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The importance of both early and delayed responses in the biological actions of nerve growth factor
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(i.e. a train of temporally structured action potentials of a certain duration) potentially greatly simplifies the analysis of complex neuronal pattern generators. Only the timing of initiation of the burst-forming potential needs to be controlled. The utilization of the same group of neurons organized in a different pattern to produce a different behavior might be particularly facilitated by the ability to synaptically or hormonally control whether individual neurons will produce a burst. So far, driver potentials have been identified in the cardiac and stomatogastric ganglia of Crustacea only. The slow potentials of stomatopod cardiac ganglion neurons have properties indicating that they are probably driver potentials24. However, in Limulus, a primitive arthropod, the motor neurons of the cardiac ganglion discharge bursts of action potentials surmounting a depolarized plateau25, but experiments have so far failed to reveal an underlying driver potential (Augustine, G., unpublished observations). The most extensively studied endogenously bursting neurons are those of the molluscs where a slowly depolarizing pacemaker potential leads to a long depolarization surmounted by overshooting action potentials. The burst-generating mechanism in molluscan endogenous bursters is confined to the soma and proximal axon, but action potential generation is an intrinsic and essential part of the burst cycle (Adams, W. B., unpublished observations). Despite the observation in molluscan bursters of square-shaped, slow depolarizations in unusual conditions following TIX treatment, it seems unlikely that driver potentials, as they have been defined for Crustacea, will be found to contribute to bursting activity in molluscs. Among the vertebrates, vasopressinergic neurons cultured from fetal mice show unusually long (30 s) Ca2+-dependent plateau potentials26. Purkinje cell dendrites show plateau potentials2’. Bursting, with underlying slow depolarization, and dependence of both the slow depolarization and of bursting on the presence of Ca2+ in mammalian thalamic neurons, make these candidates for exhibiting driver potentials28*2y. While driver or plateau potentials are not invariably associated with impulse bursting in neurons, they have been observed sufficiently frequently to suggest that they should be looked for when analysing pattern-generating neuronal networks. Certainly in arthropods they can play a major role not only
in endowing individual neurons with the ability to produce patterned output to simple inputs, but also in mediating intemeuronal co-ordination via electrotonic or non-impulse-mediated synaptic transmission. Reading list 1 Hartline, D. K. (1979) Am. Zool. 19,53-65 2 Selverston. A. I., RusseIl, D. F., Miller, J. P. and King, D. G. (1976) frog. Neurobiol. 7. 215-290 3 Russell, D. and Hartline, D. K. (1978) Science 200.45u56 4 Tazaki. K. and Cooke, I. M. (1979) /. Neurophysiol. 42, 975-999 5 Tazaki, K. and Cooke, I. M. (1979) J. Neurophysiol. 42, 1000-1021 6 Tazaki, K. and Cooke, I. M. (1979) /. Neurophysiol. 42, 1022-1047 7 Anderson, W. W. and Barker, D. (1981) 1. Exp. Zool. 216. 187-191 8 Raper. J. (1979) Science 205, W306 9 Nagy. F. and Benson, J. A. (unpublished observations) 10 Bertind, A. (1982) /. Comp. fhysiol. 149. 263-276 11 Tazaki. K. and Cooke, I. M. in Neural Control of Rhythmic Movements (Society for Experimenlal Biology, Sjfmposiwn No. 37) (Roberts, B. L. and Roberts, A., eds). Cambridge University Press, Cambridge, UK (in press) 12 Tazaki. K. and Cooke, I. M. (1983) J. Camp. Physiol. 151. 311-328 13 Tazaki, K. and Cooke, I. M. (1983) 1. Comp. Physiol. 15 1. 329-346 14 Benson, J. A. (1980) J. Exp. Biol. 87. 285-313
15 Graubard, K., Raper, J. and Hartline, D. (1980) Proc. Nail Acad. Sci. USA 77, 3733-3735 16 Hagiwara, S. (1961) Ergeb. Physiol. Biol. Chem. Exp. Pharmakol. 24. w-311 17 Miller, M. W. and Sullivan, R. E. (1981) J. Neurobiol. 12, 629-639 A. 18 Miller, M. W.. Benson, J. A. and Berlind, (1984) J. Exp. Biol. 108,97-118 19 RosseIl, D. F. (1979) Brain Res. 177, 598-602 J. P. and Selverston, A. (1982) 20 Miller, J. Neurophysiol. 48, 1378-1391 21 Barker, D. L., Kushner, P. D. and Hooper, N. K. (1979) Brain Res. 161, 9!+113 22 Nagy, F. and Dickinson, P. S. (1983) J. Exp. Biol. 105, 33-58 23 Dickinson, P. S. and Nagy. F. (1983) J. Exp. Biol. 105, 59-82 24 Watanabe, A., Obara, S. and Akiyama, T. (1%7) J. Gen. Physiol. 50, 839-862 25 Watson, W. H. and Augustine, G. J. (1982) Peptides 3,485-492 26 Legendre, P.. Cooke, 1. M. and Vincent, J.-D. (1982) J. Neurophysiol. 48, 1121-1141 27 Llini, R. and Sugimori, M. (1980) J. Physiol. (London) 305, 197-213 28 Deschenes, M., Roy, J. P. and Steriade, M. (1982) Brain Res. 239. 28%293 29 Llinb, R. and Jahnsen. H. (1982) Nanrre (London) 297,40&408 Jack A. Benson is a member of rhe Entomology Baric Research Group of the Agricultural Division. Ciba-Geigy Ltd. CH-4002 Basel, Swirzerland. Ian M. Cooke is a Professor of Zoology and the Director of the BCkt!sy Laborarory of Neurobiology, Universiry of Hawaii, Honolulu, HI 96822, USA.
