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The neurotrophic theory and naturally occurring motoneuron death
The neurotrophictheoryand naturally occurringmotoneuron death Ronald W. Oppenheim RonaldW. Oppenheimis with the Programin Cell Bio/oooyand Neuroscienceat the Departmentof Anatomy, Bowman GraySchoolof Medicine, Wake ForestUniversity, Winston-Salem, NC27103, USA.
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There is increasing evidence that target-derived molecules play a crucial role in the regulation of neuronalsurvival during development. These molecules, termed neurotrophic factors, are thought to act in specific ways as defined by the neurotrophic theory. One central tenet of the neurotrophic theory is that some neuronsin a population die because trophic molecules are available in only limited amounts during periods of naturally occurring cell death. Ddivery of trophic factor to nerve terminals could be regulated by several mechanisms, including, for example, limited production (biosynthesis) by target cells, limited release by targets, or limited uptake by pre-synaptic terminals. An examination of recent studies of motoneuron development indicates that motoneurons compete, via axona/ branching and synaptic contacts, for restrictedsites on targets that provide accessto trophic factors. According to this view, it is terminal branches and contact ('synaptic') sites that limit the regulation of neuronal survival, rather than trophic factor production.
Nonetheless, many other suspected neurotrophic factors have subsequently been identified and proposed to play a similar role to NGF, albeit for other populations of neurons 8'9. Because it was the first and most completely characterized neurotrophic factor to be identified, NGF has continued to serve as a model with which other putative neurotrophic factors are compared. Thus, unless other molecules proposed as neurotrophic factors meet certain criteria that were established originally for NGF they are often considered to be suspect1,2. One of these criteria is that target-derived neurotrophic factors are produced in limiting or subsaturating amounts. In the case of NGF, the evidence for this belief is, on the face of it, rather compelling. First, only a proportion of the NGFsensitive sensory and sympathetic ganglion cells that
A number of papers have appeared recently in the pages of this journal discussing the neurotrophic theory and its applicability to neuronal survival I-3. This theory arose out of the pioneering studies of Hamburger and Levi-Montalcini in the 1940s and 1950s on embryonic cell death, studies that ultimately led to the discovery of the prototypical neurotrophic factor, NGF4. These early studies, and the resulting discovery of a molecule (NGF) that was necessary for the survival of specific populations of embryonic neurons, established the foundation for most later conceptualizations of the ontogeny of neuron-target interactions, including the neurotrophic theory s. The neurotrophic theory provides a useful framework for understanding several features of neuronal development, including the question of why, in many populations of developing neurons, only a proportion of the original number of cells survives. It is now well known that in many regions of the CNS and PNS large numbers ( - 5 0 % ) of postmitotic neurons degenerate and die by a process of naturally occurring neuronal death 4'6. Further, it has been repeatedly demonstrated that in most populations this normal cell loss occurs during the period when neurons are establishing synaptic connections with their targets. This temporal coincidence, together with the demonstration that manipulations of the availability of putative synaptic targets alters the number of surviving innervating neurons 4, led to the proposal that neurons compete for some entity that is supplied by the targets in limiting amounts. Following the discovery of NGF, it was widely believed that this entity was, in fact, represented by the biological activity of distinct molecules derived from target cells. NGF remains the only such molecule that has been shown to meet virtually all of the criteria necessary to qualify as a specific neurotrophic factor involved in regulating the survival of discrete populations of neurons 7.
are initiallygenerated normally survive1 0 1'1 . Second, treatment of embryos or postnatal animals with exogenous sources of N G F serves to rescue most of the neurons that would normally die12-1s. And finally, the amounts of N G F present in target sites during periods of normal cell death are reported to be exceedingly small and correlated with innervation density 1 6 1'7 . Although these data are all
consistent with the notion of limited trophic factor production, they do not exclude alternative proposals for limited trophic factor availability. For instance, it is conceivable that neurons normally die because they are unable to gain access to neurotrophic molecules rather than owing to limited trophic factor production. Similarly, exogenous supplies of a neurotrophic factor may rescue neurons by circumventing the normal route of access rather than by supplementing the limiting amounts thought to be produced normally. Finally, until the amount of neurotrophic factor that is actually required by a neuron for survival is known, the mere presence of small quantities in target tissues is not in itself a compelling argument that these amounts are, in fact, sub-saturating or limiting. These considerations, together with recent evidence from studies of motoneuron survival in the chick embryo, reviewed below, underscore the need for a critical re-examination of the notion that neurotrophic factors must necessarily be produced in limiting amounts during periods of naturally occurring neuronal death.
