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Local sprouting of neurites from cultured PC12D cells in response to a concentration gradient of nerve growth factor
BRAIN RESEARCH ELSEVIER
Brain Research 656 (1994) 210-214
Short communication
Local sprouting of neurites from cultured PC12D cells in response to a concentration gradient of nerve growth factor Mamoru Sano *, Misao Iwanaga Institute for Developmental Research, Aichi Colony, Kamiya-cho, Kasugai, Aichi 480-03, Japan
Accepted 14 June 1994
Abstract
PC12D cells, a subline of PC12 cells, extend neurites very rapidly in response to NGF, even when RNA synthesis is blocked. Several minutes after the initiation of a concentration gradient of NGF from a micropipette in the vicinity of PC12D cells, clear projections emerged from cells on the side facing the micropipette while no significant changes in morphology were observed on the other side of cells. A control solution administered from a micropipette did not produce any changes in morphology. Longer exposure to the gradient of NGF of aggregates of PC12D cells increased the length of neurites extending toward the source of NGF. The observations indicate that the sprouting of neurites occurs locally in regions of PCI2D cells that are exposed to an elevated concentration of NGF. Key words: Nerve growth factor; PC12D cell; Neurite; Ruffle; Growth cone; Chemotaxis
Many studies have suggested that neurotrophic factors play a role in the establishment of appropriate neuron-target connections in the developing nervous system. Nerve growth factor (NGF) is the only molecule defined to date for which a role in the promotion of initiation and elongation of axons by sympathetic neurons has been postulated. An injection of N G F into the brain stem of the developing chick embryo evokes a marked ingrowth of sympathetic fibers from the periphery into the brain [4]. In experiments in vitro, the growth cones of sensory neurons can be shown to orient toward a source of N G F [5,8,10]. The turning response of growth cones toward cyclic nucleotides or elevated levels of Ca 2+ has also been reported [5,6]. Experiments with compartmentalized cultures of sympathetic neurons from newborn rats showed that sprouting and growth of neurites depended upon N G F in the local environment [2,3]. These rapid changes in morphology of neurons, including initiation of axons in response to NGF, may not require new gene expression. However, little is known about the molecular mechanisms that support the rapid series of events
* Corresponding author. Fax: (81) (568) 88-0829. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00709-L
induced by NGF. Most studies of the NGF-dependent outgrowth of neurites have been performed with cultured sympathetic neurons or PC12 pheochromocytoma cells. However, sympathetic ganglia have already been exposed to N G F in vivo and, thus, such experiments in vitro may actually involve the NGF-stimulated regeneration of neurites rather than their generation de novo. Furthermore, it is difficult to examine the rapid neurotropic action of N G F on cultured sympathetic neurons because N G F is essential for their basic survival as a neurotrophic factor. Although PC12 cells do not require N G F for survival, the outgrowth of neurites is one of the consequences of the fact that these cells differentiate to cells that resemble sympathetic neurons in response to NGF. Such outgrowth is not necessarily a direct consequence of exposure to NGF. Thus, neither of the systems mentioned appears to be suitable for studies of the sprouting of neurites from cell bodies in response to NGF. PC12D cells, a subclone of PC12 cells, extend neurites within several hours in response to N G F [7,11-14], even in the presence of an inhibitor of R N A synthesis [13]. By contrast, the NGF-dependent outgrowth of neurites from conventional PC12 cells requires long-term, transcriptiondependent processes [1]. Thus, PC12D cells allow us to examine the rapid sprouting of neurites that is induced
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
by N G F in a transcription-independent manner. In the present study, we examined whether sprouting of neurites might occur in regions of PC12D cells exposed to an elevated concentration of NGF. Dulbecco's modified Eagle's medium (high glucose) was obtained from G I B C O (Grand Island, NY). Fetal calf serum and horse serum were from M.A. Bioproducts (Walkersville, MD). Mouse N G F (7S form) was purified from submandibular glands of male mice [16]. Cultures of PC12D cells were maintained on 90-mmdiameter culture dishes (Falcon, Lincoln Park, N J) at 37°C in a water-saturated atmosphere of 95% air with 5% CO 2. The medium used for cell culture was Dulbecco's modified Eagle's medium supplemented 5% fetal calf serum and 10% horse serum. For the observations of cellular response to a local gradient of NGF,
211
cells were plated on 60-mm-diameter culture dishes that had been precoated with poly-L-lysine. Clumps of cells were sometimes dissociated by incubation with 1 m M E G T A in phosphate-buffered saline (PBS). After about 20 h, the medium was changed and the cells were cultured on the stage of a phase-contrast microscope D I A P H O T - T M D (Nikon, Tokyo) under conditions controlled for temperature (37°C) and for the ambient concentration of CO 2 (5%). A gradient of N G F was produced by the method of Gundersen and Barrett [5]: a solution of N G F (1 / z g / m l in PBS with 1% BSA) was steadily ejected from a micropipette controlled by a micromanipulater (NT88; Narishige, Tokyo) against the perfused flow of medium over the entire culture. The micropipette (the inner diameter of the tip was 2 - 4 / x m ) was made from a borosilicate glass
Fig. 1. Diffusion of amido black-conjugated BSA. Representative phase-contrast micrographs of the profile with cells before (a) and 30 s (b), 5 min (c), and 10 min (d) after the initiation of the steady release of a solution of dye-conjugated BSA. Scale bar = 50/.tm.
