Cell Tissue Res (1983) 228:641-648

Cell and Tissue Research 9 Springer-Verlag 1983

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture Arthur S. Tischler 1, Lloyd A. Greene 2, Paul W. Kwan 1, and Val W. Slayton 1 1 Department of Pathology Tufts University School of Medicine Boston, Massachusetts, USA; 2 Department of PharmacologyNew York University Medical Center New York, New York, USA

Summary. PC I2 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to N G F in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with N G F in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the lirst two days of exposure to N G F in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that N G F may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to N G F .

Key words: PC 12 pheochromocytoma cells - Nerve growth factor - Neurite Axonal transport

The PC 12 pheochromocytoma (Greene and Tischler 1976) is a clone of chromaffinlike cells derived from a transplantable rat adrenal medullary tumor (Warren and Chute 1972). When PC 12 cells in monolayer cultures are exposed to Nerve Growth Factor (NGF) they exhibit a number of neuron-like characteristics. These include Dr. A.S. Tischler, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA

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outgrowth o f long neuritic processes (Greene and Tischler 1976) formation o f small synaptic-like vesicles (Greene and Tischler 1976), and increased synthesis and storage o f acetylcholine (Ach) (Greene and Rein 1977). The characteristics o f the P C 1 2 line have recently been reviewed (Greene and Tischler 1982). The processes formed by PC 12 cells in m o n o l a y e r cultures have been studied morphologically both in early (Luckenbill-Edds et al. 1979) and late (Greene and Tischler 1976; Tischler and Greene 1978) stages o f outgrowth. Ultrastructural studies suggest that this outgrowth involves early sequential mobilization and reorganization o f pre-existing cytoplasmic organelles and formation o f short cytoplasmic extensions, followed by elongation and possible stabilization o f microtubules (Luckenbill-Edds et al. 1979). Processes with parallel arrays of microtubules characteristic o f neurites appear only after a lag o f several days (Greene and Tischler 1976; Luckenbill-Edds et al. 1979). The long, fully developed processes o f PC 12 cells closely resemble those formed by cultured sympathetic neurons (Bunge 1973) and by cultured adrenal chromaffin cells from some m a m m a l i a n species (Unsicker and Chamley 1977; Unsicker et al. 1978; Tischler et al. 1980). Studies on the mechanism by which N G F promotes neurite outgrowth in the PC 12 system suggest the requirement for both slow, transcription-dependent, and rapid, transcription-independent steps (Burstein and Greene 1978; Greene et al. 1980, 1982). PC 12 cells first encountering N G F exhibit slow initiation ofneurite outgrowth while cells pretrated with the factor for at least several days exhibit rapid neurite outgrowth. The rapidly-responding N G F - p r e t r e a t e d cells have been termed "primed". It has been suggested that priming by N G F is due to the selective transcription-dependent synthesis and accrual o f materials required for neurite initiation and elongation (Burstein and Greene 1978). PC 12 cells treated with N G F in spinner cultures do not form long processes detectable by light microscopy, but they do become primed by N G F so that immediate and rapid process outgrowth occurs after plating on an appropriate substrate (Greene et al. 1982). Ultrastructural examination o f PC 12 cells in spinner culture can thus shed light on the types o f cytological changes that occur u p o n priming, and which are independent o f substrate attachment and neurite outgrowth.