The importance of both early and delayed responses in the biological actions of nerve growth factor Lloyd A. Greene general principle rhal has emerged from rhe study of inrercellular signals is rhar these agents often promote both rapid!\! onserting and delayed responses. For example. insulin enhances the uptake of glucose within minufes of its application lo target cells. In conmzsl. other responses lo insulin, such as srimulation of DNA synthesis. are apparent only after larencies of tens of hours. The object of rhis article is lo review evidence rhar nerve growth factor INGF). a protein with a variety of actions on vertebrate dorsal root sensory and symparlleric neurons’-.‘. also triggers both rapid and delayed responses. In addition, we raise the issue that the highly ,asymmetric form of neurom as well as their rrophic interacrions have brought this duality of action to an especially, and perhaps uniquely, important role in the nervous system. A
Experimental systems
A major aid in studying the multiple actions of NGF is that many of them can occur in cell or tisssue culture’-‘. Two complementary in-vitro systems have
been widely employed in this regard primary cultures of ganglionic sympathetic and sensory neurons, and established clonal lines of NGF-responsive tumor cells. Of the latter, the PC12 line of rat
0 1984. Elsetier Sckncc Publisherr B.V.. Amsmdam
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TINS - March 1984
92 pheochromocytoma cells has been particularly useful’.‘. When these cells are grown in the absence of NGF they resemble their non-neoplastic counterparts, noradrenergic adrenal chromaffin cells. Exposure of PC12 cells to physiological levels of NGF causes them slowly (over a time course of days) to cease proliferation and exhibit the characteristic phenotypic properties of sympathetic neurons. In contrast to cultured NGFresponsive neurons, pheochromocytoma cells do not require NGF for the’r survival and can be obtained in a naive state without the possibility of prior irkvi’~ exposure to NGF. The PC12 cell line therefore offers the advantage that the same cell type can be compared before and after various times (both short and long) of NGP treatment. This has led to the demonstration of several new features of the action of NGF. Delayed responses to NGF Studies with the PC12 cell line have revealed a number of responses to NGF that can be detected only after latencies of at least I day. One example is the initiation of neunte outgrowth. Although NGF causes PC12 cells to undergo changes in shape and motility within an hour or so of its introduction into cultures. NGF does not promote the formation of’ processes with the lightand electron-microscopic features of neurites before a lag of about I8 h’.“.‘. It is only after -3-5 days of NGF treatment that most of the cells acquire neurites. Another example is the appearance of electrical excitability”. Prior to or within the first few days of NGF treatment it has not been possible to elicit Naf action potentials from PC12 cells. After the first few days of NGF treatment. electrical excitability becomes detectable and after several weeks of NGF exposure most of the cells can display Na+ action potentials. This slow response to NGF appears to be due to a several-fold increase in the density of sodium channels in the cell membrane”. The cessation of proliferation caused by NGF in PC12 cultures is an additional example of a delayed response. After inclusion of NGF in the culture medium the population undergoes at least one doubling before withdrawing from the cell cycle’. NGF also leads to slowly onsetting alterations in the composition of PC 12 cells. Among these changes is a 3-5fold increase in specific levels of a cell-surface component named the NGFinducible large external (NILE) glycoprotein”‘. Recent findings reveal this glycoprotein to be a widespread neuronal
marker”. Another example is a striking enhancement by NGF of the specific levels of a particular high molecular weight microtubule-associated protein (HMW MAP 1.2)“. Examples of other proteins that , undergo long-latency increases in NGF-treated PC12 cells include neuron-specific enolasei3. the 16s form of acetylcholinesterase (AChE)“‘. and binding sites for muscarinic ligands15. RNA levels also c increase in NGF-treated PC12 cells after an initial time-lag”‘. Table I lists these and several additional examples of delayed responses to NGF in cultured PC12 cells and sympathetic neurons. Delayed responses and the role of transcription The long latency with which the above types of changes become apparent suggests the potential involvement of a transcriptional step. In several cases this has been tested and it has been possible to block specific actions of NGF with inhibitors of RNA synthesis. For example. initiation of neurite outgrowth can be reversibly suppressed by inhibitors of RNA synthesis”. Other examples in which the use of inhibitors indicates a transcriptional requirement include the actions of NGF on NILE glycoprotein, AChE activity. and the HMW MAP. Transcriptionally regulated actions of NGF can occur well before the detectable onset of the delayed responses listed in Table I. Sympathetic neurons” and PC12 cellsls exhibit a large (lo-& fold) induction of ornithine decarboxylase activity which. at least in PC12 cells, is dependent on RNA synthesis. This effect is detectable within l-2 h and is maximal by 4-6 h of NGF treatment. It therefore appears that the temporal requirements for altering the transcripTABLE
I.
Examples
of
delayed
responses
to NGF
KclA Initiation Cessation Appearance Increased lnducrion Enhanced colchicinc
of ncurirc ourgrowth oC mitosis or clcctrical rxcitabiliry Icvels o(’ tyrosine hydroxylasc of the IhS form of AChE rcsistmw of microtubulcs IO
Induction of cnkcphalin receptors Increased numbers of muscarinic binding sites lncrcascd levels oi: AChE activiry NILE glycoprotcin Neuron-specific cnolasc HMW MAP I.’ Prcscncc of synaptic vesicles
-1. 6
x I9 I-l 33 34
ligandI5
