A Model of Developmental Synapse Elimination in the Central Nervous System: Possible Mechanisms and Functional Consequences

A MODEL OF DEVELOPMENTAL SYNAPSE ELIMINATION IN THE CENTRAL NERVOUS SYSTEM: POSSIBLE MECHANISMS AND FUNCTIONAL CONSEQLJENCES Ann M. Lohof, Yannick Bailly, Nicole Delhaye-Bouchaud, and Jean Mariani

Abstract ......................................................... 76 I. INTRODUCTION.. ............................................... 76 11. MECHANISMS OF SYNAPSE ELIMINATION: INFORMATION FROM DIVERSE EXPERIMENTAL SYSTEMS. ...................... 76 A. The Role of Activity. ........................................... 78 B. Differential Activity and the NMDA Receptor ....................... 79 C. ElectrophysiologicalMeasurement of Short-Term Synaptic Competition . . 81 D. Possible Signaling Mechanisms in Synapse Elimination. . . . . . . . . . . . . . . . 82 111. SYNAPSE ELIMINATION IN THE CENTRAL NERVOUS SYSTEM: THE EXAMPLE OF THE CEREBELLUM. ............................ 85 A. Climbing Fiber-Purkinje Cell Synapse Formation and Elimination . . . . . . . 85 B. MorphologicalCorrelates. ....................................... 87 Advances in Organ Biology Volume 2, pages 67-97. Copyright 8 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN:0-7623-02224

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C. The Parallel Fiber Trigger ....................................... D. Role of the Target Cell. ......................................... E. Involvement of the NMDA Receptor .............................. F. Functional Consequences of Synapse Elimination .................... IV. CONCLUDING REMARKS. ....................................... Acknowledgments ................................................ References. ......................................................

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ABSTRACT Recent research into the developmental elimination of supernumerary synapses has increased understanding of this process. We discuss in this chapter synapse elimination both at the neuromuscular junction and in the central nervous system, in terms of some possible underlying mechanisms suggested by recent studies. A well-characterized example of central nervous system synapse elimination, the climbing fiber-Purkinje cell synapse of the cerebellum, is used to explore the functional significance of synaptic regression during development.

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INTRODUCTION

The development of the nervous system is characterized by a number of regressive events. For example, neuronal death commonly adjusts the size of a population of neurons (reviewed in Oppenheim, 1991). In addition, many neurons initially produce widely-distributed axonal projections; some of these projections are removed later in development to form a mature, more restricted terminal field (Innocenti 1981, 1995; Cowan et al., 1984; Stanfield, 1984; Dehay et al., 1988). Athird regressive process is the elimination of supernumerary synapses after their overproduction earlier in development. In this chapter we will use a variety of central and peripheral synapses as examples in discussing some of the possible cellular mechanisms underlying synapse elimination. We will then describe in some detail an example of synapse elimination in the central nervous system, the climbing fiber synapse on the cerebellar Purkinje cell, considering both the cellular interactions underlying this process and its possible functional significance.

II. MECHANISMS OF SYNAPSE ELIMINATION: INFORMATION FROM DIVERSE EXPERIMENTAL SYSTEMS No single experimental system is likely to yield a complete general picture of synapse elimination. The study of elimination of central synapses can indicate some of the functional consequences of synapse elimination, and some of the relevant interactions between different cell types in a network can be examined. However,

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synapse elimination in the central nervous system is difficult to analyze, because generally a single neuron receives synapses from many axons in the mature state; it is therefore difficult to assess the number of synapses present, and whether this number changes during development. Other examples of synapse elimination may be more useful for observing and understanding the cellular and molecular interactions which underlie synaptic regression. In peripheral ganglia, for example, repeated observations in living animals of vital-dye labeled dendrites or nerve terminals have been possible (Purves et al., 1986, 1987). These studies revealed extensive rearrangement of the pre- and postsynaptic structures even in adult animals,showing that the cellular structuressupportingthe synapse are very plastic. Synapse elimination has also been demonstrated at mammalian autonomic ganglia using both morphological and electrophysiologicaltechniques (Lichtman, 1977; Lichtman and Purves 1980; Johnson and Purves, 1981). However, the developing mammalian neuromuscular junction has been the subject of the most extensive research on synapse elimination. This synapse is more accessible to experimental manipulation and physiological recordings than synapses in the central nervous system, and the analysis of the synapse elimination process is relatively easy. During the developmentof most mammalian skeletalmuscles, the number of motor axon terminals innervating a single muscle fiber is initially high; the innervating axons are then reduced in number postnatally until exactly one presynaptic nerve terminal innervates each muscle fiber (Redfern, 1970; Brown et al., 1976). While there may be a natural tendency of motor terminals to withdraw during the early postnatal period (Brown et al., 1976; but see Betz et al., 1980), there is also considerableevidence for a competitiveinteractionbetween nerve terminalsduring the synapse elimination period. The sequence of events in neuromuscular synapse elimination has been described by Balice-Gordon and Lichtman (1993), using repeated observationsof the mouse sternomastoid muscle. Presynaptic terminals and postsynaptic receptors were labelled at each observation of a neuromuscular junction, and the evolution of the synaptic morphology recorded during the process of elimination. These observations showed that the acetylcholine receptors (AChRs) underlying nerve terminal areas which are destined to be eliminated are removed in the days before nerve terminal retraction.This finding indicatesthat the postsynapticcell is actively involved in the process of synapseelimination, and that removal of the underlying AChRs may be an early step. In addition, recent work on the neuromuscular junction of mouse and snake suggests that terminals are not eliminated due to an inability to release neurotransmitter; these presynaptic terminals remain active and capable of vesicle recycling until they are eliminated (Balice-Gordon et al., 1993). Therefore the muscle cell may actively select, possibly by removal of underlying AChRs, the terminal to be eliminated.It is also possible that small random changes in the distribution of the AChRs, and the resultant changes in efficacy of the multiple terminals, produce sufficient asymmetry 'to trigger synaptic competition and elimination. These observations suggest a sequence of events beginning with