Materials and methods The experiments were conducted on two independent occasions, once with PC 12 cells from passage 10 and once with passage 24. The cells were adapted as previously described (Greene and Tischler 1982) to growth in 100 ml Wheaton spinner culture bottles (Wheaton Instruments), or in vesselsconstructed from 100ml medium bottles with stir bars suspended by stainless steel ball-chain (Ball Chain Mfg Co., Mt Vernon, NY). The culture medium was RPMI 1640 (Gibco) with l0 % heat-inactivated horse serum (K.C. Biologicals), 5 ~ofetal calf serum (K.C. Biologicals), 50 mg/ml streptomycin and 50 U ml penicillin (Gibco). After two weeks, replicate bottles were inoculated with 25ml of medium containing 106 cells/per ml, and half of the cultures were supplemented with 2.5 S mouse salivary gland NGF, prepared by the method of Mobley et al. (1976), at a concentration of 50 ng/ml. The cultures were maintained at 37~C for 14 days. Medium volume was kept constant during this period, and the medium was changed every 2 3 days. The final medium change was 24h before harvesting. At the end of the final incubation period the bottles were slowlyswirled and I ml aliquots ofmedittm were gently removed with a wide-bore pipette. The cells were then pelleted by centrifugation in BEEM capsules or microcentrifuge tubes (Fenwick et al. 1978), and the medium was removed by aspiration. The pellets were fixed overnight at 4~C in 3 ~ glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.2, and

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postfixed for I h in 1% OsOa in the same buffer.Theywerethen washed,dehydratedin a gradedethanol series followedby propyleneoxide, and embeddedin Epon. To assure objectiveassessmenteach sample was next givena codedidentityand the codeswerenot broken until after electron micrographprints had been evaluated. Ultrathin sections (80 nm) were cut through several levels of each block, and were stained with aqueous uranyl acetate and lead citrate. To confirmthat the cellshad in fact beenprimed by NGF in the spinner cultures, additional aliquots of cellscultured with and without NGF were removedfrom the spinner bottles, plated in 35 mm Falcon tissue culture dishes coated with air-dried rat tail collagen (Bornstein 1958), and incubated for 24h at 37~C in a water-saturated atmosphere of 95 % air and 5 % CO2. NGF was added to some of the cells from spinner cultures without NGF for this incubation period. The cultures were then examined by phase contrast microscopyfor process outgrowth (Burstein and Greene 1978).

Results Cells in spinner cultures with and without N G F grew in compact d u m p s up to several times the size of those in monolayer cultures (Greene and Tischler 1976). Clumps appeared to be larger in NGF-treated cultures than in controls, but considerable size overlap was observed. Virtually all cell clumps from spinner cultures with N G F for two weeks exhibited process outgrowth 24 h after being plated with N G F on a collagen substrate. In contrast, no process outgrowth was observed when cells from spinner cultures without N G F were plated with or without N G F for 24h. By these criteria, the cells treated with N G F in spinner culture were primed, while those without treatment were not primed. The ultrastructure of PC12 cells in spinner cultures without N G F was essentially the same as in monolayer cultures (Tischler and Greene 1978). The most distinctive ultrastructural feature of these cells was the presence of varying numbers of granules resembling those in chromaffin cells of immature rats (Elfvin 1967). In spinner cultures these granules tended to be smaller and sparser than in monolayer cultures (Fig. 1). Cells treated with N G F in spinner culture for two weeks were consistently distinguishable ultrastructurally from untreated cells. These NGF-treated cells lacked the long processes characteristically resulting from prolonged N G F treatment in monolayer cultures. Cells scattered throughout the cell pellets did, however, from short extensions similar to those observed in monolayer cultures within the first two days of exposure to N G F (Luckenbill-Edds et al. 1979). These extensions formed in spinner culture coursed between the perikarya of adjacent cells and along their surfaces, sometimes interdigitating with each other in the interior regions o f cell clusters. The cytoplasm of the extensions contained micro filaments, occasional microtubules and intermediate filaments, and prominent tubular reticulum. Varying numbers of mitochondria, multivesicular bodies, coated vesicles and lysosomes were also present. Ribosomes were sometimes present in proximal regions of the extensions, but tended to be absent distally. Parallel arrays of microtubules were absent. An especially striking feature of the extensions was the presence of bulbous regions which contained numerous chromaffin-type granules (Figs. 2, 3). Such structures were rarely observed in cultures not treated with N G F . Small, synaptic-like vesicles, while extremely common in PC12 cells treated with N G F in monolayer cultures (Greene and Tischler 1976; Tischler and Greene 1978) were not identified with certainty in any cells treated comparably in spinner cultures. There were no apparent differences in

