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Principles of glutamatergic synapse formation: seeing the forest for the trees
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Principles of glutamatergic synapse formation: seeing the forest for the trees Noam E Ziv* and Craig C Garner† General principles regarding glutamatergic synapse formation in the central nervous system are beginning to emerge. These principles concern the specific roles that dendrites and axons play in the induction of synaptic differentiation, the modes of presynaptic and postsynaptic assembly, the time course of synapse formation and maturation, and the roles of synaptic activity in these processes. Addresses *Rappaport Institute and the Departmentof Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion, PO Box 9649, Bat Galim, Haifa 31096, Israel; e-mail: [email protected] † Department of Neurobiology, University of Alabama at Birmingham, 1719 Sixth Avenue South, CIRC 589, Birmingham, AL 35213-0021, USA Correspondence: Noam E Ziv Current Opinion in Neurobiology 2001, 11:536–543 0959-4388/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid GABA γ-amino benzoic acid GFP green fluorescent protein NMDA N-methyl-D-aspartate PSD postsynaptic density SV synaptic vesicle VAMP vesicle-associated membrane protein
Introduction The processes underlying synapse formation within the mammalian central nervous system (CNS) have fascinated neuroscientists for many decades. Historically, attempts aimed at understanding how the CNS is ‘wired’ were based primarily on the analysis of fixed and stained tissue by means of light and electron microscopy (reviewed in [1]). Although these studies provided the infrastructure of our understanding of CNS synaptogenesis, the techniques applied were limited inherently in their ability to provide information concerning the dynamics of these processes or the cellular and molecular mechanisms involved. This has changed with the introduction of contemporary molecular, optical and electrophysiological techniques, resulting in an enormous growth in the quantity and diversity of studies devoted to this subject. Furthermore, the widely held view that synapse formation and elimination might constitute mechanisms for long-term alteration of network properties (‘learning and memory’) has instigated numerous studies concerning relationships between neuronal activity and synaptogenesis, bringing the fields of developmental neuroscience and synaptic physiology closer together. A comprehensive survey of all this work is unfortunately beyond the scope of this review. Here we focus instead on
principles that seem to be emerging from this large body of work. Specifically, we concentrate on principles concerning the genesis of glutamatergic synapses, synapses that utilize glutamate as a neurotransmitter and constitute the majority of excitatory synapses in the mammalian CNS. In doing so, we focus primarily on the cellular physiology of these processes and less on the roles of specific molecules, as this has been the topic of several recent reviews [2–7].
Contact initiation: dendrites are not passive bystanders The formation of axodendritic synapses is often described in terms reminiscent of ‘cavemen courtship’; that is, the axon led by a growth cone (the ‘caveman’ bearing a club) seeks out and finds a prospective dendritic partner (the ‘cavewoman’), thereafter inducing the formation of postsynaptic specializations (a ‘bump on the head’) and evolving into a presynaptic terminal. According to this description, axons perform active roles, whereas dendrites are relatively passive. This view is probably a carryover from studies concerning the formation of the neuromuscular junction, where the targets (muscles) are rather stationary. Dendrites, in contrast, resemble axons in the sense that they also extend growth cones, elongate and bifurcate [8]. Earlier studies [1] showed that in many regions of the developing CNS, dendritic growth occurs more or less at the same time that afferent axons arrive. Furthermore, these studies, as well as more recent reports [9,10], revealed that numerous synapses formed during these periods are located on motile dendritic structures, namely dendritic growth cones and filopodia. These observations led to the conclusion that many of the synaptogenic contacts occurring during this period are initiated by dendrites actively ‘seeking out’ axonal counterparts [1,8]. In agreement with this concept, studies carried out in vitro [11–13] and in vivo [14••,15•] confirm that dendrites, and in particular dendritic filopodia, display highly dynamic protrusive activity consistent with an active role in synaptogenic contact initiation [8,16,17]. Furthermore, it has been suggested that dendritic filopodia may initiate contacts with nearby axons, induce the formation of presynaptic terminals along axonal ‘shafts’, and thereafter differentiate into dendritic spines or pull the synapse back to the dendritic shaft [18]. Interestingly, in several systems, high-frequency synaptic stimulation [19] or repeated membrane depolarization [20] has been shown to induce the extension of numerous dendritic filopodia, some of which persist and assume spine-like morphologies that are presumed to be new synapses (see also [21,22]).
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Figure 1 Initiation of synaptogenic contacts by axons and dendrites. In this simulated ‘time-lapse sequence’, two axons (blue) and one dendrite (green) elongate, extend processes, and initiate contact sites that might evolve potentially into axodendritic synapses. Principal events: 1, 2, interactions between axonal and dendritic growth cones; 3, an axonal growth cone intersecting with a dendritic shaft; 4, 5, dendritic filopodia contacting axonal shafts; and 6, a dendritic growth cone contacting an axonal shaft.
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Thus, one emerging principle is that dendrites as well as axons are active in seeking out nearby potential synaptic partners (Figure 1). Interestingly, it has been shown recently that muscle cells in Drosophila also extend filopodia (‘myopodia’), which interact with innervating motorneuron axons [23•]. Thus, muscle cells may not be as passive as previously thought (and nor were cavewomen, in all likelihood).
Time course of individual synapse formation: a matter of hours Much of what is known on the time course of CNS synaptogenesis is based on experiments performed on low-density dissociated rat hippocampal cultures (reviewed in [24]). Within 2–3 days after plating, these neurons form axons and dendrites that establish numerous contacts. Functional synapses start appearing at days 5–6, and synaptic molecules gradually assume synaptic distributions over the following days and weeks. These observations have indicated that glutamatergic synapses form over many days, during which time synaptic molecules are recruited sequentially to developing synaptic sites [5,25]. In contrast, imaging approaches indicate that individual synapses can form within a few hours [11,12,15•,19,22,26,27]. For example, using green fluorescent protein (GFP)-tagged vesicle-associated membrane protein (VAMP)/synaptobrevin (a synaptic vesicle molecule), Ahmari et al. [28••] have shown that clusters of synaptic vesicles (SVs) become stabilized at new axodendritic contact sites, and that stimulation-evoked exocytosis and endocytosis can be demonstrated within 1 hour of contact at these sites. Similarly, Vardinon-Friedman et al. [29•] have used a retrospective immunohistochemical approach to show that new functional presynaptic boutons form within 30 min of axodendritic contact and that most new synaptic boutons are associated within 1–2 hours of their appearance with clusters of ionotropic glutamate receptors.