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multiple innervation at the endplate, the different terminals occupying approximately equal amounts of temtory; then some slight change in the density of AChRs under a synaptic site would make the two inputs functionally unequal, triggering acompetitive process which favors the stronger input. The muscle cell, via turnover or migration, then would create areas of lower AChR density under the “losing” terminals, and finally all terminals but one would withdraw and the underlying AChRs would be removed. It is not known whether eliminated synaptic terminals degenerate or are resorbed by the axon. In the central nervous system, the presence of certain types of glia has been implicated in the elimination of transient projections. In the developing cat visual cortex, macrophages appear to phagocytose groups of transitory callosal axons, although it is not clear whether these macrophages actively select the axons to eliminate or simply clear those terminals already eliminated (Innocenti et al., 1983). Glial processes also appear interposed between presynaptic terminals and a-motoneurons in the cat spinal cord during its early post-natal development, when terminals are being eliminated (Ronnevi and Conradi, 1974;Ronnevi, 1978). At the neuromuscularjunction, however, it appears that eliminated terminals are retracted into the main axon rather than degenerating, since “retraction bulbs” are found in the muscle during this process (O’Brien et al., 1978; Balice-Gordon et al., 1993). A. The Role of Activity

Increasing or decreasing the overall level of neuromuscular activity during the synapse elimination period in vivo alters the rate of synapse elimination (see Thompson, 1985, for review). Benoit and Changeux (1975) showed that tenotomy (resulting in much decreased muscle use) delayed the elimination of multiple innervation. Similar delays are produced when motor neuronal action potential conduction or neurotransmitter release are blocked (Thompson et al., 1979;Brown et al., 1981). The importance of postsynaptic activity was shown by the chronic administration of the ACh receptor blocker a-bungarotoxin (Callaway and Van Essen, 1989);muscles treated in this way had higher levels of multiple innervation. Conversely, chronic stimulation of the muscle nerve in vivo acceleratesthe synapse elimination process (O’Brien et al., 1978), depending on the pattern of stimulation (Thompson, 1983). Thus the amount of neuromuscular activity seems to regulate the rate of synapse elimination. A number of lines of experimental evidencefrom other systems support the idea that digerences in synaptic efficacy are important during remodelling of a synaptic population. If the relative synaptic activities are altered, developmentalrearrangement of synaptic afferents is disrupted. This principle is clearly seen in the mammalian visual system, where visual input (or spontaneous retinal activity earlier in development;Meister et al., 1991)during acritical period in development is required for the segregation of eye-specific zones in the thalamus and visual cortex (for reviews see Shatz, 1990; Goodman and Shatz 1993).

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Differential Activity and the NMDA Receptor

In many cases, the activation of NMDA receptors (NMDA-Rs) is implicated in the rearrangement of synaptic afferents. The NMDA-R channel requires both ligand binding and postsynaptic depolarization to open. For this reason it has been proposed to act as a “coincidencedetector,” strengtheningpresynaptic inputs which are coactive with the post-synaptic cell. Such a detector would also presumably strengthen two coactive inputs onto the same cell which cannot alone depolarize the postsynaptic cell, but can together. When both ligand binding and postsynaptic depolarizationoccur, calcium flows through the NMDA-R channel and a sequence of cytoplasmic events, whose end result is presynaptic stabilization, is thought to occur. The actual role of NMDA-R activation in developmental plasticity of synaptic afferents is not yet conclusively determined (seeFox and Daw, 1993, for a review). Nonetheless, many recent studies have focused on activity-driven mechanisms underlying the rearrangement of terminal arbors and, presumably, the functional synapses they support. One useful preparation has been the developingfrog retinotectal system, which must continuously rearrange its synapses as the retina and tectum grow so that a functional set of topographic projections is maintained. Observations of labelled retinal arbors in the live Xenopus tectum have shown directly the dynamic and rapid remodelling of these terminals (O’Rourke et al., 1994), and shown that the blockade of postsynaptic activity increased the rate of terminal arbor rearrangement. These authors suggest that the increased rearrangement reflects a decrease in stability of the retinotectal synapses. In longer-term experiments, Cline and Constantine-Paton (1989) blocked the activity of the tectal NMDA receptors in Runa pipiens during several weeks of development, then examined the precision of the retinotectal projection. Injection of a tracer into a single tectal site allowed visualization of the retinal ganglion cells projecting to that site. In animals which had experienced the blockade of tectal NMDA receptors, the single tectal site labelled a larger area of the retina than in control animals. This result indicates that the topographic ordering of the retinotectal projection had been disrupted, due to less precise targeting of the retinal terminal arbors. In the same system, this disruption of topographic organization can be clearly demonstrated if a supernumerary eye primordium is implanted at an early embryonic stage. As the animal develops, the additionalretinal afferents project onto the tectum, but segregate from the afferents of the other eye, producing a pattern of stripes superimposed on the normal retinotectal topography. The tectal stripes can be easily visualized by labelling one eye’s retinal axons. If the NMDA receptors of these tecta are chronically blocked during development, the stripe pattern does not form, indicatingthat the retinal terminals have not segregated according to their eye of origin (Cline and Constantine-Paton, 1990). These observations implicate the NMDA receptor as an activity-dependentplayer in the stabilizationof co-active synapses: multiple retinal arbors synapsing on the same tectal cells are proposed