Neuromuscular activity and trophic factor production It has been more than ten years since the first reports that the chronic blockade of neuromuscular activity in the chick embryo results in the rescue of virtually all of the spinal motoneurons that would normally degenerate and die 18-2°. Subsequent
© 1989, ElsevierSciencePublishers Ltd.(UK) 0166-2236/89/$02.00
TINS, Vol. 12, No. 7, 1989
v studies have confirmed and extended the original observations21. Although the cellular and molecular mechanisms responsible for this unexpected finding are still not known, two kinds of explanation have been offered. The first hypothesis, which I call the 'production hypothesis', proposes that the production (i.e. the biosynthesis by target cells) of a muscle (target)-derived neurotrophic factor is inversely related to muscle activity (Fig. 1). Considerable evidence now exists for the presence of an NGF-like, muscle-derived, neurotrophic factor that promotes motoneuron survival both in vitro and in vivo 22'23. Thus, according to the production hypothesis, the normal, ongoing spontaneous neuromuscular activity that characterizes the development of all vertebrate embryos 24'25 would maintain the production of this muscle-derived neurotrophic factor at levels that are limiting. It follows then that lower than normal levels of activity should increase, and higher than normal levels of activity should decrease, the production of this trophic factor. Although such perturbations of activity are known to result in the predicted changes in motoneuron survival 18-2°'26, it is not presently known whether trophic factor production is also altered as expected under these conditions.
ewpoint
However, if a muscle-derived neurotrophic factor is normally produced in sub-saturating amounts, then the 'production hypothesis' would seem to be the most plausible explanation for the known effects of altered activity on motoneuron survival. There is, in fact, some evidence showing that surgical denervation of muscle, which effectively abolishes activity, results in the increased accumulation of a muscle-derived neurotrophic factor as measured in vitro 27'28. However, these studies differ in so many important ways from the in vivo experiments on neuromuscular blockade that they offer little support for the validity of the 'production hypothesis'. By contrast, a recent study by Tanaka29, although flawed in some respects, comes closer to qualifying as a critical test of this hypothesis. Tanaka compared the effectiveness of muscle extracts obtained from skeletal muscle of normally active control chick embryos with extracts from paralysed embryos that were exposed to regimens of activity blockade similar to those known to rescue motoneurons from naturally occurring cell death in vivo. Motoneurons were retrogradely labeled in vivo at stage 29-30 (i.e. at the onset of normal cell death) with a longlived fluorescent dye (Dil) following injections into the hindlimb. The lumbar spinal cord was then
Tml~
Target C
\
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/
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/
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,
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Fig. 1. A schematic illustration of neurotrophic interactions between motoneurons and their targets according to either the "access hypothesis' (A, B) or the 'production hypothesis' (C, D), as described in the text. In the 'access hypothesis', a population of neurons compete, via axonal branches and synaptic sites in the target, for neurotrophic molecules (solid black dots in target). During normal development (A) neurons die (dashed lines) owing to limited access to neurotrophic molecules. Following activity blockade or other perturbations that increase branching and synaptic contacts (B), neurons are able to gain access to sufficient trophic factor to survive. According to the "production hypothesis', the production of trophic factors by targets is normally limited, resulting in the death of some neurons (C). Activity blockade or other perturbations that rescue neurons from death do so by virtue of increased production of trophic factors by targets (D). TINS, VoL 12, No. 7, 1989
253
removed, dissociated and grown in culture for two days. Surviving, fluorescently labeled cells that extended neurites were counted as viable motoneurons at the end of the two-day culture period. No differences were observed in the motoneuron survival activity present in the muscles of control versus curare-treated embryos. Extracts from both sources of muscle produced an equivalent doserelated increase in motoneuron survival. Against these findings, one could argue that perhaps the two-day culture period was not sufficiently long to detect differences between the survival activity of control and curare-treated muscles or that in vitro conditions are not optimal for detecting such differences, even over longer periods of culture. However, recent in vivo studies support the findings of Tanaka. We have observed no differences in motoneuron survival following treatment of chick embryos in ovo with partially purified muscle extracts from normally active versus paralysed chick and mouse embryos 3°. Both control and paralysed muscle extracts increased motoneuron survival by 30-35%. Although further in vivo studies are needed to resolve this issue, these results, together with those of Tanaka, are none the less of considerable interest in the present context since they suggest that the increased motoneuron survival observed in vivo following identical regimens of curare treatment may not be the result of an increase in the production of a neurotrophic factor. Accordingly, if motoneuron survival in vivo can be significantly increased without altering the production of a neurotrophic factor, this implies that the amount of neurotrophic factor normally available from target muscles may be limited by some other means than production or synthesis.
