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Mechanism of synapse disassembly at the developing neuromuscular junction
Mechanism of synapse disassembly at the developing neuromuscular junction Quyen T Nguyen1 and Jeff W Lichtman2 Throughout the developing nervous system of higher vertebrates, synaptic connections are concurrently being established and eliminated. The consequence of this synaptic remodeling is that axons strengthen their connections with some targets while completely disconnecting from other postsynaptic cells. The transition from multiple to single axonal innervation of skeletal muscle fibers is the most accessible example of this developmental reorganization. In muscle, the elimination of axonal input appears to be driven by a protracted competition between different axons co-innervating the same junction, with the muscle fiber as intermediary. Asynchronous synaptic activity may be the factor that differentiates the competing inputs. In some circumstances, synapses can also be lost in ways that are independent of activity. Similarities between activity-dependent and activity-independent synapse elimination provide insights into mechanisms underlying developmental synaptic reorganization.
Address
Department of Anatomy and Neurobiology, Box 8108, Washington University School of Medicine, 660 South Euclid, St Louis, Missouri 63110, USA 1e-maih [email protected] 2e-mail: [email protected] Abbreviations AChR acetylcholine receptor BDNF brain-derivedneurotrophic factor bFGF basic fibroblast growth factor CGRP calcitoningene related peptide CNTF ciliary neurotrophic factor 5-HPETE 5-hydroxy-6,8,11,14-eicosatetraenoic acid LIF leukemia inhibitory factor NMJ neuromuscularjunction NO nitric oxide NT-3 neurotrophin-3 OSM oncostatin M
TTX
tetrodotoxin
Current Opinion in Neurobiology 1996, 6:104-112
© Current Biology Ltd ISSN 0959-4388
Introduction
The nervous system disassembles many synaptic connections in early postnatal life. If there is a general purpose to this surprising phenomenon, it is not understood. Nonetheless, the nature of the change in synaptic circuitry is similar wherever it has been described. In parts of the visual system, auditory system, cerebellum, autonomic ganglia and skeletal muscle, the number of different axons that innervate individual postsynaptic cells decreases in the perinatal period [1,2]. In each of these cases, target
cells lose inputs during a defined period of perinatal life, ultimately becoming strongly innervated by relatively few (often only one) axon. Presynaptic cell death does not cause these axonal inputs to be shed, because the period of naturally occurring neuronal cell death is largely complete during embryogenesis. Rather, axons initially branch to establish relatively weak connections with many target cells, and then lose some of these branches, to concentrate more of their synapses on fewer target cells. In several different parts of the nervous system, neural activity seems an important influence on the loss, suggesting that a common mechanism may underlie synapse withdrawal wherever it occurs. One approach to understanding the cellular and molecular cascades leading to axon withdrawal is the study of simple and accessible preparations such as the neuromuscular junction (NMJ). In mammalian muscle, the retraction of motor axonal branches leaves each NMJ with only one innervating axon during early postnatal life. During embryonic life (before the transition to single innervation), however, motor neurons establish connections with a larger number of muscle cells than they will ultimately maintain. The consequence of the supernumerary connections is that each myofiber typically receives input from two or more axons, and probably no fiber is without innervation. Gradually--over the first several postnatal weeks in most rodent m u s c l e s - - t h e number of muscle cells that a given axon innervates decreases (Figure 1). There seems to be little pattern as to which muscle fibers ultimately maintain association with each axon, but the loss cannot be considered random because in the end, every fiber is innervated by precisely one axon that is capable of driving the muscle fiber to threshold. The outcome of this adjustment process gives rise to a finely tuned system adapted for the efficient integration of muscle tension as motor axons are recruited. Although there have been many proposals concerning the mechanism of synapse disassembly at the NMJ, there is as yet no consensus. Our aim in this review is to explore several themes that could constitute the basis for the underlying mechanism. These themes include the role of the pre-, postsynaptic and supporting cells as well as diffusible and substrate-bound extracellular factors in the elimination of synapses. It is likely that the cascade of events that lead to synapse loss will require understanding a number of intra- and extracellular processes. Thus, a variety of the current views that are often posited as 'alternatives' may prove to play a significant part in the complete explanation. Finally, we discuss a few non-developmental instances of synapse loss
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
105
Figure 1
Progressive transition from multiple to single innervation
•k
"k
"k © 1996 Current Opinion in Neurobiology
Diagram showing the progressive transition of mammalian skeletal muscle from multiple innervation at birth (P0) to single innervation two weeks later (P15). Initially, axons converging on the same NMJ occupy comparable synaptic areas. At one week postnatal (PT), about half of the fibers have already attained single innervation. The step-wise removal of synaptic sites occupied by withdrawing axons shows that synapse elimination is also progressive at the level of single muscle fibers; for example, compare the middle muscle fiber (*) at PO to P7 to P15. By two weeks postnatal (P15), all muscle fibers are singly innervated.
that suggest that the synapse disassembly program that occurs in development is also used in other situations where synapses need to be removed.
input, indicating that the axon losing territory continues to f u n c t i o n - - a l b e i t progressively less effectively--until it is eliminated (H Colman, J Nabekura, JW Lichtman, unpublished data).
Phenomenology
T h e sequence of events leading to nerve terminal withdrawal has been studied in living animals by microscopic monitoring of NMJs over time. These studies show that before each terminal bouton withdraws, the acetylcholine receptor (AChR) density begins to decrease postsynaptically [4,6]. T h e coordinated dismantling of pre- and postsynaptic sites is repeated multiple times until all the synaptic sites associated with one axon are removed. Once all of the pre- and postsynaptic sites associated with one axon have been eliminated, the nerve terminal resorbs back into the parent axon [5,7]. T h e sequential loss of postsynaptic AChR sites associated with one input may be instrumental in transforming the uniform receptor plaques seen at birth into the branched pattern seen two weeks later [8-11]. T h e s e observations imply three things: first, synapse elimination occurs by coordinated changes pre- and postsynaptically (Figure 2); second, that the early postsynaptic changes suggest that the muscle is an intermediary in the process; and finally, that axonal branch withdrawal is not the cause but rather the consequence of synapse elimination.
