culturing in ed. F. 0. Schmitt (The Rockfeller University Press, New dishes permits cell division and the rate of DNA synthesis is York, 1970), p. 100. much greater than in the aggregates (unpublished results). 5. Schrier, B. K., and L. Shuster, J. Neurochem., 14, 977 Thus, the glial or epithelial cells, which are present in greater (1967). 6. Blume, A., F. Gilbert, S. Wilson, J. Farber, R. Rosenberg, numbers and divide more readily, probably serve only to and M. Nirenberg, Proc. Nat. Acad. Sci. USA, 67, 786 dilute the activity of the small and slowly dividing neuronal (1970). population. 7. Austin, L., and W. K. Berry, Biochem. J., 54, 695 (1953). The ChAT activity more closely approximates the in vivo 8. Roberts, E., and D. G. Simonsen, Biochem. Pharmacol., levels than does the AChE activity (Figs. 1 and 3). A possible 12, 113 (1963). 9. Haber, B., K. Kuriyama, and E. Roberts, Science, 168, explanation may be that in the aggregates axon development 598 (1970). precedes dendritic maturation, since ChAT is localized in 10. Lowry, O., N. Rosenbrough, A. Farr, and R. Randall, J. the synaptic bouton of the axon, whereas the AChE activity Biol. Chem., 193, 26.5 (1951). is primarily in the postsynaptic membrane of the dendrite. 11. Hebb, C. O., J. Physiol., 133, 566 (1956). 12. Metzler, C. J., and D. G. Humm, Science, 113, 382 (1951). In addition to the biochemical development, the apparent 13. Roberts, E., P. Harman, and S. Frankel, Proc. Soc. Exp. segregation and alignment of cells within the aggregates sug- Med., 78, 799 (1951). gests a high degree of organization. Therefore, it is not too 14. Aghajanian, G. K., and F. E. Bloom, Brain Res., 6, 716 surprising that electron microscopic observations show the (1967). presence of numerous synapses in the aggregate cultures 15. Kuhlman, R., and 0. Lowry, J. Neurochem., 1, 173 (1956). 16. Bagdasarian, G., and D. Hulanicka, Biochim. Biophys. Acta, derived from dissociated brain cells (A. E. Vatter and N. W. 99, 367 (1965). Seeds, manuscript in preparation). Thus, the study of syn- 17. Robins, E., and I. Lowe, J. Neurochem., 8, 81 (1961). aptogenesis and its relation to the increased levels of enzymes 18. Seeds, N. W., A. G. Gilman, T. Amano, and M. W. Niren- involved in biosynthesis and berg, Proc. Nat. Acad. Sci. USA, 66, 160 (1970). 19. Benda, P., J. Lightbody, G. Sato, L. Levine, and W. in pre- and post-synaptic elements becomes possible. Sweet, Science, 161, 370 (1968). The ability of brain cell aggregates to show patterns of 20. Kao, F. T., and T. T. Puck, Proc. Nat. Acad. Sci. USA, biochemical differentiation similar to the in vivo situation 60, 1275 (1968). Downloaded by guest on October 2, 2021