Neuromuscular activity and synaptogenesis If the results of the in vitro and in vivo studies on paralysed muscle extracts 29'3° are taken at face value, then one has to postulate a mechanism other than increased trophic factor production as the basis for the increased motoneuron survival that is observed following activity blockade. In our original reports (and later) describing the in vivo effects of activity blockade on motoneuron survival 6'1s'19, we postulated that synaptic sites on myotubes represent restricted regions where motoneurons take up trophic factors and we showed that such sites were in fact increased following activity blockade 19'31. Accordingly, we have argued that a primary factor in determining whether or not a motoneuron survives is the formation of contacts or synaptic sites on myotubes, which in turn provide the motoneuron with access to a muscle-derived trophic factor. In the past ten years, there have been additional reports supporting the observation that muscles become hyper-innervated following embryonic activity blockade 32 - 35 . It has also been reported that regimens of activity blockade that reduce motoneuron death result in a marked increase in the branching of motoneuron axons within their target muscles.31 '36 . In a recent detailed 254
examination of muscle innervation and synaptogenesis following activity blockade in the chick embryo, Dahm and Landmesser35-37 found greatly enhanced nerve branching and synaptogenesis in both fast and slow hindlimb muscles during the period of normal motoneuron death. Additionally, they have found that in vivo perturbation of the function of certain adhesion molecules produced effects on axonal branching similar to those produced by activity blockade 37. Their results suggest that activity may serve to regulate the expression of certain cell adhesion molecules which in turn may control axonal branching and synaptogenesis. Activity blockade may also influence branching by means of atrophic factor, such as NGF (Ref. 5). In either case, activity blockade would promote survival by allowing more motoneurons access to a muscle-derived neurotrophic factor. As Dahm and Landmesser33 point out, neuromuscular activity may regulate motoneuron survival only to the extent that it controls intramuscular nerve branching and synaptogenesis. These data, when considered together with the results of Tanaka 29 and Houenou et a/.3°, provide an alternative to the 'production hypothesis' for explaining the increased motoneuron survival that occurs following activity blockade. This alternative, which I will call the 'access hypothesis', is of course not limited to the situation following activity blockade but can also be easily applied to the events regulating normal development (Fig. 1). According to this view, motoneurons compete for access to a target-derived trophic factor by means of axonal terminals and synapses. In contrast to the 'production hypothesis', however, sufficient amounts of a trophic factor would normally be produced by targets to support virtually all motoneurons that are generated. Thus, what is limiting is not trophic factor production, but rather the axonal branches, terminals and synaptic sites needed to gain access to the trophic factor. Any perturbations that alter branching and synaptogenesis (e.g. activity) would be expected to affect motoneuron survival without necessarily changing the production of trophic factors by target cells. One prediction of this hypothesis is that a major driving force behind the competition for access to atrophic factor is the capacity of motoneurons to branch and form contacts with myotubes. That is, motoneurons that have a greater intrinsic capacity to branch and form synapses (or to respond to extrinsic branching and synaptogenic signals) would be expected to have a competitive edge in the struggle for survival over their less well endowed neighbors. Although I have generally assumed that increased branching is correlated with increased formation of synaptic contacts, and therefore that synaptic contacts are actually the limiting parameter for survival, it should be noted that increased branching per se could contribute to survival prior to and during the early period of synaptogenesis by providing greater access to glial-derived trophic moleculesL Additionally, it is conceivable that trophic factor availTIN& Vol. 12, No. 7, 1989
v e ability could be regulated solely by the availability of synaptic sites with little, if any, need to postulate the involvement of axonal branching.
Concluding remarks A critical examination of the notion that trophic factors are p r o d u c e d in limiting amounts, has shown that this idea may not be correct for at least one population of neurons that exhibit normal cell death (i.e. spinal motoneurons). Several lines of evidence indicate that the normal survival of only a proportion of the spinal motoneurons that are initially generated during development may reflect limited access, via axonal branches and synaptic sites, to a muscle-derived neurotrophic factor rather than limited trophic factor production by target cells. If this view is correct - and at present, the evidence is admittedly still largely circumstantial - then it provides an alternative mechanism for explaining why target-derived trophic factors are available in limiting amounts. Although the arguments presented here have focused on motoneuron death, there is no reason why the access hypothesis could not also be applied to cell death of other targetdependent populations of peripheral and central neurons. Finally, in order to avoid any misunderstanding, I feel compelled to make two closing comments. First, irrespective of the specific mechanism responsible for limited availability of trophic factors, the neurotrophic hypothesis remains as the most attractive explanation for cell death and related events during development of the nervous system s . Second, the final resolution of the validity of either the access or production hypotheses will continue to depend critically on careful quantitative assessments of the levels of trophic factors produced by target cells 1'2'17.
