Electrical excitability and chemosensitivity of mouse neuroblastoma x mouse or human fibroblast hybrids

Copyright All righfs

8 1973 by Actrdemic Press, Inc. of reproduction in any form reserord

Experimental Cell Research 79 (1973) 199-212

ELECTRICAL EXCITABILITY AND CHEMOSENSITIVITY OF MOUSE NEUROBLASTOMA X MOUSE OR HUMAN FIBROBLAST HYBRIDS J. H. PEACOCK,1 F. A. McMORRW

and P. G. NELSON

Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md 20014, and Department of Biology Yale University, New Haven, Corm. 06510, USA

SUMMARY Intracellular microelectrode recording techniques were used to measure passive membrane properties, electrical excitability and chemosensitivity of mouse neuroblastoma cells and somatic cell hybrids formed between these cells and either L cells or human diploid fibroblasts. Different clones of the hybrid cells showed varying degrees of neuronal or fibroblasti; membrane-differentiated function; a selection technique involving incubation of the cells with aminopterin gave quite homogeneous non-dividing populations of cells within a given clone of the neuroblastoma x L cell hybrids. Despite relatively uniform chromosomal numbers within a given clone, the neuroblastoma x human fibroblast hybrids were morphologically and electrophysiologically heterogeneous. The possibility is considered that this may represent the effect of variable segregation of the human chromosomal complement.

In the past few years the mouse C-1300 matic cell hybrids between these very difneuroblastoma has proven to be a useful cell ferent types of cells [8-9j. culture system for the study of such neuronal In the present study, another series of characteristics as action potential genera- neuroblastoma x L cell hybrids (N x L) tion, chemosensitivity, synthesis and destruc- hybrid clones has been examined with regard tion of neurotransmitters and the formation to morphology and electrical and chemical of cell processes [l-5]. A number of clones excitability. Procedures for selection of nonof the neuroblastoma have been isolated and dividing cells have been carried out to procharacterized as to their biochemical, elec- duce populations which were as stable and trical and morphologic properties [6]. Methods homogeneous as possible. In addition, hyfor selecting stable, well differentiated non- bridization has been accomplished between dividing populations of neuroblastoma cells the mouse neuroblastoma and a human diphave been developed [7]. Enzyme deficient loid fibroblast line (N x M hybrids). We have mutants of the neuroblastoma line have been sought to address three questions: (1) Can used with mouse L cells having comple- we obtain a number of clones of N x L hymentary enzyme deficiencies to produce so- brids which are homogeneous within each clone but which exhibit a wide range of neu--robiologic properties between the different 1 Present address: Department of Neurology, Stanclones? (2) Will the properties of process ford University Medical School, Palo Alto, Calif. formation, electrical excitability and chemo94301, USA. z Present address: Department of Biology, Massachusensitivity be expressed coordinately or will setts Institute of Technology. -_ _ Boston. Mass. 02139. these properties be independently controlled? USA. Exptl Cell Res 79 (1973)