How can these two timescales (hours versus days) be reconciled? We do not think that these timescales are in conflict. Instead, they probably describe different processes. The long timescale (i.e. days), we believe, reflects the developmental time course of synaptic molecule expression in the culture systems (and perhaps in vivo as well), whereas the hour timescale reflects the period over which individual synapses are formed. Synapses will not form, however, until a minimal set of pre- and postsynaptic components becomes available. Furthermore, the state of new synapses will depend on the repertoire of available synaptic molecules at the time of their formation. If it is rich enough, new synapses will resemble more mature synapses. But if the repertoire (or abundance) of synaptic molecules is meagre, new synaptic junctions may be immature, partially functional, or not functional at all. In agreement with this concept, Renger et al. [30••] found that presynaptic boutons in very immature cultures (less than 7 days in vitro) displayed almost no capacity for evoked SV recycling, whereas those in slightly older cultures (8–12 days) did display such a capacity. The functional characteristics of the latter synapses, however, were strikingly different from those of synapses in mature cultures (more than 13 days), suggesting that synapses that form early in development undergo some type of age-dependent maturation. It is important to note, however, that concomitantly with this maturation process, many new synapses were being formed over this entire period (see also [31]). Interestingly, apart from a change in the number of docked/membrane-associated SVs, the ultrastructure of synapses formed in the youngest age group was very similar to that of synapses in older preparations. We interpret these findings to suggest that the formation of individual synaptic connections is a relatively rapid
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process, occurring over a timescale of several hours. The molecular composition and functional characteristics of such synapses depend, however, on slower developmental processes, such as the repertoire and abundance of synaptic molecules at the time at which the synapse is formed.
Induction of synaptic differentiation: bi-directional signals The formation of an axodendritic contact is only the first step in the genesis of a new synapse. During subsequent steps, the axonal and dendritic compartments at the contact site differentiate into presynaptic boutons and postsynaptic reception apparatuses, respectively. The differentiation of the presynaptic bouton includes the clustering of SVs and the formation of active zones — specialized regions of presynaptic plasma membrane containing electron-dense meshworks of cytoskeletal elements, where SVs dock and fuse [3,6]. Similarly, the differentiation of the postsynaptic reception apparatus involves the formation of the postsynaptic density (PSD) — an electron-dense, specialized cytoskeletal matrix juxtaposed to the presynaptic active zone, which serves to cluster and localize neurotransmitter receptors and other molecules to the postsynaptic membrane [2,4,32–34]. An elusive goal in the study of glutamatergic synapse formation has been the characterization of signals that trigger pre- and postsynaptic differentiation. Studies concerning the formation of the vertebrate neuromuscular junction have led to the discovery of molecules secreted by motoneurons, such as agrin, that can induce postsynaptic differentiation (reviewed in [35]). Immunohistochemical analysis of nascent glutamatergic synapses forming in vitro suggests that presynaptic differentiation often precedes postsynaptic differentiation [29•], which would be consistent with postsynaptic differentiation being induced by factors secreted by nascent presynaptic sites (but probably not agrin [36,37]; but see also [38]). This, however, cannot be the whole story, because dendrites possess an ability to induce the formation of presynaptic specializations along axonal shafts (as discussed above); thus, something — presumably on the dendritic membrane — has to trigger the formation of axonal SV release sites. Interesting candidates in this respect seem to be neuroligins — postsynaptic transmembrane adhesion molecules that bind to β-neurexins, a family of presynaptic membrane molecules [39,40]. Recently, neuroligin expressed in non-neuronal cells has been shown to induce the formation of functional presynaptic structures in pontine neurons, and soluble forms of β-neurexin have been shown to suppress synapse formation between pontine neurons and cerebellar granule cells [41••,42]. As exciting as these findings are, it remains to be demonstrated that these molecules are as ‘indispensable’ for CNS synapse formation as are agrin and its receptor component (MuSK) at the neuromuscular junction [35]. In fact, neuroligin 1 knockout mice appear to be normal [40], although other family members might compensate for the lack of this protein.
Evidence has accumulated to suggest that additional molecules such as ephrinB/EphB receptors [43•], neuronal activity-regulated pentraxin (Narp) [44], Wnt-7a [45], adhesion molecules [46,47] and others (reviewed in [7]) play certain roles in the induction of pre- or postsynaptic differentiation. For example, the binding of preaggregated ephrinB to its receptor, the tyrosine kinase EphB, was shown to promote the rapid formation of NMDA (N-methyl-D-aspartate)-type glutamate receptor clusters [43•]. Similarly, Narp, a secreted molecule expressed by a subset of hippocampal and spinal cord neurons, was shown to induce the formation of AMPA (α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid)-type glutamate receptor clusters [44]. Ultimately, it may turn out that synaptic differentiation induction involves multiple molecules or molecule families, none of which is completely indispensable. Although this tentative conclusion may prove to be wrong, the precise alignment of the active zone and tightly matching dimensions of the active zone and PSD ([6] and references therein), the diversity of molecules discovered so far, and their particular effects on pre- and postsynaptic differentiation, substantiate the concept that the induction process involves multiple bi-directional, reciprocal signals.