Synapse elimination is progressive in two ways (Figure 1). First, even though all cells in any particular skeletal muscle are multiply innervated during late embryonic life, some cells within the muscle attain single innervation earlier than others [3]. For example, in mouse sternomastoid muscle, over 90% of cells are multiply innervated at birth, half are singly innervated at one week postnatal, and by two weeks, all are singly innervated [4]. Synapse elimination is also progressive at the level of a single muscle cell. Labeling individual axons with lipophilic dyes shows multiple motor axons converging on the same NMJ initially start with comparable synaptic areas [5]. However, during the first few postnatal weeks, an increasing number of multiply innervated junctions show skewing of the synaptic area in favor of one axon over another. This implies that one of the axons is progressively losing synapses, until it no longer occupies any territory on the muscle fiber, and retracts. Recent physiological studies of multiply innervated junctions show a concomitant reduction in synaptic strength of one
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Development
multiple NMJs on their surface, and each of these is multiply innervated [17,18].
Figure 2 Synapse dismantling
Activity-dependent competition
Developmental synapse elimination is competitive in the sense that direct or indirect interactions between the axons give rise to the loss of all but one input at each junction. Because each axon converging at a NMJ has multiple synaptic contacts at that site, the competition is actually between synaptic cartels (synchronously active synapses innervating the same postsynaptic cell) rather than axons, perse [19-22]. Despite the general agreement that activity levels influence the rate of synapse elimination, there is conflicting evidence on whether the more or the less active cartel is selectively maintained. On one hand, selective stimulation of some axons during the period of developmental synapse elimination seems to cause those axons to maintain larger motor units compared to unstimulated ones [23]. Similarly, regenerating axons that are active reinnervate more muscle fibers than axons that are silenced with local tetrodotoxin ( T T X ) infusion [24]. However, opposite results have also been found: decreasing the activity of some axons (during the transition from multiple to single innervation) by a T T X cuff causes those axons to retain innervation with more muscle fibers than active axons [25,26]. In addition, during reinnervation, TTX-silenced axons can apparently displace sprouts of active axons [27].
© 1996 Current Opinion in Neurobiology
Diagram showing the coordinated sequence of synapse dismantling leading to nerve terminal withdrawal. (a) The cascade is initiated by a decrease in postsynaptic receptors (either by lateral diffusion or internalization), (b) followed by nerve terminal retraction.
T h e lack of consistent results from manipulating axonal activity may indicate the subtlety of the process. Presumably, the relevant variable is the relative activity levels of two axons co-innervating the same junction. In all these experiments, where one nerve bundle is either stimulated or paralyzed, only a minority of fibers in the muscle will be innervated by one active and one inactive axon. In addition, these experiments regulate the number of action potentials in axons, not the efficacy of the synapse. T h e biologically relevant activity parameter may be the likelihood that action potentials presynaptically give rise to postsynaptic impulses. Recent experiments that attempt to approach this problem at the level of individual junctions are described below. Activity-mediated postsynaptic changes
Activity
is c r i t i c a l l y
important
There are several lines of evidence that indicate that preor postsynaptic activity is important in synapse elimination (Table 1). Experimental attempts at raising activity levels cause muscles to complete synapse elimination earlier [12-14]. Conversely, synapse elimination at the NMJ is retarded, if not completely prevented, by experimental inhibition of activity via muscle tenotomy, deafferentation, spinal cord section, pharmacological blockade of impulse transmission (see [15]), or in activity-deficient mutants [16]. Furthermore, muscle fibers that ordinarily do not have the ability to fire action potentials (e.g. tonic fibers in reptiles) do not undergo synapse elimination: they have
As already mentioned, beneath the synapses targeted for elimination, postsynaptic specializations begin to de-differentiate before the overlying nerve terminal boutons withdraw. This transformation includes the loss of AChRs, rapsyn/43K and perhaps other molecules [4,6,28]. At the same time, the intermingled synaptic sites occupied by the surviving input remain unperturbed. This raises the question of how the postsynaptic cell simultaneously removes some synaptic sites while maintaining others nearby. One possibility is that the muscle senses differences between sites by virtue of when they are active. This would explain why in places where previously active nerve terminals are being replaced by
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
progressive myelination, AChRs are removed [4]. This would also explain why during reinnervation, re-occupied synaptic sites cause AChR density to decrease at nearby unoccupied sites in the same junction, even though denervated NMJs maintain their AChRs [29]. To test the possibility that asynchronous receptor activation is the signal that allows the muscle to selectively destabilize synaptic sites, focal postsynaptic blockade was induced by local application of et-bungarotoxin. T h e result was that the blocked regions, and subsequently their overlying nerve terminals, were eliminated, but only when a substantial part of the junction remained unblocked [30"']. T h e requirement that there be active regions for elimination to occur suggests that active regions destabilize inactive regions. T h e same mechanism could
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explain axonal competition during development if axons converging on the same muscle fiber were not firing synchronously (Figure 3): asynchronous motor neuron activation during the recruitment of motor units does occur in adults [31]; however, the firing pattern of motor axons in early postnatal life has not yet been characterized. One consequence of decreases in AChR density at the synaptic sites occupied by one axon is that these changes will themselves potentiate further differences in the relative efficacy of competing inputs. As the efficacies of competing axons diverge, activity-mediated AChR loss will be further accentuated. This kind of positive feedback probably underlies the system's remarkable efficiency at removing all traces of multiple innervation within a few weeks of birth.