Fig. 1. Electron micrograph of PC12 cells grown in spinner culture for two weeks without N G F . The cells lie singly and in small clumps. Varying numbers of chromaffin-type granules are distributed throughout the cytoplasm, as previously described in monolayer cultures without N G F (Greene and Tischler 1976; Tischler and Greene 1978). A dividing cell is present at upper right. • 4775

Fig. 2. Electron micrograph o f PC12 cells grown in spinner culture for two weeks in the presence of NGF. Short extentions in which numerous chromaffin-type granules tend to accumulate interdigitate within a clump of cells. Dividing cells were observed in cultures with and without N G F (upper right). x 4775

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Fig. 3. Higher magnification of area in center o f Fig. 2. The extensions formed after two weeks of N G F treatment in spinner cultures resemble those occurring within the first two days of exposure to N G F in monolayer cultures (Luckenbill-Edds et al. 1979). Longitudinal sections of these extensions reveal tubular reticulum (arrows), and small numbers of filaments and tubules. Bulbous distensions contain varying numbers of mitochondria, lysosomes, multivesicular bodies and coated vesicles, in addition to numerous ehromaffin-type granules. In contrast to the perikaryal cytoplasm, ribosomes are extremely rare in the cytoplasm of the extensions. Small synaptic-like vesicles are not observed, x 26000

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the appearance of the nuclei or perikaryal cytoplasm of cells treated or untreated with N G F . Such differences could, however, have been obscured by the cytological variation which has previously been noted within PC 12 cell populations (Tischler and Greene 1978; Luckenbill-Edds et al. 1979). Discussion

The present findings indicate that PC 12 cells treated with N G F in spinner cultures lack long processes but develop short extensions which contain a variety of organelles and which form bulbous distenstions filled with chromaffin-type granules. This striking centrifugal movement of organelles into peripheral structures is comparable to that which is observed within the first two days of exposure to N G F in monolayer cultures (Luckenbill-Edds et aL 1979). Such movement could possibly reflect the development of axonal transport mechanisms in response to N G F (Lasek 1980). Establishment of these mechanisms in turn may play a necessary role in N G F - p r o m o t e d priming and neurite growth. The fact that extensions were not formed by many NGF-treated cells in the spinner cultures, despite the fact that the majority of such cells appeared to be primed, suggests that the presence of extensions per se does not account for the rapid outgrowth of neurites from primed cells after plating. It is of interest that small agranular synaptic-like vesicles were extremely rare or absent in PC12 cells treated with N G F in spinner cultures, while being very numerous in cells treated comparably in monolayer cultures (Greene and Tischler 1976; Tischler and Greene 1978). Hence, the presence of such vesicles appears to require neurite outgrowth and/or attachment to a substrate. In cultured sympathetic neurons such vesicles appear to be sites of ACh storage, and increase in number with increasing ACh content (Landis 1980). Since PC 12 cells treated with N G F in monolayer cultures also show increased ACh storage (Greene and Rein 1977), the vesicles may serve the same purpose in these cells. The observations presented here indicate at the morphological level that certain responses of PC 12 cells to N G F occur irrespectively of attachment to a substrate and ofneurite outgrowth, while other responses do not. This corroborates previous findings at the biochemical and functional levels. For instance, priming (Greene et al. 1982), induction of electrical excitability and of voltage-dependent sodium channels (Rudy et al. 1982), increased levels of N I L E glycoprotein (McGuire et al. 1978) and increased total specific activity of acetylcholinesterase (Rieger et al. 1980; Lucas et al. 1980) are all promoted by N G F in spinner as well as in monolayer cultures. On the other hand, similarly to small vesicles, the 16 S form of acetylcholinesterase is increased by N G F in monolayer but not in spinner cultures (Rieger et al. 1980). Acknowledgements. The authors thank Mr. Steven Halpern for photographic work and Ms. Susan Pomales for secretarial assistance. This investigation was supported by grants PDT-171 from the American Cancer Societyand R23-CA27808 and NS 16036 from the National Institutes of Health; by the Charles A. King Trust, and the March of Dimes Birth Defects Foundation. References Bornstein MB (1958) Reconstituted rat-tail collagen used as a substrate for tissue cultures on coverslips in aximow slides and roller tubes. Lab Invest 7:134-140