Presynaptic differentiation: prefabricated precursors In principle, presynaptic differentiation and active zone assembly could be realized by the sequential, in-situ recruitment of presynaptic molecules. Two recent studies suggest, however, that active zones may be formed by the insertion of pre-assembled, macromolecular vesicular-associated complexes into the presynaptic membrane (a mechanism originally proposed by Vaughn [1]; see also [48]). In the first study [28••], GFP-tagged VAMP/synaptobrevin was reported to cluster at newly forming active zones together with other presynaptic molecules, that is, voltagedependent calcium channels, SV2, synapsin I and amphipysin as well as clusters of pleomorphic vesicular and tubular membranes thought to be cytoplasmic transport packets for presynaptic proteins. In the second study [49••], a previously unknown 80-nm dense core vesicle was purified and shown to contain specifically many active-zone components, including Bassoon, Piccolo, syntaxin, SNAP-25 and N-cadherin. Because two components of these vesicles, Bassoon and Piccolo, are nearly always found at new functional presynaptic sites [29•,49••], these findings were interpreted to suggest that these 80-nm vesicles constitute active-zone precursor vesicles, and that their fusion with the presynaptic plasma membrane might result in the rapid formation of new active zones. Presumably, the formation of new SV release sites is followed by the recruitment of SVs to these sites. Interestingly, SVs seem to be transported along axons [50,51] and recruited to new axodendritic sites [28••] in the
Principles of glutamatergic synapse formation Ziv and Garner
Figure 2 legend Synaptic differentiation by insertion of pre-assembled precursor vesicles versus sequential in situ recruitment of synaptic components. In this very simplistic simulated ‘time-lapse sequence’, presynaptic differentiation is shown to occur by the insertion of precursor vesicles containing full complements of active zone cytoskeleton complexes, which leads to the rapid formation of functional active zones. Postsynaptic differentiation is shown to occur by the sequential, in situ, recruitment of PSD scaffolding molecules followed by glutamate receptors and PSD signaling molecules. The differentiation processes are presumed to be initiated by interactions between the external aspects of axonal and dendritic membranal molecules. The time points represent the approximate time course of these processes in minutes from the point of axodendritic contact.
form of multivesicular packets, recapitulating the motif of assembly through ‘modular’ units at the presynaptic site (Figure 2).
Postsynaptic assembly: component by component In contrast to the relatively coherent picture that is emerging on presynaptic differentiation, the phenomenology of postsynaptic differentiation remains unclear. This may seem surprising considering how much has been learned on the molecular composition of the glutamatergic PSD. In spite of much uncertainty, some principles are emerging that we will discuss here. Contemporary methods such as yeast two-hybrid assays, colocalization analysis and, recently, large-scale protein complex analysis methods, such as mass spectrometry [52,53], have revealed that the glutamatergic PSD is composed of several large multimolecular complexes, each composed of dozens of molecules. These molecules fall into several categories, including glutamate receptors, scaffolding and adapter molecules, cell-adhesion molecules, cytoskeleton molecules, protein kinases/phosphatases, and other signaling molecules. One group of molecules that has received much attention is the group of scaffolding/adapter molecules that includes SAP90/PSD-95, PSD-93, SAP 102, ProSAP/Shank, SAP 97, PICK, Stargazin [54••] and many others [4,32]. Owing to their multiple binding sites for glutamate receptors on the one hand and other scaffolding/cytoskeletal molecules (and various signaling molecules) on the other, it has been postulated that these molecules might serve to build macromolecular receptor-associated signaling complexes, as well as to cluster these complexes specifically at postsynaptic sites.
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These findings might suggest that, in common with the active zone, the PSD is assembled from ‘prefabricated’ complexes; however, current data are not entirely consistent with this possibility and indicate that PSD components may be recruited sequentially to postsynaptic sites. NMDA-type and AMPA-type glutamate receptors do not seem to be part of the same core multimolecular postsynaptic complexes [53]. NMDA-type receptor 2B
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subunits are shuttled to synaptic sites on discrete transport vesicles, which contain some molecules, but not others, belonging to the multimolecular complex associated with
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NMDA receptors [55], whereas AMPA receptors seem to be recruited from diffuse pools [56,57] by rapid exocytosis and endocytosis mechanisms (reviewed in [2,58,59]). Furthermore, the recruitment of glutamate receptors to nascent synaptic sites seems to lag behind the recruitment of the scaffolding molecules SAP90/PSD-95 [29•] and ProSAP/Shank (H Vardinon-Friedman, T Boeckers, E Gundelfinger, NE Ziv, unpublished data). Finally, studies based on GFP-tagged SAP90/PSD-95 suggest that this molecule is recruited to nascent synaptic sites in a gradual manner, and not in discrete packets ([26]; and see Update). Thus, PSD assembly seems to be a multistep sequential process that occurs directly at the postsynaptic site (Figure 2).
Activity: important but not essential A key step in postsynaptic differentiation is the insertion of glutamate receptors into the postsynaptic membrane. In hippocampi of postnatal rats, as well as in other developing systems, some synapses seem to contain primarily NMDA receptors, and few or no AMPA receptors. As development proceeds, the fraction of synapses lacking AMPA receptors is reduced, but some such synapses remain, even in adult animals. These synapses may constitute the so-called ‘silent synapses’, that is, glutamatergic synapses that exhibit NMDA-, but not AMPA-receptor-mediated synaptic currents. Some researchers have proposed that excitatory synapses start out as ‘silent’ synapses and evolve into fully functional synapses by activity-dependent, NMDA-receptor-mediated, recruitment of AMPA receptors into the postsynaptic membrane (reviewed in [60,61]). Is electric activity or neurotransmitter release absolutely essential for glutamatergic synapse formation per se? The answer seems to be no. Synapses form in cultured hippocampal neurons in the presence of pharmacological agents that block spontaneous spiking activity [62] or synaptic transmission [29•,31,63]. Furthermore, mice deficient in either one of the presynaptic molecules Munc 13-1 or Munc 18-1 develop morphologically normal synapses in spite of partial [64] or complete [65••] loss of neurotransmitter release. Are electric activity or NMDA receptor activation essential for AMPA receptor recruitment to new synapses? Here, too, the answer seems to be no. AMPA receptors accumulate at new synaptic junctions when spontaneous activity and/or glutamate receptors (NMDA receptors included) are blocked [29•,31,56,62,66,67]. The recruitment of one AMPA receptor subunit, GluR4, to synapses formed in the developing rat hippocampus does seem to depend on activity and NMDA receptor activation [68•]. The expression of this subunit, however, is largely restricted to the first 4–5 postnatal days, and is reduced to less than 3% of the total AMPA receptor subunit pool by postnatal day 6 (see supplementary data for [68•]). As vigorous synaptogenesis continues beyond postnatal day 6, the general significance of these findings is currently unclear.
To confound matters further, Renger et al. [30••] have shown recently that individual immature synapses that display ‘silent-synapse’-like postsynaptic currents when stimulated presynaptically do have functional AMPA receptors — the presence of which is revealed by glutamate microiontophoresis. If this finding has general applicability, it might suggest that AMPA receptors appear at synapses much earlier during development than has been indicated previously by electrophysiological methods. Can activity regulate the AMPA receptor content (both receptor number and subunit composition) of new synapses? Apparently it can (reviewed in [61]); however, in addition to activity-dependent mechanisms, there seem to be constitutive pathways for AMPA receptor insertion into postsynaptic membranes that are insensitive to treatments that block activity-regulated AMPA receptor insertion [59,69]. It thus seems that activity or NMDA receptor activation are not indispensable for the formation of functional glutamatergic synapses. This conclusion is actually quite logical: if all synapses are initially ‘silent’, and if NMDA receptor activation is an absolute requisite for synaptic gain of function, how would neurons be excited sufficiently to activate these NMDA receptors? Although excitability in developing networks may be driven by additional mechanisms, such as γ-amino benzoic acid A receptor (GABAA)-based synaptic transmission [70], we feel that the findings described above are more congruent with the concept that AMPA receptor recruitment involves both activity-independent and activity-dependent routes, providing a satisfactory mechanism for setting up initial connections and refining them later on in a use-dependent manner.
Conclusions We have attempted to highlight general principles that seem to be emerging from the expanding body of literature concerning the formation of glutamatergic synapses. In the process, we have purposely glossed over many molecular details, aiming to focus on the ‘forest’, and not on the ‘trees’. Even so, important issues were not discussed, including among others those concerning the capacity of activity to induce new synaptic connections, the roles of activity in maintaining existing glutamatergic synapses, the roles of scaffolding molecules in PSD differentiation, the roles of active zone cytoskeleton molecules in activezone differentiation, and the mechanisms of glutamate receptor targeting to nascent synapses. Nevertheless, it is apparent that considerable progress has been made in our understanding of the cellular physiology of glutamatergic synapse formation, and we have no doubt that with time, our view of the forest will become even clearer.
Update During the time between the submission of this review and the correction of the proofs, several noteworthy studies have been published that lend further credibility to ideas
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summarized above. Of particular interest is the study by Okabe et al. [71•], on the formation of new dendritic spines and the recruitment of synaptic molecules to these new synaptic structures. By expressing molecules tagged with spectrally separable variants of GFP, the authors demonstrate that clusters of SAP90/PSD-95 can form in new dendritic filopodia/spines within ~45 min of their extension. Retrospective immunohistochemistry revealed that most new SAP90/PSD-95 clusters are associated with clusters of AMPA receptors within ~90 minutes of their appearance. Furthermore, all new SAP90/PSD-95 puncta were observed to be associated with clusters of SVs within ~60 min of their appearance. Finally, clustering of SVs (labeled by cyan fluorescent protein-tagged synaptophysin) was observed to precede the accumulation of SAP90/PSD95 at new synaptic sites. These findings provide strong support to the notion that dendritic filopodia can initiate synaptogenic contacts and thereafter evolve into dendritic spines [9–13,14••,15•,16–18], and are in good agreement with previous studies concerning the time course and temporal order of glutamatergic synapse formation [28••,29•]. A second report we wish to mention here is a study by Bresler et al. [72•], showing that GFP-tagged SAP90/PSD95 is recruited to new synaptic sites gradually from diffuse cytoplasmic pools, with kinetics fitting a single exponential with a mean time constant of ~23 min, supporting the proposition that PSD assembly occurs sequentially in situ. In agreement with previous studies [28••,29•] and the study mentioned above [71•], the authors show that SAP90/PSD-95 clusters at new axodendritic contact sites within 20–60 minutes of contact establishment, and that such sites rapidly acquire a capacity for evoked SV recycling.
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Acknowledgements
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Work in the lab of NE Ziv was supported by a grant from the Israel Science Foundation (139/98). Work in the lab of CC Garner was supported by grants from the National Institutes of Health (RO1 NS39471, PO1 AG06569). NE Ziv is a member of the Bernard Katz Minerva Center for Cell Biophysics.
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41. Scheiffele P, Fan J, Choih J, Fetter R, Serafini T: Neuroligin •• expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 2000, 101:657-669. Non-neuronal cells are engineered to express neuroligins — postsynaptic cell-adhesion molecules [40] shown previously to bind to β-neurexins [39]. These cells, but not cells expressing other molecules (N-cadherin, ephrin B1, TAG-1, agrin and L1) display the capacity to induce functional presynaptic specializations along axons of pontine neurons growing over them. Conversely, soluble forms of β-neurexin can specifically suppress this effect and suppress synapse formation between pontine neurons and cerebellar granule cells. These findings indicate that interactions between neuroligins and β-neurexins are important in the induction of presynaptic (and perhaps postsynaptic) differentiation (see also [42]).
Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D: LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 1999, 402:421-425.
28. Ahmari SE, Buchanan J, Smith SJ: Assembly of presynaptic active •• zones from cytoplasmic transport packets. Nat Neurosci 2000, 3:445-451. Green fluorescent protein tagged vesicle-associated membrane protein (VAMP–GFP) expressed in cultured hippocampal neurons is used to follow the formation of new presynaptic vesicle recycling sites. VAMP–GFP is observed to be transported along axons as discrete packets, which occasionally stabilize at new axodendritic contact sites. A capacity for evoked vesicle recycling is detected in less than 1 hour at such sites, indicating that presynaptic vesicle release sites may form quite rapidly. Surprisingly, retrospective electron microscopy reveals very few typical synaptic vesicles at such sites. Retrospective immunohistochemical analysis suggests that VAMP–GFP transport packets are associated with multiple characteristic presynaptic molecules, such as voltage-dependent calcium channels and synapsin I, leading to the suggestion that several presynaptic proteins may be transported collectively to presynaptic sites (see also [48,49••]). 29. Vardinon-Friedman H, Bresler T, Garner CC, Ziv NE: Assembly of • new individual excitatory synapses time course and temporal order of synaptic molecule recruitment. Neuron 2000, 27:57-79. Recurrent labeling with FM 4-64, a fluorescent endocytosis label, is used to detect new functional presynaptic boutons in cultures of rat hippocampal neurons, and retrospective immunohistochemistry is used to analyze the molecular composition of these putative new synaptic sites. Clusters of the presynaptic active-zone protein Bassoon are present in all new boutons, whereas clusters of postsynaptic molecules including AMPA and NMDA receptors are found, on average, ~45 min later. Time-lapse recordings reveal that functional presynaptic boutons can form at new axodendritic contact sites in less than 30 min. These findings suggest that individual glutamatergic synapses may form in 1–2 hours and indicate that presynaptic differentiation proceeds more rapidly than does postsynaptic differentiation. 30. Renger JJ, Egles C, Liu G: A developmental switch in •• neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation. Neuron 2001, 29:469-484. Developmental changes in functional characteristics of individual glutamatergic synapses are studied in preparations of cultured hippocampal neurons. The authors report that evoked postsynaptic currents elicited in immature cultures are often mediated solely by NMDA receptors, but focal glutamate iontophoresis reveals the presence of functional AMPA receptors at these very same synapses. The authors show that the types of postsynaptic receptors activated at such synapses depend on the temporal concentration profile of neurotransmitter flux and therefore propose that the functional maturation of ‘silent’ synapses may reflect the presynaptic maturation of neurotransmitter-release characteristics. 31. Gomperts SN, Carroll R, Malenka RC, Nicoll RA: Distinct roles for ionotropic and metabotropic glutamate receptors in the maturation of excitatory synapses. J Neurosci 2000, 20:2229-2237. 32. Sheng M, Pak DT: Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses. Ann New York Acad Sci 1999, 868:483-493. 33. Kennedy MB: Signal-processing machines at the postsynaptic density. Science 2000, 290:750-754. 34. Scannevin RH, Huganir RL: Postsynaptic organization and regulation of excitatory synapses. Nat Rev Neurosci 2000, 1:133-141.
42. Cantallops I, Cline HT: Synapse formation: if it looks like a duck and quacks like a duck… Curr Biol 2000, 10:R620-R623. 43. Dalva MB, Takasu MA, Lin MZ, Shamah SM, Hu L, Gale NW, • Greenberg ME: EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 2000, 103:945-956. Interactions between preformed ephrinB aggregates and EphB receptor tyrosine kinases are shown here to promote the clustering of NMDA receptors. Interactions between EphR and NMDA receptors seem to occur at the cell surface, as they are mediated by extracellular regions of these molecules. Long-term treatment of cultured hippocampal neurons with ephrinB aggregates increases the density of presynaptic as well as postsynaptic specializations. Conversely, expression of EphB variants, which block its intracellular tyrosine kinase activity, reduces the density of postsynaptic specializations. These findings suggest that ephrinB and its EphB receptors have a role in the induction of synaptic differentiation. 44. O’Brien RJ, Xu D, Petralia RS, Steward O, Huganir RL, Worley P: Synaptic clustering of AMPA receptors by the extracellular immediate-early gene product Narp. Neuron 1999, 23:309-323. 45. Hall AC, Lucas FR, Salinas PC: Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 2000, 100:525-535. 46. Brose N: Synaptic cell adhesion proteins and synaptogenesis in the mammalian central nervous system. Naturwissenschaften 1999, 86:516-524. 47.
Ranscht B: Cadherins: molecular codes for axon guidance and synapse formation. Int J Dev Neurosci 2000, 18:643-651.
48. Roos J, Kelly RB: Preassembly and transport of nerve terminals: a new concept of axonal transport. Nat Neurosci 2000, 3:415-417. 49. Zhai R, Vardinon-Friedman H, Cases-Langhoff C, Becker B, •• Gundelfinger ED, Ziv NE, Garner CC: Assembling the presynaptic active zone: characterization of an active zone precursor vesicle. Neuron 2001, 29:131-143. A previously unknown ~80 nm dense core vesicle is isolated and shown to contain specifically several components of presynaptic active zones [3,6], including Piccolo, Bassoon, Syntaxin, SNAP-25 and N-cadherin. Components of synaptic vesicles such as VAMP2, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1) are not present on this vesicle. Retrospective analysis of new synaptic vesicle recycling sites reveals that Piccolo (and Bassoon [29•]) clusters are always present at such sites. These findings indicate that presynaptic active zones may be formed by the fusion of these or similar active-zone precursor vesicles with the presynaptic plasma membrane (see also [28••,48]).
35. Hoch W: Formation of the neuromuscular junction. Agrin and its unusual receptors. Eur J Biochem 1999, 265:1-10.
50. Kraszewski K, Mundigl O, Daniell L, Verderio C, Matteoli M, De Camilli P: Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J Neurosci 1995, 15:4328-4342.
36. Serpinskaya AS, Feng G, Sanes JR, Craig AM: Synapse formation by hippocampal neurons from agrin-deficient mice. Dev Biol 1999, 205:65-78.
51. Dai Z, Peng HB: Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis. Mol Cell Neurosci 1996, 7:443-452.
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52. Walikonis RS, Jensen ON, Mann M, Provance DW Jr, Mercer JA, Kennedy MB: Identification of proteins in the postsynaptic density fraction by mass spectrometry. J Neurosci 2000, 20:4069-4080.
Li Z, Hilgenberg LGW, O’Dowd DK, Smith MA: Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice. J Neurobiol 1999, 39:547-557.
38. Bose CM, Qiu D, Bergamaschi A, Gravante B, Bossi M, Villa A, Rupp F, Malgaroli A: Agrin controls synaptic differentiation in hippocampal neurons. J Neurosci 2000, 20:9086-9095.
53. Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG: Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci 2000, 3:661-669.
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54. Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, •• Wenthold RJ, Bredt DS, Nicoll RA: Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 2000, 408:936-943. Stargazer mutant mice lack functional AMPA receptors in cerebellar granule cells. This study shows that interactions between the mutated protein stargazin and AMPA receptor subunits are critical for targeting AMPA receptor subunits to the plasma membrane, whereas interactions with synaptic PDZ molecules [4] such as PSD-95 are critical for targeting AMPA receptors to synaptic loci. 55. Setou M, Nakagawa T, Seog DH, Hirokawa N: Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 2000, 288:1796-1802. 56. Mammen AL, Huganir RL, O’Brien RJ: Redistribution and stabilization of cell surface glutamate receptors during synapse formation. J Neurosci 1997, 17:7351-7358. 57.
Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R: Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 1999, 284:1811-1816.
58. Luscher C, Nicoll RA, Malenka RC, Muller D: Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nat Neurosci 2000, 3:545-550. 59. Malinow R, Mainen ZF, Hayashi Y: LTP mechanisms: from silence to four-lane traffic. Curr Opin Neurobiol 2000, 10:352-357. 60. Feldman DE, Knudsen EI: Experience-dependent plasticity and the maturation of glutamatergic synapses. Neuron 1998, 20:1067-1071. 61. Carroll RC, Malenka RC: Delivering the goods to synapses. Nat Neurosci 2000, 3:1064-1066. 62. Craig AM, Blackstone CD, Huganir RL, Banker G: Selective clustering of glutamate and γ-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc Natl Acad Sci USA 1994, 91:12373-12377. 63. Osen-Sand A, Staple JK, Naldi E, Schiavo G, Rossetto O, Petitpierre S, Malgaroli A, Montecucco C, Catsicas S: Common and distinct fusion proteins in axonal growth and transmitter release. J Comp Neurol 1996, 367:222-234. 64. Augustin I, Rosenmund C, Sudhof TC, Brose N: Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 1999, 400:457-461. 65. Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, •• Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M et al.: Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 2000, 287:864-869. In knockout mouse deficient in Munc 18-1, all tested forms of neurotransmitter release are found to be abolished completely. In spite of the complete and permanent loss of synaptic transmission (resulting in death at birth owing to asphyxiation) brain development occurs normally. Furthermore, synapses, both central and peripheral, are readily formed and are similar ultrastructurally and functionally to those formed in control littermates. Intriguingly, normal brain assembly in the knockout mice is followed by widespread degeneration, particularly in structures formed early on, such as the brainstem. These findings suggest that synaptic transmission is not indispensable for brain development and synapse formation but is crucial for synaptic maintenance. This is corroborated by other studies, including [64], where a knockout mouse deficient in Munc-13-1, a brain-specific presynaptic phorbol-ester receptor, is generated. Gross brain development in newborn offspring (that die within hours of birth) is indistinguishable from that of control mice. Synaptic density and glutamate receptor content in cultured hippocampal neurons obtained from newborn mutant mice are similar to those in control cultures, even though more than 70% of these synapses are
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presynaptically silent. Interestingly, GABAergic synapses, as well as a small population of glutamatergic synapses, are not affected by the deletion. 66. Rao A, Craig AM: Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 1997, 19:801-812. 67.
Cottrell JR, Dube GR, Egles C, Liu G: Distribution density and clustering of functional glutamate receptors before and after synaptogenesis in hippocampal neurons. J Neurophysiol 2000, 84:1573-1587.
68. Zhu JJ, Esteban JA, Hayashi Y, Malinow R: Postnatal synaptic • potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity. Nat Neurosci 2000, 3:1098-1106. The incorporation of GluR4 (an AMPA receptor subunit) into postsynaptic sites in rat hippocampal slices is studied here using GFP-tagged GluR4, optical and electrophysiological methods. GluR4 incorporation is found to be dependent on a minimal amount of activity (spontaneous neural activity is sufficient) and on activation of NMDA receptors. Interestingly, GluR4 is subsequently replaced by a different subunit (GluR2) by a mechanism not dependent on activity nor NMDA receptors, indicating that the AMPA receptor content of glutamatergic synapses may be regulated by several pathways. 69. Lu W, Man H, Ju W, Trimble WS, MacDonald JF, Wang YT: Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 2001, 29:243-254. 70. Ben-Ari Y, Khazipov R, Leinekugel X, Caillard O, Gaiarsa JL: GABAA NMDA and AMPA receptors: a developmentally regulated ‘menage a trois’. Trends Neurosci 1997, 20:523-529. 71. Okabe S, Miwa A, Okado H: Spine formation and correlated • assembly of presynaptic and postsynaptic molecules. J Neurosci 2001, 21:6105-6114. The morphogenesis of three synaptic components — dendritic spines, PSDs and SV clusters were followed in cultured hippocampal neurons by expression of molecules tagged with spectrally separable variants of GFP, time lapse microscopy and retrospective immunohistochemistry. It is shown here that SAP90/PSD-95 clustering is spatially and temporally correlated with the extension of new dendritic filopodia/spines. In contrast, preexisting SAP90/PSD-95 clusters in dendritic shafts are preferentially eliminated without promoting spine formation. Retrospective immunohistochemistry reveals that most new SAP90/PSD-95 clusters become associated with clusters of AMPA receptors SV clusters within (on average) ~90 and ~60 minutes of their appearance, respectively. Clustering of SVs (labeled by cyan fluorescent protein-tagged synaptophysin) was observed to precede the accumulation of yellow-fluorescent protein-tagged SAP90/PSD-95 at new synaptic sites. These findings strongly support prior suggestions concerning the roles of dendritic filopodia in spine formation [9–18] and are in good agreement with other studies concerning the time course and temporal order of glutamatergic synapse formation ([28••,29•], see also [72•]). 72. Bresler T, Ramati Y, Zamorano PL, Zhai R, Garner CC, Ziv NE: The • dynamics of SAP90/PSD-95 recruitment to new synaptic junctions. Mol Cell Neurosci 2001, 18:149-167. GFP-tagged SAP90/PSD-95, time lapse confocal microscopy and cultured hippocampal neurons were used here to determine the recruitment dynamics of the PSD molecule SAP90/PSD-95 into new synaptic junctions. New SAP90/PSD-95 clusters were first observed at new axodendritic contact sites within 20–60 minutes of contact establishment. Clustering was gradual but rapid, occurring over one hour or less. Most new SAP90/ PSD-95 clusters were associated with functional presynaptic boutons as determined by labeling with FM 4-64. As discrete transport particles of SAP90/PSD-95 were not found, it was suggested that in contrast to what has been suggested for presynaptic assembly, postsynaptic assembly occurs in situ, by the sequential recruitment of PSD molecules (see also [26,71•]). Interestingly, concomitant time lapse recordings of labeled preand postsynaptic structures reveal that in culture, at least, entire synaptic structures can change their position considerably, suggesting that caution should be exercised in interpreting mobility of synaptic molecule clusters as evidence for the existence of transport particles for these molecules.
Principles of glutamatergic synapse formation: seeing the forest for the trees Noam E Ziv* and Craig C Garner† General principles regarding glutamatergic synapse formation in the central nervous system are beginning to emerge. These principles concern the specific roles that dendrites and axons play in the induction of synaptic differentiation, the modes of presynaptic and postsynaptic assembly, the time course of synapse formation and maturation, and the roles of synaptic activity in these processes. Addresses *Rappaport Institute and the Departmentof Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion, PO Box 9649, Bat Galim, Haifa 31096, Israel; e-mail: [email protected] † Department of Neurobiology, University of Alabama at Birmingham, 1719 Sixth Avenue South, CIRC 589, Birmingham, AL 35213-0021, USA Correspondence: Noam E Ziv Current Opinion in Neurobiology 2001, 11:536–543 0959-4388/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid GABA γ-amino benzoic acid GFP green fluorescent protein NMDA N-methyl-D-aspartate PSD postsynaptic density SV synaptic vesicle VAMP vesicle-associated membrane protein
Introduction The processes underlying synapse formation within the mammalian central nervous system (CNS) have fascinated neuroscientists for many decades. Historically, attempts aimed at understanding how the CNS is ‘wired’ were based primarily on the analysis of fixed and stained tissue by means of light and electron microscopy (reviewed in [1]). Although these studies provided the infrastructure of our understanding of CNS synaptogenesis, the techniques applied were limited inherently in their ability to provide information concerning the dynamics of these processes or the cellular and molecular mechanisms involved. This has changed with the introduction of contemporary molecular, optical and electrophysiological techniques, resulting in an enormous growth in the quantity and diversity of studies devoted to this subject. Furthermore, the widely held view that synapse formation and elimination might constitute mechanisms for long-term alteration of network properties (‘learning and memory’) has instigated numerous studies concerning relationships between neuronal activity and synaptogenesis, bringing the fields of developmental neuroscience and synaptic physiology closer together. A comprehensive survey of all this work is unfortunately beyond the scope of this review. Here we focus instead on
principles that seem to be emerging from this large body of work. Specifically, we concentrate on principles concerning the genesis of glutamatergic synapses, synapses that utilize glutamate as a neurotransmitter and constitute the majority of excitatory synapses in the mammalian CNS. In doing so, we focus primarily on the cellular physiology of these processes and less on the roles of specific molecules, as this has been the topic of several recent reviews [2–7].
Contact initiation: dendrites are not passive bystanders The formation of axodendritic synapses is often described in terms reminiscent of ‘cavemen courtship’; that is, the axon led by a growth cone (the ‘caveman’ bearing a club) seeks out and finds a prospective dendritic partner (the ‘cavewoman’), thereafter inducing the formation of postsynaptic specializations (a ‘bump on the head’) and evolving into a presynaptic terminal. According to this description, axons perform active roles, whereas dendrites are relatively passive. This view is probably a carryover from studies concerning the formation of the neuromuscular junction, where the targets (muscles) are rather stationary. Dendrites, in contrast, resemble axons in the sense that they also extend growth cones, elongate and bifurcate [8]. Earlier studies [1] showed that in many regions of the developing CNS, dendritic growth occurs more or less at the same time that afferent axons arrive. Furthermore, these studies, as well as more recent reports [9,10], revealed that numerous synapses formed during these periods are located on motile dendritic structures, namely dendritic growth cones and filopodia. These observations led to the conclusion that many of the synaptogenic contacts occurring during this period are initiated by dendrites actively ‘seeking out’ axonal counterparts [1,8]. In agreement with this concept, studies carried out in vitro [11–13] and in vivo [14••,15•] confirm that dendrites, and in particular dendritic filopodia, display highly dynamic protrusive activity consistent with an active role in synaptogenic contact initiation [8,16,17]. Furthermore, it has been suggested that dendritic filopodia may initiate contacts with nearby axons, induce the formation of presynaptic terminals along axonal ‘shafts’, and thereafter differentiate into dendritic spines or pull the synapse back to the dendritic shaft [18]. Interestingly, in several systems, high-frequency synaptic stimulation [19] or repeated membrane depolarization [20] has been shown to induce the extension of numerous dendritic filopodia, some of which persist and assume spine-like morphologies that are presumed to be new synapses (see also [21,22]).
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Figure 1 Initiation of synaptogenic contacts by axons and dendrites. In this simulated ‘time-lapse sequence’, two axons (blue) and one dendrite (green) elongate, extend processes, and initiate contact sites that might evolve potentially into axodendritic synapses. Principal events: 1, 2, interactions between axonal and dendritic growth cones; 3, an axonal growth cone intersecting with a dendritic shaft; 4, 5, dendritic filopodia contacting axonal shafts; and 6, a dendritic growth cone contacting an axonal shaft.
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Thus, one emerging principle is that dendrites as well as axons are active in seeking out nearby potential synaptic partners (Figure 1). Interestingly, it has been shown recently that muscle cells in Drosophila also extend filopodia (‘myopodia’), which interact with innervating motorneuron axons [23•]. Thus, muscle cells may not be as passive as previously thought (and nor were cavewomen, in all likelihood).
Time course of individual synapse formation: a matter of hours Much of what is known on the time course of CNS synaptogenesis is based on experiments performed on low-density dissociated rat hippocampal cultures (reviewed in [24]). Within 2–3 days after plating, these neurons form axons and dendrites that establish numerous contacts. Functional synapses start appearing at days 5–6, and synaptic molecules gradually assume synaptic distributions over the following days and weeks. These observations have indicated that glutamatergic synapses form over many days, during which time synaptic molecules are recruited sequentially to developing synaptic sites [5,25]. In contrast, imaging approaches indicate that individual synapses can form within a few hours [11,12,15•,19,22,26,27]. For example, using green fluorescent protein (GFP)-tagged vesicle-associated membrane protein (VAMP)/synaptobrevin (a synaptic vesicle molecule), Ahmari et al. [28••] have shown that clusters of synaptic vesicles (SVs) become stabilized at new axodendritic contact sites, and that stimulation-evoked exocytosis and endocytosis can be demonstrated within 1 hour of contact at these sites. Similarly, Vardinon-Friedman et al. [29•] have used a retrospective immunohistochemical approach to show that new functional presynaptic boutons form within 30 min of axodendritic contact and that most new synaptic boutons are associated within 1–2 hours of their appearance with clusters of ionotropic glutamate receptors.
How can these two timescales (hours versus days) be reconciled? We do not think that these timescales are in conflict. Instead, they probably describe different processes. The long timescale (i.e. days), we believe, reflects the developmental time course of synaptic molecule expression in the culture systems (and perhaps in vivo as well), whereas the hour timescale reflects the period over which individual synapses are formed. Synapses will not form, however, until a minimal set of pre- and postsynaptic components becomes available. Furthermore, the state of new synapses will depend on the repertoire of available synaptic molecules at the time of their formation. If it is rich enough, new synapses will resemble more mature synapses. But if the repertoire (or abundance) of synaptic molecules is meagre, new synaptic junctions may be immature, partially functional, or not functional at all. In agreement with this concept, Renger et al. [30••] found that presynaptic boutons in very immature cultures (less than 7 days in vitro) displayed almost no capacity for evoked SV recycling, whereas those in slightly older cultures (8–12 days) did display such a capacity. The functional characteristics of the latter synapses, however, were strikingly different from those of synapses in mature cultures (more than 13 days), suggesting that synapses that form early in development undergo some type of age-dependent maturation. It is important to note, however, that concomitantly with this maturation process, many new synapses were being formed over this entire period (see also [31]). Interestingly, apart from a change in the number of docked/membrane-associated SVs, the ultrastructure of synapses formed in the youngest age group was very similar to that of synapses in older preparations. We interpret these findings to suggest that the formation of individual synaptic connections is a relatively rapid
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process, occurring over a timescale of several hours. The molecular composition and functional characteristics of such synapses depend, however, on slower developmental processes, such as the repertoire and abundance of synaptic molecules at the time at which the synapse is formed.
Induction of synaptic differentiation: bi-directional signals The formation of an axodendritic contact is only the first step in the genesis of a new synapse. During subsequent steps, the axonal and dendritic compartments at the contact site differentiate into presynaptic boutons and postsynaptic reception apparatuses, respectively. The differentiation of the presynaptic bouton includes the clustering of SVs and the formation of active zones — specialized regions of presynaptic plasma membrane containing electron-dense meshworks of cytoskeletal elements, where SVs dock and fuse [3,6]. Similarly, the differentiation of the postsynaptic reception apparatus involves the formation of the postsynaptic density (PSD) — an electron-dense, specialized cytoskeletal matrix juxtaposed to the presynaptic active zone, which serves to cluster and localize neurotransmitter receptors and other molecules to the postsynaptic membrane [2,4,32–34]. An elusive goal in the study of glutamatergic synapse formation has been the characterization of signals that trigger pre- and postsynaptic differentiation. Studies concerning the formation of the vertebrate neuromuscular junction have led to the discovery of molecules secreted by motoneurons, such as agrin, that can induce postsynaptic differentiation (reviewed in [35]). Immunohistochemical analysis of nascent glutamatergic synapses forming in vitro suggests that presynaptic differentiation often precedes postsynaptic differentiation [29•], which would be consistent with postsynaptic differentiation being induced by factors secreted by nascent presynaptic sites (but probably not agrin [36,37]; but see also [38]). This, however, cannot be the whole story, because dendrites possess an ability to induce the formation of presynaptic specializations along axonal shafts (as discussed above); thus, something — presumably on the dendritic membrane — has to trigger the formation of axonal SV release sites. Interesting candidates in this respect seem to be neuroligins — postsynaptic transmembrane adhesion molecules that bind to β-neurexins, a family of presynaptic membrane molecules [39,40]. Recently, neuroligin expressed in non-neuronal cells has been shown to induce the formation of functional presynaptic structures in pontine neurons, and soluble forms of β-neurexin have been shown to suppress synapse formation between pontine neurons and cerebellar granule cells [41••,42]. As exciting as these findings are, it remains to be demonstrated that these molecules are as ‘indispensable’ for CNS synapse formation as are agrin and its receptor component (MuSK) at the neuromuscular junction [35]. In fact, neuroligin 1 knockout mice appear to be normal [40], although other family members might compensate for the lack of this protein.
Evidence has accumulated to suggest that additional molecules such as ephrinB/EphB receptors [43•], neuronal activity-regulated pentraxin (Narp) [44], Wnt-7a [45], adhesion molecules [46,47] and others (reviewed in [7]) play certain roles in the induction of pre- or postsynaptic differentiation. For example, the binding of preaggregated ephrinB to its receptor, the tyrosine kinase EphB, was shown to promote the rapid formation of NMDA (N-methyl-D-aspartate)-type glutamate receptor clusters [43•]. Similarly, Narp, a secreted molecule expressed by a subset of hippocampal and spinal cord neurons, was shown to induce the formation of AMPA (α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid)-type glutamate receptor clusters [44]. Ultimately, it may turn out that synaptic differentiation induction involves multiple molecules or molecule families, none of which is completely indispensable. Although this tentative conclusion may prove to be wrong, the precise alignment of the active zone and tightly matching dimensions of the active zone and PSD ([6] and references therein), the diversity of molecules discovered so far, and their particular effects on pre- and postsynaptic differentiation, substantiate the concept that the induction process involves multiple bi-directional, reciprocal signals.
Presynaptic differentiation: prefabricated precursors In principle, presynaptic differentiation and active zone assembly could be realized by the sequential, in-situ recruitment of presynaptic molecules. Two recent studies suggest, however, that active zones may be formed by the insertion of pre-assembled, macromolecular vesicular-associated complexes into the presynaptic membrane (a mechanism originally proposed by Vaughn [1]; see also [48]). In the first study [28••], GFP-tagged VAMP/synaptobrevin was reported to cluster at newly forming active zones together with other presynaptic molecules, that is, voltagedependent calcium channels, SV2, synapsin I and amphipysin as well as clusters of pleomorphic vesicular and tubular membranes thought to be cytoplasmic transport packets for presynaptic proteins. In the second study [49••], a previously unknown 80-nm dense core vesicle was purified and shown to contain specifically many active-zone components, including Bassoon, