Synapse disassembly at the neuromuscular junction

seminars in D E V E L O P M E N T A L B I O L O G Y , Vol 6, 1995: pp 195-206

Synapse disassembly at the neuromuscular junction Jeff W. Lichtman

During the early postnatal life of mammals (and analogously in other terrestrial vertebrates) the number of motor axons innervating individual muscle fibers undergoes a dramatic change: whereas at birth virtually all muscle fibers are innervated by multiple motor axons, several weeks later every muscle fiber is innervated by one axon. This transition is caused by a competitive interaction between the terminal branches of multiple axons co-innervating the same muscle fiber at the same junction. The muscle fiber seems to be the intermediary in the interaction by selectively initiating changes in the postsynaptic membrane (including loss of AchRs) at the sites occupied by one axon. The postsynaptic changes are soon followed by withdrawal of the overlying nerve terminals. The muscle seems to be able to distinguish the sites occupied by the different axons by virtue of their different activity patterns. These results may help inform on the ways in which experience alters synaptic connections throughout the developing nervous system and in the adult brain.

loses connections at the very time the brain is first being put to the test in the outside world. This has suggested to some neurobiologists that a certain degree of functional validation is necessary in order that the circuitry is appropriate for the tasks the brain will undertake in the world at large. A detailed theory of circuit selection based on an analogy with natural selection views the loss of synaptic connections as a fundamental part of brain differentiation) Although others may view this loss somewhat less globally,~ there is little doubt that many parts of the nervous system do undergo synapse loss during the later stages of development. Not only is the loss of connections widespread but it seems that the activity of the synapses is in some way linked to their survival or demise. If neural activity does regulate the maintenance of synaptic connections, then the early postnatal activity of synaptic circuits as a result of early behavior and experience would effect their ultimate fate. In this sense, understanding the developmental loss of synapses may help us understand the way in which experiences throughout life cause long term changes in the nervous system. I My aim here is to explore the phenomenon of synapse loss as it occurs at one very simple synaptic connection: the neuromuscular junction. The vertebrate neuromuscular junction has remained for decades a remarkably useful preparation precisely because of its simplicity and accessibility. Studies of the development, the structure, the molecular biology and function of this synapse surpass our knowledge of all other synapses making it an ideal site to explore poorly understood phenomena that occur in other less accessible parts of the nervous system. Developmental synaptic reorganization has been reviewed well a number of times (see especially refs 3, 4). In this paper I would like to explore several conceptual and several mechanistic issues that have guided my own attempts at understanding this phenomenon. I have worked on synapse loss since I was a graduate student during which time, interestingly, what constitutes 'understanding' has seemed to change. As a molecular analysis of virtually any biological process is now technically feasible, it has almost become de r/geur to consider such an analysis. Such has not yet taken place

Key words: synapse elimination / acetylcholine receptor / synaptic competition / electrical activity ALTHOUGH IN PRINCIPLEsynaptic circuitry could have arisen purely by the elaboration of synaptic connections, much evidence indicates that the opposing process of synapse loss also plays a major part. The coexistence of building and sculpting strategies seems to be a dominant theme throughout neural development helping to create not only appropriate synaptic circuitry but also appropriate numbers of neurons (through proliferation and cell death) and appropriate axonal trajectories (through axonal outgrowth and axonal withdrawal). In each of these cases the loss of neural elements could serve some corrective function because it occurs after a wave of building neural elements is largely complete. Thus, synaptic connections established before birth are partly undone in early postnatal life. It is interesting that such sculpting occurs postnatally. The nervous system From the Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid, St Louis, MO 63110, USA 9 Academic Press Ltd 1044-5 781/95/030195 + 12 $8.00/0 195