Table 1 Factors that may Influence synapse elimination at the neuromuscular junction. Factor Activity Total activity Activity of individual axon
Activity of postsynaptic cell
Spontaneous release
Level of factor
System
Synapse elimination
1" $
Rodent Rodent
1" $
[12,13] [15]
1" ,]. $
Rodent Rodent Rodent
$ 1" $
[23,32,34 °] [24] [25,26]
1`
Xenopus culture
$
Rodent
1" $
[35 °] [15]
1` + $
Rodent
1"
[30 °°]
$ $ 1" 1" $ 1" 1" $ 1"
Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture
1" $ T 1" 1" 1" .L
Rodent Rodent Rodent Rodent Rodent Rodent Rodent
$ 1" .[. $ $ $ 1"
[45] [49] [45-48] [45] [47] [66,67] [66,67]
1" $ 1" 1"
Rodent Rodent Rodent Rodent culture
1" $ .J. 1"
[53-55] [53-55] [50,51,52 °] [50,51,52 °]
Efficacy
References
Activity of single NMJ Regulations of efficacy NO
ATP AN5-HPETE NT-3 BDNF CNTF CGRP Trophic sustenance UF
CTNF OSM bFGF Androgens Protease and protease inhibitors CANP PNI PA
~. 1" $ 1" 1"
$ 1" 1" $ 1" 1" 1" 1"
[36] [36] [37] [38] [38] [39] [39] [40] [41 ]
hA, arachidonic acid; ATP, adenosine triphosphate; CANP, calcium-activated neutral protease; PA, plasminogen activator; PNI, protease nexin I. Other abbreviations listed at the beginning of the review.
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Development
Figure 3 Schematic showing how asynchronous receptor activation may be the signal that allows muscle to selectively destabilize s.ynaptic sites. (a) Synchronous activity. When co-innervating axons are simultaneously active, they both elicit a 'punishment' signal, but both also have a protection signal and are maintained. (b) Asynchronous activity. When one axon is active (synapse on the left) while the other is inactive (synapse on the right), the punishment signal invoked by the active axon selectively destabilizes receptors beneath the inactive axon (which lacks a protection signal), causing subsequent synapse loss of the inactive axon. (c) Inactivity. When both axons are inactive, neither the punishment signal nor the protection signal is present, and, therefore, both axons remain. Adapted from [68].
Putative mechanism of synapse loss
Punishment signal Protection signal :::::::::::::
Postsynaptic receptors
~(~
Active terminal
Inactive terminal
~ 1996 Current Opinion in Neurobiology
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
Activity-mediated retrograde messengers
Regulators of efficacy If activity differences between the inputs converging on a target cell mediate synaptic competition, then agents that modify the strength of the competing inputs will play an important role. Beginning with work from Mu-ming Poo's lab [32,33,34",35°], there is evidence to support the idea that muscle cells and/or supporting cells at the synapse may modify synaptic strength by activity-dependent release of retrogradely acting diffusible factors. Moreover, hetero- and homosynaptic effects suggest that these agents can mediate competition between axons [32,33,34°,35°]. Several candidate messengers have been proposed (Table 1). Nitric oxide (NO), a diffusible free radical implicated in various forms of neuronal plasticity has also been shown to affect developing NMJs [36]. Several other factors, including ATP, arachidonic acid (or its metabolite 5-HPETE), neurotrophin (NT)-3, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and calcitonin gene related peptide (CGRP), have also been shown to modulate synaptic activity [37-41]. Whether these changes cause long-lasting alterations in synaptic efficacy is not yet known. Nor is it yet clear if these factors play such a role at the more mature NMJs undergoing synaptic competition posmatally (i.e. more than a week after the synapses are first formed). Trophic sustenance of synapses
A long-held view ofsynaptic competition asserts that axons innervating a common target must compete with each other for a limited supply of trophic factors to maintain functional synapses. T h e idea is an extension of the well-established role of neurotrophic agents as mediators of neuronal survival. However, in this case, the survival of a synapse itself is presumed to require an adequate supply of atrophic factor. Thus, of the set of synaptic terminals established by a neuron, only those synapses receiving an adequate supply of factor locally from their postsynaptic partners will be maintained. In order for axons to compete for agents such that two axons can never co-exist for long on the same postsynaptic cell, specific proposals for activity-mediated release and uptake of these agents have been suggested [42]. Recent observations that activity effects and trophic effects may be linked, and that trophins are present and effective in the brain during times of axon withdrawal have helped legitimize this view. This competition would have to operate over relatively large areas because synaptic competition often occurs between sites located hundreds of microns apart [43,44]. T h e possibility that competition for trophic factors drives axonai loss at the NMJ has been pursued by examining the consequences of.excess or reduced putative trophic agents on the synapse elimination process. If limitation in trophic factor supply is the cause of synapse loss, then a surplus of trophic factor should cause the retention of multiple axons at a junction for as long as the surplus supply is available. Conversely, a shortage of supply should result in
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more rapid synapse elimination, perhaps causing complete denervation if the levels are scarce enough. Thus far, experimental results with putative trophic agents at the NMJ have not shown these extreme outcomes. Nonetheless, a variety of possible trophic agents seem to modify the rate of synapse elimination (Table 1). For example, daily injection of various putative trophic factors--leukemia inhibitory factor (LIF), C N T E oncostatin M (OSM) or basic fibroblast growth factor ( b F G F ) - - i n t o neonatal rodents delays, but does not prevent, the withdrawal rate of superfluous nerve terminals from skeletal muscle [45-47]. Similarly, single intramuscular injection of C N T F and bFGF into embryonic or neonatal mice can induce up to three times the normal number of multiply innervated muscle fibers two weeks after birth [48], but these effects are also transient. Conversely, LIF-deficient mice develop singly innervated muscle fibers somewhat earlier than normal, but as far as is known, no fibers lose all inputs [49].