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(3) Will hybrid clones in which a normal diploid fibroblast has been chosen as the nonneural parent show patterns of phenotypic expression similar to those exhibited by hybrids utilizing aneuploid L cells? MATERIALS AND METHODS The following cell lines were used in the present study: (a) Neuro-2A (N2A), a clone of the C-1300 mouse neuroblastoma [lo]; (b) NA, a subclone of N2A, and deficient for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Klebe, unpublished). Reversion frequency from HGPRT- to HGPRT+ is 1-2 x lo-@; (c) LM (TK-), a non-reverting thymidine kinase deficient (TK-) clone of L cells [1 I]; (d) MRC-5, a strain of normal diploid human fibroblasts isolated from the skin of an adult male. The tissue culture media used in this work consisted of 90 % Dulbecco-Vogt modified Eagle medium (DMEM) and 10% gamma globulin free newborn calf serum + 100 units/ml of penicillin + 100 &ml of streptomycin (Medium A) or 90 % DMEM and 10 % fetal calf serum with penicillin and streptomycin in concentrations of 10 units/ml and 10 pg/ml respectively (Medium B). All hybrid and parental clones had been carried through at least 3 passages in Medium B prior to electrical study. Medium B was prepared weekly, tested bacteriologically and used within 10 days. The production and characterization of the N x L and N x M hybrids will be described in detail elsewhere (McMorris. In preparation). Briefly, parental cells were mixed and allowed to attach to the surface of plastic tissue culture flasks (Falcon Plastics Co.). Fusion was induced by the addition of 1 000 hemoglutinating units of /3-propiolactone-inactivated Sendai virus, following the procedure of Klebe et al. [12]. One to two hours after fusion, cells were dispersed with trypsin and diluted into culture flasks in Medium A supplemented with 10e4 M hypoxanthine, 4 x lo--’ M aminopterin and 1.6 x 1O-6 M thymidine (HAT medium of Littlefield [13]). Both LM (TK-) and HGPRT- NA parental cells fail to grow in HAT medium because of their respective enzyme deficienties, but hybrids formed from these two lines grow because of enzyme complementation in the hybrid cells. MRC-5 cells do not have enzyme deficiencies which prevent growth in the HAT medium; they were selected against on the basis of their growth characteristics. Hybrid colonies appeared 34 weeks after fusion and well isolated clones were picked with cloning rings by the method of Puck et al. [14]. The hybrid nature of all clones was confirmed by karyotype and isozyme analyses. Both parental and hybrid forms of glucose phosphate isomerase [15] were detected in all N x L clones on starch gel electrophoresis. Similarly, all N x M clones showed both parental and hybrid forms of glucose-6-phosphate dehydrogenase [16] and of other enzymes showing easily identifiable human-mouse differences. Hybrid cell lines were maintained in the HAT selective medium except as detailed below. Exptl CeN Res 79 (1973)

For electrophysiologic study cells were dissociated with trypsin (0.25 % in Puck saline Dl) and innoculated into 60 mm (21 cm? plastic tissue culture mates (Falcon Plastic Corp.), and when in a suitable-state, were placed on a special stage of a Leitz inverted phase contrast microscope. Technique for intracellular recording and stimulation of cells in culture have been described in detail [17]. Electrical studies were made under a variety of culture conditions: (1) Cells were grown to confluency and studied within a few days, during which Medium B was changed frequently to prevent accumulation of acidic metabolites. (2) cells were plated at a density of 2 x lo8 cells/plate in DMEM lacking serum, which greatly slows cell division. (3) Cells from confluent flasks were plated at 2 x lo8 cells/plate and were maintained in Medium B + 4 x lo-’ M aminopterin. This medium kills dividing cells and selects a small f rat t’ton (about 5-10%) of the original population which was not dividing [7]. (4) Confluent cultures in 250 ml Falcon flasks were exposed to 5 000 rads of X-irradiation. This produces a population of nondividing cells. One to two weeks after X-irradiation these cells were dissociated with trypsin (as above) and plated at a density of 24 x lo5 cells/60 mm dish.

RESULTS Parental cell lines: NA and LM clones

Since neuroblastoma cells in the logarithmic phase of growth are technically difficult to study electrophysiologically and may be functionally less differentiated at least as far as electrical activity is concerned, we elected to study all cell types in the non-dividing, stationary state. For both parental cell lines used in the mouse-mouse hybrids, namely the NA mutant neuroblastoma cell (HGPRT-) and the LM fibroblast (TK-), we used Xirradiation (as described above) to produce stationary cultures. The two parental types were clearly and qualitatively different in at least four regards, as illustrated in figs 1 and 2. The neuroblastoma parent, when cell division is stopped by X-irradiation, is capable of developing an elaborate set of cell processes which extend for several hundred microns from the large cell body (fig. 1A). These cells are electrically excitable and respond to stimulating currents by generating action potentials (fig. 1C). Approx. 15% of the NA cells recorded from exhibited repe-

Electrical excitability

and chemosensitivity of cells

B.

