Control of the Na+, K+ -pump by nerve growth factor is essential to neuronal survival

Brain Research, 271 (1983) 263- 271

263

Elsevier

Control of the Na ÷,K ÷-Pump by Nerve Growth Factor is Essential to Neuronal Survival STEPHEN D. SKAPER and SILVIO VARON

Department of Biology, School of Medicine, Universityof California, San Diego, La Jolla, CA 92093 (U.S.A.) (Accepted December 14th, 1982)

Key words: Na + ,K + -pump - nerve growth factor - sensory neurons - survival

Recently we have shown that nerve growth factor ( N G F ) controls the performance of the Na +,K + - p u m p in its target ganglionic neurons in suspension cultures. In the present study, enriched neuronal preparations of embryonic day 8 (E8) chick dorsal root ganglia ( D R G ) were obtained by means of a differential attachment procedure using tissue culture plastic dishes. Neurons were routinely seeded into polyornithine-coated 16 m m culture wells in the presence of N G F . After 18 h, cultures were switched to media with or without N G F , and containing either 86Rb+ (as a tracer for K + ) or 22Na+ (as a tracer for Na ions). Over the next 12-15 h the cultures were assessed for numbers of surviving neurons and accumulated radioactivity. Cultured E8 chick D R G neurons fail to maintain their intracellular K ÷ concentration when deprived of N G F over 4 - 6 h. The NGF-deprived and K +depleted neurons reaccumulate K + within minutes of delayed N G F administration. The occurrence of this K + response in culture to added N G F parallels the response occurring in E8 neuronal suspensions, including the time of onset of irreversibility. Similar experiments performed with 22Na + indicate corresponding ionic behaviors for cultured E8 D R G neurons. These N G F controlled ionic responses in monolayer cultures occur for E7 and E 10 neurons, but not E 14 neurons and parallel the survival response to N G F of the same neurons. Blocking the pump performance by N G F deprivation leads to neuronal death. Identical resuits are obtained by addition of ouabain or omission of external K + in the presence of NGF. Partial reduction of pump performance by any one of these treatments leads to partial survival of the neuronal population in a precisely predictable manner. Therefore, control of the pump by N G F is an essential component of the N G F action on neuronal survival. INTRODUCTION

Nerve growth factor ( N G F ) is essential for the normal growth, development and maintenance of certain sensory and sympathetic neurons 5,~4. The mechanism whereby N G F exerts its effects remains to be elucidated. Such atrophic role of N G F would imply, however, that the factor acts to control some crucial function of its target neurons, in turn regulating quantitative or qualitative expressions of various cellular machineries~5-17. We have demonstrated previously that, in the absence of N G F and before irreversible changes take place, NGF-responsive sensory and sympathetic ganglionic cells in suspension lose their ability to maintain normal intracellular levels of Na + and K +2.8-1°. Addition of N G F to such factor-deprived cells results in a restoration of intracellular Na ÷ and K ÷ within minutes and in a coupled fashion. These and other data have 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

shown that N G F controls the performance of the Na +,K +-pump in its target ganglionic neurons 17. In the present report, we have examined the influence of N G F on the Na + and K + behaviors of monolayer cultures from embryonic day 8 (E8) chick dorsal root ganglionic ( D R G ) neurons. Enriched cultures of E8 neurons incubated with 86Rb+ (as a K ÷ tracer) were unable to maintain their intracellular K ÷ level when deprived of exogenous N G F over 4-6 h, but reaccumulated K ÷ within minutes of delayed N G F presentation. This K ÷ response exactly paralleled the response seen for enriched E8 neuronal suspensions. These consequences of the presence or absence of N G F were observed at E7 and El0 but not at El4, paralleling a previously observed survival response to N G F of E7 and El0, but not El5 chick D R G neurons in culture 12. Corresponding ionic behaviors with 22Na+ were also displayed. This monolayer culture system was

264 then used to examine the dose-response relationship of pump performance to that of neuronal survival under various conditions known to inhibit the Na ÷ ,K ÷ -pump. MATERIALS AND METHODS