212
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
Fig. 2. Rapid changes in morphology of PC12D cells after exposure to NGF. Phase-contrast photomicrographs of PC12D cells before (a) and 5 min (b), 30 rain (c), and 60 min (d) after the addition of N G F (50 n g / m l ) . Scale = 30 p.m.
capillary tube (Clark Electromedical Instrument, England). For examination of the gradient, BSA stained with amido black was used. When 0.25% (w/v) amido
black was added to PBS that contained 1% BSA, all the dye was precipitable by the addition of 50% ammonium sulfate. The dye conjugated with BSA was dis-
Fig. 3. Local sprouting of projections from PC12D cells exposed to N G F from a micropipette. Representative phase-contrast micrographs of P C I 2 D cells before (a) and 5 min (b) and 10 min (c) after the onset of the steady release of a solution of N G F (1 / z g / m l ) from a micropipette. Scale = 50 txm.
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
solved in PBS. A stable gradient of BSA stained with amido black was generated within 10 min in our system (Fig. 1). When N G F was added to a culture of PC12D cells, many small projections were observed around cells within 5 min and then some of these projections were rapidly extended as neurites within 60 min (Fig. 2). In a previous study, the rapid formation of ruffles around the periphery of PC12D cells in response to N G F was observed by staining of cells with rhodamine-phalloidin. Formation of ruffles was shown to be one of the earliest steps in the process that culminates in the outgrowth of neurites from PC12D cells [15]. In the present study, the changes in morphology could be monitored by phase-contrast microscopy. A gradient of NGF from a micropipette was applied to dissociated PC12D cells from a distance of about 100 mm (Fig. 3). From several to 10 min after the initiation of the gradient, clear projections emerged from cells on the same side as the micropipette, but no significant changes in morphology were observed on the contralateral side of the cells. Reproducible results were obtained by video recording of the same experiment with
213
Table 1 Rapid sprouting of projections from PC12D cells upon exposure to NGF from micropipette Emergence of projections within 10 min
NGF (1 tzg/ml) Control
positive response
on the side facing micropipette
27/34 (80%) 0/17 (0%)
21/27 (78%) 0/0
Local sprouting of projections from PC12D cells exposed to a gradient of NGF was repeatedly observed by video recording. Determinations were made as described in Materials and Methods and in the legend to Fig. 3.
many cells. By contrast, a control solution of PBS with 1% BSA from the micropipette did not produce any changes in the morphology of the cells (Table 1). These observations indicate that the sprouting of neurites occurs in localized regions of single cells that are exposed to a relatively elevated concentration of NGF. Longer exposure to the gradient of N G F of cell aggregates increased the length of neurites that extended toward the source of N G F (Fig. 4). A similar chemotactic response of the growth cones of sensory neurons
Fig. 4. Extension of neurites from PC12D cells exposed to NGF from a micropipette. Representative phase-contrast micrographs of an aggregate of PC12D cells before (a) and 4 rain (b), 2 h (c), and 3.5 h (d) after the onset of the steady release of a solution of NGF (1 /zg/ml) from a micropipette. Scale = 30/xm.