Postsynaptic proteolysis of synapses An alternative explanation for nerve terminal withdrawal or degeneration is that terminals are being destroyed or at least disconnected by the actions of proteases. Many proteases and proteases inhibitors are located at the NMJ [50]. Presently, their function is not understood. It is possible that their regulation is key in detaching nerve terminals from established synapses (Table 1). Nelson and colleagues [51,52°] have suggested a model for activitydependent postsynaptic activation of proteolysis. In their model, an activated postsynaptic cell releases proteases indiscriminately, resulting in global strength reduction of all inputs. Presynaptic activity, on the other hand, causes local release of protease inhibitors, and the conquering axons will be the ones that are active most often and can protect themselves [51]. In support of this hypothesis, Nelson and co-workers [52°] have demonstrated that several protease inhibitors (e.g. hirudin and protease nexin I) decrease the activity-dependent synaptic reduction they observed in co-cultures of myofibers and neurons in vitro. This is similar to findings of Vrbova and colleagues [12,53-55], who showed that synapse elimination during development and following reinnervation in rat skeletal muscle can be partly prevented by inhibiting proteolytic activity with inhibitors or calcium chelators. The strength of these kinds of models is that they tie electrical activity to a mechanism that could cause nerve terminal lysis. A weakness of these models is that they provide no obvious role for competition because an axon's fate is dependent only on its own activity level and resistance to proteolytic breakdown. How such a mechanism might unfailingly lead to single innervation is not yet clear. Effect of activity on supporting cells at the synapse
Schwann cells perform a wide variety of tasks related to nerve growth and maintenance [56]. Could their absence instigate synapse loss? Son and Thompson [57°°,58 °°]
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Development
have shown that Schwann cells are the first to respond to denervation or paralysis by elaborating processes upon which axons grow; these processes retract following reinnervation [59]. T h e multiple innervation seen during reinnervation is also guided by Schwann cells extending between adjacent endplates, creating bridges for axons to follow [57°',58°']. If Schwann cells are responsible for axonal sprouting, then it is possible that Schwann cells might also arbitrate sprout withdrawal. How Schwann cells could be influenced by local activity will need further investigation.
nerve terminals do not seem to maintain synaptic contact with degenerating muscle fibers for more than a day or two [65]. It is possible that variations in the activity level of motor axons between laboratory rodents (active) and laboratory frogs (not active), or temperature differences, explain the discrepancy. T h e rodent experiments suggest that postsynaptic maintenance of nerve terminal requires continual replenishment (and, therefore, synapse loss is attributable to the absence of some factor), whereas the frog work suggests that the appearance of a noxious agent may be necessary to induce synaptic detachment.
A c t i v i t y - i n d e p e n d e n t synapse e l i m i n a t i o n Synapse loss occurs in several situations besides the competitive, activity-dependent interactions already described. There are some similarities between these activity-dependent and activity-independent processes that suggest a common mechanism may underlie both.
Conclusions In two very different situations in which synapses are removed (i.e. developmental synapse elimination in muscle and following axotomy of neurons), the cellular events are remarkably similar. In both cases, disassembly of the postsynaptic apparatus precedes, and perhaps instigates, the removal of overlying nerve terminals. T h e parallels suggest that synapse elimination throughout the nervous system operates with a common mechanism. How postsynaptic changes might induce nerve terminal withdrawal is not presently known. Possible mediators include postsynaptic regulation of synaptic efficacy, trophic factor availability, proteolytic activity, and glial cells.
Axotomy causes the withdrawal of presynaptic input Axotomy of various CNS and PNS neurons causes weakening of their afferent input and ultimately synapse loss. In particular, stimulation of the presynaptic input to the axotomized neuron shows weakened synaptic transmission [60]. T h e physiology of synapses that are in the process of being eliminated following axotomy in the ciliary ganglion of chicks has been studied in detail, showing that they have reduced quantal size and content. These are the very same changes seen at NMJs undergoing synapse elimination during development (H Colman, J Nabekura, JW Lichtman, unpublished data). T h e reduced quantal size in both cases is thought to be caused by a decrease in postsynaptic sensitivity ([61]; H Colman, J Nabekura, JW Lichtman, unpublished data), probably secondary to a decrease in number of postsynaptic receptors [62]. In axotomy and in development, the changes in quantal size occur before terminal bouton detachment, suggesting that decreased postsynaptic receptor density may be an early step in the cascade leading to synaptic loss. Thus, in two very different situations where synapses are removed, the same sequence of events occurs, suggesting a common cellular program.
Acknowledgements Work done in JW Lichtman's laboratory was supported by grants from the National Institutes of Health and the lvluscular Dystrophy Association.
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Hantai D, Rao JS, Festoff BW: Serine proteases and serplns: their possible roles In the motor system. Rev Neurol (Paris) 1988, 144:680-687.
30. ••
Balice-Gordon RJ, Lichtman JW: Long-term synapse loss induced by focal blockade of postsynaptic receptors. Nature 1994, 372:519-524. Irreversible activation blockade of some receptors within a singly innervated neuromuscular junction induces loss of those receptors and subsequent withdrawal of the overlying nerve terminal. This experiment suggests that asynchronous receptor activation may be key in postsynaptic mediation of synapse withdrawal. 31.
HennemanE, Somjen G, Carpenter DO: Excitability end inhibitility of motomeurons of different sizes. J Neurophysiol 1965, 28:599-620.
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Liu Y, Raids RD, Fitzgerald S, Festoff BW, Nelson PG: Proteolytlc activity, synapse elimination, and the Hebb synapse. J Neurobio11994, 25:325-335.
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Lo YJ, Poo M-m: Activity-dependent synaptlc competition in vitro: heterosynaptlc suppression of developing synapses. Science 1991,254:1019-1022.
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Liu Y, Fields RD, Festoff BW, Nelson PG: Proteolytlc action of thrombin Is required for electrical activity-dependent synapse reduction. Proc Nat/Acad Sci USA 1994, 91:103OO-10304.
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Several protease inhibitors are shown to suppress activity-dependent synaptic reduction in an in vitro culture system of young neurons and myocytes. 53.
Connold AL, Evers JV, Vrbova G: Effect of low calcium and protease Inhibitors on synapse elimination during postnatal development in the rat soleus muscle. Dev Brain Res 1986,
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Cotman CW, Nieto-Sampedro M, Harris EW: Synapse replacement in the nervous system of adult vertebrates. Physio/Rev 1981, 61:684-784.
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Brenner HR, Martin AR: Reduction in scetylcholine sensitivity of axotomized ciliary ganglion cells. J Physio/(Lond) 1976, 260:159-175.