f W. Lichtman be elicited by each axon), was decreasing d u r i n g early postnatal life (Figure 1). T h e experiments o f Brown et al also suggested that competition between axons was a likely motive force for elimination. T h e y f o u n d that few if any fibers were completely denervated d u r i n g the period o f synapse loss arguing that the elimination was controlled in a way that always prevented the elimination o f all o f the axons converging o n a muscle fiber. This implied that the axons were not operating i n d e p e n d e n t l y o f each other, otherwise occasionally, at least, all the axons innervating a muscle fiber should have b e e n eliminated. It is n o t so clear what function is served by synapse elimination at the n e u r o m u s c u l a r junction. T h e r e is little evidence to suggest that the process designed simply to correct gross errors o f innervation. T h e m o t o r n e u r o n a l connections being eliminated are appropriate in terms o f originating in the correct m o t o r n e u r o n pool ( r e m e m b e r the eliminated axons are still maintaining connections with o t h e r muscle fibers in the muscle). O n the o t h e r hand, synapse selection does seem to sharpen the projection pattern to some degree 9 In some muscles, for example, there is evidence indicating that the m o t o r units are m o r e h o m o g e n e o u s in terms o f muscle fiber type after the elimination is complete (see for example r e f 12) suggesting that the loss o f connections might help establish m o t o r units in which all the fibers are the same type. But this can not be the only purpose o f this process because it also occurs very robustly even in muscles in which the vast majority o f fibers are o f the same type. Similarly the process does n o t seem to serve to give an axon a spatially distinct territory in a muscle. Although, in some muscles, m o t o r units do obey c o m p a r t m e n t a l boundaries and evidence suggests that these boundaries are s h a r p e n e d by some elimination process (see for example ref 13), this however does not seem the major function o f the elimination because it occurs in all skeletal muscles even in ones in which n o c o m p a r t m e n t s are visible. Moreover, the process occurs t h r o u g h o u t the fibers o f a muscle not only at c o m p a r t m e n t a l borders. For functional reasons it is plausible that m o t o r units n e e d to be distributed in a particular way and synapse loss selects which fibers an axon remains in contact with to optimize that distribution. This possibility while n o t excluded seems unlikely because in many muscles, m o t o r units are nearly randomly intermixed with the m o t o r units o f o t h e r axons with n o obvious p a t t e m to the location o f the fibers contacted by o n e axon. laA5 This suggests that there is n o a-priori plan in terms o f

for synapse loss in development. Thus for the moment, whatever little we do u n d e r s t a n d about this p h e n o m e n o n is based m o r e on how we imagine cells to behave rather than how we envision molecules are acting. Almost certainly this cellular framework will soon give way to a molecular o n e (as has already o c c u r r e d for the converse process o f synapse formation). But in this p a p e r I will try to take stock o f where I think we are mainly in cellular terms.

Conceptual matters r

or error eorreaion?

Neuroanatomists s's in the early part o f this century had n o t e d using silver stains that developing neuromuscular junctions were occasionally contacted by m o r e than one m o t o r axon. Cajal 5 considered these cases (which he called 'erreurs 6volutives des terminaisons' - - probably m e a n i n g developmental errors) as part o f a general trend in young animals (and also following regeneration o f nerves) for accessory nerve branches to be withdrawn. In Tello's 6 detailed descriptions o f n e u r o m u s c u l a r d e v e l o p m e n t he p r o p o s e d that some attracting stimulus emanates from the sites o f n e u r o m u s c u l a r junctions that attract o t h e r axons ('fibras accesorias') to those sites. H e did not however suggest why such connections are transient. Perhaps because the extra connections were clearly e p h e m e r a l and there was no good rationale for these 'errors' in the first place, a general awareness o f a change in the innervation o f muscle fibers did not occur until 1970 when P. Redfern 9 rediscovered the p h e n o m e n o n by finding with intracellular recording that rat diap h r a g m muscle fibers are virtually always innervated by m o r e than one axon at birth whereas these same fibers are innervated by only o n e m o t o r axon just two weeks later. H e suggested that naturally occurring death o f m o t o r neurons perhaps explained the loss o f innervation in developing muscle. Six years later however, n e u r o n a l death as an explanation o f loss o f innervation was ruled out when M. Brown, J. Jansen and D. Van Essen 1~ (see also ref 11) showed that the n u m b e r o f m o t o r axons innervating a muscle r e m a i n e d constant during early postnatal life. Rather, their results showed that the connections being lost must have c o m e from the m o t o r axons remaining in the muscle because the n u m b e r o f muscle fibers contacted by each m o t o r axon (as measured by the p r o p o r t i o n o f the total muscle contraction that could 9

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Figure 1. Diagram showing the change in innervation of individual neuromuscular junctions (black ovals) by individual motomeurons (MN) that occurs between birth (left) and several weeks later (right). There is a loss of axonal convergence as axonal branches are eliminated from each junction leaving every muscle fiber singly innervated. This change also means that there is a reduction in axonal divergence because each motomeuron ultimately contacts fewer muscle fibers than it had innervated at birth.