20 1

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“Lh 29 Sep’71 200/~M Fig. 1. Abscissa: sec. (A) NA neuroblastoma cell, X-irradiated; (B 1-5) Intracellularly recorded responses to short pulses of acetylcholine delivered to the cell surface at the locations indicated in (A); (C) Action potential (lower trace) elicited by intracellularly applied current pulse (upper trace).

titive firing of action potentials either by penetration of the cell with the microelectrode (presumably due to critical injury depolarization) or during prolonged stimulation by currents applied through the microelectrode. The neuroblastoma parent cells were responsive to iontophoretically applied acetylcholine and both depolarizing (excitatory) and hyperpolarizing (inhibitory) responses were seen. Fig. 1B shows that both types of responses could be elicited from the same cell and that in this case a definite topographical organization of the responses was seen. Acetylcholine applied to the cell body

elicited a depolarizing response (D response) and acetylcholine (ACh) applied to any of the three cell processeselicited a hyperpolarizing response (H response). Combined D-H responses were obtained with ACh application to the region of the soma-processjunctions. Electrical or mechanical stimulation of this or other NA cells did not elicit the electrical response characteristic of LM and other L cells (see below). The LM (TK-) parent produced few if any processes under any culture conditions that we have used, and of over 100 L cells studied, we have never seen any evidence of action Exptl Cell Res 79 (1973)

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IO June ‘71 Fig. 2. Abscissa: C, F, set; B, E, msec; ordinate: B, E, membrane potential, mV. Morphology and electrical responses of neuroblastoma and L cells. (A) Microphotograph of NA neuroblastoma cells incubated in Medium B and at confluency; (B) Responses intracellularly recorded from cell shown in (A) to depolarizing (B,) and hyperpolarizing (B8) current pulses. Note action potential elicited by depolarizing current; (C), Same cell as (A) and (B). Hyperpolarizing voltage change (upper trace) elicited by pulse of acetylcholine applied iontophoretically (lower trace) from a pipette located just outside the cell being recorded from. ACh pulse about 40 nA; (0) LM (TK-) L cell mutant in Medium B and at confluency; (E) Responses as in (B) but showing only passive responses to depolarizing current; (F) Hyperpolarizing activation response in same cell as (D). Upper trace is the voltage record while the lower trace shows stimulating current (about 30 nA) and small current pulses used to measure cell resistance. Note slow hyperpolarization following the large stimulating pulse of current and the decreased cell resistance accompanying the hyperpolarization which is indicated by the decrease in amplitude of the voltage change produced by the small pulses of current indicated on current trace.

Expti Cell Res 79 (1973)

Electrical excitability

potential generation or delayed rectification. The LM (TK-) L cell clone, as well as other strains of L cells, exhibits an active electrical response to mechanical and electrical stimulation [18]. This consists of a large 3-5 set increase in membrane potential which is accompanied by an increase in the cell membrane permeability, largely to potassium ions. We have termed this the hyperpolarizing activation or H. A. response. This response is largely lacking in neuroblastoma cells, although we have seen some features of the response in a few percent of the several hundred neuroblastoma cells from which we have recorded. No depolarizing responsesto iontophoretically applied acetylcholine have been obtained from L cells, but in 20 % of these cells a membrane hyperpolarization can be elicited by iontophoretically applied ACh [19]. The X-irradiation selection technique results in cells with a wide variety of morphologic variants in the neuroblastoma cultures and it seemed desirable to compare the NA and LM lines in another condition. When cells are allowed to grow to confluency, cell division is greatly reduced compared with culture in the logarithmic phase of growth. Electrical activity of the NA cells is substantially reduced relative to the X-irradiated cultures, but the qualitative electrophysiologic differences between the NA and LM clones is maintained (fig. 2). Here cell morphology of the two cell types is not very different (fig. 2A, D) but some degree of action potential generation is still present in the NA line (fig. 2B) and absent in the LM (TK-) line (fig. 2E). The H.A. response occurs in the LM (TK-) line (fig. 2F) but not in the NA line (not shown) and acetylcholine responses occur in the NA line (fig. 2C) but not in the LM cell (not shown). Note the substantial difference in the time course of the ACh response in the neuroblastoma cell

and chemosensitivity of cells

203

as compared with the H.A. response in the L cell. Both NA and LM (TK-) cells were incubated in Medium B containing 4 x lo-’ M aminopterin. With the N-18 clone of the mouse neuroblastoma this has proven to be a satisfactory method of obtaining a stable population of electrically excitable cells with richly developed cell processes.A negligible portion of the NA and LM (TK-) cells survived this procedure, however, so that no studies of these cells could be done under these conditions. Neuroblastoma x L cell hybrids

A major purpose of the present work was to examine different lines of NA x LM (TK-) hybrid cells. Eight lines of cells which had been colony cloned from the hybridization flasks have been examined 15 to > 50 generations after fusion. In sharp contrast to the parental lines, a substantial number of cells of all hybrid lines survived incubation in Medium B +4 x IO7 M aminopterin and a stable population of well developed cells could be obtained for comparative study of the different hybrid lines. Although the aminopterin selection technique resulted in a homogeneous population of cells within each of several of the clones, the clones were strikingly different from one another. This is illustrated in fig. 3 where cells from early passage (about 15 generations after fusion) cultures of clone NL-I-8 and NL-I-3 are compared. The cells from NL-1-8 exhibit large plump refractile cell bodies and a rich proliferation of cell processes (fig. 3A). All of the cells recorded from had electrically excitable membranes and examples of action potentials recorded from 4 different cells are shown in fig. 3B. By contrast the cells from NL-I-3 are flatter, have few or no processesand very weak action potential generation, if any. The subExptl Cell Res 79 (1973)

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,.-’ \ ,,,,I’ 3

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50

I 100

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Morphologic and electrical characteristics of two different strains of NA x LM (TK-) hybrids. (A), Examples of a neuron-like hybrid line NL-I-8 after incubation in aminopterin (4 x lo-’ M) containing DMEM -t 10 % FCS for 10 days; (B1-4) Four examples of action potentials generated by different cells of the NL-I-8 clone in response to stimulating current pulses; (C) Examples of more fibroblastic hybrid line NL-I-3 under culture conditions similar to (A); (01-4) Four examples of nearly passive behavior of 4 different cells from clone NL-I-3 when stimulated by depolarizing pulses of current. Note great similarity of examples in (B) and in (D) and substantial difference between (B) and (D). Calibration pulses at beginning of all traces in B, D represent 1OmV.

stantial delayed rectification (the sag in the membrane voltage records in fig. 34-J confirm that these are indeed hybrid cells, since L cells do not exhibit delayed rectification (fig. 24. Table 1 lists some quantitative electrophysiologic data from early passagecultures of a small population of cells from these two lines. These populations were selected as representing the most stable and technically Exptl Cell Res 79 (1973)

best quality recordings and to reflect the highest degree of electrical activity of which the lines were capable. The membrane time constant, which is a function of membrane specific resistivity and is a sensitive indicator of cell damage was similar in the two groups of cells. Membrane potential was lower and cell resistance was higher in NL-I-3 than in NL-I-8. We have taken the maximum rate of rise of the action potential as a quantitative