tional machinery cannot entirely account for the time-lag in appearance of the delayed responses. Mechanisms other than transcriptional control may also underlie certain delayed actions of NGF. For instance, high concentrations of NGF bring about specific increases in the number of molecules of tyrosine hydroxylase in sympathetic neurons”.‘“. Studies with sympathetic neurons in culture indicate that this response is not affected by inhibitors of transcription and may involve translational control’“. Early responses to NGF In addition to its delayed actions. NGF triggers a set of responses that can be detected with latencies ranging from minutes to hours. Features of these responses include independence from protein and RNA synthesis and the potential to be expressed locally and independently of the cell body. A point that must be stressed is that such responses will be distinguished here from those events which are causal steps in the mechanism of action of NGF. That is, this discussion will focus mainly on early actions of NGF that are biologically important end-points rather than on the underlying mechanistic steps. Neurite regetteration and the j~ritnittg’ model Promotion of neurite regeneration appears to be an example of a rapidly onsetting response to NGF. When NGF-responsive sympathetic and sensory neurons are -first cultured in the presence of the factor, they begin to grow neurites soon after attachment to the substrate; by 1 day in vitro these neurites may reach hundreds of micrometers in length’. This NGF-dependent production of processes is not blocked by inhibition of RNA synthesis and therefore appears to be independent of transcription’“. These outgrowth chardcteristics are quite distinct from those of the delayed initiation of neurites in PC12 cultures which, as noted above, has a time-lag of about 1 day, proceeds slowly [--SO pm day-‘), and is sensitive to inhibitors of transcription”,‘. Such differences in outgrowth characteristics appear to be attributable to the past history of the target cells. Ganglionic neurons which are cultured from embryos or neonates are in most cases likely to have had prior it?-viva exposure to NGF, and their in-vitro behavior probably represents regeneration rather than initiation of neurites. Thus, by the
93
TINS - March I984 time these neurons are cultured, they may have already had the opportunity for long-latency responses to NGF which are lacking in ‘nai’ve’ PC12 cells. To test the effect of pre-exposure to NGF, PC12 cells have been treated with NGF for l-2 weeks and then divested of their neurites by mechanical shearing. When these ‘primed’ cells were replated in the presence of NGF they behaved like neurons. That is, regeneration commenced shortly after attachment, the neurites elongated at a rapid rate (reaching 200-300 km in length within 24 h), and outgrowth was now insensitive to inhibitors of RNA synthesis”.‘. Such findings have led to the proposal of a ‘priming’ model in which it is suggested that both early and delayed responses to NGF play critical roles in neurite outgrowth”. In this scheme, the delayed response brings about specific, transcription-dependent changes which confer upon the cell the potential to grow neurites. These might include. for example, increased synthesis of specific MAPS”. These changes, however, are not sufficient to bring about neurite growth. For this to happen. a fast, transcription-independent response to NGF must also occur. This model accounts for the above data as follows. (i) Initiation of neurites by nai’ve PC12 cells is slow and requires transcription because it is dependent on the delayed response to NGF. (ii) Cultured neurons and ‘primed’ PC12 cells exhibit transcriptionindependent neurite outgrowth because they have already undergone the delayed response. Outgrowth by such cells is rapid since it is governed by the shortlatency response to NGF. (iii) Responsive neurons with prior exposure to NGF and ‘primed’ PC12 cells do not produce neurites if NGF is absent. This occurs because neurite outgrowth requires that NGF must be present to trigger a fast response. Rapid actions of NGF on growth cones Several types of approaches have indicated that the short-latency response that affects neurite outgrowth may include local actions on growth cones”-“. For example, Gundersen and Barrett’” demonstrated that the growth cones of cultured sensory neurons could be induced to turn to, and follow, a local gradient of NGF concentration which was produced by releasing NGF from a micropipette. This action occurred within 20 min. Such findings have been reinforced and extended by recent scanning electron microscope (EM) and timelapse light microscope (LM) experiments
responses be triggered in the growth cones of neurites which have been completely severed from their cell bodies. Such Rek findings regarding rapid, local actions of NGF on growth cone movement are Regulation of growth cone shape, consistent with the possibility that periI I. 22. 23. 25 motility and locomotion pheral targets may promote the inMaintenance of neuronal survival 2. 20. 21. 26 Regulation of the uptake of growth of neurites by releasing NGF”-“. nutrients and anabolic precursors 26, 2X. 2Y. In summary, both early and delayed 30 Activation of tyrosine hydroxylase actions of NGF appear to be involved in neurite outgrowth. The delayed actions may cause the synthesis of material with the growth cones of neurite-bearing required to construct and/or stabilize PC12 cells and cultured neurons”.“. The growth cones have been observed the neurite, while the rapidly onsetting under three conditions: during conactions may promote the motility of growth cones which in turn leads to tinuous exposure, after withdrawal, and neurite extension. after readdition of NGF. Observations made by time-lapse recordings of the continuously treated growth cones were Additional examples of early responses to NGF consistent with previous studies. The The maintenance of neuronal survival growth cones have a flattened, spread configuration; extend numerous shortand structural integrity appears to be a second example of a short-latency, tranlived, motile finger-like projectioris; and scription-independent biological action undergo locomotion and extension. of NGF. When cultured embryonic Within a few hours of NGF withdrawal the growth cones round up, lose their sympathetic and sensory neurons are deprived of NGF. structural and biomotile projections, and cease locomotion. chemical signs of deterioration appear Readdition of NGF initiates a rapid within hours2.6.26 and cell death follows recovery. Within two min the formation of motile projections is apparent and by soon afterwards. These effects can be about 20 min the growth cones begin to rapidly reversed by restoration of NGF. reflatten and resume locomotion. Scan- The observations”’ that NGF can proning EM observations25 confirm these mote neurite outgrowth by transcripchanges and reveal an additional facet. tionally blocked neurons also indicate that the ability of NGF to sustain survival Growth cones (but not cell bodies) of NGF-exposed PC12 cells and rat is independent of RNA synthesis. In sympathetic neurons possess numerous addition, the studies of Campenot” ruffles, which disappear within a few demonstrate that the capacity of NGF to support the structural integrity of the hours of NGF deprivation and reappear distal portions of neurites is expressed within 30 s of re-exposure to NGF. Investigations on chick embryo sensory locally. These influences of NGF may neurons by Gundersen and Barrett” as be particularly important in regulating well as by recent scanning electron microneuron survival and number during scope studies (Connolly, J., Seeley, P. J. development. Thus, the