214
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
to NGF has been reported [5,8]. However, no turning of growing neurites toward the source of NGF was observed in the present study (not shown). Our results simply showed the enhanced growth of neurites on the side of cells closer to the source of NGF. The studies of neurons from dissociated dorsal root ganglia by Letourneau [8] and Rutishauser and Edelman [10] demonstrated that the sites of initiation of nerve fibers on cell bodies were not consistently determined by a gradient of NGF. Because of the lengthy incubation of neurons in a gradient of N G F in such studies, it was unclear whether the first sites of initiation of nerve fibers were affected by the gradient. Observations by Campenot indicated that the concentration of N G F in the local environment of neurites exerts control over their local growth, independent of other regions of each neuron [2]. In conformity with these observations, the present results provide the first demonstration that the sprouting of neurites from cell bodies might be controlled within a localized region of a cell that is exposed to a high concentration of the neurotrophic factor. The sprouting of axons is rapid but may involve very complex processes, such as reconstitution of membrane and cytoskeletal proteins and changes in the adhesion of the cell surface to substratum. Studies on axon elongation have been performed for the most part with primary cultures of neurons that are composed of heterogeneous populations of cells. Such populations are not optimal for biochemical studies. In PC12D cells, rapid changes in morphology and the subsequent outgrowth of neurites occur in response not only to NGF but also to dibutyryl cAMP and to basic fibroblast growth factor [7,13]. The variant cell line should be very useful in attempts to clarify the molecular mechanism of the NGF-induced sprouting of neurites from cell bodies.
[1] Burstein, D., and Greene, L.A., Evidence for RNA synthesisdependent and independent pathways in stimulation of neurite outgrowth by nerve growth factor, Proc. Natl. Acad. Sci. USA, 75 (1978) 6059-6063.
[2] Campenot, R.B., Local control of neurite development by nerve growth factor, Proc. Natl. Acad. Sci. USA, 74 (1977) 4516-4519. [3] Campenot, R.B., Local control of neurite sprouting in cultured sympathetic neurons by nerve growth factor, Det~. Brain Res., 37 (1987) 293-301. [4] Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 237 (1987) 1154-1162. [5] Gundersen, R.W. and Barrett, J.N., Neuronal chemotaxis: chick dorsal-root axons turn toward high concentrations of nerve growth factor, Science, 206 (1979) 1079-1080. [6] Gundersen, R.W. and Barrett, J.N., Characterization of the turning response of dorsal root neurites toward nerve growth factor, J. Cell Biol., 87 (1980) 546-554. [7] Katoh-Semba, R., Kitajima, S., Yamazaki, Y. and Sano, M., Neuritic growth from a new subline of PC12 pheochromocytoma cells: cyclic AMP mimics the action of nerve growth factor, J. Neurosci. Res., 17 (1987) 36-44 [8] Letourneau, P.C., Chemotactic response of nerve fiber elongation to nerve growth factor, Dec. Biol., 66 (1978) 183-196. [9] Lohof, A.M., Quillan, M., Dan, Y. and Poo, M.-m., Asymmetric modulation of cytosolic cAMP activity induces growth cone turning, Z Neurosci., 12 (1992) 1253-1261. [10] Rutishauser, U. and Edelman, M., Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture, J. Cell Biol., 87 (1980) 370-378. [11] Sano, M., Nishiyama, K. and Kitajima, S., Nerve growth factordependent protein kinase that phosphorylates microtubule-asociated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells, J. Neurochem., 55 (1990) 427-435. [12] Sano, M., Katoh-Semba, R., Kitajima, S. and Sato, C., Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells, Brain Res., 510 (1990) 269-276. [13] Sano, M. and Kitajima, S., Activation of microtubule-associated protein kinase in PC12 D cells in response to both fibroblast growth factor and epidermal growth factor and concomitant stimulation of the outgrowth of neurites, J. Neurochem., 58 (1992) 837-844. [14] Sano, M., Chromatographic resolution and characterization of a nerve growth factor-dependent kinase that phosphorylates microtubule-associated proteins 1 and 2 in PC12 cells, J. Neurochem., 59 (1992) 1263-1272. [15] Sano, M. and Iwanaga, M., Requirement for specific protein kinase activities during the rapid redistribution of F-actin that precedes the outgrowth of neurites in PC12D cells, Cell Str. Func., 17 (1992) 341-350. [16] Varon, S., Nomura and Shooter, E.M., The isolation of the mouse nerve growth factor proteins in a high molecular weight form, Biochemistry, 6 (1967) 2202-2209.
Brain Research 656 (1994) 210-214
Short communication
Local sprouting of neurites from cultured PC12D cells in response to a concentration gradient of nerve growth factor Mamoru Sano *, Misao Iwanaga Institute for Developmental Research, Aichi Colony, Kamiya-cho, Kasugai, Aichi 480-03, Japan
Accepted 14 June 1994
Abstract
PC12D cells, a subline of PC12 cells, extend neurites very rapidly in response to NGF, even when RNA synthesis is blocked. Several minutes after the initiation of a concentration gradient of NGF from a micropipette in the vicinity of PC12D cells, clear projections emerged from cells on the side facing the micropipette while no significant changes in morphology were observed on the other side of cells. A control solution administered from a micropipette did not produce any changes in morphology. Longer exposure to the gradient of NGF of aggregates of PC12D cells increased the length of neurites extending toward the source of NGF. The observations indicate that the sprouting of neurites occurs locally in regions of PCI2D cells that are exposed to an elevated concentration of NGF. Key words: Nerve growth factor; PC12D cell; Neurite; Ruffle; Growth cone; Chemotaxis
Many studies have suggested that neurotrophic factors play a role in the establishment of appropriate neuron-target connections in the developing nervous system. Nerve growth factor (NGF) is the only molecule defined to date for which a role in the promotion of initiation and elongation of axons by sympathetic neurons has been postulated. An injection of N G F into the brain stem of the developing chick embryo evokes a marked ingrowth of sympathetic fibers from the periphery into the brain [4]. In experiments in vitro, the growth cones of sensory neurons can be shown to orient toward a source of N G F [5,8,10]. The turning response of growth cones toward cyclic nucleotides or elevated levels of Ca 2+ has also been reported [5,6]. Experiments with compartmentalized cultures of sympathetic neurons from newborn rats showed that sprouting and growth of neurites depended upon N G F in the local environment [2,3]. These rapid changes in morphology of neurons, including initiation of axons in response to NGF, may not require new gene expression. However, little is known about the molecular mechanisms that support the rapid series of events
* Corresponding author. Fax: (81) (568) 88-0829. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00709-L
induced by NGF. Most studies of the NGF-dependent outgrowth of neurites have been performed with cultured sympathetic neurons or PC12 pheochromocytoma cells. However, sympathetic ganglia have already been exposed to N G F in vivo and, thus, such experiments in vitro may actually involve the NGF-stimulated regeneration of neurites rather than their generation de novo. Furthermore, it is difficult to examine the rapid neurotropic action of N G F on cultured sympathetic neurons because N G F is essential for their basic survival as a neurotrophic factor. Although PC12 cells do not require N G F for survival, the outgrowth of neurites is one of the consequences of the fact that these cells differentiate to cells that resemble sympathetic neurons in response to NGF. Such outgrowth is not necessarily a direct consequence of exposure to NGF. Thus, neither of the systems mentioned appears to be suitable for studies of the sprouting of neurites from cell bodies in response to NGF. PC12D cells, a subclone of PC12 cells, extend neurites within several hours in response to N G F [7,11-14], even in the presence of an inhibitor of R N A synthesis [13]. By contrast, the NGF-dependent outgrowth of neurites from conventional PC12 cells requires long-term, transcriptiondependent processes [1]. Thus, PC12D cells allow us to examine the rapid sprouting of neurites that is induced
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
by N G F in a transcription-independent manner. In the present study, we examined whether sprouting of neurites might occur in regions of PC12D cells exposed to an elevated concentration of NGF. Dulbecco's modified Eagle's medium (high glucose) was obtained from G I B C O (Grand Island, NY). Fetal calf serum and horse serum were from M.A. Bioproducts (Walkersville, MD). Mouse N G F (7S form) was purified from submandibular glands of male mice [16]. Cultures of PC12D cells were maintained on 90-mmdiameter culture dishes (Falcon, Lincoln Park, N J) at 37°C in a water-saturated atmosphere of 95% air with 5% CO 2. The medium used for cell culture was Dulbecco's modified Eagle's medium supplemented 5% fetal calf serum and 10% horse serum. For the observations of cellular response to a local gradient of NGF,
211
cells were plated on 60-mm-diameter culture dishes that had been precoated with poly-L-lysine. Clumps of cells were sometimes dissociated by incubation with 1 m M E G T A in phosphate-buffered saline (PBS). After about 20 h, the medium was changed and the cells were cultured on the stage of a phase-contrast microscope D I A P H O T - T M D (Nikon, Tokyo) under conditions controlled for temperature (37°C) and for the ambient concentration of CO 2 (5%). A gradient of N G F was produced by the method of Gundersen and Barrett [5]: a solution of N G F (1 / z g / m l in PBS with 1% BSA) was steadily ejected from a micropipette controlled by a micromanipulater (NT88; Narishige, Tokyo) against the perfused flow of medium over the entire culture. The micropipette (the inner diameter of the tip was 2 - 4 / x m ) was made from a borosilicate glass
Fig. 1. Diffusion of amido black-conjugated BSA. Representative phase-contrast micrographs of the profile with cells before (a) and 30 s (b), 5 min (c), and 10 min (d) after the initiation of the steady release of a solution of dye-conjugated BSA. Scale bar = 50/.tm.
212
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
Fig. 2. Rapid changes in morphology of PC12D cells after exposure to NGF. Phase-contrast photomicrographs of PC12D cells before (a) and 5 min (b), 30 rain (c), and 60 min (d) after the addition of N G F (50 n g / m l ) . Scale = 30 p.m.
capillary tube (Clark Electromedical Instrument, England). For examination of the gradient, BSA stained with amido black was used. When 0.25% (w/v) amido
black was added to PBS that contained 1% BSA, all the dye was precipitable by the addition of 50% ammonium sulfate. The dye conjugated with BSA was dis-
Fig. 3. Local sprouting of projections from PC12D cells exposed to N G F from a micropipette. Representative phase-contrast micrographs of P C I 2 D cells before (a) and 5 min (b) and 10 min (c) after the onset of the steady release of a solution of N G F (1 / z g / m l ) from a micropipette. Scale = 50 txm.
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
solved in PBS. A stable gradient of BSA stained with amido black was generated within 10 min in our system (Fig. 1). When N G F was added to a culture of PC12D cells, many small projections were observed around cells within 5 min and then some of these projections were rapidly extended as neurites within 60 min (Fig. 2). In a previous study, the rapid formation of ruffles around the periphery of PC12D cells in response to N G F was observed by staining of cells with rhodamine-phalloidin. Formation of ruffles was shown to be one of the earliest steps in the process that culminates in the outgrowth of neurites from PC12D cells [15]. In the present study, the changes in morphology could be monitored by phase-contrast microscopy. A gradient of NGF from a micropipette was applied to dissociated PC12D cells from a distance of about 100 mm (Fig. 3). From several to 10 min after the initiation of the gradient, clear projections emerged from cells on the same side as the micropipette, but no significant changes in morphology were observed on the contralateral side of the cells. Reproducible results were obtained by video recording of the same experiment with
213
Table 1 Rapid sprouting of projections from PC12D cells upon exposure to NGF from micropipette Emergence of projections within 10 min
NGF (1 tzg/ml) Control
positive response
on the side facing micropipette
27/34 (80%) 0/17 (0%)
21/27 (78%) 0/0
Local sprouting of projections from PC12D cells exposed to a gradient of NGF was repeatedly observed by video recording. Determinations were made as described in Materials and Methods and in the legend to Fig. 3.
many cells. By contrast, a control solution of PBS with 1% BSA from the micropipette did not produce any changes in the morphology of the cells (Table 1). These observations indicate that the sprouting of neurites occurs in localized regions of single cells that are exposed to a relatively elevated concentration of NGF. Longer exposure to the gradient of N G F of cell aggregates increased the length of neurites that extended toward the source of N G F (Fig. 4). A similar chemotactic response of the growth cones of sensory neurons
Fig. 4. Extension of neurites from PC12D cells exposed to NGF from a micropipette. Representative phase-contrast micrographs of an aggregate of PC12D cells before (a) and 4 rain (b), 2 h (c), and 3.5 h (d) after the onset of the steady release of a solution of NGF (1 /zg/ml) from a micropipette. Scale = 30/xm.