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to be stabilized, by an NMDA-dependent mechanism, if they are active at the same -time (a case much more likely for retinal axons originating in the same region of the same eye) and destabilized if they are not co-active. By this mechanism, retinal axon terminals could sort themselves into an appropriate topographic projection. Similar examples of activity- or NMDA- dependent afferent sorting have been shown in mammalian visual systems. Shatz and Stryker (1988) showed that, in the fetal cat, infusion at the optic chiasm of tetrodotoxin to block action potential activity prevented the segregation of retinal afferent terminals into their eye-specific laminae in the lateral geniculatenucleus (LGN). In addition, in the ferret LGN, the later segregation of retinal afferents into cell-type-specific sublaminae is disrupted by thalamic infusion of the NMDA receptor antagonist APV (Hahm et al., 1991). In this latter case it is not the cosegregation of co-active terminals from the same eye that is affected but the further restriction of terminal arbors from two types of retinal ganglion cells (on-center and off-center) into the appropriate sublaminae.NMDA receptor activation also seems to be required for the formation of a topographically-correctretino-collicular organization from an initially disordered projection. Chronic administration of APV to the rat superior colliculus during the period of large-scale remodelling of afferents resulted in the persistence of elaborate terminal arbors in topographically inappropriate locations (Simon et al., 1992). Further, during induced rearrangement of thalamic afferents to the rat primary sensory cortex, chronic APV treatment prevents the formation of a functionally-appropriatepattern (Schlaggar et al., 1993). In the developing cat visual cortex, the continuous infusion of APV during monocular deprivation results in electrophysiologicalabnormalities of the cortical neurons: specifically, the ocular dominance shift which normally results from monocular deprivation does not occur (Bear et al., 1990).The role of postsynaptic activity in the segregation of eye-specific afferents was further shown in the developing cat cortex by continuous infusion of a GABA-A receptor agonist, muscimol (Hata and Stryker, 1994). During monocular deprivation, muscimol infusion prevented the expansion of cortical territory devoted to the active eye which is normal when one eye’s inputs are inactive. Instead, cortical territory devoted to the inactiveeye expanded, indicatingthat in the absence of postsynaptic activity an inactive afferent is actually preferentially stabilized. Further evidence for the role of synaptic efficacy in the long-term structural changes which underlie synapse elimination has come from repeated observation of the mouse sternomastoid muscle during the period of synapse elimination in viva Synaptic competition can be induced, after normal developmental synapse elimination is complete, by local blockade of the ACh receptors underlying a terminal region; this local blockade prevents the overlying nerve terminal region from having any electrophysiological effect on the muscle cell. When a-bungarotoxin was applied to a small region of a neuromuscular junction to irreversibly block the AChRs, the AChRs and the nerve terminal regions overlying the blocked zone were eliminated over the next few days (Balice-Gordon and Lichtman, 1994).

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If the ACh receptors underlying the entire terminal were blocked no terminal withdrawal occurred, indicating that the difference in efficacy of the terminal regions was the important factor, not the overall level of activity. This artificial activity-dependent competition could be induced between different regions of the same presynaptic terminal, indicating that the mechanisms determining whether a terminal region will be maintained or eliminated must be highly localized at the synapse. Physiological recordings at these neuromuscularjunctions, made during the process of developmental synapse elimination, suggest that a loss of synaptic strength in one input does occur during this period (Nabekuraand Lichtman, 1989; Colman and Lichtman, 1993), supporting the idea that these changes in relative synaptic efficacy may be important in determining which terminals are stabilized and which are eliminated. Attempts to demonstrate a direct relationship between differential synaptic activity and synapse stabilization have given conflicting results. Magchielse and Meeter (1986) used co-cultures of chick ciliary ganglia and muscle cells to study the effects of activity on multiple innervation. They found that “phasic” (intermittent high frequency) stimulation reduced multiple innervation, while the same amount of stimulation administered continuously at lower frequency produced no change. If only one of two ganglia in a culture was phasically stimulated, innervations made by the non-stimulated ganglion were preferentially eliminated. However, there is also evidence for inactive synapses to be preferentially stabilized. Using blockade of a small proportion of motor axons in vivo during the period of synapse elimination, Callaway et al. (1987, 1989) showed that these inactive afferents had larger motor unit sizes than in controls, meaning that fewer of their synapses had been eliminated. This difference outlasted the time of effective activity blockade (Callaway et al., 1989). Other studies have found no evidence for preferential maintenance of synapses based on differential activity. Nelson et al. (1993), using a culture system, found that neuromuscular synapsesmade by two groups of afferents were equally eliminated when either or both sets of afferents were stimulated.The authors propose that activity of the postsynapticcell produces a generalized tendency toward synapse withdrawal, and that spatial factors are more important than differential activity in determining which synapse is eliminated. C.

EledrophysiologicalMeasurement of Short-term Synaptic Competition

A complement to these observations of activity effects on synapse elimination are studies of activity-dependent changes in synapticefficacy which could possibly initiate the elimination of some synapses. Inhibitory interactionsbetween competing axon terminals at multiply-innervated muscle fibers have been recorded electrophysiologically in vivo. Recording from neonatal rat lumbrical muscle and separately stimulating two innervating nerves, Betz et al. (1989) found that when the nerves were stimulated in succession the response elicited by the second stimulus was gradually suppressed. This synaptic suppression did not require

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depolarization of the muscle membrane, but it did require a close temporal relationship between the two stimuli; when the delay between the stimuli was increased the suppression did not occur. These measures of physiological competition provide a possible mechanism for longer-term synaptic competition and selective stabilization, if a less physiologically-effective innervating terminal is more likely to be retracted. A possible short-term electrophysiological basis for synapticcompetition based on activity has also been studied in culture. Lo and Po0 (1991) used cultured embryonic Xenopus neurons and muscle cells to study competition between two neurons innervating the same muscle cell. When one of the neurons was preferentially stimulated at high frequency, the synaptic efficacy of the other neuron was reduced. This heterosynaptic suppression could also be elicited by electrophoretic application of ACh to a singly-innervated muscle cell, if the ACh application was sufficiently close in time to the synaptic stimulation (Dan and Poo, 1992a), indicating that current flow through the ACh receptor channels in the muscle membrane is sufficient to suppress the efficacy of a less-active innervating nerve terminal. D. Possible Signalling Mechanisms in Synapse Elimination

The signals between developing synaptic partners during synaptic competition and elimination remain unclear. It is possible that the post-synaptic cell “chooses” a terminal to be maintained simply by removing transmitter receptors underlying the other terminals, rendering them functionally ineffective; something of this nature seems likely to occur at the neuromuscularjunction (see above). Displaced receptors could then migrate to sites under the competing terminal, strengthening its physiological effect. The signalling which instructs the “losing” terminals to retract is still unknown. An activity-dependent balance between protease and protease-inhibitor is one possible signalling mechanism. O’Brien et al. (1978) showed that ACh-treatmentof muscles elicited release of proteolytic enzymes, and Connold et al. (1986) demonstrated that neuromuscularsynapse elimination in vivo could be reduced by chronic application of protease inhibitor or calcium chelators, suggesting the involvement of a calcium-activatedprotease. Liu et al. (1992), using neuromuscularsynapses in culture, showed that protease inhibitor prevents stimulation-inducedsynapse elimination in this system; evidence of a specific role for thrombin was later provided (Liu et al., 1994a).The same authors (Liu et al., 1994b;Nelson et al., 1995)propose that postsynaptic activity produces the release of protease which acts to reduce the efficacy of all presynaptic inputs. A strongly active presynaptic terminal would induce the release of a protease inhibitor, possibly from surrounding glial cells (review in Nelson et al. 1995), which would locally protect the active terminal. Another possibility is the competition of innervating terminals for a limited amount of trophic factor supplied by the target. This type of trophic interaction is

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a well-supported mechanism for target-dependent neuronal survival during naturally-occurring cell death (Oppenheim, 1991);the amount of target determinesthe number of innervating neurons which survive the cell-death period, and extracts derived from the target can be shown to support the survival of the appropriate neurons in v i m . Atrophic factor acting by a similar mechanismto mediate synapse elimination would have to both encourage synaptic stabilization by its presence and selectively affect the preferred terminal (Changeux and Danchin, 1976). If the preferred terminal is the most physiologically effective one, then activity-dependent postsynaptic release and presynaptic uptake of the factor would suffice. There is some experimental support for the existence of a general activity-dependent release process from a target cell. Dan and Po0 (1992b) demonstrated calcium-dependent secretion in cultured muscle cells: increases in the intracellular calcium level, such as would be triggered by synaptic activation, produced increased quantal secretion of a neurotransmitter loaded into the cell. This finding raises the possibility that such a calcium-dependent release mechanism could normally operate to provide a retrograde signal to the innervating nerve terminals. If the target cell secreted, in an activity-dependent manner, a trophic factor which could selectively affect the physiologically-effectiveterminals (by having an effect only on active terminals, for example), this trophic factor could determine the maintenance of one innervating terminal in preference to the others (reviewed in Dan and Poo, 1994). (Figure 1) Evidence is accumulatingthat some known neurotrophic factors may have roles in synaptic plasticity and elimination as well as in neuronal survival.For example, the cytokines CNTF and LIF have been implicated in the regulation of the timing or extent of neuromuscular synapse elimination (English and Schwartz, 1992, 1993;Jordan, 1993; Gurney and Kwon, 1993; Kwon et al., 1994).In the rat visual cortex, an oversupply of nerve growth factor (NGF) prevents the ocular dominance shift which normally follows monocular deprivation, supporting the involvement of this trophic factor in the functional segregation of visual input (Maffei et al., 1992). An over-supply of some neurotrophins (the family of factors comprising NGF, BDNF, "I-3 and NT-4/5) in the cat visual cortex interferes with the developmental segregation of axon terminals into ocular dominance columns, an activity-dependent process (Cabelli et al., 1995). Finally, BDNF has been shown to increase the complexity of retinal axon arborization in the Xenopus tectum, presumably reflecting increases in synapseformation or stabilization(Cohen-Cory and Fraser, 1995). In addition, a number of recent studies indicate that some neurotrophic factors can directly affect synaptic activity and plasticity (reviewed by Thoenen, 1995). Neurotrophins and CNTF can produce increases in the release of neurotransmitter, measured either electrophysiologically at the synapse or biochemically (Lohof et al., 1993; Knipper et al., 1994; Lessmann et al., 1994; Kim et al., 1994; Kang and Schuman, 1995; Stoop and Poo, 1995), and hippocampal long-term potentiation is impaired in mice deficient for BDNF (Korte et al., 1995). Interestingly, there is

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Figure 1. Possible mechanisms for retrograde signaling at developing synapses. Presynaptic transmitter release activates the postsynaptic cell, increasing levels of Ca2+ and/or other second messengers. This in turn results in the increased production or release of a retrograde factor, which may be membrane permeant or contained in vesicles. Binding to presynaptic receptors for the factor would then affect synaptic stabilization by mechanisms still unknown. Adapted from Dan and Pool 1994.

now evidence that hippocampal neurons can release NGF and BDNF in an activity-dependent manner (Bliichl and Thoenen, 1995; Griesbeck et al., 1995); this activity-dependentrelease differs from constitutiverelease in that it takes place throughout the neuronal processes, including dendrites (Thoenen, 1995). Thus NGF could act as a retrograde neuromodulator,in the manner described above (and as discussed by Dan and Poo, 1994). The interactions between neuronal activity, the release of neurotrophic factors, and synapticpiasticity and elimination is likely to be complex, but the further study of these interactions should yield interesting insights into the mechanisms and determinantsof developmental synapse elimination.

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SYNAPSE ELIMINATION IN THE CENTRAL NERVOUS SYSTEM: THE EXAMPLE OF THE CEREBELLUM

Although the overproduction and subsequent pruning of axon terminals has been demonstrated in a number of systems, particularly visual systems, electrophysiological analysis of synapse elimination is possible in only a few central synaptic populations. One successful study of this type described the maturation of a synaptic population in the chick auditory system (Jackson and Parks, 1982). Early in deveIopment (E13), an average of four cochlear nerve axons innervate each neuron of the nucleus magnocellularis. Four days later, the mean number of innervating axons has been reduced to 2.2, and this number remains stable at later stages. The electrophysiologically-measureddecrease in functional synapses is accompanied by decreased branching of cochlear axons as they enter the nucleus, whereas the number of axons in the nerve does not change. In many other studies of the central nervous system, morphological tools are used to study synaptic changes. Morphological analysis of certain areas within the central nervous system has been applied to show the ultrastructure of synaptic populations during the process of formation and elimination (Landis et al., 1989; Missler et al., 1993), to follow the changes in synaptic density over time (Bourgeois and Rakic, 1993), or to demonstrate that true elimination of synapses accompanies the refinement of axonal projections (Campbell and Shatz, 1992). A.

Climbing Fiber-Purkinje Cell Synapse Formation and Elimination

The Purkinje cell of the cerebellum offers a relatively simple opportunity to study developmental synapse elimination in the central nervous system using both morphological and electrophysiological techniques. The most extensive studies have been done in the rodent, but the same events have been demonstrated in the rabbit (Barragan and Delhaye-Bouchaud, 1980) and are likely to occur also in the ferret (Benoit et al., 1987). This process of synapse elimination occurs postnatally, the cellular populations of the cerebellum are well-described, and electrophysiological analysis is not difficult. In the adult, the cerebellar Purkinje cells (PCs) receive two classes of excitatory synapse (Figure 2A). Each Purkinje cell is contactedby a single axon of the inferior olivary neurons, the climbingfibers (CFs). The PC also receives inputs from many parallel fibers, the axons of the abundant granule cells. These patterns of synapticconnectivity develop during the first three postnatal weeks in the rat. The climbing fibers make functional synapses on the PCs by posttlatal day 3 (Crepel, 1971); both morphological and physiological evidence show that these immature, more-branched (O’Learyet al., 1971) climbing fibers multiply-innervatesingle PCs (Crepel et al., 1976;Triller and Sotelo, 1980) (Figure 2B). Elimination of all but one CF synapse occurs during the second post-natal week (Mariani and Changeux, 1980a, 1981; Crepel et al., 1981); this developmentalchange can be measured by recording synaptic potentials from the

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Figure 2. Innervation of rat cerebellar Purkinje cells (PC) by climbingfibers (CF) and parallelfibers (PF). A. Innervation in the normal adult

cerebellum. Each Purkinje cell is innervated by one CF and by many PFs. B. In the immature cerebellum, several CFs contact a single PC; the supernumerary CFs will later be eliminated. The granule cell precursors are dividing in the external germinal layer (EGL) and the parallel fibers have not yet fully developed. C.In an agranular adult cerebellum, the granule cell precursors have been eliminated by early postnatal X-irradiation (or, in the mouse, by genetic mutation). Multiple CFs are maintained on a single PC. Mossy fibers (MF)which normally synapse on the granule cells make aberrant synapses on the PCs.

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Purkinje cell, either those occurring spontaneously or those in response to electrical stimulation of the climbing fiber pathway (Figure 3). Early in postnataI development the synaptic potentials have more than one size, indicating that they are produced by more than one climbing fiber. In the normal adult, similar recordings show synaptic potentials of only one size. Between postnatal days 5 and 15, therefore, the number of climbing fibers synapsing on a single Purkinje cell decreases from 3 or 4 to 1 single fiber. At the same time, the CF synapses appear to translocate from the PC soma to the dendrites; this translocation is unlikely to be a cause of synapse elimination, however, as the two characteristicsof synapse number and synaptic location can be experimentally dissociated (Mariani, 1983; Sotelo, 1990). The single remaining climbing fiber also elaboratesits terminations on the PC such that the number of synaptic boutons actually increases despite the reduction in number of innervating fibers (Larramendi et al., 1989; discussion in Chedotal and Sotelo, 1992, 1993). Figure 4 shows two different stages of CF innervation on a PC. It should be noted that this reduction occurs from a redundant state in which a mean number of 3.5 CFs impinge on each PC. This mean value is identical to the one found in the developing neuromuscular junction; and in other examples of synapse elimination the ratio between the maximal number of presynaptic axons and the final number is also equal to 3.5 or to a fraction of 3.5. These constant values suggest that the synapticredundancy at its maximum is limited by topological constraints and can be formally described or simulated as an isostatic random stacking of hard spheres, following the laws of physics of disordered media (Waysand and Mariani, 1989; Eddi et al., 1995, in press). These constraints may also act during the regression phase, although this has yet to be established (Eddi et al., 1996). The elimination of climbing fiber synapses occurs independently of cell death of the inferior olivary neurons. Although a moderate amount of postnatal cell death does occur in this nucleus, it is too small to account for the decrease in climbing fiber synapses, and occurs mostly before the phase of synaptic regression (review in Bourrat and Sotelo, 1984; Mariani and Delhaye-Bouchaud, 1987; DelhayeBouchaud et al., 1985; Armengol and Lopez-Roman, 1992).

B. Morphological Correlates Some attempts have been made to find morphological correlatesof the different states of the climbing fiber synapses. Landis et al. (1989) used electron microscopic techniques to examine the structure of the CF synapses during the fust postnatal weeks, when synapses are translocating from the PC soma to the dendrites and when supernumerary synapses are being eliminated. An aggregate of particles is present on the postsynaptic face of the mature axo-dendritic CF-PC synaptic junction (Landis and Reese, 1974); this aggregate is generally not present at the immature somatic synapses. The particle aggregates were, however, found associ-

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Figure3. lntracellular recordings from Purkinje cells showingclimbing fiber excitatory postsynaptic potentials (EPSP). A schematic diagram of the experimental preparation is shown between A and B; Purkinje cells are impaled with a microelectrode in the cerebellum and climbing fiber responses elicited by stimulation of the inferior olivary nucleus (ION). A. The typical all-or-none climbing fiber EPSP in the mature cerebellum, occurring either spontaneously (Al) or after stimulation of the ION (AZ).This cell is considered to be innervated by only one climbing fiber because the spontaneous EPSPs are all of one size and because increasing the intensity of ION stimulation does not change the evoked EPSP.size. B. The stepwise variations in climbing fiber EPSP size found in multi-innervated Purkinje cells. Several sizes of spontaneous EPSP are recorded (Bl), and increasing the intensity of ION stimulation activates multiple climbing fibers, producing evoked EPSPs of different sizes (BZ). Scale bars: A l , B1, 5 ms, 5 mV; A2,B2, 10 ms, 10 mV. From Mariani and Delhaye-Bouchaud, 1987.