local availability and Greene, L. A., unpublished obseror absence of NGF may determine vations) suggest a somewhat different whether a given neuroblast will either pattern in which NGF rapidly affects the perish, or, survive and mature. (See, for example, the work of Kessler et al.” in formation of filopodia. rather than of ruffles. Rapid regulation of membrane which administration of NGF to rat ruffling or of filopodial extension may embryos caused an increase in number therefore contribute to the mechanism of ectopic catecholamine-containing by which NGF controls growth cone neuroblasts, perhaps by promoting their locomotion. survival.) At the local level in the periThere is evidence that the rapid phery, the presence or absence of NGF actions of NGF on growth cones can be could also regulate whether or not triggered locally and independently of neurites are maintained and encouraged RNA and protein synthesis. By means to grow into various potential targets”-“. of a culture system in which the distal Varon and Skaperlh and colleagues portions of neurites and their cell bodies have shown that NGF regulates the memcan each be exposed to separate media, brane NA+/K+ pump of certain cultured Campenot” demonstrated that NGF chick embryo sympathetic and sensory can locally regulate neurite maintenance neurons. Pump activity is impaired and extension. Also, in the time-lapse when NGF is withdrawn and is rapidly recording studies described above”, (within seconds) restored when NGF is responses to NGF readdition could re-added. These workers have provided TABLE II. Examples to NGF
of early
biological
TINS - March 1984
94 evidence to support the hypothesis that this rapid action may represent the mechanism by which NGF supports neuronal survivalz6. Another early response promoted by NGF is the transport of nutrients and precursors such as amino acids. sugars and nucleic acids’.‘“.‘“. In ganglionic neuronal cultures, the uptake of these molecules falls after NGF deprivation and is rapidly restored after NGF readdition”; in PC12 cultures, NGF rapidly elevates transport over a basal level”. This type of early action may represent a general anabolic response to NGF which in turn promotes the overall striking hypertrophic actions of the facto?.‘“. The effects of NGF on nutrient transport have, been suggested to be governed as a consequence of the action of NGF on the Na+/‘K+ pump”6.‘Y. Activation of tyrosine hydroxylase is a last example that can be offered of an acute response to NGF that has biological consequences. As noted above. NGF causes delayed regulation of the number of tyrosine hydroxylase molecules in sympathetic neurons. Recent studies have uncovered an additional rapidly expressed effect of NGF on this enzyme”“. When PC12 cultures (either nai’ve or pre-treated with NGF and then deprived of NGF for several hours) are exposed to NGF. the activity of their tyrosine hydroxylase is significantly increased. Since this effect occurs within 20 min and is unaffected by the presence of protein synthesis inhibitors. it appears to be due to activation of pre-existing enzyme rather than to enhanced synthesis. Although the occurrence of such an activation in viva has not yet been tested, if it does occur it could provide a means for NGF to rapidly and locally alter the synthesis of catecholamines and. thereby, noradrenergic transmission. This is especially attractive in light of the possibility that target organs for sympathetic neurons may be local sources of NGF”. The mechanism of activation of tyrosine hydroxylase by NGF could involve phosphorylation. Tyrosine hydroxylase can be activated by phosphorylation” and it has been shown that NGF promotes a rapid increase in the extent to which this enzyme is phosphorylated in PC12 culture?. Possible biological significance of early and delayed responses to NGF This review has emphasized that NGF possesses both early and delayed actions. The delayed responses appear to involve the cellular synthetic machinery while the early responses appear to be independent of effects on synthesis
and may be locally triggered. What might be the general biological significance of this dual system of action? One view is that each class of responses plays a different set of roles. The delayed actions of NGFmay induce and/or regulate the specific differentiated functions of NGF-responsive cells. That is, NGF may turn on or modulate the expression of specific neuronal programs. During development this type of action may regulate the differentiation of precursor cells into mature, post-mitotic sympathetic neurons. In developed neurons, long-term responses to NGF may accommodate demands of the periphery for increased levels of neurotransmitter or for additional innervation. Such responses may also play a major role in regulating the synthesis of materials required for neuronal repair and regeneration. While delayed, synthesis-dependent responses to NGF may regulate or modulate various general aspects of neuronal function. thev cannot alone provide for rapid. local&ed interaction of neurons with their surroundings. Since neurons are highly polarized in shape, their synthetic and genetic apparatuses are generally far removed from the sites at which these cells contact their peripheral targets. This means a considerable delay (of days) between binding of NGF and the promotion of peripheral long-term responses to the factor. Also. such delayed responses are likely to affect all or large portions of the neuron. Furthermore, slow synthesisdependent actions would not be responsive to rapid fluctuations in NGF levels. The short-latency responses. in contrast, may provide for local interactions between sympathetic and sensory neurons and their peripheries. Thus. for example. local fluctuations in NGF concentration (as provided. for instance. by target organs) could vield rapid, spatially restricted alterations-in neurotransmitter synthesis or of neurite outgrowth and maintenance. By means then of its two classes of actions - short latency and delayed - NGF may exert multiple levels of control over the development. local peripheral interactions. general function and repair of its neuronal targets. Acknowledgements Helpful discussions with Drs David Buntein. Mark Black. Alcmene Chalazonitis. James Connolly, Steven Green and P. John Seeleyon the issuesdiscussedhere are gratefully acknowledged. Reading list I Greene. L. A. and Anntc. Rev. Newosci.
Shooter. E. 3. 353-W
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3. 37.-l-! E. and Greene. L. A. (1978) Proc. NOI/ Acad. Sci. USA 75. 6U5Y41~~3 Grcenc. L. A.. Burstein. D. E. and Black.
Burstein. D.
M. M. (1982) Del,. Biol. Yl. 3(l.!-316 Dichtcr. M. A.. Tischlcr. A. S. and Grccnc. L. A. (1977) Nworrtw fLo,~r/o,i) 268. 501-50-1 9 Rudy, B., Kirschenbaum. B. and Greene. L. A. (1982) IO McGuire. J. A. V. (1978) I I Salton. S. Greene. L.