Electrical excitability

and chemosensitivity of cells

205

Table 1. Comparison of electrical properties of 2 N x L hybrid clones

Clone

Resting membrane potential (-mV

Membrane time constant (mse4

Input resistance WW

Maximum action potential rise rate w/v

NL-I-3 NL-I-8

23 37

15 16

51 28

2 32

measure of electrical activity. This was 16 were relatively small. The different classesof times higher in NL-I-8 than in NL-I-3. Over clones exhibited very different rates of rise 90 ‘X0of the cells in NL-I-8 exhibited well of the action potentials and incidences of developed action potentials, while none of repetitive firing of action potentials. These the cells in these NL-I-3 cultures exhibited indices of electrical excitability correlated well greater activity than that shown in fig. 3 D,-,. with the morphologic property. An inverse The hybrid lines could be grouped into relationship was seen with regard to the three rather distinct categories. Five of the fibroblastic characteristic of H.A. generation. lines were relatively homogeneous and re- H.A. responseswere seenin the neuronal class sembled the neuroblastoma parent as far as of hybrid clones, but this was present in electrical and morphologic characteristics only 10% of these cells while 2/3 of the cells were concerned. These will be termed ‘neu- of the fibroblastic line of cell exhibited the ronal’ lines, Two of the lines were eachhet- response. This latter incidence is nearly as erogeneous, with some cells in each resemb- high as in the LM line itself. Depolarizing ling the NA parent and some the NL (TK-) responses to acetylcholine were seen only in parent, both morphologically and electro- the neuronal lines, and the failure to find any physiologically. The remaining two lines were acetylcholine D responses in the fibroblastic homogeneous and resembled the LM (TK-) hybrid lines is consistent with their low parent. capability of generating action potentials. We have assembled data with regard to The ACh elicited hyperpolarization in LM the morphologic and electrophysiologic (TK-) cells was similar to that of the electriccharacteristics of the NA and LM (TK-) par- ally produced H.A. response and both were ents and the hybrid lines in table 2. The substantially longer than the ACh-elicited clones of hybrids have been grouped as de- hyperpolarization in neuroblastoma cells. A scribed above into neuronal, mixed and comparison of the latency, total duration and fibroblastic classes. The average number of duration at l/2 maximum amplitude for the cell processes which were longer than one various responses is provided in table 3. It cell diameter was taken as an index of mor- seems likely that the nature of the ACh phologic differentiation and the clones varied response in L cells may be quite different significantly in this regard. It is clear that from that in neuroblastoma cells. neuronal lines have the largest number of cell processesper cell with less in the mixed Neuroblastoma x human fibroblast hybrids clones and still less in the fibroblastic lines. The MRC-5 line of human fibroblasts conDifferences in resting membrane potential sisted of very flat, thin, rapidly growing cells Exptl Cell Res 79 (1973)

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Table 2. Electrophysiologic No. of processes > 1 cell diameter

Clone

Neuroblastoma parents N2A & NA 3.3 No. of cells 15

properties

Resting membrane potential ( - mV)

of neuroblastoma, L cell and hybrid cell lines

Maximum dv/dt W/S)

Repetitive firing ( %I

36 45

18

20

32 119 26 30 27 37

28

21 64

0

Hyperpolarizing activation ( %)

D

6

0

16

Acetylcholine response ( X)

8

D-H

H

11 36

17

0 83 0

18

Total cells

45

N x L Hybrids

2.7 87 1.2 27 0.5 27

Neuronal No. of cells Mixed No. of cells Fibroblast No. of cells

22 54

8

12 16

1

0 5

11 45 55 13 67 24

10

97 64

0

0

119 28 18

0

0 18

30 0 37

L cell parent

LM No. of cells

co.5 27

0

15

20

0

64

64

Maximum dv/dt refers to the maximum rate of change of the membrane potential with respect to time during the rising phase of the action potential evoked with depolarizing current pulses. D, D-H and H acetylchohne responses are depolarizing, combined depolarizing-hyperpolarizing and hyperpolarizing responses respectively.

which we were unable to study electrophysiologically because of technical limitations. Non-dividing cells could not be selected by the aminopterin technique and cells in confluent cultures were too thin for satisfactory impalement. Two lines of hybrids formed between the NA neuroblastoma and the MRC-5 human diploid fibroblasts have been examined. These hybrid lines also produced a stable populaTable 3. Hyperpolarizing

NA & NL Hybrids Acetylcholine H.A.

tion of non-dividing cells when incubated in Medium B +4 x lo-’ M aminopterin and the electrophysiologic studies have been done on such preparations. The cells are heterogeneous both morphologically and electrophysiologically; the diversity of morphologic types seenon a single plate of a N x M hybrid line is illustrated in fig. 4. A hybrid cell which exhibited full expression of neuroblastomalike electrophysiologic properties is shown in

response time course Latency (se4

Total duration (W

Duration at l/2 amplitude (set)

0.6 1

3 7

1.7 5

8 7

4.2 1.0

;

5.0 5.8

3:

No. of cells

L Cells H.A. Acetylcholine Exptl Cell Res 79 (1973)

Electrical excitability

and chemosensitivity of cells

201

200 pM Fig. 4. Examples of diverse morphologic types found on the same plate of mouse (neuroblastoma) x human (fibroblast) hybrids (N x M). 14 -- 731801

Exptl Cell Res 79 (1973)

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&4

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Fig. 5. Abscissa: sec. Morphology and electrical response of mouse (NA) x human (fibroblast) hybrid NM VII-lo. Culture was in Medium B + aminopterin, 4 x 1O-7M. (A) Microphotograph of cell whose responses are shown in (B-D); (B l-4) Responses to pulses of applied acetylcholine at positions on cell surface corresponding to the location of the traces. (C, 0) Repetitive generation of action potentials interrupted by hyperpolarization (upper traces) produced by ACh pulse (ca. 30 nA) (lower traces). (0) was recorded 9 h after (C). Time marks in B-D represent 1 sec. Voltage calibration in B applies to C, D.

figs 5 and 6. This cell generated repetitive action potentials for extended periods and the cell was tested over a 12 h period in the chamber of the electrophysiologic apparatus. Its electrical properties were little changed over this time period (fig. 5C, D). This cell developed depolarizing responsesto ACh applied to the cell body and combined D-H responsesto ACh applied to the cell process. The D-response was largely blocked by iontophoretically applied d-tubocurarine (fig. 6). Depolarizing responses were seen in 16 % of Expti Cell Res 79 (1973)

the cells of the NM hybrid lines tested and either an H or D response to ACh was seen in 31 % of the cells tested. Some 16 % of the cells developed H.A. responses upon adequate stimulation and in one cell both an action potential and an H.A. response was seen. Responsiveness to acetylcholine was not always correlated with well-developed action potential generation nor with a neuronal morphology although this generally was the case. Fig. 7 shows a flat NA x MRC-5 hybrid cell with no processes. This cell ex-

Electrical excitabifity

hibited only a slight active response to depolarizing current (fig. 7 C) although delayed rectification was prominent (upward trace in fig. 78) and the response was clearly not that of a fibroblast. This cell did respond to acetylcholine with a hyperpolarizing response (fig. 7 E) but did not exhibit an H.A. response (fig. 70). The heterogeneity of form and function noted above in the N x M hybrids could have a number of causes. One cause might be that the hybrid lines are of multicellular origin. The origin of each N X M clone from a flask with only one large thriving colony and the karyotypic uniformity among cells of these hybrids minimizes but does not rule out this possibility. The observed heterogeneity could be due to a variable degree of differentiation of genetically homogeneouspopulation. Finally, the possibility exists that the hybrid cells are segregating out human chromosomes bearing regulatory genes specifically affecting one or more components of neuronal function. Confirmation of this possibility awaits electrophysiological and detailed karyotypic analysis of stable subclones. It is interesting that the two N x M hybrids examined, NM-V-II and NM-VII-IO, are more homogeneous with respect to chromosome number than N x L hybrid clones which were functionally more homogeneous. Comparison of standard deviations indicate a tighter distribution of chromosome number in the N x M than in the N x L clones (table 4). DISCUSSION It is clear that hybridization of neuroblastoma cells with non-neuronal lines [8] can generate a number of hybrid cell lines each of which is quite homogeneous. The properties of the different hybrid lines may differ markedly, one from the other, with regard to a number of neurobiologic properties (tables 1, 2; fig.

and chemosensitivity of ceffs 209 B.

A. CURRENT

]lnA

1 I

Fig. 6. Abscissa: sec.

Effect of iontophoreticallv applied n-tubocurarine on depolarization produced-by-l&se of acetylcholine in cell illustrated in fig, 5. In (A) and (B). line 1 represents current being passed’ through intracellular electrode to measure cell membrane resistance; line 2 represents transmembrane potential. First deflection is a 10 mV, 10 msec calibration pulse; first downward deflection is reponse to current pulse shown on line 1, and the following upward deflection is depolarization evoked by a pulse of acetylcholine. Line 3 records applied acetylcholine pulse; line 4 represents applied d-Tubocurarine (dTC) and line 5 is a 1 set time calibration. Series (A) shows ACh response elicited during an application of dTC and (B) shows large response elicited in absence of applied d-Tubocurarine. Artifacts occur on voltage trace on (A) at onset and termination of dTC pulse.