Materials

HEBM: Eagle's basal medium (BME, Gibco) supplemented with 26.4 mM NaHCO 3, 33.3 mM D-glucose, 2 mM L-glutamine, and 100 units/ml penicillin. LEBM: HEBM modified to contain 2.6 mM NaHCO3 and 20 mM HEPES. HEBMFCS: HEBM with 10% (v/v) fetal calf serum (Irvine Scientific). LEBM-FCS: LEBM with 10% fetal calf serum. Rubidium-86 (carrier-free, 86RbC1, 1-8 mCi/mg, 1-5 mCi/ml) and sodium22 (carrier-free, 9.7 /~g Na/ml, 0.97 mCi/ml) were purchased from Amersham, Arlington Heights, III. Bovine serum albumin and HEPES were from Sigma Co., St. Louis, MO. Fiberglass filter discs (Whatman G F / A , 2.4 cm diameter) were obtained from Reeve Angel Co., Clifton, NJ. Nerve growth factor, 7S form, was prepared from the submaxillary glands of adult male mice j8. Concentrations of N G F were expressed in Biological Units (BU)/ml, where 1 BU/ml is the minimum concentration of 7S N G F providing for maximal survival of DRG neurons in culturO 1.19.All chemicals were reagent grade. Measurement o f N a ÷ and K + movements in monolayer cultures

Dorsal root ganglia from E7-E14 White Leghorn chick eggs were dissociated in HEBM-FCS as described previously ~9. Enriched neurons were prepared by means of a selective attachment procedure over a plastic surface, for 2 h in the absence o f N G F 2. The resulting cell suspension, containing predominantly unattached neurons (75-80% of all unattached cells) was centrifuged at 400 g for 5 min, and the pellet suspended in HEBM-FCS to 1.2 x 1@ neurons/ml. Multiwell plates (Falcon), containing 24 16-mm wells previously coated with polyornithine (0.5 ml/well of a 0.1 m g / m l solution in 15 mM borate, pH 8.4), were seeded with 0.5 ml of the neuronal suspension (60,000 neurons). All

culture wells contained 10 BU N G F / m l . Following 18 h incubation at 37 °C in a humidified atmosphere of 5% CO2-95% air, the cultures were washed 3 times with 0.5 ml HEBM-FCS without NGF. For the variable K + experiments, the washes used K +-free medium and the serum was first dialyzed for 24 h against K+-free HEBM. Individual wells then received 0.5 ml HEBM-FCS with or without 10 BU N G F / m l , and with the appropriate radioisotope (7.5 /~Ci/m122Na+ or 0.5/~Ci/m186Rb+). At different times, cultures were processed for determination of both neuronal numbers and accumulated radioactivity, as follows. The medium was removed and the cultures rapidly washed 3 times with LEBM containing 20 mM HEPES and 0.5% (w/v) bovine serum albumin. The cultures were fixed with 2% paraformaldehyde in phosphatebuffered saline and quantitatively evaluated by direct neuronal counts under phase microscopylg. The fixative was removed and saved: Cells were treated with 0.5 ml 10% (w/v) trichloroacetic acid (TCA) for 10 min, and the cells washed once with 0.5 ml 10% TCA. Both TCA extracts and the fixative solution were combined and counted for radioactivity by liquid scintillation techniquesv. Replicate culture wells lacking N G F received N G F at sampling times, and were processed as above following an additional 30-60 min incubation. In some instances, neurons were seeded directly with the addition of radioisotope in the presence or absence of N G F and processed as above, at the different times. Measurement o f N a + and K + movements in cell suspensions

Enriched suspensions of embryonic D R G neurons were prepared in HEBM-FCS as described in the preceding section. Following centrifugation, cell pellets were suspended in LEBM-FCS to 3.5 x 1@ neurons/ml. 86Rb+ o r :2Na+ was added at 0.5 ~Ci/ml or 7.5 ~Ci/ml, respectively. Replicate 0.1 ml aliquots were set up in 12 x 75 mm tubes containing 10 BU N G F / m l , or no NGF. All samples were incubated in a 37 °C shaking water bath. At different times during the course of the incubations, cells were transferred to G F / A filters by adding to

265 the tubes 2 ml o f L E B M containing 20 m M H E P E S and 0.5% bovine serum albumin. Filters were washed with an a d d e d 2 ml o f this m e d i u m 5 times, u n d e r suction, dried and c o u n t e d 7. Replicate aliquots o f cells deprived o f N G F received the factor at these times, and were sampled 3060 min later. Filter blanks consisted o f m e d i u m without cells but with radioisotope, carried through as were the cell samples.