1. Newosci. 2. 1405-1411 C.. Grccnc. L. A. and Furano. Cell IS. 357-365 R. J.. Richter-Landsbcrg. C.. A. and Shclanski. M. L. (IYS3)
J. Netrrosci. 3. JJI4.54 I2 Greene. L. A.. Licm. R. K. H. and Shclanski. M. L. ( 1983) J. Cell Biol. 96. lb-S.1 I3 Vinores. S. A.. Marangos. P. J.. Parma. A. M. and Guroff. G. (IYXI) J. Nerrrochem. 37. 597-w 14 Rieger. F.. Shclanski. M. L. and Grccnc. L. A. (1980) Del,. Rio/. 76. 238-(-7J3 I5 Jumhlatt. J. E. and Tischlcr. A. S. (1981) No/we fLorrdon) 2Y7. 152-15-l I6 Gunniy. P. W.. Landrcth. G. E.. Lay. P.. Ingnatius. M. and Shooter. E. M. (IYSI) J. Newosci. 36X-370 I7
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MacDonnell. P. C.. Nagaiah. K.. Lakshmanan. J. and Guroff. G. ( lY77) Proc. Nut/ Ac&. Sci. USA 74. 4681-46X4 Grcenc. L. A. and McGuirc. J. C. (IYlS) N[lrrtre fLo/xfon) 276. IYI-I94 Hclti. F.. Gnahn. H.. Schwab. M. E. and
Thocnen. H. (lY82) J. Nertrmci. 20 Partlow. L. M. and Larrahcc. J. Newochem. IX. 2lOl-2118
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Campenor. R. B. (lY82) Do,. Biol. Y3. 13-71 Griffin. C. G. and Lctourncau. P. C. (IYS(1) J. Cell Rio/. 86. 1%lhl 23 Gunderscn. R. W. and Barrctr. J. N. (19X0) 1. Cell Biol. 87. 546-554 74 Sceley. P. J. and Greene. Nud Accrd. Sci. USA X0. 15 Connolly. J. L.. Scclcy. and Greene. L. A. ( 1982) 26 Varon. S. and Skapcr. Riochem. Sci. 8. 22-25
L. A. (IYX3) Proc. 278Y-2793 P. J.. Green. S. A. J. C‘e// Rio/. Y5. 2(1-h S. D. (IYS.7) 7rc11rl.y
27 Kessler. J. A.. Cochard. P. and Black. I. B. ( 1970) Numre fLorrc/on) 2X0. IJI-IJ?. 2x McGuire. J. C. and Greene. L. A. (lY70) J. Biol. Chern. 254. 33hZ-3307 29 Skaper. S. D. and Varon. 172. 302-313
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30 Grcenc. L. A.. Scclcy. P. J.. Rukcnstcin. A.. DiPiaua. M. and Howard. A. J. Nwrochem. (in press) Meligcni. J. A.. Hayock. J. W.. W. F. and Waymirc. J. C. (lY82) C‘llWl. 257. I xxI x4(1 Halegoua, S. and Patrick, J. (1980) 571-581 Black. M. and Greene. Rio/. 95. 379-3X6 Inoue. C/fern.
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Lloyd A. Greene i.y Prqfimor of Pharmtrco/r),y~ or die New York Univeni~y Medicrd Cemer. SC/IOO/ of Medicine. New York, NY IOOl6. USA.
TINS - March 1984
(i.e. a train of temporally structured action potentials of a certain duration) potentially greatly simplifies the analysis of complex neuronal pattern generators. Only the timing of initiation of the burst-forming potential needs to be controlled. The utilization of the same group of neurons organized in a different pattern to produce a different behavior might be particularly facilitated by the ability to synaptically or hormonally control whether individual neurons will produce a burst. So far, driver potentials have been identified in the cardiac and stomatogastric ganglia of Crustacea only. The slow potentials of stomatopod cardiac ganglion neurons have properties indicating that they are probably driver potentials24. However, in Limulus, a primitive arthropod, the motor neurons of the cardiac ganglion discharge bursts of action potentials surmounting a depolarized plateau25, but experiments have so far failed to reveal an underlying driver potential (Augustine, G., unpublished observations). The most extensively studied endogenously bursting neurons are those of the molluscs where a slowly depolarizing pacemaker potential leads to a long depolarization surmounted by overshooting action potentials. The burst-generating mechanism in molluscan endogenous bursters is confined to the soma and proximal axon, but action potential generation is an intrinsic and essential part of the burst cycle (Adams, W. B., unpublished observations). Despite the observation in molluscan bursters of square-shaped, slow depolarizations in unusual conditions following TIX treatment, it seems unlikely that driver potentials, as they have been defined for Crustacea, will be found to contribute to bursting activity in molluscs. Among the vertebrates, vasopressinergic neurons cultured from fetal mice show unusually long (30 s) Ca2+-dependent plateau potentials26. Purkinje cell dendrites show plateau potentials2’. Bursting, with underlying slow depolarization, and dependence of both the slow depolarization and of bursting on the presence of Ca2+ in mammalian thalamic neurons, make these candidates for exhibiting driver potentials28*2y. While driver or plateau potentials are not invariably associated with impulse bursting in neurons, they have been observed sufficiently frequently to suggest that they should be looked for when analysing pattern-generating neuronal networks. Certainly in arthropods they can play a major role not only
in endowing individual neurons with the ability to produce patterned output to simple inputs, but also in mediating intemeuronal co-ordination via electrotonic or non-impulse-mediated synaptic transmission. Reading list 1 Hartline, D. K. (1979) Am. Zool. 19,53-65 2 Selverston. A. I., RusseIl, D. F., Miller, J. P. and King, D. G. (1976) frog. Neurobiol. 7. 215-290 3 Russell, D. and Hartline, D. K. (1978) Science 200.45u56 4 Tazaki. K. and Cooke, I. M. (1979) /. Neurophysiol. 42, 975-999 5 Tazaki, K. and Cooke, I. M. (1979) J. Neurophysiol. 42, 1000-1021 6 Tazaki, K. and Cooke, I. M. (1979) /. Neurophysiol. 42, 1022-1047 7 Anderson, W. W. and Barker, D. (1981) 1. Exp. Zool. 216. 187-191 8 Raper. J. (1979) Science 205, W306 9 Nagy. F. and Benson, J. A. (unpublished observations) 10 Bertind, A. (1982) /. Comp. fhysiol. 149. 263-276 11 Tazaki. K. and Cooke, I. M. in Neural Control of Rhythmic Movements (Society for Experimenlal Biology, Sjfmposiwn No. 37) (Roberts, B. L. and Roberts, A., eds). Cambridge University Press, Cambridge, UK (in press) 12 Tazaki. K. and Cooke, I. M. (1983) J. Camp. Physiol. 151. 311-328 13 Tazaki, K. and Cooke, I. M. (1983) 1. Comp. Physiol. 15 1. 329-346 14 Benson, J. A. (1980) J. Exp. Biol. 87. 285-313
15 Graubard, K., Raper, J. and Hartline, D. (1980) Proc. Nail Acad. Sci. USA 77, 3733-3735 16 Hagiwara, S. (1961) Ergeb. Physiol. Biol. Chem. Exp. Pharmakol. 24. w-311 17 Miller, M. W. and Sullivan, R. E. (1981) J. Neurobiol. 12, 629-639 A. 18 Miller, M. W.. Benson, J. A. and Berlind, (1984) J. Exp. Biol. 108,97-118 19 RosseIl, D. F. (1979) Brain Res. 177, 598-602 J. P. and Selverston, A. (1982) 20 Miller, J. Neurophysiol. 48, 1378-1391 21 Barker, D. L., Kushner, P. D. and Hooper, N. K. (1979) Brain Res. 161, 9!+113 22 Nagy, F. and Dickinson, P. S. (1983) J. Exp. Biol. 105, 33-58 23 Dickinson, P. S. and Nagy. F. (1983) J. Exp. Biol. 105, 59-82 24 Watanabe, A., Obara, S. and Akiyama, T. (1%7) J. Gen. Physiol. 50, 839-862 25 Watson, W. H. and Augustine, G. J. (1982) Peptides 3,485-492 26 Legendre, P.. Cooke, 1. M. and Vincent, J.-D. (1982) J. Neurophysiol. 48, 1121-1141 27 Llini, R. and Sugimori, M. (1980) J. Physiol. (London) 305, 197-213 28 Deschenes, M., Roy, J. P. and Steriade, M. (1982) Brain Res. 239. 28%293 29 Llinb, R. and Jahnsen. H. (1982) Nanrre (London) 297,40&408 Jack A. Benson is a member of rhe Entomology Baric Research Group of the Agricultural Division. Ciba-Geigy Ltd. CH-4002 Basel, Swirzerland. Ian M. Cooke is a Professor of Zoology and the Director of the BCkt!sy Laborarory of Neurobiology, Universiry of Hawaii, Honolulu, HI 96822, USA.