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Rotter A, Birdsall NJM, Burgen ASV, Field PM, Smolen A, Raisman, G: Muscarinic receptors in the central nervous system of the rat. IV. A comparison of the effects of axotomy and deafferentation on the binding of [3H]propylbenzilylcholine mustard and associated synaptic changes in the hypoglossal and pontine nuclei. Brain Res Rev 1979, 1:207-224. Sanes JR, Marshall LM, McMahan UJ: Reinnervation of muscle fiber basal lamina after removal of myofibers. J Cell Biol 1978,
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Vrbova G, Fisher TJ: The effect of inhibiting the calcium activated neutral protease, on motor unit size after partial denervation of the rat soleus muscle. Eur J Neurosci 1989, 1:616-625.
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Zhu PH, Vrbova G: The role of calcium in the elimination of polyneuronal innervation of rat soleus muscle fibres. Eur J Neurosci 1992, 4:433-437.
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Aguayo AJ, David S, Bray GM: Influences of the glial environment on the elongation of axons after injury: transplantation studies in adult rodents. J Exp Biol 1981, 95:231-240.
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Son YJ, Thompson WJ: Schwann cell processes guide regeneration of peripheral axons. Neuron 1995, 14:125-132. annotation [58"].
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Son YJ, Thompson WJ: Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells. Neuron 1995, 14:133-141. These two studies [57"',58"'] give a nice dynamic analysis of axonal regeneration on Schwann cell processes that extend in response to nerve injury. These glial processes appear to precede nerve terminal sprouting, suggesting that Schwann cells may play an important role in inducing and guiding axonal regeneration. 59.
Reynolds ML, Woolf C J: Terminal Schwann cells elaborate extensive processes following denervaUon of the motor endplate. J Neurocytol 1992, 21:50-66.
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DunaevskyA, Connor EA: Long-term maintenance of presynaptlc function in the absence of target muscle fibers. J Neurosci 1995, 15:6137-6144. A study using activity-dependent uptake of fluorescent dye to demonstrate that in frogs, presynaptic activity can be maintained for months in the absence of a viable postsynaptic muscle fiber. 65.
Rich MM, Lichtman JW: Motor nerve terminal loss from degenerating muscle fibres. Neuron 1989, 3:677-688.
66.
Jordan CL, Letinsky MS, Arnold AP: The role of gonadal hormones in neuromuscular synapse elimination in rats. I. Androgen delays the loss of multiple innervation in the levator ani muscle. J Neurosci 1989, 9:229-238.
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Jordan CL, Letinsky MS, Arnold AP: The role of gonadal hormones in neuromuscular synapse elimination in rats. II. Multiple innervatlon persists In the adult levator anl muscle after juvenile androgen treatment J Neurosci 1989, 9:239-247.
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Address
Department of Anatomy and Neurobiology, Box 8108, Washington University School of Medicine, 660 South Euclid, St Louis, Missouri 63110, USA 1e-maih [email protected] 2e-mail: [email protected] Abbreviations AChR acetylcholine receptor BDNF brain-derivedneurotrophic factor bFGF basic fibroblast growth factor CGRP calcitoningene related peptide CNTF ciliary neurotrophic factor 5-HPETE 5-hydroxy-6,8,11,14-eicosatetraenoic acid LIF leukemia inhibitory factor NMJ neuromuscularjunction NO nitric oxide NT-3 neurotrophin-3 OSM oncostatin M
TTX
tetrodotoxin
Current Opinion in Neurobiology 1996, 6:104-112
© Current Biology Ltd ISSN 0959-4388
Introduction
The nervous system disassembles many synaptic connections in early postnatal life. If there is a general purpose to this surprising phenomenon, it is not understood. Nonetheless, the nature of the change in synaptic circuitry is similar wherever it has been described. In parts of the visual system, auditory system, cerebellum, autonomic ganglia and skeletal muscle, the number of different axons that innervate individual postsynaptic cells decreases in the perinatal period [1,2]. In each of these cases, target
cells lose inputs during a defined period of perinatal life, ultimately becoming strongly innervated by relatively few (often only one) axon. Presynaptic cell death does not cause these axonal inputs to be shed, because the period of naturally occurring neuronal cell death is largely complete during embryogenesis. Rather, axons initially branch to establish relatively weak connections with many target cells, and then lose some of these branches, to concentrate more of their synapses on fewer target cells. In several different parts of the nervous system, neural activity seems an important influence on the loss, suggesting that a common mechanism may underlie synapse withdrawal wherever it occurs. One approach to understanding the cellular and molecular cascades leading to axon withdrawal is the study of simple and accessible preparations such as the neuromuscular junction (NMJ). In mammalian muscle, the retraction of motor axonal branches leaves each NMJ with only one innervating axon during early postnatal life. During embryonic life (before the transition to single innervation), however, motor neurons establish connections with a larger number of muscle cells than they will ultimately maintain. The consequence of the supernumerary connections is that each myofiber typically receives input from two or more axons, and probably no fiber is without innervation. Gradually--over the first several postnatal weeks in most rodent m u s c l e s - - t h e number of muscle cells that a given axon innervates decreases (Figure 1). There seems to be little pattern as to which muscle fibers ultimately maintain association with each axon, but the loss cannot be considered random because in the end, every fiber is innervated by precisely one axon that is capable of driving the muscle fiber to threshold. The outcome of this adjustment process gives rise to a finely tuned system adapted for the efficient integration of muscle tension as motor axons are recruited. Although there have been many proposals concerning the mechanism of synapse disassembly at the NMJ, there is as yet no consensus. Our aim in this review is to explore several themes that could constitute the basis for the underlying mechanism. These themes include the role of the pre-, postsynaptic and supporting cells as well as diffusible and substrate-bound extracellular factors in the elimination of synapses. It is likely that the cascade of events that lead to synapse loss will require understanding a number of intra- and extracellular processes. Thus, a variety of the current views that are often posited as 'alternatives' may prove to play a significant part in the complete explanation. Finally, we discuss a few non-developmental instances of synapse loss
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
105
Figure 1
Progressive transition from multiple to single innervation
•k
"k
"k © 1996 Current Opinion in Neurobiology
Diagram showing the progressive transition of mammalian skeletal muscle from multiple innervation at birth (P0) to single innervation two weeks later (P15). Initially, axons converging on the same NMJ occupy comparable synaptic areas. At one week postnatal (PT), about half of the fibers have already attained single innervation. The step-wise removal of synaptic sites occupied by withdrawing axons shows that synapse elimination is also progressive at the level of single muscle fibers; for example, compare the middle muscle fiber (*) at PO to P7 to P15. By two weeks postnatal (P15), all muscle fibers are singly innervated.
that suggest that the synapse disassembly program that occurs in development is also used in other situations where synapses need to be removed.
input, indicating that the axon losing territory continues to f u n c t i o n - - a l b e i t progressively less effectively--until it is eliminated (H Colman, J Nabekura, JW Lichtman, unpublished data).