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Effect of NGF on the maintenance o f high intracellular K + bv chick DRG neurons in monolayer culture In the experiments illustrated in Fig. 1, the intracellular m o v e m e n t s o f K ÷ were studied by the use o f the K + analog 86Rb+. W e have previously shown 2 that 86Rb+ m o v e m e n t s are a valid indicator o f N a + , K + - p u m p p e r f o r m a n c e . First using a suspension o f enriched D R G neurons, both equilibration and s u b s e q u e n t behavior o f 86Rb+ were c o m p a r e d in the c o n t i n u o u s presence and in the absence o f N G F (Fig. IA). In the presence o f N G F the neurons reached equilibration with the 86Rb+ after a b o u t 90 min and m a i n t a i n e d a constant intracellular K + concentration t h r o u g h o u t the 10 h pulse period. In the absence o f N G F , the neurons began to equilibrate with 86Rb+ but failed to reach the N G F supported level, reflecting the d e v e l o p m e n t o f a decreased N a + , K * - p u m p c o m p e t e n c e during this time, as well as the preceding 2.5 h period (during both o f which N G F was absent). It should be kept in mind that at the time 86Rb÷ was presented to the cells, they had already been deprived o f N G F for 2.5 h in the course o f the neuronal e n r i c h m e n t procedure. T h e n e u r o n s continued to lose K ÷ during the first 4 h o f the incubation with 86Rb+ (total incubation time without N G F = 6.5 h), with a stable low value m a i n t a i n e d after this time. T h e N G F - d e p r i v e d and K ÷ - d e p l e t e d neurons r e a c c u m u l a t e d K ÷ with minutes u p o n delayed N G F presentation, reaching the K ÷ levels seen in the N G F - m a i n tained neurons during the first 6 h. B e y o n d this time, the ability o f N G F to effect complete reversal o f the ionic defect steadily diminished,

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8SRb+ Pulse (hours) Fig. 1. Equilibration and retention of 86Rb+ by E8 chick DRG neurons under the influence of NGF. A: suspensions. Dissociated DRG cells were first subjected to a 2.5 h period (prior to 86Rb+ pulse) of NGF deprivation for selective attachment. At time 0 enriched neurons were incubated in LEBM-FCS (3.5 x 105/ml)with 86RbCI (0.5 ~tCi/ml) and 10 BU/ml NGF (0) or no NGF (©). At different times NGF (10 BU/ml) was added to replicate samples of NGF-deprived cells (A) for 30 min of additional incubation. Radioactivity accumulated was measured as described under Methods. B:monolavers. Enriched neurons were cultured for 18 h with NGF (10 BU/ml). At that time they were switched to fresh medium with (0) or without (©) NGF, and pulsed with 0.5 /~Ci/ml 86RbCI. Replicate cultures of NGF-deprived cells received NGF at the same sampling times (&) and were processed 30 min later. Each point represents the average of duplicate measurements from two experiments. until by 10 h (12.5 h o f total incubation) very little reversibility remained. These results were similar to what had been o b s e r v e d previously for unfractionated cell dissociates o f E8 chick D R G over a m o r e limited (7 h) time span l°. C o r r e s p o n d i n g experiments were then carried

266 out using monolayer cultures of enriched E8 chick D R G neurons, which had been allowed to attach in the presence of N G F for 18 h before receiving fresh medium without or with NGF. At the time of medium change, 86Rb+ was added. In the presence of N G F , neurons accumulated S6Rb + t o reach equilibrium again within 90 min, while NGF-deprived neurons lost their K + contents over the following 6 h and reaccumulated it within minutes, if once again supplied with NGF. These K ÷ responses to exogenous N G F in monolayer culture parallel the responses seen for enriched E8 neuronal suspensions, including the time at which irreversibility of the K + defect first becomes detectable. Absolute values of 86Rb+ accumulation were very similar for neurons both in monolayers and in suspensions. Also, the slopes of the lines representing development of: (i) the defect in intracellular K + control, and (ii) the time course of irreversibility of the K + defect were similar both to each other and across the two systems (monolayer culture or cell suspension). Similar experiments carried out with 22Na+ indicated corresponding ionic behaviors (data not shown).