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Figure 4. Multiple (A) and single (B)climbing fiber (CF) innervation of Purkinje cells

(PC) during postnatal synaptogenesis in the cerebellar cortex of the rat. Above, anterograde tracing of CFs with fluorescent carbocyanine I. Below, schematics of PC innervation by CFs as labeled above. (A) Postnatal day 8 (Pa). Multiple CF innervation of the PC forms a perisomatic “nest.”250X. (B) P I 3. Single CF innervation leaving the “capuchon”stage on the apical soma toward the primary dendrite arborization of the PC. 350X. Bar = 25 pm. ated with some of the synapses on the apical soma or proximal dendrites. These aggregates seem therefore to be preferentially associated with more mature, stable synapses; it is possible that the aggregates mark those synapses which are longlived or which will be maintained. Another possible morphologicalcorrelate of synapse maturation is the presence of the ecto-enzyme 5’-nucleotidase. The activity of this adenosine-producing enzyme is detectable within the synaptic clefts of excitatory cerebellar synapses

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transiently during their development (Schoen et al., 1991). During normal development, 5’-nucleotidase activity i s found at the early somatic CF-PC synapses and not at the later-developing dendritic synapses. Bailly et al. (1990) found that the 5’-nucleotidase activity at CF-PC synapses persisted in mature rat cerebella which had been induced to maintain multiple innervations (by X-irradiation; see below). Inthe abnormal multiply-innervatedcerebella, 5’-nucleotidase activity was present irrespective of synapse location. Figure 5 shows examples of electron micrographs

Figure 5. Electron micrographs of climbing fiber (CF) synapses on Purkinje cell (PC) dendritic spines after enzyme cytochemistry for 5’-(ecto)nucleotidase (5”) in the molecular layer of the cerebellar cortex of a normal adult tat (A) and of an adult rat X-irradiated at P5 (B). (A) The synaptic cleft (arrowheads) between a presynaptic CF varicosity and a postsynaptic PC spine is devoid of opaque reaction product, which sparsely decorates glial membranes in the normal adult cerebellum. Indicated are two 5”-negative parallel fiber synapses on PC spines (*). (B) In contrast, after postnatal X-irradiation, the asymmetrical synapses made by this CF varicosity on three PC spines show intense 5”-specific labeling (arrowheads). Note also the 5” labeling of the postsynaptic density of a non-innervated PC spine (*) surrounded by glia. 31 200X. Bar = 0.5p.m.

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taken from normal adult and X-irradiated, multiply-innervated cerebella. In the normal hdult rat cerebellum, synapticclefts do not contain 5’-nucleotidaseactivity, whereas this activity persists at synapses in the X-irradiated case. Thus when synapse elimination does not occur, a marker of synaptic immaturity in the rat cerebellum is retained. C.

The Parallel Fiber Trigger

A number of lines of evidence indicate that the establishment by the granule cells of the parallel fiber synapses is a critical heterosynaptic event in triggering elimination of the supernumerary climbing fiber synapses. The formation of these synapses occurs just as the CF elimination is beginning. Multiple climbing fiber innervationspersist when Purkinje cells develop in the absence of granule cells, as occurs in two mouse mutants, weaver and reeler (Crepel and Mariani, 1976;Siggins et al., 1976; Mariani et al., 1977;Puro and Woodward, 1977; Mariani, 1982). The multiple innervation also persists in the stuggerer mutant, where the parallel fibers are present but fail to form synaptic contacts with the Purkinje cells (Mariani and Changeux, 1980b; Crepel et al., 1980). Further evidencefor the importanceof the granulecells comes from experiments done in adult ferrets infected postnatally with a mink enteritis virus (Benoit et al., 1987),which produces ataxia and cerebellar hypoplasia. The viral infection has an anti-mitotic effect on the cells of the external germinal layer which normally give rise to the granule cells. In these animals the parallel fiber synapses on the Purkinje cells are therefore absent. Intracellular recordings from the Purkinje cells of these animals during graded stimulation of the climbing fiber pathway reveal synaptic potentials of more than one size, indicating remaining innervation by more than one climbing fiber. Another line of investigation into the role of the granule cells has employed directed X-irradiation of the cerebellum in early postnatal rats (Woodward et al., 1974; Crepel et al., 1976b; Delhaye-Bouchaud et al., 1978; Benoit et al., 1984; Bailly et al., 1988; Mariani et al., 1990) (see Figure 2C). Irradiation in the first postnatal week destroys the proliferating granule cells, and the timing of X-irradiation producing the largest effect on multiple innervation indicates that a relatively small group of the earliest granule cells to “settle” and develop can produce this heterosynaptic effect (Delhaye-Bouchaud et al., 1978). Figure 6 shows the morphology of an X-irradiated cerebellum compared to a control. The X-irradiated cerebellum is hypoplasic, due to the much-reduced number of granule cells. Morphological investigation of the climbing fibers in these irradiated cerebella shows that multiple CFs maintain contact with a single PC (Sotelo, 1981). Figure 7 shows dye-labeling of climbing fibers in an irradiated cerebellum; multiple CFs can be seen innervating a single PC.Physiological studies indicate that in at least some cases these multiple CFs contact the same dendritic segment (Crepel and Delhaye-Bouchaud, 1979). In addition, analysis of the electrophysiological char-

Figure 6. (A) Cerebellar atrophy in an adult rat X-irradiated at P5. Compare to the normal adult cerebellum in (6).Sagittal section in the median vermis, stained with cresyl violet. Arrowheads point to lobule VIII. 1OX. Bar = 1mm.

Figure 7. Persistence of multiple climbing fibers on a Purkinje cell during agranular development of the X-irradiated rat cerebellum. PI 3. Delayed somato-dendritictransfer of CFs, which still multiply-innervate the basal PC somata. Compare with the apical climbing monoinnervation of the PC soma at the same age in a normal cerebellum (Figure 4B). Bar = 25 pm. 84

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acteristics of the climbing-fibersynapses on the Purkinje cells has shown not only that the supernumerary synapses are maintained, but also that the functional somatotopic responses of the Purkinje cells are disrupted (see Functional Consequences below). D. Role of the Target Cell

Recent evidence also indicates that the Purkinje cell must reach a certain developmental state before the formation of the parallel fiber synapses can trigger eliminationof the supernumeraryCF synapses (Rabacchi et al., 1992a).The lurcher mouse mutation is illustrativeof the importanceof the target in synapseelimination. Lurcher is a mutation which acts directly on the PCs to produce their death during early postnatal life and causes secondary loss of granule cells and inferior olivary neurons (presumably due to the loss of target). During early postnataldevelopment, the lurcher Purkinje cells receive synaptic input both from the climbing fibers and from parallel fibers originating from a roughly normal number of granule cells (Caddy and Biscoe, 1979). Up to postnatal day 8, no morphological defects in the PCs or their synaptic contacts are visible (Dumesnil-Bousezand Sotelo, 1992),but 50% of the Purkinje cells then die during the second postnatal week (Caddy and Biscoe, 1979). The surviving lurcher Purkinje cells retain multiple climbing fiber innervation,probably until they die and certainly until after the time when synapse elimination is complete in normal cerebella (Rabacchi et al., 1992a). Since the density of parallel fiber synapses on the Purkinje cells is roughly normal during this period (Dumesnil-Bousez and Sotelo, 1992), these retained supernumerary climbing fiber inputs cannot be attributed to the absence of the granule cell trigger. In addition, work with adult chimeric animals, in which only wild-type Purkinje cells could have survived, showed that those cells were singly innervated, even in chimeras that were almost entirely mutant. The most conspicuous difference between these chimeras and the lurcher mutant cerebellum is the genotype of the Purkinjecells; thus this result suggests that the Purkinjecell with a lurcher genotype is unable to respond to the parallel fiber trigger which would otherwise initiate synapse elimination. The analysis of the lurcher mutant strengthens the argument that the Purkinje cell must be intrinsically competentto respond to the parallel-fiber trigger, and actively contributes to the process of initiating synapse elimination. Further evidence for the importance of the Purkinje cell target in directing synapse elimination comes from work done with cerebellar grafts. Sotelo and Alvarado-Mallart (1986) showed that embryonic cerebellar cells can be successfully integrated into adult cerebella which lack Purkinje cells due to the pcd (Purkinje cell degeneration)mutation. The grafted embryonic Purkinje cells seem to undergo relatively normal development in this abnormal environment; they proliferate, migrate into an approximately appropriate location, and develop dendritic trees (Sotelo et al., 1990). Most interestingly, the host climbing fibers and parallel fibers make synaptic contacts with the grafted F’urkinje cells. The morpho-

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logical development of these grafted Purkinje cells and their synapses thus seems to be governed by the Purkinje cells themselves (Sotelo et al., 1990). In addition, the process of climbing fiber synapse elimination in these cerebella follows a pattern very similar to that seen in normal development: electrophysiological recordings from the grafted Purkinje cells (Gardette et al., 1990) show that the Purkinje cells are transiently multiply-innervated, then monoinnervated after several more days of development. Thus the morphological and electrophysiological analysis of cerebella with grafted embryonic Purkinje cells indicates that the adult host climbing fibers and parallel fibers respond to the presence of the Purkinje cell target and follow a developmental process determined by the Purkinje cell. E.

Involvement of the NMDA Receptor

Elimination of supernumerary climbing fiber synapses seems also to be a phenomenon requiring activation of the NMDA-type glutamate receptor (NMDAR). Synaptic activity is known to be important in several cases of synapse elimination and regression of axon collaterals, and activation of the NMDA-R seems to be specifically important in several systems (as discussed above). The effects of NMDA-R blockade on the elimination of climbing fiber-Purkinje cell synapses were described by Rabacchi et al. (1992b). Using a technique in which a substance can be slowly released from a polymer matrix, these authors provided chronic administration of the NMDA receptor antagonist APV on the surface of the cerebellar vermis during the period of synapseelimination.This treatment resulted in the maintenance of multiple climbing fiber synapses on the Purkinje cells, as measured by electrophysiological recording. The gross morphology of the cerebellum and the health of the granule cells were not visibly affected, as shown in Figure 8. The critical site of activation of NMDA receptors is not known; these receptors are present transiently on the Purkinje cells themselves during early postnatal period (Dupont et al., 1987;Krupa and Crepel, 1990;Rosenmund et al., 1992),and on the granulecells as well (Howe et al., 1991). If the granule cells are the important NMDA-R activation site, the reported effects of chronic APV application could be due to a resulting modification of granule cell activity. If, on the other hand, the critical site of NMDA-R activation is on the Purkinje cells, this receptor could mediate the detection and stabilization of the climbing fiber synapsewhose activity is concomitant with the parallel fiber inputs. The involvement of another glutamate receptor subunit has recently been implicated in synapseelimination: mice mutant for the 62 subunit retain multiple climbing fiber synapses on their Purkinje cells, although interpretation of this finding is complicated by the fact that parallel fiber synapse formation is reduced in these mice (Kashiwabuchi et al., 1995). F.

