Chemically defined requirements for the survival of cultured 8-day chick embryo ciliary ganglion neurons

Brain Research, 302 (1984) 281-290

281

Elsevier BRE 10062

Chemically Defined Requirements for the Survival of Cultured 8-day Chick Embryo Ciliary Ganglion Neurons STEPHEN D. SKAPER, IVAN SELAK, MARSTON MANTHORPE and SILVIO VARON

Department of Biology, School of Medicine, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) (Accepted October 25th, 1983)

Key words: serum-free culture - - ciliary neurons - - survival - - trophic agents

We have previously demonstrated that both peripheral and central neurons from embryonic chick and newborn mouse can be maintained in a serum-free defined culture medium containing the appropriate neuronotrophic agent and the N1 supplement consisting of insulin, transferrin, putrescine, progesterone and selenite. In the present studies we have examined the short-term survival requirements of 8-day embryonic chick ciliary ganglion (CG) neurons. By comparing CG neuronal survival in our standard culture medium, Eagle's Basal Medium (EBM), with several other commercially available basal media, we have established that CG neurons also have specific requirements for pyruvate, serine and iron (Fe3+), in addition to their trophic factor (Ciliary Neuronotrophic Factor, CNTF) and the N1 supplement. The data suggest the existence of 3 subsets of CG neurons differing in their essential needs, namely: (1) those supported by glucose in the absence of pyruvate, (2) those requiring exogenous pyruvate but not serine or Fe3÷, and (3) those which need pyruvate, serine and Fe3÷. The minimal effective concentration of pyruvate could be decreased by a factor of 50 in the concurrent presence of serine and Fe3÷. Serine was also a limiting element in the survival of some of these CG neurons. The Fe3÷ concentration required by the same neurons was considerably diminished with the availability of transferrin, perhaps reflecting an increased Fe3÷ transmembrane transport efficiency. Insulinwas found to be the only N1 ingredient required for the survival of CG neurons. Insulin was a constant requirement for all 3 subsets of CG neurons, even when cultured in the total absence of glucose (but presence of pyruvate).

INTRODUCTION

insulin, transferrin, putrescine, progesterone and selenite 4. Serum-free, N l - s u p p l e m e n t e d medium can

Investigations of nerve cells in dissociated, monolayer cultures have stressed the critical d e p e n d e n c e of both n e u r o n a l survival and neurite production/

support the survival of all types of peripheral n e u r o n s examined thus far, provided their appropriate neuronotrophic factor is also supplied4,16, 23. In addition,

maintenance on the composition of the culture medium and substratum 23. In addition to the ingredients of a basal m e d i u m (nutrients, ions, vitamins), media for neuronal cultures have traditionally comprised undefined supplements from various sources such as embryonic extracts, placental fluid or serum24. A t least with regard to peripheral neurons, specific macromolecular agents, or n e u r o n o t r o p h i c factors, have also been recognized as necessary for n e u r o n a l survival and are added to the m e d i u m in crude or partially purified forms 21. Over the past several years it has been shown that the serum r e q u i r e m e n t can be met by the use of a defined s u p p l e m e n t N1, a mixture of

a variety of central nervous system n e u r o n s can be supported by N1 when seeded at high densities or when supplied with certain conditioned medial, 15. More detailed investigations with dorsal root 17 and sympathetic ganglionic n e u r o n s 13 from the chick embryo have revealed that the 5 constituents of the N1 supplement are not all needed and that different combinations are in fact required by different neurons or by n e u r o n s of different embryonic ages. In the present report we have extended such analyses to the n e u r o n s of 8-day (E8) chick embryo ciliary ganglia (CG). We find that C G n e u r o n s require only insulin, among the 5 N1 ingredients. Insulin (or N1),

Correspondence: S. D. Skaper, Department of Biology, M-001, University of California, San Diego, La Jolla, CA 92093, U.S.A. Tel. (619) 452-3738. 0006-8993/84/$03.00 t~) 1984 Elsevier Science Publishers B .V.