Ionic responses to NGF as a function of embryonic age The dependence on N G F by chick D R G neurons varies with embryonic age. Recently we have shown ]2.~3 that developmental changes in NGF-dependence also apply to the ionic responses. Exogenous N G F becomes less and less needed (but not less effective) for ionic control in suspensions of intact and dissociated DRG, as embryonic age increases. The monolayer culture system was, therefore, further examined using D R G neurons from several embryonic ages. For these experiments it was important to be able to use the neuronal cultures directly on the seeding day. Preliminary experiments showed that E8 D R G neurons in serum-containing medium could also be seeded directly into the polyornithine-coated culture wells with or without N G F , and pulsed at that time with radioisotope. These cultures can already be processed for cell numbers and radioactivity accumulated after a minimum attachment period of 3 h, as described un-

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Fig. 2. Effect of embryonic age on Na + behaviors of chick DRG neurons in monolayer culture. Enriched neuronal preparations from E7, El0 and El4 DRG were seeded into 16 mm wells (60,000/well) in HEBM-FCS. Some cultures received, at time zero, N G F (10 BU/ml) without or with ouabain (0.2 mM). Replicate NGF-deprived cultures were supplied with N G F after 6 h, and processed 30 min later. All cultures were pulsed with 22NaCI (7.5,uCi/ml) at time zero. Each point represents the average of triplicate measurements from each of two separate experiments.

der Methods for cultures examined only after the first 18 h. Fig. 2 summarizes the results of experiments using 22Na+ as tracer for the total 6 h period in the presence or absence of NGF. Some cultures with N G F also received ouabain (0.2 mM) at time 0, while some NGF-free cultures received N G F at 6 h and were processed 30 min later. After 6 h of N G F deprivation, E7 and El0 neurons accumulated 2 2.5 times more 22Na+ than did corresponding NGF-treated cultures. Administration of N G F at this time resulted in a return of intracellular Na ÷ concentrations to normal levels. Intracellular Na + increased 2-3-fold between E7 and El0 in both NGF-maintained and NGF-deprived neurons, reflecting an increase

267 in intracellular volume during this development period 13. Neurons from El4 D R G showed little further age-related increase in their Na ÷ content. They also showed no increase in intracellular Na + when deprived of N G F . At each age, ouabain treatment of NGF-supplemented cultures caused a 2-3-fold increase in 22Na+ accumulation, indicating the continued presence of an Na ÷ accessible space sensitive to ouabain (Na ÷,K ÷-pump controlled). These consequences of the presence or absence of N G F parallel a previously observed 12 survival response to N G F of chick D R G neurons at these very embryonic ages.

Effect of N G F deprivation on neuronal death and Na ÷ accumulation in monolayer cultures In these experiments, enriched E8 chick D R G neurons were seeded on a polyornithine substratum in HEBM-FCS plus NGF. After 18 h the cultures were washed free of N G F and fed with fresh medium lacking N G F . Over the next 21 h the course of neuronal survival was followed, as was the Na ÷ content measured with the tracer, 22Na+. As illustrated in Fig. 3, neuron number remained constant for the first 9 h, after which it declined to 50% of the starting value by 15 h, and to only 5% by 21 h. The accumulation of22Na +,

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Fig. 3. Neuronaldeath and Na+ accumulationin monolayer cultures of E8 chick DRG neurons deprived of NGF. Enriched neurons were cultured for 18 h in 16 mm wells (60,000/well) in HEBM-FCSwith 10 BU/ml NGF. The cultures were washed extensivelywith NGF-free medium,and fed with fresh medium containing22NaC1(7.5/.tCi/ml). At the times shown cultures were analyzed for neurons/well (A), cpm/well (rq), and cpm/105 neurons (0) as described under Methods. Each point represents the average of 3-4 measurementsfrom each of two experiments.

expressed as cpm/culture, leveled off between 9 and 12 h, and declined from 12 h onward, paralleling closely the decline in the number of neurons between 12 h and 21 h. When expressed as cpm per 1@ neurons, 22Na+ accumulation also increased in a linear manner for 9 h in parallel with the total Z2Na+ content of the culture (since neuron number remained constant), and reached a constant level by 12 h, which it maintained throughout the rest of the time. Maximum cpm per neuron, then, was reached coincidentally with the first measurable decline in neuron number and was not affected by the subsequent decline itself. These data clearly demonstrate that the development of the defect in intracellular Na ÷ control precedes, and is independent of, the death of the cultured neurons.