Selected references 1 Barde, Y-A. (1988) Trends Neurosci. 11,343-346 2 Davies,A. M. (1988) Trends Neurosci. 11,243-244 3 Johnson, E. M., Jr, Taniuchi, M. and DiStefano, P. S. (1988) Trends Neurosci. 11,299-304 40ppenheim, R. (1981) in Studies in Developmental Neurobiology: Essays in Honor of Viktor Hamburger (Cowan, W. M., ed.), pp. 74-133, Oxford University Press 5 Purves, D. (1988) Body and Brain: A Trophic Theory of Neural Connections Harvard University Press 6 Hamburger, V. and Oppenheim, R. (1982) Neurosci. Comment. 1, 39-55 7 Levi-Montalcini, R. (1987)Science 237, 1154-1162 8 Berg, D. (1984) Annu. Rev. Neurosci. 7, 149-160 9 Barde, Y-A., Edgar, D. and Thoenen, H. (1983) Annu. Rev. Physiol. 45, 601-612 10 Wright, L., Cunningham, T. and Smolen, A. (1983) J. Neurocytol. 12,727-738 11 Hamburger, V., Brunso-Bechtold, J. and Yip, J. (1981) J. Neurosci. 1, 60-71 12 Levi-Montalcini, R. and Hamburger, V. (1953) J. Exp. Zool. 123, 233-287 13 Gorin, P. and Johnson, E. (1979) Proc. Natl Acad. Sci. USA 76, 5382-5386 14 Levi-Montalcini, R. and Cohen, S. (1956) Proc. NatlAcad. Sci. USA 42,695-699 15 Oppenheim, R., Maderdrut, J. and Wells, D. (1982)J. Comp. Neurol. 210, 174-189 TINS, Vol. 12, No. 7, 1989
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Korsching, S. and Thoenen, H. (1988) Dev. Biol. 126, 40-46 Davis, A. etal. (1987) Nature 326, 353-358 Pittman, R. and Oppenheim, R. (1978) Nature 271,364-366 Pittman, R. and Oppenheim, R. (1979) 1 Comp. Neurol. 187, 425-446 Laing, N. and Prestige, M. (1978) J. Physiol (London) 282, 33-34P Oppenheim, R. (1985) Trends Neurosci. 8, 487-493 Oppenheim, R., Haverkamp, L., Prevette, D., McManaman, J. and Appel, S. (1988) Science 240, 919-922 Dohrmann, U., Edgar, D., Sendter, M. and Thoenen, H. (1986) Dev. Biol. 118, 209-221 Oppenheim, R. (1982) in Current Topics in Developmental Biology: Neural Development (Hunt, R. K., ed.), pp. 257-309, Academic Press Hamburger, V. (1963) (9. Rev. Biol. 38, 342-365 Oppenheim, R. and Nunez, R. (1982) Nature 295, 57-59 Hill, M. and Bennett, M. (1986) Dev. Brain Res. 24,305-312 Henderson, C. (1988) in Plasticity of the Neuromuscular System (Evered, D. and Whelan, J., eds), pp. 172-191, John Wiley & Sons Tanaka, H. (1987) Dev. Biol. 124, 347-357 Houenou, L., Prevette, D. and Oppenheim, R. Soc. Neurosci. Abstr. 15 (in press) Oppenheim, R. and Chu-Wang, I. (1983) in Somatic and Autonomic Nerve-Muscle Interactions (Burnstock, G., ed.), pp. 58-107, Elsevier Ding, R., Jansen, J., Laing, N. and Tonnesen, H. (1983) J. Neurocytol. 12,887-919 Oppenheim, R. (1984) Dev. Biol. 101, 35-39 Oppenheim, R. etal. (1986) Dev. BioL 114, 426-436 Dahm, L. and Landmesser,L. (1988) Soc. Neurosci. Abstr. 14, 823 Dahm, L. and Landmesser,L. (1988) Dev. Biol. 130,621-644 Landmesser, L., Dahm, L., Schultz, K. and Rutishauser, U. (1988) Dev. Bio1130, 645-670
po n% Acknowledgements Supportedby NIH 6rant NS20402. The developmentof the ideasexpressedhere benefitedgreatly from the comments andsuggestionsof ViktorHamburger, LucienHouenou, EugeneJohnson,Jeff Lichtmanand Dale Purves. l alsothank YvesBardefor his commentsduring the reviewprocess.
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252
There is increasing evidence that target-derived molecules play a crucial role in the regulation of neuronalsurvival during development. These molecules, termed neurotrophic factors, are thought to act in specific ways as defined by the neurotrophic theory. One central tenet of the neurotrophic theory is that some neuronsin a population die because trophic molecules are available in only limited amounts during periods of naturally occurring cell death. Ddivery of trophic factor to nerve terminals could be regulated by several mechanisms, including, for example, limited production (biosynthesis) by target cells, limited release by targets, or limited uptake by pre-synaptic terminals. An examination of recent studies of motoneuron development indicates that motoneurons compete, via axona/ branching and synaptic contacts, for restrictedsites on targets that provide accessto trophic factors. According to this view, it is terminal branches and contact ('synaptic') sites that limit the regulation of neuronal survival, rather than trophic factor production.
Nonetheless, many other suspected neurotrophic factors have subsequently been identified and proposed to play a similar role to NGF, albeit for other populations of neurons 8'9. Because it was the first and most completely characterized neurotrophic factor to be identified, NGF has continued to serve as a model with which other putative neurotrophic factors are compared. Thus, unless other molecules proposed as neurotrophic factors meet certain criteria that were established originally for NGF they are often considered to be suspect1,2. One of these criteria is that target-derived neurotrophic factors are produced in limiting or subsaturating amounts. In the case of NGF, the evidence for this belief is, on the face of it, rather compelling. First, only a proportion of the NGFsensitive sensory and sympathetic ganglion cells that
A number of papers have appeared recently in the pages of this journal discussing the neurotrophic theory and its applicability to neuronal survival I-3. This theory arose out of the pioneering studies of Hamburger and Levi-Montalcini in the 1940s and 1950s on embryonic cell death, studies that ultimately led to the discovery of the prototypical neurotrophic factor, NGF4. These early studies, and the resulting discovery of a molecule (NGF) that was necessary for the survival of specific populations of embryonic neurons, established the foundation for most later conceptualizations of the ontogeny of neuron-target interactions, including the neurotrophic theory s. The neurotrophic theory provides a useful framework for understanding several features of neuronal development, including the question of why, in many populations of developing neurons, only a proportion of the original number of cells survives. It is now well known that in many regions of the CNS and PNS large numbers ( - 5 0 % ) of postmitotic neurons degenerate and die by a process of naturally occurring neuronal death 4'6. Further, it has been repeatedly demonstrated that in most populations this normal cell loss occurs during the period when neurons are establishing synaptic connections with their targets. This temporal coincidence, together with the demonstration that manipulations of the availability of putative synaptic targets alters the number of surviving innervating neurons 4, led to the proposal that neurons compete for some entity that is supplied by the targets in limiting amounts. Following the discovery of NGF, it was widely believed that this entity was, in fact, represented by the biological activity of distinct molecules derived from target cells. NGF remains the only such molecule that has been shown to meet virtually all of the criteria necessary to qualify as a specific neurotrophic factor involved in regulating the survival of discrete populations of neurons 7.
are initiallygenerated normally survive1 0 1'1 . Second, treatment of embryos or postnatal animals with exogenous sources of N G F serves to rescue most of the neurons that would normally die12-1s. And finally, the amounts of N G F present in target sites during periods of normal cell death are reported to be exceedingly small and correlated with innervation density 1 6 1'7 . Although these data are all
consistent with the notion of limited trophic factor production, they do not exclude alternative proposals for limited trophic factor availability. For instance, it is conceivable that neurons normally die because they are unable to gain access to neurotrophic molecules rather than owing to limited trophic factor production. Similarly, exogenous supplies of a neurotrophic factor may rescue neurons by circumventing the normal route of access rather than by supplementing the limiting amounts thought to be produced normally. Finally, until the amount of neurotrophic factor that is actually required by a neuron for survival is known, the mere presence of small quantities in target tissues is not in itself a compelling argument that these amounts are, in fact, sub-saturating or limiting. These considerations, together with recent evidence from studies of motoneuron survival in the chick embryo, reviewed below, underscore the need for a critical re-examination of the notion that neurotrophic factors must necessarily be produced in limiting amounts during periods of naturally occurring neuronal death.
Neuromuscular activity and trophic factor production It has been more than ten years since the first reports that the chronic blockade of neuromuscular activity in the chick embryo results in the rescue of virtually all of the spinal motoneurons that would normally degenerate and die 18-2°. Subsequent
© 1989, ElsevierSciencePublishers Ltd.(UK) 0166-2236/89/$02.00
TINS, Vol. 12, No. 7, 1989
v studies have confirmed and extended the original observations21. Although the cellular and molecular mechanisms responsible for this unexpected finding are still not known, two kinds of explanation have been offered. The first hypothesis, which I call the 'production hypothesis', proposes that the production (i.e. the biosynthesis by target cells) of a muscle (target)-derived neurotrophic factor is inversely related to muscle activity (Fig. 1). Considerable evidence now exists for the presence of an NGF-like, muscle-derived, neurotrophic factor that promotes motoneuron survival both in vitro and in vivo 22'23. Thus, according to the production hypothesis, the normal, ongoing spontaneous neuromuscular activity that characterizes the development of all vertebrate embryos 24'25 would maintain the production of this muscle-derived neurotrophic factor at levels that are limiting. It follows then that lower than normal levels of activity should increase, and higher than normal levels of activity should decrease, the production of this trophic factor. Although such perturbations of activity are known to result in the predicted changes in motoneuron survival 18-2°'26, it is not presently known whether trophic factor production is also altered as expected under these conditions.
ewpoint
However, if a muscle-derived neurotrophic factor is normally produced in sub-saturating amounts, then the 'production hypothesis' would seem to be the most plausible explanation for the known effects of altered activity on motoneuron survival. There is, in fact, some evidence showing that surgical denervation of muscle, which effectively abolishes activity, results in the increased accumulation of a muscle-derived neurotrophic factor as measured in vitro 27'28. However, these studies differ in so many important ways from the in vivo experiments on neuromuscular blockade that they offer little support for the validity of the 'production hypothesis'. By contrast, a recent study by Tanaka29, although flawed in some respects, comes closer to qualifying as a critical test of this hypothesis. Tanaka compared the effectiveness of muscle extracts obtained from skeletal muscle of normally active control chick embryos with extracts from paralysed embryos that were exposed to regimens of activity blockade similar to those known to rescue motoneurons from naturally occurring cell death in vivo. Motoneurons were retrogradely labeled in vivo at stage 29-30 (i.e. at the onset of normal cell death) with a longlived fluorescent dye (Dil) following injections into the hindlimb. The lumbar spinal cord was then
Tml~
Target C
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\
/
\
/
\
/
\
,
/
1
Fig. 1. A schematic illustration of neurotrophic interactions between motoneurons and their targets according to either the "access hypothesis' (A, B) or the 'production hypothesis' (C, D), as described in the text. In the 'access hypothesis', a population of neurons compete, via axonal branches and synaptic sites in the target, for neurotrophic molecules (solid black dots in target). During normal development (A) neurons die (dashed lines) owing to limited access to neurotrophic molecules. Following activity blockade or other perturbations that increase branching and synaptic contacts (B), neurons are able to gain access to sufficient trophic factor to survive. According to the "production hypothesis', the production of trophic factors by targets is normally limited, resulting in the death of some neurons (C). Activity blockade or other perturbations that rescue neurons from death do so by virtue of increased production of trophic factors by targets (D). TINS, VoL 12, No. 7, 1989
253
removed, dissociated and grown in culture for two days. Surviving, fluorescently labeled cells that extended neurites were counted as viable motoneurons at the end of the two-day culture period. No differences were observed in the motoneuron survival activity present in the muscles of control versus curare-treated embryos. Extracts from both sources of muscle produced an equivalent doserelated increase in motoneuron survival. Against these findings, one could argue that perhaps the two-day culture period was not sufficiently long to detect differences between the survival activity of control and curare-treated muscles or that in vitro conditions are not optimal for detecting such differences, even over longer periods of culture. However, recent in vivo studies support the findings of Tanaka. We have observed no differences in motoneuron survival following treatment of chick embryos in ovo with partially purified muscle extracts from normally active versus paralysed chick and mouse embryos 3°. Both control and paralysed muscle extracts increased motoneuron survival by 30-35%. Although further in vivo studies are needed to resolve this issue, these results, together with those of Tanaka, are none the less of considerable interest in the present context since they suggest that the increased motoneuron survival observed in vivo following identical regimens of curare treatment may not be the result of an increase in the production of a neurotrophic factor. Accordingly, if motoneuron survival in vivo can be significantly increased without altering the production of a neurotrophic factor, this implies that the amount of neurotrophic factor normally available from target muscles may be limited by some other means than production or synthesis.