Functional Consequences of Synapse Elimination

The functional reasons for synapse elimination are not known, but several possibilities can be proposed. Some theories proposed that the purpose of synapse

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Figure 8. Representative sagittal sections of cerebellar vermis of P18 rats treated with D,L-APV (B)or with the inactive stereoisomerL-AW (A) delivered in vivo from implanted

microsources since P5. There are no apparent morphological differences between the two treatments, but synapse elimination is disrupted (see text). Thionin staining. 1OX. Bar = 1 mm. From Rabacchi et al., 1992b. elimination was to remove synapses which had formed in the wrong target field; this now seems unlikely to be the primary function since growing axons generally make synapses in the appropriate target area due to growth-cone guidance mechanisms (Landmesser, 1978; Eisen et al., 1986). Perhaps synapses are overproduced to insure that no target cell lacks a contact; then competition results in a reduction to the mature synaptic relationships. In many studies of the visual system, it has become clear that the refinement of afferent projections to a target population segregates these afferents into functionally-appropriatedomains. Work in the rat cerebellum has shown that a similar development of functional domains parallels the elimination of supernumerary climbing fiber synapses. In the mature cerebellum, afferents in the climbing fiber pathway are organized into precise sagittal bands and subdivided into microzones, which are considered to be the functional data processing units in the cerebellum (Oscarsson, 1979). Anatomically, the sagittal bands are present even in very young polyinnervated cerebella (Dupont et al., 1981; Sotelo et al., 1984), but the anatomical methods do not show the presence or absence of the functional microzones. Electrophysiological comparisons have been made between normal adult rats and rats irradiated postnatally to retain multiple innervations, to determine the possible significance of synapseelimination in the refinement of functionalproperties in the cerebellum. Cerebellar maps showing the climbing fiber-mediated responses of Purkinje cells

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to peripheral Stimulation were constructed based on the location and depth of athe cells recorded. In an initial experiment using electrical stimulation of the periphery, no substantial differences in the response characteristics of the Purkinje cells were found between the controls and poly-innervated cerebella (Mulle et al., 1987; Mariani et al., 1987). This observation indicates that the overall topographic organization of the inputs to the cerebellum remained intact, and that the X-irradiation procedure does not adversely affect the olivary neurons which give rise to the climbing fibers. A more precise method of stimulation, mechanical movement of the vibrissae, gave a clearer picture of the differences between groups, showing that the synapse elimination process seems to modify the inputs within the already-organized structure. In normal adult rats, Purkinje cells showing climbing fiber responses elicited by mechanical stimulation of the contralateral vibrissae are generally restricted to a functional microzone located 200pm from the midline in the contralateral lobule VII (Thomson et al., 1989). In animals which were X-irradiated during early postnatal life to retain multiple climbing fiber innervations, the Purkinje cells responsive to vibrissal stimulation were no longer restricted to the normal microzone: responsive cells were found over a much wider lateral range (Piat et al., 1991; Fuhrman et al., 1994). More than 50%of the cells recorded in the polyinnervated cerebella responded to vibrissal stimulation, as opposed to only 15%in the normal cerebella. These results indicate that the functional segregation of climbing fiber inputs was disrupted in the polyinnervated cerebella, and strongly suggests that synapse elimination is normally important in the restriction of these climbing fiber synapses to provide the correct functional responses to peripheral stimulation. The question then arose as to whether synapse elimination could participate in another level of segregation, that of the ipsilateral and contralateral inputs. In the normal adult, the olivocerebellar pathway is entirely crossed (Campbell and Armstrong, 1983), but a transient ipsilateral pathway is present during postnatal development (Sherrard and Bower, 1986;Lopez-Roman et al., 1993,1994).Figure 9 shows a schematicdiagram of the pathways mediating the cerebellar response to vibrissal stimulation. In recordings from normal adult rats, responsive Purkinje cells could generally be activated only by contralateral vibrissal stimulation. In X-irradiated (polyinnervated)rats, Purkinje cells responsive to either ipsilateral or contralateral stimulation were found (Fuhrman et al., 1995). In addition, some of the responsive cells could be bilaterally-driven, while no bilaterally-driven Purkinje cell was found in the control cerebella. These experiments suggest that one function of elimination of supernumerary climbing fiber synapses is to remove inappropriate ipsilateral inputs and to refine the climbing fiber projections such that restricted functional microzones are formed. Sensory information from the vibrissae is very important in allowing the animal to determine the location of objects in the environmentand to coordinateappropriatemotor responses;thus the synapse elimination process, by segregatingipsilateral and contralateralinputs and

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Figure 9. Schematic drawing of the projections from the vibrissal system to the vermis of the rat cerebellum. Solid lines show the normal pathway of sensory information. The dashedline shows the transient ipsilateralclimbingfiber pathwaywhich is presentduring development. The apparent maintenance of this ipsilateral pathway in X-irradiated

cerebella, as demonstrated by climbing fiber-mediated responses to vibrissal stimuli, suggests that one function of synapse elimination in this system is to remove the functionally-inappropriateipsilateralinputs. ION: inferior olivary nucleus. MAO: medial accessory olive. From Fuhrman et al., 1995.

restricting inputs to appropriate cerebellar target regions, may play an important role in the development of normal responses to external stimuli.

IV. CONCLUDING REMARKS Both the cellular mechanisms and the functional significanceof synapse elimination remain incompletely described. Different experimental systems are likely to contribute differentpieces of information leading to a generalized model of synapse

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elimination. Work on the easily-accessible neuromuscular junction, for example,

' or on in vitro models is more likely to address the molecular signaling mechanisms

between cells during the synapseelimination process. Studiesin the central nervous system can help elucidatethe functionalconsequences of synapse elimination.The cerebellar Purkinje cell, which we have discussed here in some detail, may provide a model in which both functional significanceand celIular mechanisms of synapse elimination can be studied.

ACKNOWLEDGMENTS We are grateful to Dr. Yves Fuhrman for help with the figures. Work on this subject in the laboratory of the authors has been supported by the CNRS, INSERM, and the European Community (projectBARNEF, grant BM41CT941378).A.M.L. thanks the Human Science Frontier Program Organization for post-doctoral support.

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