3). A five-fold range of variation in the degree of cell process formation was seen in different hybrid lines and more than a 20fold difference in electrical excitability. A similarly large variation in the expression of an electrical response characteristic of L cells was seen between clones (table 2). In a previous study of the changes in membrane potential elicited by iontophoretically acetylcholine, only about one-third of the neuroblastoma cells exhibited any responsiveness [20]. This result is confirmed in the present study. The incidence of ACh responses in the hybrid lines varied from zero to approximately the same incidence as in the parent neuroblastoma line. Despite the fact that ACh responsiveness is not seen in the Exptl Cell Res 79 (1973)

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(El, C, E) (left) mV; (right) nA. Morphology and electrical responses of N x M hybrid, NM VII-lo. (A) Photomicrograph of cell recordings from which are shown in (B-D); (B) Upper traces are currents passed through impaling microelectrode to hyperpolarize and depolarize the cell. Lower traces show membrane potential elicited by those currents. No action potential occurs, but marked asymmetry of voltage response demonstrates marked delayed rectification in this cell; (C) In the presence of steady hyperpolarizing current a depolarizing current pulse still does not elicit an action potential. (D) Strong electrical stimulation (lower trace) does not elicit an H.A. response (upper trace). (E) Acetylcholine pulses (lower trace) elicit hyperpolarizing responses (upper trace).

majority of neuroblastoma cells, this property is highly differentiated one in a small minority of the cells. Two types of receptors may be synthesized in the same cell, as indicated by differential blockade of the D Exptl Cell Res 79 (1973)

and H responses by curare and atropine [20]. The interaction of ACh with these receptors produces very different changes in membrane permeability and so elicits either depolarization or hyperpolarization of the

Electrical excitability Table 4. Chromosome counts of hybrid lines Chromosome number

S.D.

Clone

No. of cells

NM-V-II NM-VII-10 NL-3 NL-7A

91 92 114 151

4 4 7 10

34 30 49 41

membrane. These receptors and the mecbanism responsible for the membrane perme-

ability changes are assembled in a spatially highly organized fashion on the surface of the cell so that, as in fig. 1, cell processes respond to ACh differently than does the cell body. In no case of well over 100 N x L hybrid cells tested were D-H responses to ACh seen. This may well represent a sampling problem, since the incidence of compound responses in a large series of neuroblastoma cells was only 5-10 %. Alternatively, if the compound response represents the most differentiated state of the neuroblastoma line, the failure to find such responses may represent some lack of this differentiation in the hybrids. As shown in table 2 there was in general a coordinate expression of the neuroblastoma or 1, cell characteristics in the different lines of hybrid cells. Thus, process-rich cells tended to be electrically excitable, to be lacking the fibroblastic electrical response and to have a relatively high probability of responding to acetylcholine. In the mixed group of clones a high degree of heterogeneity in cell properties was seen, but in these cells also neuronal properties tended to be expressed together in a given cell or to be uniformly unexpressed. This coordinate expression of neuronal phenotypes in hybrid cells will be discussed further in a future publication (F. A. McMorris. In preparation). The two lines of mouse neuroblsstoma x human fibroblast hybrids were heterogeneous.

and chemosensiticity of cells

211

The chromosomal and enzyme analyses establish the hybrid nature of these lines and electrical evidence for both parental type; confirms this. It is an attractive hypothesis that the different cell types exhibit functional differences due to differential loss of chromosomes. Single cell cloning of these lines with correlative electrophysiologic and karyotypic analysis of the subclones will be of great interest in these lines. The full expression of the neuroblastoma characteristic occurred in some cells of the mouse-man hybrids. A high degree of electrical excitability and combined D and H responses to ACh were seen with a topographically organized distribution of the D and H receptors. The ACh response was seen in one cell that had very weak electrical excitability and a rather typically fibroblastic morphology. This raises the possibility that some segregation of neuronal properties might occur in the hybrid line which would greatly facilitate the study of the molecular basis for the various properties. We thank Mrs Marie Neal for excellent technical assistance in this work. F. A. M. thanks Dr Frank H. Ruddle for helpful discussions, encouragement and support. This research was supported by NIH Grant 5ROl-GM-09966 to F. H. Ruddle. Portions of the material uresented here have been oresented bv F. A. M. as p&t of the thesis requirement for the -degree of Doctor of Philosophy at Yale University. Present address of J. H. P. is Department of NeuroIogv. Stanford Universitv Medical School. Palo Alto, Calif. Present address bf F. A. M. is I&partm:nf of Biology, Massachusetts Institute of Technology, Boston, Massachusetts. -.

I

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