The importance of both early and delayed responses in the biological actions of nerve growth factor Lloyd A. Greene general principle rhal has emerged from rhe study of inrercellular signals is rhar these agents often promote both rapid!\! onserting and delayed responses. For example. insulin enhances the uptake of glucose within minufes of its application lo target cells. In conmzsl. other responses lo insulin, such as srimulation of DNA synthesis. are apparent only after larencies of tens of hours. The object of rhis article is lo review evidence rhar nerve growth factor INGF). a protein with a variety of actions on vertebrate dorsal root sensory and symparlleric neurons’-.‘. also triggers both rapid and delayed responses. In addition, we raise the issue that the highly ,asymmetric form of neurom as well as their rrophic interacrions have brought this duality of action to an especially, and perhaps uniquely, important role in the nervous system. A
Experimental systems
A major aid in studying the multiple actions of NGF is that many of them can occur in cell or tisssue culture’-‘. Two complementary in-vitro systems have
been widely employed in this regard primary cultures of ganglionic sympathetic and sensory neurons, and established clonal lines of NGF-responsive tumor cells. Of the latter, the PC12 line of rat
0 1984. Elsetier Sckncc Publisherr B.V.. Amsmdam
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TINS - March 1984
92 pheochromocytoma cells has been particularly useful’.‘. When these cells are grown in the absence of NGF they resemble their non-neoplastic counterparts, noradrenergic adrenal chromaffin cells. Exposure of PC12 cells to physiological levels of NGF causes them slowly (over a time course of days) to cease proliferation and exhibit the characteristic phenotypic properties of sympathetic neurons. In contrast to cultured NGFresponsive neurons, pheochromocytoma cells do not require NGF for the’r survival and can be obtained in a naive state without the possibility of prior irkvi’~ exposure to NGF. The PC12 cell line therefore offers the advantage that the same cell type can be compared before and after various times (both short and long) of NGP treatment. This has led to the demonstration of several new features of the action of NGF. Delayed responses to NGF Studies with the PC12 cell line have revealed a number of responses to NGF that can be detected only after latencies of at least I day. One example is the initiation of neunte outgrowth. Although NGF causes PC12 cells to undergo changes in shape and motility within an hour or so of its introduction into cultures. NGF does not promote the formation of’ processes with the lightand electron-microscopic features of neurites before a lag of about I8 h’.“.‘. It is only after -3-5 days of NGF treatment that most of the cells acquire neurites. Another example is the appearance of electrical excitability”. Prior to or within the first few days of NGF treatment it has not been possible to elicit Naf action potentials from PC12 cells. After the first few days of NGF treatment. electrical excitability becomes detectable and after several weeks of NGF exposure most of the cells can display Na+ action potentials. This slow response to NGF appears to be due to a several-fold increase in the density of sodium channels in the cell membrane”. The cessation of proliferation caused by NGF in PC12 cultures is an additional example of a delayed response. After inclusion of NGF in the culture medium the population undergoes at least one doubling before withdrawing from the cell cycle’. NGF also leads to slowly onsetting alterations in the composition of PC 12 cells. Among these changes is a 3-5fold increase in specific levels of a cell-surface component named the NGFinducible large external (NILE) glycoprotein”‘. Recent findings reveal this glycoprotein to be a widespread neuronal
marker”. Another example is a striking enhancement by NGF of the specific levels of a particular high molecular weight microtubule-associated protein (HMW MAP 1.2)“. Examples of other proteins that , undergo long-latency increases in NGF-treated PC12 cells include neuron-specific enolasei3. the 16s form of acetylcholinesterase (AChE)“‘. and binding sites for muscarinic ligands15. RNA levels also c increase in NGF-treated PC12 cells after an initial time-lag”‘. Table I lists these and several additional examples of delayed responses to NGF in cultured PC12 cells and sympathetic neurons. Delayed responses and the role of transcription The long latency with which the above types of changes become apparent suggests the potential involvement of a transcriptional step. In several cases this has been tested and it has been possible to block specific actions of NGF with inhibitors of RNA synthesis. For example. initiation of neurite outgrowth can be reversibly suppressed by inhibitors of RNA synthesis”. Other examples in which the use of inhibitors indicates a transcriptional requirement include the actions of NGF on NILE glycoprotein, AChE activity. and the HMW MAP. Transcriptionally regulated actions of NGF can occur well before the detectable onset of the delayed responses listed in Table I. Sympathetic neurons” and PC12 cellsls exhibit a large (lo-& fold) induction of ornithine decarboxylase activity which. at least in PC12 cells, is dependent on RNA synthesis. This effect is detectable within l-2 h and is maximal by 4-6 h of NGF treatment. It therefore appears that the temporal requirements for altering the transcripTABLE
I.