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Bunge MB (1973) Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J Cell Biol 56:713 775 Burstein DE, Greene LA (1978) Evidence for both RNA-synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor. Proc Natl Acad Sci USA 75:6059 6063 Elfvin L-G (1967) The development of the secretory granules in the rat adrenal medulla. J Ultrastruct Res 17:45 62 Fenwick EM, Fajdiga PB, Howe NBS, Livett BG (1978) Functional and morphological characterization of isolated bovine adrenal medullary cells. J Cell Biol 76:12-30 Greene LA, Rein G (1977) Synthesis, storage and release of acetylcholine by a noradrenergic pheochromocytoma cell line. Nature 268:349 351 Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:24242428 Greene LA, Tischler AS (1982) PCI2 pheochromocytoma cultures in neurobiological research. In: Fedoroff S, Hertz L (eds) Advances in cellular neurobiology. Academic Press, New York, p 373 Greene LA, Burstein DE, Black MM (1980) The priming model for the mechanism of action of nerve growth factor: evidence derived from clonal PC12 pheochromocytoma cells. In: Giacobini E, Vernadakis A, Shahar A (eds) Tissue culture in neurobiology. Raven Press, New York 1980, p 313 Greene LA, Burstein DE, Black MM (1982) The role of transcription dependent priming in nerve growth factor-promoted neurite outgrowth. Dev Biol 91:305-316 Landis SC (1980) Developmental changes in the neurotransmitter properties of dissociated sympathetic neurons: A cytochemical study of the effects of medium. Dev Biol 77:349-361 Lasek RH (1980) Axonal transport: A dynamic view of neuronal structures. Trends Neurosci 3:87-91 Lucas CA, Czlonkowska A, Kreutzberg GW (1980) Regulation of acetylcholinesterase by nerve growth factor in the pheochromocytoma PC 12 cell line. Neurosci Lett 18:333 337 Luckenbill-Edds L, Van Horn C, Greene LA (1979) Fine structure of initial outgrowth of processes induced in a pheochromocytoma cell line by nerve growth factor. J Neurocytol 8:493-511 McGuire JC, Greene LA, Furano AV (1978) NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells. Cell 15:357 365 Mobley WC, Schenker A, Shooter EM (1976) Characterization and isolation ofproteolytically modified nerve growth factor. Biochemistry 15:5543-5551 Rieger F, Shelanski ML, Greene LA (1980) The effects of nerve growth factor on acetylcholinesterase and its multiple forms in cultures of rat PC12 pheochromocytoma cells: Increased total specific activity and appearance of the 16 S molecular form. Dev Biol 76:238-243 Rudy B, Kirschenbaum B, Greene LA (1982) Nerve growth factor-induced increase in saxitoxin binding to rat PC 12 pheochromocytoma cells. J Neurosci 2:1405-1411 Tischler AS, Greene LA (1978) Morphologic and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab Invest 39:77-89 Tischler AS, DeLellis RA, Biales B, Nunnemacher G, Carraba V, Wolfe HJ (1980) Nerve growth factor induced neurite outgrowth from normal human chromaffin cells. Lab Invest 43:399-409 Unsicker K, Chamley JH (1977) Growth characteristics of postnatal rat adrenal medulla in culture. Cell Tissue Res 177:247 268 Unsicker K, Krisch B, Otten U, Thoenen H (1978) Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc Natl Acad Sci (USA) 75: 3498-3502 Warren S, Chute RN (1972) Pheochromocytoma. Cancer 29:327-331

Accepted October 5, 1982