The derivation and characterization of neuronal cell lines from rat and mouse brain

Bra/n Research, 135 (1977) 25-36 Elsevier/North-Holland Biomedi~tl Press

25

THE DERIVATION AND CHARACTERIZATION OF NEURONAL CELL LINES FROM RAT AND MOUSE BRAIN

KAREN BULLOCH, WILLIAM B. STALLCUP and MELVIN COHN

The Salk Institute for Biological Studies, P.O. Box 1809, San Diego, Calif. 92112 (U.S.A.) (Accepted February 2nd, 1977)

SUMMARY

This study shows that permanent cell lines can be established from rat and mouse brain by direct tissue culture methodology without the aid of exogenous chemical or viral transforming agents. These cells were derived from specific areas of the brain, such as the cerebellum and hippocampus, at chosen times during fetal and neonatal development. Success in establishing neuronal cell lines was dependent upon the use of selection pressures designed to keep the background of giial cells and fibroblasts at a minimum. These manipulations included care in the choice and processing of the original tissue, utilization of cytotoxic anti-glial sera, and continuous manual isolation of cells with neuronal morphology. Slow-growing nerve cells were thus allowed to adapt spontaneously to culture with a minimum of competition from faster-adapting cell types. Many of these cell lines are judged to be neuronal on the basis hitheir electrical excitability and their characteristic surface antigens. The cells respond positively in a sodium flux assay which has been shown to correlate well with the ability to generate an action potential, and also express one or more of three antigens previously found to be specific for nerve cells.

INTRODUCTION

The most successful way of establishing cell lines from the nervous system in tissue culture has required the induction of tumors followed by the derivation of cloned cell lines from these tumors. This approach has the disadvantage that the origins of the cell lines are largely unknown. This paper deals with another approach, namely the direct culture of cells taken at specific times from weU-defined regions of the normal brain. The ultimate goal of this approach is the derivation of cell lines at will from known neural cell types.

26 We owe to Druckrey and his colleagues the development of the methodology for inducing neural tumors in ratsS, ts. Their technique of administering carefully titrated doses of nitrosoethyl urea transplacentally to fetal rats at specific times resulted in the specific induction of central nervous system neoplasms. They estabfished that there were differences in the susceptibility of rat strains to this induction procedure (BDIX rats being a particularly sensitive strain) and that the resultant tumors were of several morphological types, usually gliomas and ependymomas. Schubert et aL found that BDIX rat brain tumors derived by the Druckrey method could easily be put into tissue culture, and they established a large number of cloned cell lines from independently derived tumors TM. This permitted an analysis of several parameters which have been extremely useful in characterization of these cells: electrophysiology, receptors, secreted products, neurotransmitters, and surface antigens. Two of these have been of primary importance in our own studies: an assay of electrical excitability which can be reliably used to define neuronal cells, and antisera toward cell surface antigens which not only distinguish nerve cells from glial cells but also subdivide each of these two groups of cells. There is a good correlation between the ability of a cell to generate an action potential (as assayed electrophysiologically) and the ability of veratridine to stimulate sodium uptake into that cell through special voltage dependent channels in the membraneS,is, x4. Thus one can readily assay for excitable cells by comparing the uptake of 22Na+ in the presence and absence of veratridine. With regard to surface antigens, properly absorbed rabbit antisera against chosen BDIX cell lines allowed us to define 3 nerve-specific antigens NI, N2, and N3, and two glial specific antigens, GI and G2, which are useful in classifying the collection of cell lines t4. More important, as shown by absorption experiments using normal brain tissue, these antisera are reactive with po?ulations of cells that arise -~ different times and places in the developing rat brain xs. 3 his allows us to take a first step toward matching various cell lines to their counterparts in the nervous system and to begin mapping the appearance and disappearance of various cell types. Since the precise anatomical origins of the cell lines derived through the tumor induction program are not known, it is possible that the library of cells thus obtained may not provide a broad sampling of the entire central nervous system. For this reason we undertook the derivation of cell lines directly from normal brain tissue which was well-defined in terms of both the anatomy and the age of the animal. In this way we could enlarge our cell library, and subsequently our library of ceilsurface markers, in an orderly fashion. This p~per describes the tissue culture techniques we have used to derive neuronal and giial cell lines from normal rat and mouse brain. It also reports our initial attempts to classit~ these cells using both the sodium uptake assay and the specific surface antigens to distinguish major cell types. In choosing BDIX rats for our initial work, we thought the rapid adaptability of the BDIX tumors to culture might be a trait that would also apply to normal tissues, and it was our hope that cell lines derived through spontaneous adaptation to culture rather" than by chemically induced transformation might retain more of their 'normal' characteristics. In the event that BDIX rats should prove unsuitable we also began