282 however, does not replace serum unless a dialyzable serum fraction is also supplied. The dialyzable serum fraction can in turn be replaced by a mixture of pyruvate, serine and ferric ions. The latter three ingredients are shown to differentially support 3 subsets of the E8 CG neurons. MATERIALS AND METHODS

Solutions and media HEBM: the basic culture medium consists of Eagle's basal medium (EBM) supplemented with 2.6 × 10-2 M NaHCO3, 3.3 × 10-~ M D-glucose, 2.0 × 10-3 M L-glutamine, and 100 units/ml penicillin. The serum-free supplement N1 consists of insulin (8.3 x 10-7 M), transferrin (6.2 x 10-8 M), putrescine (1.0 × 10-4 M), progesterone (2.0 × 10-8 M), and sodium selenite (3.0 x 10-8 M). Other media used were DMEM (Dulbecco's Modified Eagle's Medium) Ham's F12, Leibowitz L15, RPMI-1640, and MEM (Eagle's Minimal Essential Medium), all of which media were supplemented with 2 mM L-glutamine and 100 units/ml penicillin. Additional medium supplementations will be described in the text when used. Polyornithine hydrobromide (PORN) was prepared in 1.5 x 10-2 M borate buffer, pH 8.4 at 0.1 mg/ml. The eye-derived Ciliary Neuronotrophic Factor (CNTF) was extracted and partially purified from selected El5 chick intraocular tissues 9. One trophic unit (TU) of CNTF activity is defined as that amount in one ml of medium supporting one-half the maximal number of E8 chick CG neurons after 24 h in culture. Neuronal culture conditions Ciliary ganglia from E8 chick were dissected and dissociated in EBM supplemented with 1% (w/v) ovalbumin, using previously described procedures s. The cell suspension was diluted to 40,000 cells/ml (about 20,000 neurons/ml) in the culture medium to be tested. In some cases an enriched population of neurons was prepared by means of a selective attachment procedure initially described for chick sympathetic ganglion 22 and dorsal root ganglion 14 cells. Briefly: (i) ganglionic dissociates were exposed to a tissue culture plastic surface for 2 h; (ii) the medium, containing unattached neurons ( > 85% neuronal purity) was centrifuged at 300 g for 4 min; and (iii) the

cell pellet was then resuspended in the test medium to give 20,000 neurons/ml. Culture plates containing 96 wells (6 mm diameter) were coated with polyornithine, followed by exposure to a source of polyornithine-binding neuritepromoting factor (PNPF-PORN substratum) 10,u. This substratum enhances neurite outgrowth and makes easier the quantitation of neurons in these cultures. Wells received 50 #l of basal medium containing fetal calf serum (FCS), the full N1 supplement, or various combinations of the N1 components or selected nutrients, all present at twice the desired final concentration. Unless otherwise specified, all wells also contained a final concentration of 20 TU/ml CNTF. All wells were then seeded with 50 jxl of cell suspension (1000 neurons). Modifications to this protocol will be described in the text when used. The cultures were incubated for 24 h at 37 °C, fixed, and quantitatively evaluated by direct neuronal counts, as previously described 25.

Materials Sources of materials were: EBM and DMEM from Grand Island Biological Company, Grand Island, NY; MEM, F12, L15 and RPMI-1640 from the Core Culture Facility of the University of California, San Diego; FCS from Irvine Scientific Company, Irvine, CA; trypsin from Nutritional Biochemicals, Cleveland, OH; penicillin from Parke-Davis, Detroit, MI; bovine crystalline insulin, human transferrin (90% iron-flee), progesterone, putrescine dihydrochloride, ovalbumin, pyruvate, serine, glycine, L-glutamine and polyornithine hydrobromide (30,000 MW) from Sigma, St. Louis, MO; sodium selenite from ICN Pharmaceuticals, Plainview, NY. RESULTS

Serum-versus Nl-supplementation of EBM and other basal media Our standard basal medium, Eagle's Basal Medium (EBM), used in conjunction with serum (with or without the N1 supplement) permitted about 80% of the E8 CG neurons seeded to be maintained for at least 24 h, provided their macromolecular trophic factor CNTF was also available (Table I). N1 was not adequate for full support and less than half of the maximal (serum-supported) survival was achieved.

283 TABLE I

Survival of ciliary ganglion neurons in different basal media with different supplementations 6 mm microtiter culture wells were seeded with 1000 E8 CG neurons with the test media shown below, as described under Methods. Cell counts were determined after 24 h. FCS was present at 10% (v/v), and FCS dialyzate at 1% (v/v). CNTF = chick eye-derived ciliary neuronotrophic factor. Numbers represent the mean of 3 cultures from each of two experiments.