Examples
of
delayed
responses
to NGF
KclA Initiation Cessation Appearance Increased lnducrion Enhanced colchicinc
of ncurirc ourgrowth oC mitosis or clcctrical rxcitabiliry Icvels o(’ tyrosine hydroxylasc of the IhS form of AChE rcsistmw of microtubulcs IO
Induction of cnkcphalin receptors Increased numbers of muscarinic binding sites lncrcascd levels oi: AChE activiry NILE glycoprotcin Neuron-specific cnolasc HMW MAP I.’ Prcscncc of synaptic vesicles
-1. 6
x I9 I-l 33 34
ligandI5
tional machinery cannot entirely account for the time-lag in appearance of the delayed responses. Mechanisms other than transcriptional control may also underlie certain delayed actions of NGF. For instance, high concentrations of NGF bring about specific increases in the number of molecules of tyrosine hydroxylase in sympathetic neurons”.‘“. Studies with sympathetic neurons in culture indicate that this response is not affected by inhibitors of transcription and may involve translational control’“. Early responses to NGF In addition to its delayed actions. NGF triggers a set of responses that can be detected with latencies ranging from minutes to hours. Features of these responses include independence from protein and RNA synthesis and the potential to be expressed locally and independently of the cell body. A point that must be stressed is that such responses will be distinguished here from those events which are causal steps in the mechanism of action of NGF. That is, this discussion will focus mainly on early actions of NGF that are biologically important end-points rather than on the underlying mechanistic steps. Neurite regetteration and the j~ritnittg’ model Promotion of neurite regeneration appears to be an example of a rapidly onsetting response to NGF. When NGF-responsive sympathetic and sensory neurons are -first cultured in the presence of the factor, they begin to grow neurites soon after attachment to the substrate; by 1 day in vitro these neurites may reach hundreds of micrometers in length’. This NGF-dependent production of processes is not blocked by inhibition of RNA synthesis and therefore appears to be independent of transcription’“. These outgrowth chardcteristics are quite distinct from those of the delayed initiation of neurites in PC12 cultures which, as noted above, has a time-lag of about 1 day, proceeds slowly [--SO pm day-‘), and is sensitive to inhibitors of transcription”,‘. Such differences in outgrowth characteristics appear to be attributable to the past history of the target cells. Ganglionic neurons which are cultured from embryos or neonates are in most cases likely to have had prior it?-viva exposure to NGF, and their in-vitro behavior probably represents regeneration rather than initiation of neurites. Thus, by the
93
TINS - March I984 time these neurons are cultured, they may have already had the opportunity for long-latency responses to NGF which are lacking in ‘nai’ve’ PC12 cells. To test the effect of pre-exposure to NGF, PC12 cells have been treated with NGF for l-2 weeks and then divested of their neurites by mechanical shearing. When these ‘primed’ cells were replated in the presence of NGF they behaved like neurons. That is, regeneration commenced shortly after attachment, the neurites elongated at a rapid rate (reaching 200-300 km in length within 24 h), and outgrowth was now insensitive to inhibitors of RNA synthesis”.‘. Such findings have led to the proposal of a ‘priming’ model in which it is suggested that both early and delayed responses to NGF play critical roles in neurite outgrowth”. In this scheme, the delayed response brings about specific, transcription-dependent changes which confer upon the cell the potential to grow neurites. These might include. for example, increased synthesis of specific MAPS”. These changes, however, are not sufficient to bring about neurite growth. For this to happen. a fast, transcription-independent response to NGF must also occur. This model accounts for the above data as follows. (i) Initiation of neurites by nai’ve PC12 cells is slow and requires transcription because it is dependent on the delayed response to NGF. (ii) Cultured neurons and ‘primed’ PC12 cells exhibit transcriptionindependent neurite outgrowth because they have already undergone the delayed response. Outgrowth by such cells is rapid since it is governed by the shortlatency response to NGF. (iii) Responsive neurons with prior exposure to NGF and ‘primed’ PC12 cells do not produce neurites if NGF is absent. This occurs because neurite outgrowth requires that NGF must be present to trigger a fast response. Rapid actions of NGF on growth cones Several types of approaches have indicated that the short-latency response that affects neurite outgrowth may include local actions on growth cones”-“. For example, Gundersen and Barrett’” demonstrated that the growth cones of cultured sensory neurons could be induced to turn to, and follow, a local gradient of NGF concentration which was produced by releasing NGF from a micropipette. This action occurred within 20 min. Such findings have been reinforced and extended by recent scanning electron microscope (EM) and timelapse light microscope (LM) experiments
responses be triggered in the growth cones of neurites which have been completely severed from their cell bodies. Such Rek findings regarding rapid, local actions of NGF on growth cone movement are Regulation of growth cone shape, consistent with the possibility that periI I. 22. 23. 25 motility and locomotion pheral targets may promote the inMaintenance of neuronal survival 2. 20. 21. 26 Regulation of the uptake of growth of neurites by releasing NGF”-“. nutrients and anabolic precursors 26, 2X. 2Y. In summary, both early and delayed 30 Activation of tyrosine hydroxylase actions of NGF appear to be involved in neurite outgrowth. The delayed actions may cause the synthesis of material with the growth cones of neurite-bearing required to construct and/or stabilize PC12 cells and cultured neurons”.“. The growth cones have been observed the neurite, while the rapidly onsetting under three conditions: during conactions may promote the motility of growth cones which in turn leads to tinuous exposure, after withdrawal, and neurite extension. after readdition of NGF. Observations made by time-lapse recordings of the continuously treated growth cones were Additional examples of early responses to NGF consistent with previous studies. The The maintenance of neuronal survival growth cones have a flattened, spread configuration; extend numerous shortand structural integrity appears to be a second example of a short-latency, tranlived, motile finger-like projectioris; and scription-independent biological action undergo locomotion and extension. of NGF. When cultured embryonic Within a few hours of NGF withdrawal the growth cones round up, lose their sympathetic and sensory neurons are deprived of NGF. structural and biomotile projections, and cease locomotion. chemical signs of deterioration appear Readdition of NGF initiates a rapid within hours2.6.26 and cell death follows recovery. Within two min the formation of motile projections is apparent and by soon afterwards. These effects can be about 20 min the growth cones begin to rapidly reversed by restoration of NGF. reflatten and resume locomotion. Scan- The observations”’ that NGF can proning EM observations25 confirm these mote neurite outgrowth by transcripchanges and