27 parallel experiments with cells taken from Lewis rats and C573L/6J mice. We were particularly interested in the mouse cultures for two reasons. First, the genetics of the mouse are understood in more detail at present than those e$ the rat. A knowledge of genetics might allow us to make more specific antisera by immunization across small strain differences rather than across wide species differences. In addition, we would be able to study the genetics of any interesting traits expressed by the cultured mouse cells. Second, there are a number of neurological mutants of the mouse H, which would not only provide us with the means of making very selective antisera, but would also enable us to attempt reconstitution experiments with our cultured cells. MATERIALS AND METHODS

Sodium uptake ~9"Na+ uptake assays were performed as previously describeda,~a,~4. Veratridine was obtained from K and K Chemicals, ouabain and scorpion venom (ScTX) (Tityus serrulatus) from Sigma, and the ~eNa+ from Amersham Searle.

Antisera All of the antisera used in this work (anti-Bg, anti-C6B, anti-B35, anti-BI03) have been described previously, as have the radioimmune binding assays used for determining the presence of the specific antigens NI, N2, N3, GI and G2 on cell surfaces 14. The cytotoxic anti-glial sera were prepared by absorbing the anti-B9 and anfi-C6B glial sera with the neuronal cell lines BS0, BI03, and B35. As previously reported, in the presence of complement these absorbed antisera effectively cause lysis of a number of glial cell types without affecting the viability of neuronal ones. These two antisera were centrifuged at 20,000 × g for 10 min, frozen, thawed, and passed through 0.22/~m Millipore filters (Millipore Corporation, Bedford, Mass.) to assure the removal of any viable cells remaining from the absorption process. The sera were diluted to 1/3 strength and mixed in equal proportions for use in the antigliai selection prodecure described below.

Culture of new cell lines Inbred colonies of BDIX rats, Lewis rats, and C57BL/6J mice are maintained at the Salk Institute. Pregnant animals between 9 and 15 days" gestation were anesthetized with ether, their abdomens washed with 70% ethanol and the embryos removed individually under sterile conditions. Fetuses were prepared one at a time in order to minimize neural cell death in the remaining embryos. The fetuses were decapitated, and various areas of the brain were dissected. When neonatal tissue was cultured, animals less than 10 h old were used. The methodology used for dissociation of the tissue into single cells was essentially that of Hemminki~. Similar samples from the various fetuses were pooled in cold Hemminld's solution, where external blood vessels and debris were removed. The cleaned pieces were put into a dish containing cold Hemminki's solution -k 0.1% trypsin, where the tissue was gently teased into smaller pieces with forceps. A Pasteur pipette, cut to 1.5 mm bore, was used to dis-