Medium used

Neurons~well + CNTF

--CNTF

EBM EBM + N1 + FCS EBM + N1 EBM + N1 + dialyzedFCS EBM + N1 + FCS dialyzate

0

0

790 350 330 610

0 0 0 0

DMEM + N1 F12 + N1 L15 + N1

800 880 860

0 0 0

MEM + N1 RPMI-1640 + N1

0 0

0 0

Use of serum that had been extensively dialyzed against EBM did not restore maximal survival, while the serum dialyzate itself was able to enhance survival considerably even when tested at a 10-fold lower concentration than the undialyzed serum. Thus: (1) E8 CG neurons require additional supplements beside the N1 constituents; (2) serum provides such supplements in the form of dialyzable molecules; and (3) CNTF remains a constant requirement for the survival of E8 CG neurons. Different commercial media differ considerably in their basal ingredients. Since the missing serum supplements were dialyzable, they might fortuitously occur as regular constituents of basal media other than EBM. We, therefore, proceeded to test 5 other basal media. All media were supplemented with N1 in the presence or absence of CNTF. The results are also shown in Table I. In the absence of serum (but with N1 and CNTF), 3 of these media (DMEM, F12 and L15) proved fully competent to provide maximal support. In contrast, the other two media (MEM and RPMI-1640) failed to support even the partial neuronal survival elicited by EBM. All basal media, when supplemented with serum, supported the same maximal survival (data not shown). In all cases omission of CNTF resulted in the complete loss of neurons.

Since all 6 basal media have completely defined compositions, it became possible to identify those ingredients which, alone or in combination, would confer full competence to the fully or partially deficient media.

Identification o f specific basal medium ingredients required for CG neuronal survival The analysis presented here has been focused primarily on DMEM composition. This basal medium contains several ingredients at 4-fold higher levels than MEM or EBM, but none in greater excess. DMEM also contains the following 4 ingredients not present in MEM or EBM (although some are found in the RPMI-1640): pyruvate (1.0 x 10-3 M), glycine (4.0 x 104 M), serine (4.0 x 104 M, and ferric ions (Fe 3+, 2.5 x 10-7 M). All 4 components are also present in the other two competent media, F12 and L15. They became, therefore, prime candidates for further investigation. The experimental approach consisted in testing the 3 deficient basal media (MEM, EBM, RPMI-1640) in the presence of the 4 candidates, singly or in various combinations (but always in the presence of N1 and CNTF). Table II summarizes the relevant results, expressed as survival relative to serum-supplemented cultures. Note the already recognized distinction of EBM as the only one of the 3 deficient media capable of partial support (about one-third of maximal) in the absence of additional ingredients. The full combination of pyruvate, glycine, serine and Fe 3+ did have profound effects on the competence of all 3 media. It supported 70% of maximal survival in MEM, brought EBM to full competence, but conferred to RPMI-1640 only 55--60% support capability. It appears, therefore, that these 4 ingredients include critical ones to CG neuronal survival. Further analyses established a number of additional points. Glycine, serine and ferric ions made no contributions to any one medium when added individually, in pairs (not shown) or all 3 together - - as long as pyruvate was omitted. The two incompetent media (MEM, RPMI-1640) remained fully incompetent, and the partially competent one (EBM) maintained its partial support of CG neurons. Pyruvate, on the other hand, proved to be a necessary but apparently not sufficient ingredient. MEM competence rose from 0 to 35-40% and EBM competence rose

284 TABLE II

Relevance of specific basal medium ingredientsfor the survival of ciliary ganglionic neurons 6 mm microtiter culture wells were seeded with 1000 E8 CG neurons in the various test media supplemented as shown, and described in detail in Methods. Analysis was carried out after 24 h. Serum-supported survival (= 100%) = 850 neurons. All media were supplemented with N1 and CNTF. Pyruvate (PYR) = 1.0 x 10-3 M, glyeine (GLY) = 4.0 x 10-4 M, serine (SER) = 4.0 × 10-4 M, Fe3+ = 2.5 x 10-7 M. Each value was taken from 3 cultures in each of two experiments.

Media used

% Neuronal survival

Serum supplementation

No additives

MEM

EBM

RPMI-1640

100 0

100 36

100 0

PYR +

GLY +

SER +

Fe3÷

70

100

56

--

GLY --GLY +

-SER -SER +

--Fe3+ Fe3+

0 0 0 0

37 36 38 39

0 0 0 0

-GLY

---Fe3+ -Fe3÷

37 38 42 39 42 36

70 68 66 70 66 67

57 54 60 61 58 69

Fe3+

69

96

62

-

-

--PYR PYR + PYR PYR PYR + PYR +

-GLY + GLY

--SER -SER --

PYR

--

SER +

-

-

from 35 to 70% when pyruvate was added alone. No

Analysis of the N1 requirement

further i m p r o v e m e n t was achieved by pairing pyru-

The question of which N1 constituents are n e e d e d

vate with each of the other 3 test ingredients, nor by

by E8 C G neurons had originated this entire study.

supplying it in combination with both glycine and se-

Pyruvate, serine and Fe 3÷ were added together with

rine or glycine and Fe 3+. H o w e v e r , the combination

C N T F to either E B M (to support subsets A + B + C)

of pyruvate, serine and Fe 3+ did provide the same in-

or M E M (for subsets B + C). These two m ed i a were

creases in c o m p e t e n c e as had the full combination of

then supplemented with c o m p l e t e N1, or its individu-

the 4 ingredients. With the RPMI-1640, the 55--60%

al constituents alone or in various combinations.

survival achieved with the 4 ingredients was equally

Table III summarizes the results. Omission of N1 re-

achieved by the addition of the pyruvate alone (or in any other combination tested).

suits in no survival. C o m p l e t e N1 supports the expected numbers of neurons, about 600 with M E M

These data suggest 3 subsets of C G neurons differing in their essential needs, namely: subset A, sup-

and 850 with E B M (the difference being the subset A). Insulin, presented alone, achieved the same re-

ported by E B M but not by M E M or RPMI-1640, representing about one-third of the viable neurons seed-

sults. Ciliary neurons did not require transferrin (unlike the previous findings with both dorsal root 17 and

ed, subset B, requiring exogenous pyruvate but not

sympathetic 13 ganglionic neurons), selenite (unlike

serine or Fe 3÷, and comprising a n o th e r third of the

sympathetic neurons), putrescine or progesterone.

neurons, and subset C which needs both pyruvate and the additional presence of serine and Fe 3÷, and ac-

C G neuron subsets were next examined. Cells were

counts for the remaining third of the viable C G neurons; it is added to subset B in M E M and to subsets A

seeded in E B M (subset A ) , M E M plus pyruvate (subset B), M E M plus pyruvate, serine and Fe 3÷

and B in E B M .

(subsets B + C), or in E B M plus pyruvate, serine and

The requirements for insulin by each of the three

285 TABLE III

Analysis o f N l constituents needed for CG neuronal survival E8 CG neurons were seeded at 1000 per well as described under Methods. Basal medium consisted of MEM or EBM supplemented with 1.0 x 10-3 M pyruvate, 4.0 x 10-4 M serine, 2.5 x 10-7 M Fe a+, and 20 TU/ml CNTF. These media were further supplemented with the indicated N1 constituents. Neuronal survival was determined at 24 h. Values represent the mean of three cultures from each of two experiments.

Basal medium + CNTF + (PYR + SER + Fe3÷)

Neurons~well

No N1 supplementation Complete N 1 Insulin (I) Transferrin (T) I+T I + T + selenite (Se) T + Se + putrescine + progesterone

MEM

EBM

0 590 600 0 640 600 0

0 810 860 0 860 790 0

Fe 3+ (which supports all three subsets). All cultures received CNTF, and insulin was supplied in serial dilutions to define the 50% effective doses (ED50) in each of these systems. Fig. 1 shows the dose-response curves obtained. For direct comparison, the maximal net effect of insulin (survival at maximal doses less survival at highly diluted levels) was equated to 100% in each case. The ED50 was the same in all cases, about I x 10-7 M. Thus, all 3 neuronal subsets require insulin at the same concentrations.

Pyruvate was added at serial dilutions to two different media, both containing CNTF and NI: E B M (subsets A + B), and EBM plus serine and Fe 3+ (subsets A + B + C). The results are shown in Fig. 2. The 3 subsets, postulated on the basis of earlier experiments, are confirmed here. Omission from and addition to EBM of pyruvate, in combination with serine and Fe 3+ yield the two plateaus and single baseline for neuronal survival. These values, in turn, provide the following information on neuronal subsets. Subset A is displayed by the two EBM curves, regardless

\

60

c

E "x

Quantitation of the pyruvate requirement

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30" •

A

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//

A

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3OO

10 Insulin

told

- dilution

Fig. 1. Titration of the insulin requirement for survival of CG neurons in different serum-free supplemented media. One thousand ciliary ganglion neurons were seeded into 6 mm wells (see Methods) containing a constant level of 20 TU/ml CNTF and two-fold serial dilutions of insulin made in EBM (O), MEM plus pyruvate (©), MEM plus pyruvate, serine and Fe 3+ (A), or EBM plus pyruvate, serine and Fe 3+ (&). A line reflecting the average of all the conditions was drawn to connect the points. One-half maximal survival was achieved at an insulin concentration of 9.2 x 10-s M (the one-fold dilution represents 8.3 × 10-7 M insulin).

ol ~'

~

1'o Pyruvate

Iold

,~2 -

I

~o3

dilution

Fig. 2. Titration of the pyruvate requirement for survival of CG neurons in different serum-free supplemented media. One thousand ciliary ganglion neurons were seeded into 6 mm wells containing a constant level of 20 TU/mi CTF, N1 and two-fold serial dilutions of pyruvate made in EBM (O) or EBM plus serine and Fe 3+ (&). One-fold dilution of pyruvate equals 1.0 x 10-3 M.

286 of the presence or absence of serine plus Fe 3÷, when pyruvate (not present in E B M ) is no longer available to rescue the other subsets. In the present experiments, subset A was 240 neurons/well. Subset B can be calculated from the E B M curve, by subtracting from its plateau (590) the baseline value (240) corresponding to subset A. The difference of 350 neurons/ well represents subset B. Subset C, which requires serine and Fe 3÷ as well as pyruvate, can also be calculated. Subtraction of the E B M plateau (subsets A + B = 590) from that of the E B M plus serine and Fe 3÷ curve (subsets A + B + C = 900) yields a value for subset C of 310 neurons/well. The pyruvate concentrations n e e d e d by E8 C G neurons vary dramatically between subsets B and C or when subset B is examined with and without serine and Fe 3+. In the E B M curve, pyruvate is n e e d e d for subset B at 1.0 × 10-3 M to achieve maximal effect and ceases to be effective at about a 5-fold dilution. The EDs0 for subset B, then, is 4.5 x 10--4 M under these conditions. The inclusion in E B M of serine and Fe 3÷ adds subset C to the surviving population but also retains subset B. Both, however, are maximally supported by pyruvate concentrations as low as 4.0 x 10-5 M and no longer s u p p o r t e d only when pyruvate has been diluted to about 3 x 10-6 M. In the presence

80o 60-

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40

=E 20

I

lO

10 2

103

of serine plus F e 3+, then, the EDs0 of the B as well as the C subsets has decreased to 1.0 x 10-5 M. It is known that serine can be generated intracellularly from pyruvate. W e explored, therefore, the possibility that subset B does have endogenous requirements for serine similar to those of subset C but, unlike the latter, can meet them through an excess exogenous supply of pyruvate. In addition to the conditions examined in Fig. 2, we provided C G cells with M E M plus serine or E B M plus serine (the omission of Fe 3+ prevents the survival of subset C). The results are presented in Fig. 3 as percent of maximal net survival induced by pyruvate at serial dilutions. The additional presence of serine reduced the pyruvate requirements quite dramatically. The EDs0 for pyruvate measured in the absence of serine (curve A ) was, with both M E M and E B M , the 4.5 x 10 -4 M expected for subset B. The presence of serine shifted the pyruvate EDs0 to 4.5 and 2.3 x 10- 5 M in E B M and M E M , respectively. The difference between the two media remains to be investigated. The serine-induced shifts, however, were not fully reaching the EDs0 measurable with either m e d i u m when Fe 3÷ was also present, namely 1.0 x 10-5 M. It appears, therefore, that the additional availability of Fe 3÷ further enhances the performance of the Fe3÷-independent subset B beside adding subset C to the surviving neuronal population. Aside from that Fe 3÷ role, the data of Fig. 3 add two new points of information: (1) subset B may need the bulk of exogenous pyruvate as a substrate for the generation of e n d o g e n o u s serine, and only a small fraction of it for o t h e r critical purposes - - possibly for the generation of acetyl C o A ; and (2) subset C differs from A and B not only by its requirement for exogenous Fe 3÷ but also by its inability to utilize pyruvate as a potential source of endogenous serine.