reveal an additional facet. tionally blocked neurons also indicate that the ability of NGF to sustain survival Growth cones (but not cell bodies) of NGF-exposed PC12 cells and rat is independent of RNA synthesis. In sympathetic neurons possess numerous addition, the studies of Campenot” ruffles, which disappear within a few demonstrate that the capacity of NGF to support the structural integrity of the hours of NGF deprivation and reappear distal portions of neurites is expressed within 30 s of re-exposure to NGF. Investigations on chick embryo sensory locally. These influences of NGF may neurons by Gundersen and Barrett” as be particularly important in regulating well as by recent scanning electron microneuron survival and number during scope studies (Connolly, J., Seeley, P. J. development. Thus, the local availability and Greene, L. A., unpublished obseror absence of NGF may determine vations) suggest a somewhat different whether a given neuroblast will either pattern in which NGF rapidly affects the perish, or, survive and mature. (See, for example, the work of Kessler et al.” in formation of filopodia. rather than of ruffles. Rapid regulation of membrane which administration of NGF to rat ruffling or of filopodial extension may embryos caused an increase in number therefore contribute to the mechanism of ectopic catecholamine-containing by which NGF controls growth cone neuroblasts, perhaps by promoting their locomotion. survival.) At the local level in the periThere is evidence that the rapid phery, the presence or absence of NGF actions of NGF on growth cones can be could also regulate whether or not triggered locally and independently of neurites are maintained and encouraged RNA and protein synthesis. By means to grow into various potential targets”-“. of a culture system in which the distal Varon and Skaperlh and colleagues portions of neurites and their cell bodies have shown that NGF regulates the memcan each be exposed to separate media, brane NA+/K+ pump of certain cultured Campenot” demonstrated that NGF chick embryo sympathetic and sensory can locally regulate neurite maintenance neurons. Pump activity is impaired and extension. Also, in the time-lapse when NGF is withdrawn and is rapidly recording studies described above”, (within seconds) restored when NGF is responses to NGF readdition could re-added. These workers have provided TABLE II. Examples to NGF
of early
biological
TINS - March 1984
94 evidence to support the hypothesis that this rapid action may represent the mechanism by which NGF supports neuronal survivalz6. Another early response promoted by NGF is the transport of nutrients and precursors such as amino acids. sugars and nucleic acids’.‘“.‘“. In ganglionic neuronal cultures, the uptake of these molecules falls after NGF deprivation and is rapidly restored after NGF readdition”; in PC12 cultures, NGF rapidly elevates transport over a basal level”. This type of early action may represent a general anabolic response to NGF which in turn promotes the overall striking hypertrophic actions of the facto?.‘“. The effects of NGF on nutrient transport have, been suggested to be governed as a consequence of the action of NGF on the Na+/‘K+ pump”6.‘Y. Activation of tyrosine hydroxylase is a last example that can be offered of an acute response to NGF that has biological consequences. As noted above. NGF causes delayed regulation of the number of tyrosine hydroxylase molecules in sympathetic neurons. Recent studies have uncovered an additional rapidly expressed effect of NGF on this enzyme”“. When PC12 cultures (either nai’ve or pre-treated with NGF and then deprived of NGF for several hours) are exposed to NGF. the activity of their tyrosine hydroxylase is significantly increased. Since this effect occurs within 20 min and is unaffected by the presence of protein synthesis inhibitors. it appears to be due to activation of pre-existing enzyme rather than to enhanced synthesis. Although the occurrence of such an activation in viva has not yet been tested, if it does occur it could provide a means for NGF to rapidly and locally alter the synthesis of catecholamines and. thereby, noradrenergic transmission. This is especially attractive in light of the possibility that target organs for sympathetic neurons may be local sources of NGF”. The mechanism of activation of tyrosine hydroxylase by NGF could involve phosphorylation. Tyrosine hydroxylase can be activated by phosphorylation” and it has been shown that NGF promotes a rapid increase in the extent to which this enzyme is phosphorylated in PC12 culture?. Possible biological significance of early and delayed responses to NGF This review has emphasized that NGF possesses both early and delayed actions. The delayed responses appear to involve the cellular synthetic machinery while the early responses appear to be independent of effects on synthesis
and may be locally triggered. What might be the general biological significance of this dual system of action? One view is that each class of responses plays a different set of roles. The delayed actions of NGFmay induce and/or regulate the specific differentiated functions of NGF-responsive cells. That is, NGF may turn on or modulate the expression of specific neuronal programs. During development this type of action may regulate the differentiation of precursor cells into mature, post-mitotic sympathetic neurons. In developed neurons, long-term responses to NGF may accommodate demands of the periphery for increased levels of neurotransmitter or for additional innervation. Such responses may also play a major role in regulating the synthesis of materials required for neuronal repair and regeneration. While delayed, synthesis-dependent responses to NGF may regulate or modulate various general aspects of neuronal function. thev cannot alone provide for rapid. local&ed interaction of neurons with their surroundings. Since neurons are highly polarized in shape, their synthetic and genetic apparatuses are generally far removed from the sites at which these cells contact their peripheral targets. This means a considerable delay (of days) between binding of NGF and the promotion of peripheral long-term responses to the factor. Also. such delayed responses are likely to affect all or large portions of the neuron. Furthermore, slow synthesisdependent actions would not be responsive to rapid fluctuations in NGF levels. The short-latency responses. in contrast, may provide for local interactions between sympathetic and sensory neurons and their peripheries. Thus. for example. local fluctuations in NGF concentration (as provided. for instance. by target organs) could vield rapid, spatially restricted alterations-in neurotransmitter synthesis or of neurite outgrowth and maintenance. By means then of its two classes of actions - short latency and delayed - NGF may exert multiple levels of control over the development. local peripheral interactions. general function and repair of its neuronal targets. Acknowledgements Helpful discussions with Drs David Buntein. Mark Black. Alcmene Chalazonitis. James Connolly, Steven Green and P. John Seeleyon the issuesdiscussedhere are gratefully acknowledged. Reading list I Greene. L. A. and Anntc. Rev. Newosci.
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Lloyd A. Greene i.y Prqfimor of Pharmtrco/r),y~ or die New York Univeni~y Medicrd Cemer. SC/IOO/ of Medicine. New York, NY IOOl6. USA.