28

aggregate the pieces by gentle pipeting. The suspension was next placed in a small Erlenmeyer flask and sha',~en for 1 rain in a 36 °C water bath, after which aggregates were again dispersed by pipeting. Additional shaking for 5 rain at 36 °C, followed by further dispersion, was carried out before the cells were returned to a shaker platform for a final 30 rain. Two volumes of cold Dulbecco's Modified Eagles medium (DME) + 20% fetal calf serum (FCS) were then adch~d to the suspension, which was passed through a sterile Nitex 210 mesh filter. The filtered cells were pelleted at 500 rpm for 7 rain and washed twice with DME -I- 20% FCS. At this point the cells were either put directly into culture in tissue culture dishes in DME + 20% FCS -I- 4 × 10-s M glutamine, or subjected to the first step of the anti-glial selection. For this selection the cells were gently suspended in 0.1 ml of balanced salt solution (B$$) to which an equal volume of the pooled cytotoxic antiglial sera (see above) was added. This mixture was incubated at 37 °C for 5 rain with occasional swirling, after which 0.1 ml of 30% guinea pig complement (GIBCO) was added. After a 4S-rain incubation at 37 °C with occasional swirling the suspension was centrifuged at 500 rpm for 7 rain. The cell pellet was resuspended in 0.1 ml of BSS, and the cytotoxic selection was repeated a second time to increase the efficiency of killing. The cells were finally centrifuged, washed, and plated in tissue culture dishes in DME -t- 20% FCS + 4 × 10-s M glutamine. Since our absorbed rabbit anti-rat ~ r a bave not proved to be very cross-reactive with mouse cells, either in the radioimmune binding assay or in cytotoxic tests, after an initial unsuccessful attempt in which very few cells were lysed, use of the anti-glial selection was discontinued in the case of the mouse cultures. When it was necessary to remove attached cells from the culture dishes for passaging, the cultures were covered with Viokase (GIBCO) diluted 1:1 with DME and incubated at 37 °C until the cells began to come free. The cells could then be removed gently from the dish, washed free of the Viokase, and replated as desired. In order te remove only a small cluster of cells from a confluent culture, we used the technique of ring subculturing. The area of interest was isolated within an 8 mm diameter ring dipped in sterile silicon grease to insure a good seal with the surface of the dish. The enclosed cells were then treated with diluted Viokase and removed. Cells were cloned by one of two methods. In some cases cells were plated on fragments of sterile cover slips. Fragments with a single attached cell were selected microscopically and transferred to individual tissue culture dishes. In most cases cells were cloned by plating a dilute cell suspension in Falcon microtiter wells. Wells containing single cells were observed, and when the cells reached sufficient density the clones were passaged.

Histocompatibility typing Cultured cells from the Lewis rat could be distinguished from BDIX cells by means of an antiserum raised against histocompatibility antigens. BDIX rats were immunized over a period of two months by weekly injections of Lewis spleen cells. The best of the resulting antisera had a titer of 11400 in a hemagglutination assay 9 using Lewis red blood cells. Cultured neural cells were tested for reactivity with this

29 antiserum in the following way. One ml of antiserum at a 1150 dilution was absorbed with 2 × l0 Tcells at room temperature for 30 rain. The cells were removed by centrifugation and the serum was retested for ability to agglutinate Lewis red blood cells. A positive absorption is one in which the titer of the antiserum is reduced from 11400 to a significantly lower value. Lewis cells would be expected to be positive while BDIX cells should be negative. Tumorigenicity tests In order to test the tumorigenicity of our cultured cell lines, we injected animals subcutaneously with 106 cells. The behavior of the new cell lines was compared to that of established BDIX cell lines such as B35 and BI03. These two cell lines produced tumors 2 cm in diameter within 3 weeks when injected into BDIX rats, but failed to produce tumors in Lewis and Buffalo rats or in C57BL/6J mice (tumors did appear in nude mice). RESULTS