Neuromuscular activity and synaptogenesis If the results of the in vitro and in vivo studies on paralysed muscle extracts 29'3° are taken at face value, then one has to postulate a mechanism other than increased trophic factor production as the basis for the increased motoneuron survival that is observed following activity blockade. In our original reports (and later) describing the in vivo effects of activity blockade on motoneuron survival 6'1s'19, we postulated that synaptic sites on myotubes represent restricted regions where motoneurons take up trophic factors and we showed that such sites were in fact increased following activity blockade 19'31. Accordingly, we have argued that a primary factor in determining whether or not a motoneuron survives is the formation of contacts or synaptic sites on myotubes, which in turn provide the motoneuron with access to a muscle-derived trophic factor. In the past ten years, there have been additional reports supporting the observation that muscles become hyper-innervated following embryonic activity blockade 32 - 35 . It has also been reported that regimens of activity blockade that reduce motoneuron death result in a marked increase in the branching of motoneuron axons within their target muscles.31 '36 . In a recent detailed 254
examination of muscle innervation and synaptogenesis following activity blockade in the chick embryo, Dahm and Landmesser35-37 found greatly enhanced nerve branching and synaptogenesis in both fast and slow hindlimb muscles during the period of normal motoneuron death. Additionally, they have found that in vivo perturbation of the function of certain adhesion molecules produced effects on axonal branching similar to those produced by activity blockade 37. Their results suggest that activity may serve to regulate the expression of certain cell adhesion molecules which in turn may control axonal branching and synaptogenesis. Activity blockade may also influence branching by means of atrophic factor, such as NGF (Ref. 5). In either case, activity blockade would promote survival by allowing more motoneurons access to a muscle-derived neurotrophic factor. As Dahm and Landmesser33 point out, neuromuscular activity may regulate motoneuron survival only to the extent that it controls intramuscular nerve branching and synaptogenesis. These data, when considered together with the results of Tanaka 29 and Houenou et a/.3°, provide an alternative to the 'production hypothesis' for explaining the increased motoneuron survival that occurs following activity blockade. This alternative, which I will call the 'access hypothesis', is of course not limited to the situation following activity blockade but can also be easily applied to the events regulating normal development (Fig. 1). According to this view, motoneurons compete for access to a target-derived trophic factor by means of axonal terminals and synapses. In contrast to the 'production hypothesis', however, sufficient amounts of a trophic factor would normally be produced by targets to support virtually all motoneurons that are generated. Thus, what is limiting is not trophic factor production, but rather the axonal branches, terminals and synaptic sites needed to gain access to the trophic factor. Any perturbations that alter branching and synaptogenesis (e.g. activity) would be expected to affect motoneuron survival without necessarily changing the production of trophic factors by target cells. One prediction of this hypothesis is that a major driving force behind the competition for access to atrophic factor is the capacity of motoneurons to branch and form contacts with myotubes. That is, motoneurons that have a greater intrinsic capacity to branch and form synapses (or to respond to extrinsic branching and synaptogenic signals) would be expected to have a competitive edge in the struggle for survival over their less well endowed neighbors. Although I have generally assumed that increased branching is correlated with increased formation of synaptic contacts, and therefore that synaptic contacts are actually the limiting parameter for survival, it should be noted that increased branching per se could contribute to survival prior to and during the early period of synaptogenesis by providing greater access to glial-derived trophic moleculesL Additionally, it is conceivable that trophic factor availTIN& Vol. 12, No. 7, 1989
v e ability could be regulated solely by the availability of synaptic sites with little, if any, need to postulate the involvement of axonal branching.
Concluding remarks A critical examination of the notion that trophic factors are p r o d u c e d in limiting amounts, has shown that this idea may not be correct for at least one population of neurons that exhibit normal cell death (i.e. spinal motoneurons). Several lines of evidence indicate that the normal survival of only a proportion of the spinal motoneurons that are initially generated during development may reflect limited access, via axonal branches and synaptic sites, to a muscle-derived neurotrophic factor rather than limited trophic factor production by target cells. If this view is correct - and at present, the evidence is admittedly still largely circumstantial - then it provides an alternative mechanism for explaining why target-derived trophic factors are available in limiting amounts. Although the arguments presented here have focused on motoneuron death, there is no reason why the access hypothesis could not also be applied to cell death of other targetdependent populations of peripheral and central neurons. Finally, in order to avoid any misunderstanding, I feel compelled to make two closing comments. First, irrespective of the specific mechanism responsible for limited availability of trophic factors, the neurotrophic hypothesis remains as the most attractive explanation for cell death and related events during development of the nervous system s . Second, the final resolution of the validity of either the access or production hypotheses will continue to depend critically on careful quantitative assessments of the levels of trophic factors produced by target cells 1'2'17.
Selected references 1 Barde, Y-A. (1988) Trends Neurosci. 11,343-346 2 Davies,A. M. (1988) Trends Neurosci. 11,243-244 3 Johnson, E. M., Jr, Taniuchi, M. and DiStefano, P. S. (1988) Trends Neurosci. 11,299-304 40ppenheim, R. (1981) in Studies in Developmental Neurobiology: Essays in Honor of Viktor Hamburger (Cowan, W. M., ed.), pp. 74-133, Oxford University Press 5 Purves, D. (1988) Body and Brain: A Trophic Theory of Neural Connections Harvard University Press 6 Hamburger, V. and Oppenheim, R. (1982) Neurosci. Comment. 1, 39-55 7 Levi-Montalcini, R. (1987)Science 237, 1154-1162 8 Berg, D. (1984) Annu. Rev. Neurosci. 7, 149-160 9 Barde, Y-A., Edgar, D. and Thoenen, H. (1983) Annu. Rev. Physiol. 45, 601-612 10 Wright, L., Cunningham, T. and Smolen, A. (1983) J. Neurocytol. 12,727-738 11 Hamburger, V., Brunso-Bechtold, J. and Yip, J. (1981) J. Neurosci. 1, 60-71 12 Levi-Montalcini, R. and Hamburger, V. (1953) J. Exp. Zool. 123, 233-287 13 Gorin, P. and Johnson, E. (1979) Proc. Natl Acad. Sci. USA 76, 5382-5386 14 Levi-Montalcini, R. and Cohen, S. (1956) Proc. NatlAcad. Sci. USA 42,695-699 15 Oppenheim, R., Maderdrut, J. and Wells, D. (1982)J. Comp. Neurol. 210, 174-189 TINS, Vol. 12, No. 7, 1989
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Korsching, S. and Thoenen, H. (1988) Dev. Biol. 126, 40-46 Davis, A. etal. (1987) Nature 326, 353-358 Pittman, R. and Oppenheim, R. (1978) Nature 271,364-366 Pittman, R. and Oppenheim, R. (1979) 1 Comp. Neurol. 187, 425-446 Laing, N. and Prestige, M. (1978) J. Physiol (London) 282, 33-34P Oppenheim, R. (1985) Trends Neurosci. 8, 487-493 Oppenheim, R., Haverkamp, L., Prevette, D., McManaman, J. and Appel, S. (1988) Science 240, 919-922 Dohrmann, U., Edgar, D., Sendter, M. and Thoenen, H. (1986) Dev. Biol. 118, 209-221 Oppenheim, R. (1982) in Current Topics in Developmental Biology: Neural Development (Hunt, R. K., ed.), pp. 257-309, Academic Press Hamburger, V. (1963) (9. Rev. Biol. 38, 342-365 Oppenheim, R. and Nunez, R. (1982) Nature 295, 57-59 Hill, M. and Bennett, M. (1986) Dev. Brain Res. 24,305-312 Henderson, C. (1988) in Plasticity of the Neuromuscular System (Evered, D. and Whelan, J., eds), pp. 172-191, John Wiley & Sons Tanaka, H. (1987) Dev. Biol. 124, 347-357 Houenou, L., Prevette, D. and Oppenheim, R. Soc. Neurosci. Abstr. 15 (in press) Oppenheim, R. and Chu-Wang, I. (1983) in Somatic and Autonomic Nerve-Muscle Interactions (Burnstock, G., ed.), pp. 58-107, Elsevier Ding, R., Jansen, J., Laing, N. and Tonnesen, H. (1983) J. Neurocytol. 12,887-919 Oppenheim, R. (1984) Dev. Biol. 101, 35-39 Oppenheim, R. etal. (1986) Dev. BioL 114, 426-436 Dahm, L. and Landmesser,L. (1988) Soc. Neurosci. Abstr. 14, 823 Dahm, L. and Landmesser,L. (1988) Dev. Biol. 130,621-644 Landmesser, L., Dahm, L., Schultz, K. and Rutishauser, U. (1988) Dev. Bio1130, 645-670
po n% Acknowledgements Supportedby NIH 6rant NS20402. The developmentof the ideasexpressedhere benefitedgreatly from the comments andsuggestionsof ViktorHamburger, LucienHouenou, EugeneJohnson,Jeff Lichtmanand Dale Purves. l alsothank YvesBardefor his commentsduring the reviewprocess.
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