Phenomenology
T h e sequence of events leading to nerve terminal withdrawal has been studied in living animals by microscopic monitoring of NMJs over time. These studies show that before each terminal bouton withdraws, the acetylcholine receptor (AChR) density begins to decrease postsynaptically [4,6]. T h e coordinated dismantling of pre- and postsynaptic sites is repeated multiple times until all the synaptic sites associated with one axon are removed. Once all of the pre- and postsynaptic sites associated with one axon have been eliminated, the nerve terminal resorbs back into the parent axon [5,7]. T h e sequential loss of postsynaptic AChR sites associated with one input may be instrumental in transforming the uniform receptor plaques seen at birth into the branched pattern seen two weeks later [8-11]. T h e s e observations imply three things: first, synapse elimination occurs by coordinated changes pre- and postsynaptically (Figure 2); second, that the early postsynaptic changes suggest that the muscle is an intermediary in the process; and finally, that axonal branch withdrawal is not the cause but rather the consequence of synapse elimination.
Synapse elimination is progressive in two ways (Figure 1). First, even though all cells in any particular skeletal muscle are multiply innervated during late embryonic life, some cells within the muscle attain single innervation earlier than others [3]. For example, in mouse sternomastoid muscle, over 90% of cells are multiply innervated at birth, half are singly innervated at one week postnatal, and by two weeks, all are singly innervated [4]. Synapse elimination is also progressive at the level of a single muscle cell. Labeling individual axons with lipophilic dyes shows multiple motor axons converging on the same NMJ initially start with comparable synaptic areas [5]. However, during the first few postnatal weeks, an increasing number of multiply innervated junctions show skewing of the synaptic area in favor of one axon over another. This implies that one of the axons is progressively losing synapses, until it no longer occupies any territory on the muscle fiber, and retracts. Recent physiological studies of multiply innervated junctions show a concomitant reduction in synaptic strength of one
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Development
multiple NMJs on their surface, and each of these is multiply innervated [17,18].
Figure 2 Synapse dismantling
Activity-dependent competition
Developmental synapse elimination is competitive in the sense that direct or indirect interactions between the axons give rise to the loss of all but one input at each junction. Because each axon converging at a NMJ has multiple synaptic contacts at that site, the competition is actually between synaptic cartels (synchronously active synapses innervating the same postsynaptic cell) rather than axons, perse [19-22]. Despite the general agreement that activity levels influence the rate of synapse elimination, there is conflicting evidence on whether the more or the less active cartel is selectively maintained. On one hand, selective stimulation of some axons during the period of developmental synapse elimination seems to cause those axons to maintain larger motor units compared to unstimulated ones [23]. Similarly, regenerating axons that are active reinnervate more muscle fibers than axons that are silenced with local tetrodotoxin ( T T X ) infusion [24]. However, opposite results have also been found: decreasing the activity of some axons (during the transition from multiple to single innervation) by a T T X cuff causes those axons to retain innervation with more muscle fibers than active axons [25,26]. In addition, during reinnervation, TTX-silenced axons can apparently displace sprouts of active axons [27].
© 1996 Current Opinion in Neurobiology
Diagram showing the coordinated sequence of synapse dismantling leading to nerve terminal withdrawal. (a) The cascade is initiated by a decrease in postsynaptic receptors (either by lateral diffusion or internalization), (b) followed by nerve terminal retraction.
T h e lack of consistent results from manipulating axonal activity may indicate the subtlety of the process. Presumably, the relevant variable is the relative activity levels of two axons co-innervating the same junction. In all these experiments, where one nerve bundle is either stimulated or paralyzed, only a minority of fibers in the muscle will be innervated by one active and one inactive axon. In addition, these experiments regulate the number of action potentials in axons, not the efficacy of the synapse. T h e biologically relevant activity parameter may be the likelihood that action potentials presynaptically give rise to postsynaptic impulses. Recent experiments that attempt to approach this problem at the level of individual junctions are described below. Activity-mediated postsynaptic changes
Activity
is c r i t i c a l l y
important
There are several lines of evidence that indicate that preor postsynaptic activity is important in synapse elimination (Table 1). Experimental attempts at raising activity levels cause muscles to complete synapse elimination earlier [12-14]. Conversely, synapse elimination at the NMJ is retarded, if not completely prevented, by experimental inhibition of activity via muscle tenotomy, deafferentation, spinal cord section, pharmacological blockade of impulse transmission (see [15]), or in activity-deficient mutants [16]. Furthermore, muscle fibers that ordinarily do not have the ability to fire action potentials (e.g. tonic fibers in reptiles) do not undergo synapse elimination: they have
As already mentioned, beneath the synapses targeted for elimination, postsynaptic specializations begin to de-differentiate before the overlying nerve terminal boutons withdraw. This transformation includes the loss of AChRs, rapsyn/43K and perhaps other molecules [4,6,28]. At the same time, the intermingled synaptic sites occupied by the surviving input remain unperturbed. This raises the question of how the postsynaptic cell simultaneously removes some synaptic sites while maintaining others nearby. One possibility is that the muscle senses differences between sites by virtue of when they are active. This would explain why in places where previously active nerve terminals are being replaced by
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
progressive myelination, AChRs are removed [4]. This would also explain why during reinnervation, re-occupied synaptic sites cause AChR density to decrease at nearby unoccupied sites in the same junction, even though denervated NMJs maintain their AChRs [29]. To test the possibility that asynchronous receptor activation is the signal that allows the muscle to selectively destabilize synaptic sites, focal postsynaptic blockade was induced by local application of et-bungarotoxin. T h e result was that the blocked regions, and subsequently their overlying nerve terminals, were eliminated, but only when a substantial part of the junction remained unblocked [30"']. T h e requirement that there be active regions for elimination to occur suggests that active regions destabilize inactive regions. T h e same mechanism could
107
explain axonal competition during development if axons converging on the same muscle fiber were not firing synchronously (Figure 3): asynchronous motor neuron activation during the recruitment of motor units does occur in adults [31]; however, the firing pattern of motor axons in early postnatal life has not yet been characterized. One consequence of decreases in AChR density at the synaptic sites occupied by one axon is that these changes will themselves potentiate further differences in the relative efficacy of competing inputs. As the efficacies of competing axons diverge, activity-mediated AChR loss will be further accentuated. This kind of positive feedback probably underlies the system's remarkable efficiency at removing all traces of multiple innervation within a few weeks of birth.