During the first two or three days after the primary dissociation of tissue and the initial anti-glial selections, relatively few cells adhered to the culture dish. For this reason the cultures were left undisturbed without any change of medium. Thus non-adhering cells were not lost and, moreover, the medium seemed to be favorably conditioned by a high cell density. Single cells were rarely seen to adhere tightly to the culture dish. Usually, the cells would form aggregates which settled from suspension and adhered to the culture dishes by the end of the first week. During the ensuing weeks the cultures were maintained without passaging simply by replacing the medium weekly. By avoiding passaging and by allowing cultures to become contact inhibited, we hoped to retard the proliferation of fibroblasts and glial cells, which normally overwhelm slow-growing nerve cells. On such confluent feeder layers, neuronal cells might have time to adapt to culture and begin active division. On the occasions when rat cultures were passaged they were re-subjected to the anti-glial selection procedure in order to reduce the non-neuronal population in the new dish. When areas of cells with nerve-like morphology were observed, they were selected out and subcuitured by one of two methods: (a) ring subculturing (Methods) or (b) a very brief, gentle treatment with Viokase which removed only the top layer of loosely adhering nerve-like cells. In this way the background of unwanted cell types was further minimized. The resulting subcultures often l~st their neuronal morphology and took on the appearance of undifferentiated, flattened cells. However, as the cultures were maintained over a period of several months, loci of cell proliferation were noted in which cells began to re-express neuronal morphology (see Fig. l). These colonies were again subcuitured after isolating them in a metal ring as described above. Often cells were seen to go through several cycles of this appearance and disappearance of neuronal morphology before they fmally adapted permanently to culture. Once the morphological characteristics of a culture became stable, cells were cloned and maintained as permanent cell lines. In all cases, cells at the various

30

Fig. i. Colony formation. This phase contrast photomicrograph illustrates the colony formations found in primary cultures after 3 months, in the lower half of the figure can be seen the typical layer of flat cells which covers the surface of the tissue culture dish. In the upper half of this photograph is the edge of a colony of small spindle-shaped cells which have proliferated on top of the confluent monolayer. Colonies of this type were selected, subcultured, and eventually cloned as described in Methods. × 100. stages of adaptation, as well as the established cell lines, were stored in liquid nitrogen. Table I presents a list of the cloned cell lines derived from our program to date. It includes information regarding the area o f the brain from which the explants were made, the age of the animals used, the number of anti-glial selections made during the initial weeks of culture, and the tumorigenicity of the established cell line. Some examples of these cell lines are shown in Fig. 2.

Strain and species markers Many studies in which two or more populations of cultured cells are mixed require t h a t t h e different cell types be characterized by easily recognizable markers. Cell fusion and studies of cell-cell interaction are two types of experiments in which it is often necessary to confirm the identity of the respective cells a posteriori. In the case of BDIX and Lewis rat cells the difference in histocompatibility type provides us with a means of distinguishing the cells. An antiserum raised in BDIX rats against Lewis spleen cells recognizes the Lewis histocompatibility antigens. As shown in Table II, Lewis spleen cells and LCII cl B cells both have the capacity

31 TABLE I

Origin o f cell lines Cell line

XKM cl I XKC cl 7 ZKC cl 2 CK2D cl E CKSW c116 CKMD cl 5 SKC cl 5 BE cli I BEc117 LCI! cl B LCII cl 3 R aC cl 3 /~C cl AS ~HC cl 2 ~HC T3

Strain

BDIX rat BDIX rat BDIX rat BDIX rat BDIX rat BDIX rat BDIX rat BDIX rat BDIX rat Lewis rat Lewis rat C57BL/6J mouse C57BL/6J mouse C57BL/6J mouse C57BL/6J mouse

Age

Area o f brain

Number o f anti-glial selections

2nd fetal week 2nd fetal week 2nd fetal week 2nd fetal week 2nd fetal week 2nd fetal week 10 h postnatal 3rd fetal week 3rd fetal week 3rd fetal week 3rd fetal week 10 h postnatal 3rd fetal week 3rd fetal week 3rd fetal week

Medulla Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum Cerebellum whole brain whole brain Cerebellum Cerebellum Cerebellum Cerebellum Hippocampus Hippocampus

4 4 5 4 4 4 2 0 0 3 3 0 0 0 0

Tumorigenicity a

÷ + ± -~+ + --~ -~b -÷ ~

---

s When injected into syngeneic host as described in Methods. b When re-established in tissue culture these tumors exhibited the same morphology as the original cultures and retained the ability to give a positive response in the sodium uptake assay.

Fig. 2. Morphology of cell lines. These phase contrast photomicrographs show examples of the morphologies expressed by some of the established cell lines. A: BE d 11; B: ZKC cl 2; C: XKM cl 1; D: aC ¢1 3; E:/~HC el 2; F: SKC cl 5. × 100.

32 TABLE H

Histocompatibility typing A BDIX antiserum asainst Lewis spleen cells was absorbed as described in Methods with the cells indicated under Absorption. These absorbtxl antisera had activities as listed under Hemagglutination titer. Normal BDIX serum had no (ktoctabh~ agglutinating activity at a dilution of 1/10.

Absorption

Hemagglutination titer

None

1/400

BDIX spleen

1/400

BI03 Lewisspleen LCII cl B

1/400 1/50 1/100

to absorb activity from this antiserum, while BDIX spleen cells and BI03 cells lack this capacity. The histocompatibility difference is confirmed by tumorigenicity studies. LCil cl B cells produce tumors in Lewis rats, while BDIX cell lines produce tumors only in BDIX rats. The C57BL/6J cell lines are characterized by their typical mouse karyotype7. In addition, there are histocompatibility differences between the rat and mouse cell lines. One of the mouse cell lines, aC cl 3, produces tumors in C57BL/6J mice. The rat cells fail to do so.

Excitability of cells We had previously found an extremely good correlation between the ability of a cell to generate an action potential and its ability to take up 2~Na÷ in the presence of veratridinet3,14. We have also found that, at least in the limited number of cases tested, it is possible to distinguish nerve cells from muscle cells on the basis of their response to scorpion venom~ Scorpion venom affects the way in which excitable cells interact with veratridine4. In nerve cells scorpion venom may cause an increase in the binding affinity for veratridine or it may cause both an increase in binding affinity and a large increase in the maximum rate of sodium uptake. In muscle cells scorpion venom does not alter the affinity for veratridine, but do~s cause increases in the rate of sodium uptake 17. Consequently, the sodium uptake assay was our first criterion for screening potential neuronal cell lines. Table III shows the rate of sodium uptake stimulated by veratridine and scorpion venom in a number of the newly selected cell lines. Some previously existing cell lines are included for comparison: B35 and BI03 are examples of rat central neurons, B9 and C6B are rat glial cells, L6 is a rat muscle cell line and C1300 cl NI8 is a mouse neuroblastoma. It is apparent that many of the new rat cell lines are comparable to the established rat nerve cell lines in their behavior: they have low but reproducible responses to veratridine and large responses to veratridine plus scorpion venom. None of the apparently excitable rat cell lines exhibit the insensitivity of the L6 rat muscle cells to scorpion venom. As noted in the accompanying manuscript xT, the XKM cell line is unique among the rat cells in that scorpion venom does not

33 TABLE III

Properties of cell lines Sodium flux assays were performed as described previouslya,13,14. Veratridine was used at a concentration o f 2 x 10-4 M. Crude scorpion venom (ScTX) was dissolved in 0.01 M phosphate buffer, pH 7.4, and used at a concentration of 100 pg/ml. Uptake values are given as moles of ~='Na+ per min per mg of cellular protein.

Rate Of~2Na +uptake

Neuronal antigens

Cellline

NI

Veratridine ScTX + Veratridine

C1300cl NI8

4.6 x 10-s

I.,6 1335 BI03 B9 C6B

7.0 × 10 -9 i . 8 x 10- 9 3.1 × 10 -9 < 10- t° < 10 - l °

XKMcll 8 . 0 x 10-9 XKCcl7 1.1 × 10- 9 C K S W d 16 2.6 × 10-9 C K 2 D d E 2.9 × 10-9 ZKCd2 1.9 × 10-° C K M D d 5 3.0 × 10- 0 SKCcl 5 1.6 x 10-9 SKC d N 3.2 x 10-9 B E e f II < 10- to B E d 17 < I0 -I° LCIIclB 1.9 x 10- e LCI! ¢! 3R 1.2 x 10- 9 aCcl3 1.9 x 10- s /~C el AS 1,2 x 10- 9 /~HCI 2 < 10-l° ~ H C d T 3 < 10-1°

8.0 1.0 1.5 4.5

x x x x

i.2 1.0 4A 3.0 2.1 2.6 1.6 3.0

× × × × x x × x

1.8 1.6 3.9 7.2

x x x x

IO-sk 10-s" 3400 10-s 20,000" 10-s 22,000" 3000 4000 10-as 21,000" 10-a 22,000" 10-a 19,000" 10-s 18,000" !0 -s 21,000" 10-s 21,500" 10-s 3800 10 -s 3509 4000 2500 10- s 2750 10- s 2300 10- sa 10- 0

Glial antigens

N2

N3

GI

G2

3100 15,000" 16,000" 2000 2500 14000* 13,000" 17,000" 16,500" 14,500" 13,400" 2400 2150 2000 2500 2800 2200

2100 12,000" 3000 2000 2500 13,000" 13,500" 8300* 2000 10,000" 3000 2700 2250 2000 2200 2300 2175

15,200" 2700 2500 2100 14,000" 1900 2000 2500 1900 1800 2300 2200 1950 3000 15,000" 2300 2500

2300 2000 2500 10,500" 3000 2700 2200 2600 2500 2600 2000 2350 2100 15,000' 9500 1900 2000

s Although scorpion venom does not greatly inca'ease the rate of veratridineAnduced sodium uptake, it does increase the apparent affinity for veratridine by a factor of 4 or 5 (ref. 17). b Scorpion venom has only a small effect on the rate of veratridine-induced sodium uptake, and has no observable effect on the binding constant for veratridine t~. Radioimmune binding assays were performed as described previously ~4. Values shown here represent the counts per minute bound to each cell line when tested with specific antiserz against the five antigens, N I , N2, N3, G I , and G2. * represents values which are significantly above background, while unmarked values represent background binding.

greatly increase the rate of uptake seen with veratridine alone. It does, however, increase the affinity of the cells for veratridine. Among the mouse cell lines this latter behavior is found in both the C1300 neuroblastoma and the aC cl 3 cell lines - that is, binding affinity is increased, while Vmax is raised only slightly. On the other hand, ~C el AS behaves more like the rat nerves, having a much higher Vmax in the presence of scorpion venom.

Surface antigens We have previously shown that there are at least 3 antigens, NI, N2 and N3,

34 which appear to be characteristic of nerve cell linesx4. NI was found on all 6 BDIX neuronal cell lines tested, while N2 and N3 were expressed by 4 and 2 of these cell lines, respectively. Two other antigens, GI and G2, were found only on giial cells. The new cell lines have also been screened using these antisera, and the results are given in Table IH. Some of the previously existing cell lines are included for comparison. These antisera do not appear to cross-react well with mouse tissues, and therefore have not proven useful in deafing with the new mouse cell fines. Table HI shows that the nerve-specific antigens NI and N2 are expressed by most of the excitable rat cell lines. In addition, 4 of the new cell lines express the N3 antigen. Two of the non-excitable cell lines, BE cl 11 and BE cl 17, express glialspecific antigens. DISCUSSION

We believe there are three key factors involved in our successful establishment of neuronal cell lines: (a) we selected areas of the fetal and neonatal brain in which neurogenesis was still occurring. The cerebellum and hippocampus are areas in which nerve cells continue to proliferate even after the birth of the animalX,~,ta; (b) we used cytotoxic selections against glial cell types to enrich our cultures for nerve cells. The usefulness of this technique in dealing with the rat cells is illustrated by the fact that in the one case in which no selections were made, only giial cell types were obtained (BE cl 11 and BE cl 17). Nevertheless, the anti-giial treatment is not absolutely essential. Due to the ineffectiveness of the antisera in dealing with mouse cells, the mouse neurons were established without the aid of the anti-giial sera. This emphasizes the importance of the third factor, the culture methods themselves; (¢) the cultures were handled in such a way as to maximize the survival of neuronal cell types. As outlined in detail in the Methods section, primary cultures were given ample time to adhere to the dish in order to avoid loss of potential nerve cells during media changes. Frequent pass~g,:=g . f the entire culture was avoided during these early stages in order to keep rapidly-growing fibroblasts and giial c~,!s from overwhelming the more slowly-adapting nerve cells. Instead, selections for desired cell types with neuronal morphology were repeatedly made by either removing small areas of the culture isolated within a small metal ring or by gently removing only the top layer of loosely adhering nerve-like cells. Finally, the expression of 'differentiated' neuronal morphology seemed to be a reversible process. In many cases cells were seen to undergo several cycles of expression of this morphology before they eventually reached a stable state. Therefore, care was taken to maintain such cultures until the morphological characteristics did become stable. Clearly, in many cases this adaptation to culture involved some type of in vitro transformation of the cells, as evidenced by the ability of many of the cells to produce tumors in syngeneic animals. We cannot identify the mechanism or cause of this transformation, but it is apparently a common phenomenon in dealing with many types of cultured rodeat cellss. At present we have two criteria for defining nerve cells in culture: (a) electrical excitability and (b) nerve-specific surface antigens. Many of the cell lines we have

35 established meet both ~'-fthese criteria. With regard_ to the first point, the cells respond positively in a sodium uptake assay which has proved to be a good measure of electrical excitability. The ability of veratridine to open certain voltage-dependent sodium channels on a cell has been shown to correlate very well with the ability of that cell to generate an action potentialS,13,14. In addition, scorpion venom and veratridine interact with these channels in a highly cooperative manner 4, and the combined action of these two toxins produces very large sodium fluxes in our new rat cell lines. At the toxin concentrations used, this behavior is characteristic of rat nerve but not rat muscle cells1~. As for the second point, the new cell lines express antigens previously found to be characteristic of neuronal cell lines14,15. Most of the new BDIX cells that are excitable express both NI and N2, and four of them also express N3. Most of our efforts have been directed toward selection of cells from the cerebellum. The distinctive cell types of the cerebellum and the existence of cerebeilar mutants make this area of the brain particularly attractive. In addition, we have one cell line from the medulla (XKM cl 1) that has clear neuronal properties. Hippocampal cell lines, although very neuronal in appearance (see Fig. 2,/~HC cl 2), have thus far failed to respond in our sodium flux assays. This assay is somewhat limited in that it measures only sodium uptake, and it is still possible that ionic currents might be carried in these cells by other ions such as calcium. Some of the rat cell lines (SKC cl 5, SKC cl N, LCII cl B and LCII cl 3R) which respond positively in the sodium uptake assay fail to react with the neuronal antisera. Thus we cannot unambiguously answer the question of whether these cells are nerve or muscle. We can say, however, that their behavior toward veratridine and scorpion venom more closely resembles that of other rat nerve cells than that of rat muscle cells and that they lack the GI antigen which was found on the L6 and 1344 muscle cell lines ~4. It would be somewhat naive to suppose that NI, N2, and N3 are the only nerve.specific antigens, and it is our hope that these four cell lines may represent new types of nerve cells not heretofore studied in culture. One immediate advantage of our new cell lines is that we know something of their anatomical point of origin, i.e. cerebellum, medulla, etc. Also, it appears that some of the lines have characteristics not found in previously existing lines. For example, ZKC cl 2 lias a unique surface determinant involved in cell-cell adhesion. This determinant is lacking on other neuronal cells studied TM. With regard to electrical properties, the XKM ci 1 cells have sodium channels with characteristics which are unique among the rat cells. ~C cl AS cells appear to be unique in this regard among the mouse linestT. Most important to us is the question of whether these cells express new antigens not yet found on other cell lines. We hope that this information will be provided by antisera prepared against cell lines such as XKM, ZKC and especially the rat and mouse cell lines that do not express any of the presently known antigens. ACKNOWLEDGEMENTS

This work was supported by NIH Grants AI05875 and AI00430 to M.C. and by National Foundation Basil O'Connor Starter Grant 5-60 and National Science Foundation Grant BNS 76-01548. to W.B.S.