Pyruvate fold-dilution

Fig. 3. Pyruvate requirement of the different neuronal subsets present in the ciliary ganglion. Data similar to those of Fig. 2 were plotted as a percent of the maximal net effect of pyruvate, i.e. the number of neurons surviving in response to a given concentration of pyruvate as a percentage of the maximal number which can survive in response to the optimal pyruvate concentration. The subsets defined by the presence of pyruvate (+ serine) in the presence or absence of iron are described in the text. A: no additions (0,0). B, C: serine, 4.0 × 10-4 M (FI,m). D: serine, 4.0 x 10-4 M plus 2.5 x 10-7 M Fe j+ (A,&). Closed symbols, MEM; open symbols, EBM. All cultures contained 20 TU/ml CNTF and the N1 supplement. One-fold dilution of pyruvate equals 1.0 x 10-3 M.

Quantitation of the serine requirement Media containing 1.0 x 10-3 M pyruvate plus Fe 3+ will support subset B in the absence of serine and add subset C only in the additional presence of serine, thereby permitting quantitation of the serine requirement by subset C (for its survival). Conversely, use of pyruvate at 1.0 x 10-4 M in the absence of Fe 3÷ will not support subset B (see Fig. 3) unless serine is a d d e d (and not support subset C even with serine), thereby permitting quantitation of the serine

287 ,

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A "~

80

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40'

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4o.

20.

~

20.

S lo Serine

,

I

8

Ioo

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103

i

t lO 4

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I 10 5

fold-dilution Fe 3+

Fig. 4. Serine requirements of the ciliary ganglion neuronal subsets. Titration curves for serine were used to plot the percent of maximal net survival effect of serine as a function of sefine concentration, as done for pyruvate in Fig. 3. MEM plus pyruvate (1.0 x 10-3 M) plus Fe 3+ (2.5 x 10-7 M), (O); EBM plus pyruvate (1.0 x 10-3 M) plus Fe 3+ (2.5 × 10-7 M), (O); EBM plus pyruvate (1.0 x 10--4 M), (A); MEM plus pyruvate (1.0 x 1 0 a M ) , (A). All cultures contained 20 TU/ml CNTF and N1. One-fold dilution of serine equals 4.0 x 1oa M.

requirement by subset B (for the reduction of its pyruvate requirement). These experimental conditions were applied to both M E M and E B M over a 100-fold range of serine dilutions from its concentration in D M E M of 4.0 × 10-4 M. Fig. 4 shows the results expressed as percent of maximal net effect (after subtraction of the neurons not requiring serine). Unlike some of the previous experiments, these showed identical behaviors in the two media, thereby excluding differential influences on the serine requirements. For subset C (high pyruvate plus Fe3+), the serine EDs0 was 3.3 x 10-5 M. For subset B (low pyruvate, no Fe3+), the serine EDs0 was 6.2 × 10-5 M, indicating a two-fold higher serine requirement by subset B than subset C. Minimal serine concentrations for optimal support were 1.0 and 1.9 x 10-~ M, respectively, in the two above situations.

Quantitation of the Fe3+ requirement We have determined, thus far, that Fe 3+ (beside pyruvate and serine) is required for the survival of subset C of these neurons. The ED50 for Fe 3÷ in both M E M and E B M containing pyruvate and serine (and CNTF) was next defined. Both media were supplemented either with insulin and transferrin or with insulin alone. Ferric ions were present at dilutions covering 4 orders of magnitude relative to their D M E M concentration of 2.5 x 10-7 M. The results are shown in Fig. 5, in the usual terms of percent net maximal

fold - dilution

Fig. 5. Iron requirement for ciliary ganglion neuronal survival in the presence and absence of exogenous transferrin. Titration

curves for the Fe3÷ requirement of CG neurons were determined in the presence (&,A) and absence (O,©) of transferrin, 6.2 x 10-8 M. Serum-free EBM (open symbols) and MEM (solid symbols) were used after supplementation with pyruvate (1.0 x 10-3 M) and serine (4.0 x 10-4 M), as well as insulin (8.3 x 10-7 M) and CNTF (20 TU/ml). A ten-fold dilution of Fe3÷ equals 2.5 x 10-8 M.

effects to permit direct comparison of subset C behaviors in the two basal media. As with serine both media yielded identical results, excluding differential media influences on the Fe 3+ requirements by subset C. In the presence of insulin alone, the minimal requirement for optimal support was found to be at about a 70-fold dilution of the D M E M concentration, yielding an EDs0 for Fe 3+ of 1.5 × 10-9 M. When transferrin was used concurrently with insulin, the EDs0 was reduced another 50-fold, to 3.0 × 10-11 M. Thus, while not required for C G neuronal survival, transferrin was an important contributor to the behavior of subset C. It remains to be determined whether transferrin reduces the requirement for exogenous Fe 3+ by (i) delivering to the medium Fe 3+ bound to it in amounts corresponding to the exogenous Fe 3+ reduction, or (ii) acting more traditionally as a Fea+-carrier across the cell membrane. DISCUSSION

Operationally, it seems possible to distinguish 3 subsets within the cultured C G neuronal population on the basis of in vitro requirements (see Table 4). It remains speculation whether the 3 operationally defined CG neuronal subsets represent discrete subclasses of ciliary ganglionic neurons (at least two are known to exist) or reflect heterogeneities in em-

288 TABLE IV Summary of evidence for 3 subsets of ciliary ganglion neurons with different survival requirements in monolayer cultures Culture medium

Additive(s)

% Maximal neuronal survival

Data from Table

Subset(s) supported

EBM + CNTF + N1

None PYR PYR,Ser,Fe 3+ PYR,Ser,Fe 3÷

35 70 96 100

1 2 2 3

A A+B A+B+C A + B+ C

None PYR PYR,Ser,Fe 3÷ PYR,Ser,Fe 3÷

0 37 69 77

1 2 2 3

None B B+C B+ C

EBM + CNTF + Insulin MEM + CNTF + N1 MEM + CNTF + Insulin

bryonic development or mechanical damage. (i) Subset A is supported, in the presence of CNTF and insulin, by E B M and not by M E M or RPMI1640. It does not require, therefore, any additional ingredients. It represents about one-third of the neurons seeded. (ii) Subset B requires CNTF, insulin and pyruvate and no additional basal ingredient. It comprises grossly another third of the neuronal population. Pyruvate supports this subset in M E M in apparent isolation from the other subsets and adds it in E B M to the EBM-supported subset A. (iii) Subset C also requires pyruvate, but needs the additional presence of serine and Fe 3÷. It accounts for the remaining third of the E8 CG neurons. It is added to subset B in M E M and to subsets A and B in EBM. Using differential survival requirements within the E8 chick CG cultures, we defined their quantitative requirements for insulin, pyruvate, serine and/or Fe 3÷. The following main conclusions can be summarized: (i) Pyruvate is effective at 50-fold lower concentrations in the presence of a minimum concentration of serine and Fe 3÷. (ii) Serine is also a limiting element for the survival of these neurons, since it must be either supplied exogenously or generated endogenously from excess pyruvate. (iii) Exogenous Fe3+ also influences the requirement for exogenous pyruvate, implying some cellular restriction in Fe3÷-dependent processes utilizing pyruvate. (iv) Ferric ions are needed in picomolar amounts if

transferrin is also available, possibly to increase Fe 3÷ transmembrane transport efficiency. (v) Insulin is a constant requirement for all CG neurons, even in the total absence of glucose (when pyruvate is present). Previous studies from this and other laboratories have described serum-free, chemically defined media for the culture of sensory4,17-19 and sympathetic 5,6,13,19,23 ganglionic neurons, and also a variety of central neuronslA2,1L The CG neuronal system is the first one where insulin is a sufficient single substitute for the N1 supplement. Insulin appears to be critical for both survival and proliferative growth of a large variety of cultured cells 2. The requirement for the C G neuronal trophic agent, CNTF, cannot be superceded by insulin. The specific role(s) of insulin in the present in vitro system remains unknown. The minimal concentration of insulin needed for optimal neuronal survival was about 3 × 10-7 M, which is nearly 1000-fold higher than that usually described to exist in (human) plasma. Several alternative explanations can be proposed, among them: (1) an unknown trace contaminant, rather than insulin itself, is responsible for the effect, (2) insulin mimics a different hormone by binding with much lower affinity to the latter's receptors, or (3) insulin inactivation proceeds so rapidly in the culture medium (a half-life of 10-30 min is reported in several textbooks) that the 1000-fold excess is needed to secure adequate levels over the 24 h assay period. A n attempt was made to explore this last possibility, based on the premise that medium collected from a 24 h culture should no longer contain insulin levels adequate to support a subsequent culture. The results

289 (not shown) deny such an expectation: 'used' and fresh insulin media titrated on new cultures with EDs0's of 1.2 and 0.9 x 10-7 M, respectively. A similar experiment, extended to test cultures carried for 48 instead of 24 h, yielded EDs0's of 1.4 x 10-7 M for the '24 h-used' and 1.7 x 10-7 M for the 'fresh' insulin media. Subset A has been defined as that which survives in EBM (plus N1 and CNTF) but not MEM (or RPMI1640) and does not require pyruvate, serine or Fe 3÷ supplementation. The differential support by EBM could reflect (1) the presence in EBM of promoting constituents, missing in MEM or RPMI-1640, or (ii) the presence in MEM or RPMI-1640 of inhibitory constituents, either missing in E B M or offset by EBM components. Our EBM contains biotin (not present in MEM), and a 6-fold higher glucose concentration than does MEM. Omission of biotin from EBM did not eliminate its competence for subset A and, conversely, addition of biotin to MEM failed to make this medium competent. Reduction of glucose in EBM from 33.3 mM to 5.6 mM (MEM level), and the converse increase of MEM glucose to 33.3 mM also failed to alter the effects of the two basal media (data not shown). No other EBM constituents suggest themselves as obvious candidates for the EBM preferential behavior. In the present study one-third of the E8 C G neurons appear to survive without exogenous pyruvate (subset A), although 70% of them do require pyruvate. The requirement for exogenous pyruvate needs to be examined with regard to the survival (and neuritic performance) of other neurons: (i) from different peripheral neural tissues (sensory, sympathetic ganglia), (ii) from ganglionic neurons of different developmental ages and from different animal species, and (iii) from central neural source tissues. This last

REFERENCES 1 Barbin, G., Selak, I., Manthorpe, M. and Varon, S., Use of central neuronal cultures for the detection of neuronotrophic agents, Neuroscience, in press. 2 Barnes, D. and Sato, G., Methods for growth of cultured cells in serum-free medium, Anal. Biochem., 102 (1980) 255-270. 3 Blass, J. P., Kark, R. A. P. and Menon, N. K., Low activities of the pyruvate and oxoglutarate dehydrogenase complexes in five patients with Friedreich's ataxia, N. Eng. J. Meal., 295 (1976)62-67.

category is particularly cogent, in light of the requirement of low molecular weight trophic agents already uncovered with several avian and rodent CNS neurons1, 26. Future investigations may explore the possibility the pyruvate requirements are exacerbated in pathological situations, using in vitro and in vivo models. Aberrations in pyruvate metabolism and pyruvate-metabolizing enzymes have already been described for several neuropathies3,7, 20. These data suggest that most E8 CG neurons - and, eventually, all other neurons found to require exogenous pyruvate for survival - - do not utilize glucose efficiently enough to meet their needs for endogenous pyruvate. A defective glucose utilization, in turn, may reflect defects in glucose transport or in the glycolytic pathway. Were this to be a general case for neurons, pyruvate may prove to be a critical link in glia-neuron interrelations and one that could be altered when either the neuronal or the glial performances might be impaired by pathological processes. Pyruvate, glycine and serine are interconnected by a number of pathways, and the additional requirement for serine by some CG neurons may have a basis in some such interconnections. Neuronal requirements for basal medium ingredients may eventually help understand the mechanism of action of the macromolecular neuronotrophic factors, as well as of the N1 constituents which are also essential for neuronal survival. ACKNOWLEDGEMENTS This work was supported by a grant from F I D I A Pharmaceutical. Ivan Selak is Aspirant du FNRS (Belgium), University of Li6ge, School of Medicine, Department of Neurology and Histology.

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