Table 1 Factors that may Influence synapse elimination at the neuromuscular junction. Factor Activity Total activity Activity of individual axon
Activity of postsynaptic cell
Spontaneous release
Level of factor
System
Synapse elimination
1" $
Rodent Rodent
1" $
[12,13] [15]
1" ,]. $
Rodent Rodent Rodent
$ 1" $
[23,32,34 °] [24] [25,26]
1`
Xenopus culture
$
Rodent
1" $
[35 °] [15]
1` + $
Rodent
1"
[30 °°]
$ $ 1" 1" $ 1" 1" $ 1"
Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture Xenopus culture
1" $ T 1" 1" 1" .L
Rodent Rodent Rodent Rodent Rodent Rodent Rodent
$ 1" .[. $ $ $ 1"
[45] [49] [45-48] [45] [47] [66,67] [66,67]
1" $ 1" 1"
Rodent Rodent Rodent Rodent culture
1" $ .J. 1"
[53-55] [53-55] [50,51,52 °] [50,51,52 °]
Efficacy
References
Activity of single NMJ Regulations of efficacy NO
ATP AN5-HPETE NT-3 BDNF CNTF CGRP Trophic sustenance UF
CTNF OSM bFGF Androgens Protease and protease inhibitors CANP PNI PA
~. 1" $ 1" 1"
$ 1" 1" $ 1" 1" 1" 1"
[36] [36] [37] [38] [38] [39] [39] [40] [41 ]
hA, arachidonic acid; ATP, adenosine triphosphate; CANP, calcium-activated neutral protease; PA, plasminogen activator; PNI, protease nexin I. Other abbreviations listed at the beginning of the review.
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Development
Figure 3 Schematic showing how asynchronous receptor activation may be the signal that allows muscle to selectively destabilize s.ynaptic sites. (a) Synchronous activity. When co-innervating axons are simultaneously active, they both elicit a 'punishment' signal, but both also have a protection signal and are maintained. (b) Asynchronous activity. When one axon is active (synapse on the left) while the other is inactive (synapse on the right), the punishment signal invoked by the active axon selectively destabilizes receptors beneath the inactive axon (which lacks a protection signal), causing subsequent synapse loss of the inactive axon. (c) Inactivity. When both axons are inactive, neither the punishment signal nor the protection signal is present, and, therefore, both axons remain. Adapted from [68].
Putative mechanism of synapse loss
Punishment signal Protection signal :::::::::::::
Postsynaptic receptors
~(~
Active terminal
Inactive terminal
~ 1996 Current Opinion in Neurobiology
Synapse disassembly at the developing neuromuscular junction Nguyen and Lichtman
Activity-mediated retrograde messengers
Regulators of efficacy If activity differences between the inputs converging on a target cell mediate synaptic competition, then agents that modify the strength of the competing inputs will play an important role. Beginning with work from Mu-ming Poo's lab [32,33,34",35°], there is evidence to support the idea that muscle cells and/or supporting cells at the synapse may modify synaptic strength by activity-dependent release of retrogradely acting diffusible factors. Moreover, hetero- and homosynaptic effects suggest that these agents can mediate competition between axons [32,33,34°,35°]. Several candidate messengers have been proposed (Table 1). Nitric oxide (NO), a diffusible free radical implicated in various forms of neuronal plasticity has also been shown to affect developing NMJs [36]. Several other factors, including ATP, arachidonic acid (or its metabolite 5-HPETE), neurotrophin (NT)-3, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and calcitonin gene related peptide (CGRP), have also been shown to modulate synaptic activity [37-41]. Whether these changes cause long-lasting alterations in synaptic efficacy is not yet known. Nor is it yet clear if these factors play such a role at the more mature NMJs undergoing synaptic competition posmatally (i.e. more than a week after the synapses are first formed). Trophic sustenance of synapses
A long-held view ofsynaptic competition asserts that axons innervating a common target must compete with each other for a limited supply of trophic factors to maintain functional synapses. T h e idea is an extension of the well-established role of neurotrophic agents as mediators of neuronal survival. However, in this case, the survival of a synapse itself is presumed to require an adequate supply of atrophic factor. Thus, of the set of synaptic terminals established by a neuron, only those synapses receiving an adequate supply of factor locally from their postsynaptic partners will be maintained. In order for axons to compete for agents such that two axons can never co-exist for long on the same postsynaptic cell, specific proposals for activity-mediated release and uptake of these agents have been suggested [42]. Recent observations that activity effects and trophic effects may be linked, and that trophins are present and effective in the brain during times of axon withdrawal have helped legitimize this view. This competition would have to operate over relatively large areas because synaptic competition often occurs between sites located hundreds of microns apart [43,44]. T h e possibility that competition for trophic factors drives axonai loss at the NMJ has been pursued by examining the consequences of.excess or reduced putative trophic agents on the synapse elimination process. If limitation in trophic factor supply is the cause of synapse loss, then a surplus of trophic factor should cause the retention of multiple axons at a junction for as long as the surplus supply is available. Conversely, a shortage of supply should result in
109
more rapid synapse elimination, perhaps causing complete denervation if the levels are scarce enough. Thus far, experimental results with putative trophic agents at the NMJ have not shown these extreme outcomes. Nonetheless, a variety of possible trophic agents seem to modify the rate of synapse elimination (Table 1). For example, daily injection of various putative trophic factors--leukemia inhibitory factor (LIF), C N T E oncostatin M (OSM) or basic fibroblast growth factor ( b F G F ) - - i n t o neonatal rodents delays, but does not prevent, the withdrawal rate of superfluous nerve terminals from skeletal muscle [45-47]. Similarly, single intramuscular injection of C N T F and bFGF into embryonic or neonatal mice can induce up to three times the normal number of multiply innervated muscle fibers two weeks after birth [48], but these effects are also transient. Conversely, LIF-deficient mice develop singly innervated muscle fibers somewhat earlier than normal, but as far as is known, no fibers lose all inputs [49].
Postsynaptic proteolysis of synapses An alternative explanation for nerve terminal withdrawal or degeneration is that terminals are being destroyed or at least disconnected by the actions of proteases. Many proteases and proteases inhibitors are located at the NMJ [50]. Presently, their function is not understood. It is possible that their regulation is key in detaching nerve terminals from established synapses (Table 1). Nelson and colleagues [51,52°] have suggested a model for activitydependent postsynaptic activation of proteolysis. In their model, an activated postsynaptic cell releases proteases indiscriminately, resulting in global strength reduction of all inputs. Presynaptic activity, on the other hand, causes local release of protease inhibitors, and the conquering axons will be the ones that are active most often and can protect themselves [51]. In support of this hypothesis, Nelson and co-workers [52°] have demonstrated that several protease inhibitors (e.g. hirudin and protease nexin I) decrease the activity-dependent synaptic reduction they observed in co-cultures of myofibers and neurons in vitro. This is similar to findings of Vrbova and colleagues [12,53-55], who showed that synapse elimination during development and following reinnervation in rat skeletal muscle can be partly prevented by inhibiting proteolytic activity with inhibitors or calcium chelators. The strength of these kinds of models is that they tie electrical activity to a mechanism that could cause nerve terminal lysis. A weakness of these models is that they provide no obvious role for competition because an axon's fate is dependent only on its own activity level and resistance to proteolytic breakdown. How such a mechanism might unfailingly lead to single innervation is not yet clear. Effect of activity on supporting cells at the synapse
Schwann cells perform a wide variety of tasks related to nerve growth and maintenance [56]. Could their absence instigate synapse loss? Son and Thompson [57°°,58 °°]
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Development
have shown that Schwann cells are the first to respond to denervation or paralysis by elaborating processes upon which axons grow; these processes retract following reinnervation [59]. T h e multiple innervation seen during reinnervation is also guided by Schwann cells extending between adjacent endplates, creating bridges for axons to follow [57°',58°']. If Schwann cells are responsible for axonal sprouting, then it is possible that Schwann cells might also arbitrate sprout withdrawal. How Schwann cells could be influenced by local activity will need further investigation.
nerve terminals do not seem to maintain synaptic contact with degenerating muscle fibers for more than a day or two [65]. It is possible that variations in the activity level of motor axons between laboratory rodents (active) and laboratory frogs (not active), or temperature differences, explain the discrepancy. T h e rodent experiments suggest that postsynaptic maintenance of nerve terminal requires continual replenishment (and, therefore, synapse loss is attributable to the absence of some factor), whereas the frog work suggests that the appearance of a noxious agent may be necessary to induce synaptic detachment.
A c t i v i t y - i n d e p e n d e n t synapse e l i m i n a t i o n Synapse loss occurs in several situations besides the competitive, activity-dependent interactions already described. There are some similarities between these activity-dependent and activity-independent processes that suggest a common mechanism may underlie both.
Conclusions In two very different situations in which synapses are removed (i.e. developmental synapse elimination in muscle and following axotomy of neurons), the cellular events are remarkably similar. In both cases, disassembly of the postsynaptic apparatus precedes, and perhaps instigates, the removal of overlying nerve terminals. T h e parallels suggest that synapse elimination throughout the nervous system operates with a common mechanism. How postsynaptic changes might induce nerve terminal withdrawal is not presently known. Possible mediators include postsynaptic regulation of synaptic efficacy, trophic factor availability, proteolytic activity, and glial cells.
Axotomy causes the withdrawal of presynaptic input Axotomy of various CNS and PNS neurons causes weakening of their afferent input and ultimately synapse loss. In particular, stimulation of the presynaptic input to the axotomized neuron shows weakened synaptic transmission [60]. T h e physiology of synapses that are in the process of being eliminated following axotomy in the ciliary ganglion of chicks has been studied in detail, showing that they have reduced quantal size and content. These are the very same changes seen at NMJs undergoing synapse elimination during development (H Colman, J Nabekura, JW Lichtman, unpublished data). T h e reduced quantal size in both cases is thought to be caused by a decrease in postsynaptic sensitivity ([61]; H Colman, J Nabekura, JW Lichtman, unpublished data), probably secondary to a decrease in number of postsynaptic receptors [62]. In axotomy and in development, the changes in quantal size occur before terminal bouton detachment, suggesting that decreased postsynaptic receptor density may be an early step in the cascade leading to synaptic loss. Thus, in two very different situations where synapses are removed, the same sequence of events occurs, suggesting a common cellular program.
Acknowledgements Work done in JW Lichtman's laboratory was supported by grants from the National Institutes of Health and the lvluscular Dystrophy Association.
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Nerve terminal retraction from basal lamina ghosts To understand whether synapse elimination is due to the absence of some normally supplied agent or the presence of some noxious stimulus, it is useful to consider the fate of synapses when the postsynaptic cell is removed. In frogs, axons regenerating onto cell-free basal lamina of a killed muscle fiber re-occupy the site of the original endplate, but are apparently not stably maintained in the absence of a viable postsynaptic cell and eventually fall off after several weeks [63]. Dunaevsky and Connor [64°°], however, showed that if the nerve is left intact, then contact between nerve terminal and basal lamina in the absence of a muscle fiber can be maintained for a substantially longer time. In contrast, in mice, motor
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