36 REFERENCES I Altman, J., Postnatal growth and differentiation of the mammalian brain, with implications for a morphological theo~ of memory. In O. C. Quartan, T. Melnichuk, and F. O. Schmitt (Eds.), The Neuroscicnce& a Study Program, Rockefeller University Press, New York, 1967, pp. 723-743. 2 Angevine, J. B., Critical cellular events in the shaping of neural centers. In F. O. Schnfitt (Ed.), The Neuroscicnces, Second Study Program, Rockefeller University Press, New York, 1970, pp. 62-72. 3 Catterall, W. A. and NJrenher8, M., Sodium uptake associated with activation of action potential ionophores of cultured neuroMastoma and muscle cells, Proe. nat. Acad. Sci. (Wash.), 70 (1973) 3759-3763. 4 Catterall, W.A., Cooperative activation of action potential Na + ionophores by neurotoxins, Proc. nat. Acod. SH. (Wash.), 72 (1975) 1782-1786. 5 Druckrey, H., Preussman, R. and Ivankovic, S., N-Nitrnso compounds in organotropic and transplam~ental carcinogenesis, Ann. N.Y. Acod. Sci., 163 (1969) 676-695. 6 Hemminki, K., Preparation of viable and morphologically intact cells from newborn rat brain, Exp. Cell Res., 75 (1972) 379-384. 7 Elsu, T. C and Benirschke, K. (Eds.), An Atlas of Mammalian Chromosomes, Vol. 1, SpringerVerlag, Berlin, 1967, p. 17. 8 Moorhead, P. S., How long can one safely work with cell lines?, In Vitro, 10 (1974) 143-148. 9 Rubiustein, P. and Kaliss, N., H-2 typing with the polyvinylpyrrolidone (PVP) method, Transplantation, 17 (1974) 121. 10 Schubert, D., Heinemann, $., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J. H., Culp, W. and Brandt, B. L., Clonal cell lines from the rat central nervous system, Nature (Lond.), 249 (1974) 224-227. I I Sidman, R. L., Green, M. C. and Appel, S. H., Catalog o/the Neurological Mutants o/the Mouse, Harvard University Press, Cambridge, Mass., 1965. 12 Sidman, R. L., Cell proliferation, migration and interaction in the developing mammalian central nervous system. In F. O. Schmitt (Ed.), The N~'aroselences, Second Study Program, Rockefeller University Press, New York, 1970, pp. 100-107. 13 Stallcap, W. B. and Cohn, M., Electrical properties ofa clonal cell line as determined by measurement of ion fluxes, Exp. Cell Res., 98 (1976) 277-284. 14 Stallcup, W. B. and Cohn, M., Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system, Exp. Cell Res., 98 (1976) 285-297. 15 Stallcup, W. B., Nerve and glial-specifu: antigeus on cloned neural cell lines. In Z. Hall, R. Kelly and C. F. Fox (Eds.), Progress in Clinical and Biological Research, Vol. 15, Cellular Nearobiology, Alan R. Liss, Inc., New York, 1977, in press. ! 6 Stallcup, W. B., Specificity of adhesion between cloned neural cell lines, Brain Research, 126 (1977) 475-486. 17 Stallcup, W. B., Comparative pharmacology of voltage-dependent sodium channels, Brain Research, 135 (1977) 37-53. 18 Wechsler, W., Kleihues, P., Matsumoto, S., Zulch, K.J., lvankovic, S., Preussmann, R. and Druckrey, H., Pathology of experimental neurogenic tumo1~,chemically induced during prenatal and postnatal life, Ann. IV. Y. Aead. Sci., 159 (1969) 360-408.