Neuronal survival under conditions which block the Na +,K +-pump If activity of the Na ÷ ,K + -pump is essential to neuronal survival, then blocking the pump performance by means other than N G F deprivation (such as ouabain or the lack of external K + ) should also lead to neuronal death and do so even in the presence of NGF. Fig. 4 shows the survival in the presence of N G F (upper curve) and the death in its absence (lower curve, open circles) of neurons from E8 chick D R G cultured in monolayer, following an initial period with N G F of 18 h. When N G F is supplied together with 0.2 mM ouabain or with a medium lacking K +, neuronal cell death develops over the next 30 h with the very same time course as if the cultures were deprived of N G F in the absence of ouabain or the presence of external K +. The common feature of the 3 death-inducing treatments is a blocked pump performance. Thus, neuronal survival requires the ongoing performance of the Na ÷,K+-pump, and the N G F action to support the pump is essential for neuronal survival. Correlations between neuronal survival and Na +,K ÷-pump performance If pump performance controls neuronal survival, then partial reduction of pump performance should lead in a predictable way to partial survi-

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blocking the Na + ,K + -pump. Enriched neurons were seeded on polyornithine-coated 35 mm plastic dishes with 10 BU/ml of NGF. After 18 h the cultures were washed 3 times with NGF-free medium (K +-free medium for the no-K + experiment) and refed with medium containing: 10 BU/ml N G F (O); 10 B U / m l of N G F with 0.2 mM ouabain ( x ) or K+-free medium (A); no N G F (©). The number of surviving neurons was determined at selected intervals over a 48 h period after refeeding. Values represent the average of duplicate cultures from each of two experiments.

val of the neuronal population. The full (and fully reversible) effect of N G F deprivation on pump performance requires about 6 hours 8.1° (at which time no reduction in cell numbers is yet observable), and that on neuronal survival requires about 24 h (Fig. 4). Using K ÷ retention (86Rb+ as tracer) as a measure of pump activity, monolayer cultures of enriched neurons from E8 chick DRG were exposed to 86Rb+ in culture media containing varying concentrations of NGF, or maximal amounts (10 BU/ml) of N G F plus varying concentrations of ouabain, or maximal N G F in the presence of decreasing concentrations of K ÷. After 6 h and 24 h the cultures were processed for determination of neuronal cell numbers and radioactivity retained. The relative degree of pump control was found to be the same at 6 h and 24 h, even though neuronal survival greatly differed at these two times in all but the fully supported cultures (see Fig. 3). One such experiment, with varying concentrations of NGF, is shown in Fig. 5. Neuronal survival (measured as remaining cells per well) and pump performance (measured as 86Rb+ cpm per 1@ neurons) both exhibited overlapping sigmoidal-shaped behaviors in response to NGF. Other experiments, in which maximal N G F concen-

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Fig. 5. Effect of N G F concentration on neuronal survival and Na ÷,K ÷-pump performance. Enriched neurons from E8 chick DRG were cultured for 18 h with 10 B U / m l NGF. Cultures were then washed with NGF-free medium and refed with medium containing 0.5/~Ci 86Rb+/ml and variable concentrations of N G F (0-1.0 BU/ml). After 6 h and 24 h at 37 °C, the cultures were analyzed for numbers of neurons present and 86Rb+ cpm retained by the cells. Each point represents the average of two or three cultures from each of two separate experiments.

trations were used with variable ouabain or external K ÷, yielded similar relationships for pump performance and neuronal survival. Neuronal survival and pump performance from experiments such as that illustrated in Fig. 5 were then expressed as percentages of the difference between highest support (NGF, K ÷, no ouabain) and lowest support (no NGF, or high ouabain, or no K ÷). The results are illustrated in Fig. 6. Panel A demonstrates the direct proportionality between neuronal survival at 24 h and the extent to which pump performance was retained at 6 h when the concentration of N G F available to the culture was varied between 0 and I BU/ml. The calculated slope of 1.!0 compares to an idealized slope of 1.00 with a very strong coefficient of correlation (r = 0.98). Similar plots were derived by using optimal concentrations of N G F in the presence of 0-50 ~M ouabain (panel B) or of 0.03-10 mM KCI (panel C). Such plots gave, respectively, slopes of 1.04 (r = 0.96) and 0.94 (r = 0.97). These results verified the proposition that the degree of ultimate neuronal survival can be predicted several hours earlier by the degree of pump inhibition regard-

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less of the manner by which such inhibition has been imposed. DISCUSSION

Previous studies from this laboratory 2.8-m have shown that, in D R G and sympathetic ganglionic cells, N G F regulates the intracellular levels of Na ÷ and K ÷ by regulating the performance of the Na+,K+-pump. Those experiments were carried out on suspensions of unfractionated ganglionic dissociates in a simple buffered medium with salts and bovine serum albumin. For the purpose of examining the consequences for neuronal survival of alterations in Na+,K+-pump performance over longer time periods, it was necessary to establish a system capable of displaying both the ionic pump and the cell survival, NGF-dependent behaviors already described. In the present studies, we show that Na ÷ and K ÷ movements in enriched E8 chick D R G neurons cultured in monolayers are also controlled by NGF. The time course of development of the reversible ionic defect and the time at which irreversibility of ionic control is first observable were the same for E8 neurons whether examined as monolayer or as suspension cultures, thereby demonstrating both the generality of these NGF-related behaviors and the suitability of monolayer ganglionic cultures for their further investigation. The validity of these monolayer Fig. 6. Correlations between Na +,K +-pump performance and neuronal survival. Data were derived from curves like those described in Fig. 5. Control cultures received medium with 10 B U / m l N G F , 5.5 mM KC1 and no ouabain. The following experimental modifications were made: panel A, 01.0 B U / m l NGF, 5.5 mM KCI, no ouabain;panel B, 0-50 #M ouabain, 10 B U / m l NGF. 5.5 mM KCI; panel C, 0.0310 mM KC1, 10 B U / m l NGF, no ouabain. In the case of the variable K + experiment, the serum used had been dialyzed 24 h against K +-free HEBM. After 6 h and 24 h of incubation at 37 °C, the cultures were analyzed for numbers of neurons present and 86Rb + cpm retained by the cells. Pump performance and neuronal survival were calculated as a percent of the maximal difference between control and experimental cultures for each point. The slope of each line was determined from a linear regression analysis (see text for values of slope and coefficient of correlation). Each point represents the average of 2 or 3 cultures from each of two independent experiments.

270 cultures for the study of NGF-regulated ionic behaviors is further demonstrated by the experiments with neurons derived from DRG of different embryonic ages. The development of a Na ÷ defect in the absence of NGF, and its rapid reversal following delayed N G F presentation, occurs with neurons from E7 and El0, but not from El4 DRG - as already found for both K ÷ and Na ÷ with the corresponding cell suspensions t2. The ionic dependence on N G F by the DRG neurons changes with their developmental age along the same temporal pattern as does the neuronal dependence on N G F for their survival in culture. The experiments addressing the question of a correlation between Na÷,K÷-pump and survival effects of N G F clearly show that neuronal survival requires the performance of the Na÷,K+-pump. In turn, we have previously shown that pump performance requires the presence of N G F at its optimal biological concentrations. We can, therefore, conclude that control of the pump by N G F is an essential component of the N G F action on neuronal survival. This is not to say that the pump control by N G F is the first step in the mechanism of action of NGF. Nor can one state at present whether the action of N G F on the Na + ,K +-pump is the sole mechanism by which N G F controls neuronal survival. The demonstration that N G F control of the pump is a causal link to N G F control of survival defines two main questions for future investigations: (1) which of the immediate consequences of pump activity - changes in intracellular ionic concentrations, transmembrane REFERENCES 1 Becker, R. U., Mechanisms of growth Control Thomas, New York, 1981. 2 Boonstra, J., Skaper, S. D. and Varon, S., Regulation of Na +,K +-pump activity by nerve growth factor in chick embryo dorsal root ganglion cells, J. cell Physiol., 113 (1982) 28-34. 3 Chalazonitis, A. and Fischbach, G. D., Elevated potassium induces morphological differentiation of dorsal root ganglionic neurons in dissociated cell culture, Develop. Biol., 78 (1980) 173- 183. 4 Jaffe, L. and Nuccitelli, R., Electrical controls of development, Ann. Rev. Biophys. Bioenging., 6 (1977) 445476.

ionic gradients, or ATP consumption - are the ones essential for survival, and (2) what are the sequential steps leading from the N G F encounter with its receptor to the restoration and support of Na ÷ ,K ÷-pump activity. The monolayer system will permit one to ascertain the relevance for neuronal survival of the ionic imbalances which develop during N G F deprivation by: (i) mimicking such ionic imbalances in the presence of N G F and examining whether they also lead to neuronal death, and (ii) attempting to compensate for the ionic imbalances and ascertaining whether neuronal survival can be thus maintained in the absence of NGF. Conversely, the cell suspension system appears best suited for the investigation of the molecular events preceding pump control by NGF. The data reported here may have more general implications than with regard to the N G F phenomenon alone. One can speculate that monovalent cations may serve as 'second messengers' for several other agents regulating cellular behaviors via binding to surface receptors. Monovalent cations have, in fact, been noted to regulate other neuronal behaviors, such as maturation3 and shifts in transmitter mode z°, as well as various behaviors in other cells including cell proliferation 6, process elongation 4, and general growth and repair ~. ACKNOWLEDGEMENTS

This work was carried out with partial support from NINCDS Grant NS- 16349.

5 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 6 Rozengurt, E. and Mendoza, S., Monovalent ion fluxes and the control of cell proliferation in cultured fibroblasts, Ann. NYAcad. ScL, 339 (1980) 175- 190. 7 Skaper, S. D. and Varon, S., Nerve growth factor action on 2-deoxy-D-glucose transport in dorsal root ganglionic dissociates from chick embryo, Brain Research, 163 (1979) 89- 100. 8 Skaper, S. D. and Varon, S., Properties of the Na + exclusion mechanism controlled by Nerve Growth Factor in chick embryo dorsal root ganglionic neurons, J. Neurochem., 34 (1980) 1654- 1660. 9 Skaper, S. D. and Varon, S., Maintenance by nerve growth factor of the intracellular sodium environment in

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spinal sensory and sympathetic cells, Brain Research, 197 (1980) 379-389. Skaper. S. D. and Varon, S., Nerve growth factor influences potassium movements in chick embryo dorsal root ganglionic cells, Exp. Cell. Res., 131 (1981)353-361. Skaper, S. D. and Varon, S., Three independent biological assays for nerve growth factor: no measurable activity in human sera, Exp. Neurol., 76 (1982) 655-665. Skaper, S. D. and Varon, S., Ionic behaviorsand nerve growth factor dependence in developing chick ganglia. II. Studies with purified neurons of dorsal root ganglia, Develop. BioL, in press. Skaper, S. D., Selak, I. and Varon, S., Ionic behaviors and nerve growth factor dependence in developing embryonic chick ganglia. I. Studies with intact dorsal root ganglia, Develop. Brain Res., 3 (1982) 41 9-428. Varon, S., Nerve growth factor and its mode of action, Exp. Neurol., 48 (1975) 75 92. Varon, S. and Adler, R., Nerve growth factors and control of nerve growth, Curr. Topics Develop. Biol., 16 (1980) 207- 252.

16 Varon, S. and Bunge, R. P., Trophic mechanisms in the peripheral nervous system, Ann. Rev. Neurosci., 1 (1978) 327-361. 17 Varon, S. and Skaper, S. D., Nerve growth factor and neuronotrophic functions. In G. Burnstock, G. Urbova and R O'Brien (Eds.), Somatic and Autonomic Nerve-Muscle Interactions, Elsevier North-Holland, Amsterdam, in press. 18 Varon, S., Nomura, J., Perez-Polo, J. R. and Shooter, E. M., The isolation and assay of the Nerve Growth Factor proteins. In R. Fried (Ed.), Methods and Techniques of Neurosciences, Marcel Dekker, New York, 1972, pp. 203-229. 19 Varon, S., Skaper, S. D. and Manthorpe, M., Trophic activities for dorsal root and sympathetic ganglionic neurons in media conditioned by Schwann and other peripheral cells, Develop. Brain Res., 1 ( 1981) 73- 87. 20 Walicke, P. A., Campenot, R. B. and Patterson, P. H., Determination of transmitter function by neuronal activity, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 5767-5771.