axonal b r a n c h i n g that is b e c o m i n g manifest by the elimination-process. A r a n d o m distribution o f the fibers remaining in a m o t o r unit may also indicate that the o u t c o m e o f the elimination process on o n e muscle fiber has little impact o n the o u t c o m e o f the process o n a neighboring fiber. What then might synapse elimination be doing for an animal? O n e thing is clear: there is a strong t e n d e n c y for twitch skeletal muscle fibers in all terrestrial vertebrates to be singly innervated. T h e fact that these same fibers seem to be universally innervated by m o r e than o n e axon at an early stage suggests that we n e e d to explain b o t h the necessity for polyneuronal innervation early as well as single innervation later on. It is possible that the early polyneuronal innervation is some kind o f insurance that guarantees that each fiber gets innervated by at least o n e axon. If this is correct then the subsequent synapse loss is a mechanism to assure that the fibers are singly innervated. This raises the obvious questior o f what advantage single innervation confers. T h e answer may be related to the fact that in vertebrates muscle strength is increased in part by the recruitm e n t o f addition m o t o r a x o n s J 6'a7 Thus, in o r d e r to

have the ability to grade the tension o f a muscle contraction in a fine series o f ste~s it is necessary to have a n u m b e r o f m o t o r units with non-overlapping targets. If a muscle fiber was co-innervated by multiple m o t o r axons then recruiting that fiber a second or third time after it was already contracting due to the acdons o f the first recruited m o t o r axon would add no additional tension to the muscle and thus these additional synapses would be wasted. This inability to add force by additional r e c r u i t m e n t stems ultimately from the fact that vertebrate twitch skeletal muscles are non-integrating. T h e n e u r o m u s c u l a r j u n c t i o n has a high safety factor - - a single axon can consistendy drive a muscle fiber to threshold even with repetitive firing. In those vertebrate skeletal muscles that d o integrate by contracting, as a function o f the d e g r e e o f depolarization (as occurs in tonic fibers in reptiles), multiple innervation is maintained t h r o u g h o u t life. 17,18

Is synaptic competition the necessary and sufficient stimulus for synapse loss? W h e n Brown et alm tested the idea that axons are 197

f W. Lichtman vated. I n d e e d subthreshold innervation may be one consequence o f unusually large m o t o r units following partial denervation. 1~ Thus, it may be that in the experiments where the n u m b e r o f m o t o r units is r e d u c e d synapse elimination is in fact completely prevented but the strength o f some o f the connections is so weak the fibers are innervated in an inadequate subthreshold way. Such weakly innervated fibers may even die as a result o f inadequate innervation (just as chronically denervated fibers do) but it is quite possible they do not u n d e r g o synapse elimination. From a theoretical standpoint intrinsic withdrawal is also difficult to reconcile with the absence o f any denervated fibers during n o r m a l development. If an axon's likelihood o f remaining at an endplate is decreased by the presence o f o t h e r axons, it would seem likely that at least occasionally the forces p r o m o t i n g intrinsic withdrawal and synaptic competition would work in parallel causing b o t h axons to be eliminated from the same j u n c t i o n yet apparently that never happens. In my view at least, without direct evidence showing the last remaining axon being removed from a muscle fiber, competition between axons may be the necessary and sufficient stimulus for developmental synapse loss leading to single innervation o f muscle fibers.

involved in some form o f competition they surprisingly obtained results that were at odds with the view that competition was driving synapse elimination. When they experimentally partially denervated the soleus muscle in neonates to drastically diminish the n u m b e r o f axons to a very small n u m b e r they f o u n d that m o t o r unit sizes as estimated by tension measurements still decreased. Thus, even when the final n u m b e r o f innervating axons was only two or three (normally 25-30 axons innervate the soleus) and it was unlikely that most muscle fibers were contacted even transiently by m o r e than one axon, they still f o u n d that m o t o r n e u r o n s s e e m e d to contract the size o f their m o t o r units during development. This result was used to argue that axons may have an intrinsic tendency to withdraw some o f their connections even in the absence o f competition from o t h e r axons vying for the same fibers. This idea makes some intuitive sense: as muscles grow, the size o f individual m o t o r nerve terminals enlarges (see for example ref 19), thus the ability o f a m o t o r axon to synthesize synaptic machinery may be stretched b e y o n d its capacity if all the connections it began with are maintained. O n the o t h e r hand, when a muscle is partially d e n e r r a t e d axons can still sprout to innervate additional fibers suggesting that ordinarily axons are not working at the limits o f their capacity. T h e possibility that axons intrinsically lose connections in the absence o f competition has b e e n re-examined several times (reviewed in ref 3) with the mixed result suggesting (in the soleus at least) there seems to be both some decrease in m o t o r unit size that can n o t be a c c o u n t e d for by competition as well as an ability o f c o m p e t i n g axons to cause m o t o r units to shrink in size as multiple innervation is removed, s'4 Although the idea of intrinsic withdrawal is viable the evidence for it is indirect. Additionally, there is a technical caveat about the tension m e a s u r e m e n t technique used to estimate the size o f m o t o r units that could cause misleading conclusions. Because m o t o r unit size is measured by the tension elicited by stimulation o f an axon, inputs that are too weak to drive a muscle fiber to threshold will be missed, a~ This means that as muscles grow some terminals may grow to keep pace with the enlarging fibers they innervate while o t h e r terminals f r o m the same axon may be unable to keep pace (especially if the m o t o r axon is n o t being eliminated by competition f r o m a n o t h e r axon). T h e result being that as fibers grow the measured size o f a m o t o r unit would decrease due to a decrease in the n u m b e r o f fibers that are driven to threshold r a t h e r than the n u m b e r o f fibers inner-

Direct or indirect competition? Competition, however, is a term with m o r e than one definition. 21-2s Broadly speaking, competition occurs when individuals (i.e. competitors) vie to obtain something that, in the long run, c a n n o t be shared or divvied up. Thus, at the n e u r o m u s c u l a r j u n c t i o n , the axons c o m p e t e for the exclusive innervation o f the muscle fiber. But there are two r a t h e r different ways that such competition could occur. At o n e e x t r e m e axons could be involved in what o n e might call 'direct' competition m e a n i n g that by their actions each attempts to obtain exclusive control over something essential. This something might be a t r o p h i c factor in short supply or synaptic sites. In direct competition the acquisition o f the agent by o n e axon must in some way tip the balance in its favor by either directly weakening its o p p o n e n t s ' ability to acquire the ~factor o r m o r e indirectly weakening its o p p o n e n t by increasing its own ability to acquire additional amounts o f an agent that is in limited supply. Many examples o f competition in biology (such as competition for ecological niches or between populations o f n e u r o n s vying for survival) may actually be just such a direct battle between entities that are trying in essence 198

Synapse disassembly to limit their opponents ability to obtain some essential commodity. Indeed one of the simplest conceptions of direct competition is that axons are attempting to acquire the lion's share of a t r o p h i c factor (for synaptic maintenance) Supplied by the target cell. The increasing availability of putative candidate molecules has stimulated investigators to begin to evaluate their roles in synapse elimination by either adding extra factor or recently by studying synapse elimination in mice lacking the gene. 24'25 At the other extreme the competition can be completely 'indirect' such that the competitors are not trying to weaken their opponents or strengthen themselves. In fact the competitors may be entirely unaffected by the competition and quite oblivious of the fact that they are even involved in a competition - - at least until the decision time at the end of the process. In such indirect competitions there is always a third party [judging' the competitors. In this kind of competition the actions of the winning competitor do not themselves have any effect on the competitive behavior of other individuals that have the same aim (e.g. a male bird showing its plumage or singing to court a female does not adversely effect the singing ability or plumage of other male birds) but ultimately influences the fate of the competitor. For example, one axon by its activity could attempt to 'convince' a muscle fiber to choose it as the sole source of innervation without effecting the activity patterns of other axons innervating the same muscle fiber but nonetheless leading to their elimination. Because the competitive interaction is mediated by a third party, there may be no obvious link between the actions of the competitors (e.g. presynaptic activity) and the ultimate result (e.g. single innervation). This is different from direct competition where the process (e.g. competing for a t r o p h i c factor) and the result (e.g. axonal withdrawal due to lack of trophic factor) seem more connected in a causal way. Thus, in indirect competition, a motor axon need not have any competitive strategy to win a muscle fiber; its normal behavior may lead to that outcome simply because the muscle fiber has a choosing strategy. Understanding such competitions then, requires that the emphasis shift towards the judges and away from the competitors as the mechanism of competition may be more related to what the judges are doing than the actions of the competitors. Between these two extremes, of course, are a range of competitive possibilities that have attributes of both indirect and direct competition. For example, a third party may be an intermediary that can strengthen and

weaken the competitors during the process as well as choose the winner. Synapse loss at the neuromuscular junction may occur in this intermediate way (see later).

Does the postsynaptic cell play a pivotal role in synapse loss? In synapse elimination a number of lines of evidence do point to the possibility that the postsynaptic target cell may be the judge (and executioner). One line of research that suggests this is studies of synapse elimination in non-muscle systems. In the late 1970's several investigators began to see an analogous synaptic reorganization in the pattern of connections to neurons (see refs 26,27 for review). Studies in the cerebellum ~8'29 and autonomic parasympathetic ganglias~ showed a decrease in the number of innervating axons converging on neurons in early postnatal life that was remarkably analogous to events at the neuromuscular junction. In rodents, both the climbing fiber input to the cerebellar Purkinje cells and preganglionic innervation to submandibular ganglion cells underwent a transition from multiple innervation in early postnatal life to predominantly single innervation several weeks later. However, the loss of axonal convergence is not limited to those situations where postsynaptic cells end up with only one innervating axon. Developmental studies of multiply innervated ganglion cells show~ that they lose a fraction of their innervating axons in development also. 31'32 These neuronal examples suggested that the postsynaptic cell regulates the number of converging axons that are maintained after elimination is complete. Intracellular dye injection has been used to correlate the dendritic morphology with the number of innervating axons, sa's4 A striking correspondence exists between the number of axons the neuron is ultimately innervated by and the number of dendrites the neuron possesses. This has led to the suggestion that dendrites might serve as protected spadai domains each of which buffers an axon from competitive interactions with inputs on other dendrites, as Interestingly, ganglion cells that are singly innervated in maturity often have no dendrites. 30 '36 '3 7 The role of dendrites in mitigating synaptic competition may have less to do with spatial domains than with the electrical properties of the underling membrane. Muscle fibers that are permanently innervated by multiple axons differ from twitch fibers (which become singly innervated) by being unable to generate action potentials. 15'17'18 Many dendrites are also inexcitable. These 199

f W. Lichtman e x p e r i m e n t s thus show that the target cell can have an influence o n the process that causes synapse loss.

interspersed a m o n g the terminals o f o t h e r axons confined to the same j u n c t i o n . Because o f this anatomical a r r a n g e m e n t , the terminals that will be

Mechanistic issues

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How are presynaptic axons physically removed?

Figure 2. Diagram showing the inferred sequence of events that leads to synapse elimination at one neuromuscular juncdon (based on refs 19, 40, 50). At birth two axons contact the same AChR plaque on the muscle fiber surface. Each axon has elaborated several bouto~s which are intermingled. Over the next 10 days, there is a gradual change in the number of boutons each axon maintains. One input (dashed axon, possessing white boutons) loses territory in a step-wise way. The other input (solid axon, possessing black boutons) gradually increases its area. The territories lost by one axon do not seem to be captured by the other axon. Rather, the growth of one axon is accomplished by intercalary expansion as it enlarges to keep pace with an expanding receptor plaque (which itself is stretching as the muscle fiber membrane enlarges to keep pace with the growth of the animal). The boutons lost by the other axon are matched postsynaptically by loss of AChRcontaining sites. This postsynaptic AChR loss may cause perforations and cleavages in the receptor plaque. Synapse elimination is complete when the final bouton of the losing axon is eliminated and the now withered axon withdraws from the muscle fiber.

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Although there have b e e n s o m e suggestions that synapses can be silenced without physical or structural changes (i.e. 'functional synaptic suppression', r e f 38), silver stains a n d o t h e r anatomical techniques leave no d o u b t that the transition f r o m multiple to single innervation at the n e u r o m u s c u l a r j u n c t i o n is a c c o m p a n i e d by the actual structural removal o f o n e or m o r e axons, a9'4~ Several investigators have n o t e d axonal endings that are bulb-like at j u n c t i o n s underg o i n g synapse elimination. Riley coined the t e r m retraction b u l b to convey the idea that the a x o n was withdrawing f r o m the muscle fiber, a9 S o m e investigators n o t e d products o f d e g e n e r a t i o n at y o u n g junctions, 41 b u t it is now generally assumed that axons withdraw r a t h e r than degenerate. 42'4s To get a sense how the c o m p e t i n g inputs are spatially deployed, we have used lipophillic dyes to label the m e m b r a n e s o f the c o m p e t i n g axons different colors. 4~ This a p p r o a c h provides a detailed picture o f the anatomical changes that o c c u r to nerve terminals as an a x o n withdraws (Figure 2). T h e s e studies show that the terminals o f the axons that multiply innervate a n e u r o m u s c u l a r j u n c t i o n are interdigitated: the synaptic endings o f each a x o n are

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Synapse disassembly Are there postsynaptic changes associated with synapse loss?

eliminated must often be immediately adjacent to the terminals that will be maintained. This implies that the process that leads to elimination is a highly local process. T h e loss does not to a p p e a r to be a sudden calamity for an axon's ramification on a muscle fiber because the loss o f synapses appears to o c c u r gradually. Whereas at birth each axon typically has the same n u m b e r o f synaptic boutons contacting a muscle fiber, at later stages there is typically a large difference in the n u m b e r o f boutons from two axons converging at the same endplate. T h e protracted nature o f the process perhaps means that each synaptic site is u n d e r g o i n g synapse elimination somewhat i n d e p e n d ently. After all the terminals have withdrawn the axon then withdraws with a smaI1 bulb at its end. By this time, the axon appears thin or withered in comparison to the axon remaining at the junction. An i m p o r t a n t question is w h e t h e r axon withdrawal should be viewed as simply the ultimate c o n s e q u e n c e o f an axon having lost all its synapses or r a t h e r should the p h e n o m e n o n be viewed in some sense the o t h e r way round: that synapse loss is the c o n s e q u e n c e not the cause o f an axon's decision to d e p a r t (i.e. only after the axon has 'decided' to terminate its association with the muscle do the synapses begin to be removed). It seems likely that at least from the functional standpoint, an axon b r a n c h is still normal in that it is capable o f releasing neurotransmitter in response to n e r v e stimulation even after some o f its synapses have withdrawn (22,43,44 and Colman et a~ unpublished). Intracellular recording in mouse muscle shows that over the period o f synapse loss in the first two weeks there is a gradually increasing disparity in the quantal c o n t e n t o f the c o m p e t i n g inputs. T h e elimination process seems to be highly selective allowing the synaptic sites o f o n e axon to be maintained while all the synaptic sites o f the o t h e r axon are removed. In this sense the fate o f synaptic terminals is linked to the p a r e n t axon. We have previously used the term 'synaptic cartel '21'29'4~ to convey the p r e s u m e d intent o f a set o f synapses derived from o n e axon to m o n o p o l i z e (i.e. exclusively innervate) the target. O n e c o m m o n feature o f members o f a synaptic cartel besides their cytoplasmic contiguity is that they are electrically synchronously active. It is possible that c o o r d i n a t e d n e u r o t r a n s m i t t e r release at all the synapses associated with the terminals o f o n e axon is the property that binds their fates t o g e t h e r (see later). This would imply that if multiple axons were synchronously active their synapses would n o t c o m p e t e b u t r a t h e r f o r m a larger cartel.

T h e study of Brown et a~ 1~ already m e n t i o n e d , also includes an e x p e r i m e n t in which the soleus was implanted with a foreign nerve causing some muscle fibers to be innervated at two spatially separated sites along their length (see also refs 45,46). T h e result o f this e x p e r i m e n t was that synapse elimination still ensued causing the elimination o f either the foreign or native axon but only so long as the two sites were less than 1 m m apart. To account for this result the authors discussed the possibility of some kind o f signal 'passing f r o m the muscle to the synapses u p o n it'. T h e y suggested two putative mechanisms: 'a signal from muscle that actively i n d u c e d the removal o f unwanted terminals' or 'synapses might c o m p e t e for some substance provided, in limited amounts by the muscle fiber and n e e d e d for the survival o f the synapse'. Each o f these alternatives raise the possibility that there may be postsynaptic changes (either the presence of an inhibitory substance or the absence o f some required factor) at the sites on the muscle fiber where synapses are lost. To study the changes that o c c u r over the course o f synapse elimination several years ago we developed m e t h o d s that p e r m i t t e d viewing o f the same synaptic j u n c t i o n multiple times over many days. 47'4a We used this approach first to follow the loss o f multiple innervation in adult animals after nerve regeneration. 49 Axonal sprouts multiply innervate many muscle fibers d u r i n g the first few weeks following the nerve's return. Eventually these fibers retract leaving muscle fibers singly innervated once again. In this study we f o u n d g o o d evidence that the postsynaptic cell u n d e r g o e s changes that are co-localized with the sites o f synapse elimination. Thus r a t h e r than seeing the remaining axon occupy the sites vacated by the withdrawing axon we observed that these sites b e c a m e p e r m a n e n t l y uninnervated. W h e n the postsynaptic distribution o f acetylcholine receptors (AChRs) was stained with fluorescently tagged a-bungarotoxin, we f o u n d that the sites that had lost nerve terminals had also lost all evidence o f AChRs. To d e t e r m i n e the c h r o n o l o g y o f the pre- a n d postsynaptic losses we followed junctions over the several days in which synapses were eliminated. We were surprised to find that AChR disappearance began before there was evidence o f nerve terminal loss f r o m the same sites. Thus at the time nerve terminal staining was still robust, the density o f AChRs in the postsynaptic m e m b r a n e was b e c o m i n g obviously low. Besides the 201

J. w. Lichtman vital dye studies, evidence that the nerve was still physically intact at sites losing AChRs includes the temporary maintenance of a 'railroad track' pattern in the acetylcholinesterase at sites losing AChRs, and staining with antibodies to several synaptic vesicle antigens (Colman and Lichtman, in preparation). These results make a strong case for the idea that the axon lingers for a while at sites that have begun to lose AChRs. However, several weeks after nerve regeneration there is again perfect correspondence between the nerve and receptor pattern even though AChR areas have disappeared, indicating that sites that lose AChRs without exception eventually go on to lose their nerve terminals. The loss of AChRs itself was temporally related to the time of return of the nerve following regeneration which could be delayed by double nerve crush or nerve cut. Thus, we found that the loss of postsynaptic AChRs was not induced by denervation but rather by a process that begins only after the nerve returns to the muscle. Thus, the innervation instigates the postsynaptic changes and the postsynaptic changes may then instigate the loss of the innervation. Because these nerve regeneration studies were done in an adult muscle it was important to see whether the elimination of multiple innervation during normal development h a p p e n e d in a similar fashion. We used a similar in-situ approach to follow neonatal synapse loss. s~ These studies also showed a precocious loss of receptor staining at sites of nerve terminal loss (Figure 3). From the in-situ studies of synapse loss in adult and neonatal muscle we conclude that the postsynaptic membrane is permanently changed at sites of synapse loss. Moreover, the earliest evidence that a site will ultimately lose its synaptic connection is postsynaptic. Taken at face value these results imply that a retrograde message instigates the process. This view is strengthened by electrophysiological findings showing that during the process of synapse elimination synaptic release comprising very weakly effective quanta can be observed when stimulating one of the axons multiply innervating a neuromuscular junction (refs 43,44 and Colman and Lichtman, unpublished). These very weak synaptic events may be due to the release of normal amounts of acetylcholine over postsynaptic sites that have already lost a significant n u m b e r of AChRs.

What is the mechanis~n of synapse disassemb~ ? A cellular mechanism that fully accounts for this developmental p h e n o m e n o n must not only explain 202

the result but also the way in which it occurs. Given the pre- and postsynaptic changes it would seem that the withdrawal of axons is associated with the wholesale dismantling of the synapse. Synapse disassembly is presumably part a causal chain of events beginning with some signal emanating from the competing synaptic cartels and ending with the withdrawal of a withered axon after each of its synapses has systemadcally been dismanded. As with other developmental reorganizations there have been many theories that plausibly give the final outcome of single innervation (see for example refs 51-54). My own thinking has been influenced by the involvement of the postsynaptic cell, in particular: how can the muscle fiber so precisely discriminate between the synapses of the axon that will remain and the interspersed synapses of the axons that will be eliminated. In other words what aspects of the competing nerve terminals are different e n o u g h that the postsynaptic cell could discem that difference? One candidate is that the activity patterns of the different axons are different. It is well known that activation of motor units occurs by recruitment. 16 This means that as tension is established in a muscle the n u m b e r of motor axons that are activated increases. If such a recruitment strategy was already functional in early postnatal life then that would mean that the different axons that co-innervate the same muscle fiber would n o t activate the underlying AChRs at the same time. On the other h a n d once all of the receptor sites were occupied by one axon the AChRs t h r o u g h o u t the j u n c t i o n would be synchronously active. This then is a plausible cue for the muscle fiber to use to distinguish between the multiple inputs. Indirect competition between inputs on the basis of their postsynaptic activity profiles also has one very interesting property. If the muscle fiber is mediating competition based in some way on the activity patterns of each axon then the early loss of AChP,s associated with the sites of one axon will have the effect of weakening that axon's ability to influence the muscle fiber in its favor by postsynaptic activity. Because we knew that adult muscle fibers still retained the ability to eliminate multiple innervation 49 we attempted to fool an adult muscle fiber into 'thinking' it was multiply innervated by desynchronizing the activity of a patch of the AChR density by focally blocking that region of the postsynaptic m e m b r a n e with a saturating dose of a-bungarotoxin. 55 The result of this experiment was that focally blocked sites lost AChR density over several days and the overlying nerve terminal withdrew from that site with a slight delay. Importantly, blockade of all of the

Synapse disassembly

EMBRYONK

P~

Figure 3. Diagram showing the inferred sequence of events that underlies the loss of boutons and receptor sites during synapse elimination. Embryonic junctions are multiply innervated and contain relatively few boutons. By birth many junctions are larger with more synaptic sites but still multiply innervated. The step-wise loss of boutrons (Figure 2) begins with changes in the density of AChRs in the postsynaptic membrane underlying one input. Subsequently, the overlying bouton is eliminated. This sequence is repeated multiple times until the junction is left with boutons from one axon only and AChRs are restricted to the remaining synaptic sites. Between the end of the synapse elimination process and adulthood, the junction grows by intercalary expansion rather than by the addition of new sites of nerve muscle contact.

j u n c t i o n or even relatively large areas ( > 50%) caused no loss o f pre or postsynaptic sites. T h e s e e x p e r i m e n t s show that synapticaUy active regions o f the post-

synaptic cell, if they give rise to a sufficient a m o u n t o f postsynaptic activation, can cause the demise o f inactive sites a n d thus provide the r u d i m e n t s o f a 203

J. w. Lichtman

Inactive

tive

V

local activity "protection" signal

retrograde acting "punishment" signal causes elimination cal activity "punishment" signal

Figure 4. Overview ofa putadve mechanism underlying synaptic competition between boutons that are not activadng the postsynaptic AChRs synchronously. When the input on the left is active it initiates two signals within the postsynaptic cell. Neurotransmitter release initiates a local protection signal (1) which prevents the AChRs that it activated from being lost. In addition, a punishment signal (2) induced by the nerve activity, destabilizes AChRs that are inactive and thus unprotected. This punishment signal leads to the loss of AChRs and other postsynaptic molecules. A consequence of this postsynaptic loss is a retrograde signal (3) which ultimately causes the overlying nerve terminal to be eliminated.

plausible mechanism to account for competition between synaptic cartels (Figure 4) at a j u n c t i o n and also competition between m o r e distant sites. 56 For postsynaptic activity elicited by one axon to destabilize synapses not active at the same time, a m i n i m u m o f three separate signaling processes would seem to be necessary. First the activity must locally protect the postsynaptic m e m b r a n e associated with the active site from whatever deleterious factors it uses to cause AChRs to disappear elsewhere. This protection must even protect AChRs that are inactive in the active region because AChRs that are blocked with ct-bungarotoxin in regions where most receptors are unblocked are n o t eliminated, ss Thus r e c e p t o r activation per se does not seem to be the property that confers 'protection' o n AChRs in the active region. Rather some change in the e n v i r o n m e n t associated with the ensemble activity o f many receptors is suggested. Second, some factor must originate with the local activity that causes the inactive postsynaptic areas to lose all their AChRs and also the 43K protein. 5~ This is presumably a signal that can spread but is presently limited to distances u n d e r 1ram.

Lastly, some signaling mechanism must exist to inform the nerve terminal over the postsynaptic sites that are disappearing to also withdraw. Experiments in which AChRs are caused to disappear by different mechanisms also lead to nerve terminal loss 58'59 perhaps suggesting a role o f the AChR in synaptic maintenance. T h e nature o f n o n e o f the signals is known although a n u m b e r o f likely candidates have b e e n proposed. 2~'55'6~

Conclusions Future studies o f synapse disassembly will almost certainly be directed at r e d u c i n g this cellular phen o m e n o n into the myriads o f molecular cascades that underlie it. T h e disassembly o f synapses during d e v e l o p m e n t is probably ripe for this kind o f analysis although to my m i n d it will be quite challenging for several reasons. T h e interaction that leads to synapse elimination is highly dynamic and spatially restricted m e a n i n g that the molecular basis o f the process is perhaps m o r e a result o f changes i n d u c e d by chemical

204

Synapse disassembly r e a c t i o n s o f a fixed set o f m o l e c u l e s t h a n by the e x p r e s s i o n o f n e w genes. S e c o n d , t h e i n t e r a c t i o n involves at least t h r e e d i f f e r e n t cells (at least two c o m p e t i t o r s a n d t h e m u s c l e fiber) - - it will n o t b e s t r a i g h t f o r w a r d to u n r a v e l the c h a i n o f causality if s i m u l t a n e o u s c h a n g e s are o c c u r r i n g i n d i f f e r e n t cells. O n t h e o t h e r h a n d i n s o m e relatively c r u d e way o n e might imagine that the skeleton of a m e c h a n i s m ( m i n u s the m o l e c u l e s ) is a l r e a d y i n h a n d . It will b e i n t e r e s t i n g to test this c e l l u l a r c o n c e p t i o n o f s y n a p s e loss at o t h e r s y n a p t i c sites u n d e r g o i n g t h e s a m e k i n d of changes.

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