214
M. Sano, M. lwanaga /Brain Research 656 (1994) 210-214
to NGF has been reported [5,8]. However, no turning of growing neurites toward the source of NGF was observed in the present study (not shown). Our results simply showed the enhanced growth of neurites on the side of cells closer to the source of NGF. The studies of neurons from dissociated dorsal root ganglia by Letourneau [8] and Rutishauser and Edelman [10] demonstrated that the sites of initiation of nerve fibers on cell bodies were not consistently determined by a gradient of NGF. Because of the lengthy incubation of neurons in a gradient of N G F in such studies, it was unclear whether the first sites of initiation of nerve fibers were affected by the gradient. Observations by Campenot indicated that the concentration of N G F in the local environment of neurites exerts control over their local growth, independent of other regions of each neuron [2]. In conformity with these observations, the present results provide the first demonstration that the sprouting of neurites from cell bodies might be controlled within a localized region of a cell that is exposed to a high concentration of the neurotrophic factor. The sprouting of axons is rapid but may involve very complex processes, such as reconstitution of membrane and cytoskeletal proteins and changes in the adhesion of the cell surface to substratum. Studies on axon elongation have been performed for the most part with primary cultures of neurons that are composed of heterogeneous populations of cells. Such populations are not optimal for biochemical studies. In PC12D cells, rapid changes in morphology and the subsequent outgrowth of neurites occur in response not only to NGF but also to dibutyryl cAMP and to basic fibroblast growth factor [7,13]. The variant cell line should be very useful in attempts to clarify the molecular mechanism of the NGF-induced sprouting of neurites from cell bodies.
[1] Burstein, D., and Greene, L.A., Evidence for RNA synthesisdependent and independent pathways in stimulation of neurite outgrowth by nerve growth factor, Proc. Natl. Acad. Sci. USA, 75 (1978) 6059-6063.
[2] Campenot, R.B., Local control of neurite development by nerve growth factor, Proc. Natl. Acad. Sci. USA, 74 (1977) 4516-4519. [3] Campenot, R.B., Local control of neurite sprouting in cultured sympathetic neurons by nerve growth factor, Det~. Brain Res., 37 (1987) 293-301. [4] Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 237 (1987) 1154-1162. [5] Gundersen, R.W. and Barrett, J.N., Neuronal chemotaxis: chick dorsal-root axons turn toward high concentrations of nerve growth factor, Science, 206 (1979) 1079-1080. [6] Gundersen, R.W. and Barrett, J.N., Characterization of the turning response of dorsal root neurites toward nerve growth factor, J. Cell Biol., 87 (1980) 546-554. [7] Katoh-Semba, R., Kitajima, S., Yamazaki, Y. and Sano, M., Neuritic growth from a new subline of PC12 pheochromocytoma cells: cyclic AMP mimics the action of nerve growth factor, J. Neurosci. Res., 17 (1987) 36-44 [8] Letourneau, P.C., Chemotactic response of nerve fiber elongation to nerve growth factor, Dec. Biol., 66 (1978) 183-196. [9] Lohof, A.M., Quillan, M., Dan, Y. and Poo, M.-m., Asymmetric modulation of cytosolic cAMP activity induces growth cone turning, Z Neurosci., 12 (1992) 1253-1261. [10] Rutishauser, U. and Edelman, M., Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture, J. Cell Biol., 87 (1980) 370-378. [11] Sano, M., Nishiyama, K. and Kitajima, S., Nerve growth factordependent protein kinase that phosphorylates microtubule-asociated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells, J. Neurochem., 55 (1990) 427-435. [12] Sano, M., Katoh-Semba, R., Kitajima, S. and Sato, C., Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells, Brain Res., 510 (1990) 269-276. [13] Sano, M. and Kitajima, S., Activation of microtubule-associated protein kinase in PC12 D cells in response to both fibroblast growth factor and epidermal growth factor and concomitant stimulation of the outgrowth of neurites, J. Neurochem., 58 (1992) 837-844. [14] Sano, M., Chromatographic resolution and characterization of a nerve growth factor-dependent kinase that phosphorylates microtubule-associated proteins 1 and 2 in PC12 cells, J. Neurochem., 59 (1992) 1263-1272. [15] Sano, M. and Iwanaga, M., Requirement for specific protein kinase activities during the rapid redistribution of F-actin that precedes the outgrowth of neurites in PC12D cells, Cell Str. Func., 17 (1992) 341-350. [16] Varon, S., Nomura and Shooter, E.M., The isolation of the mouse nerve growth factor proteins in a high molecular weight form, Biochemistry, 6 (1967) 2202-2209.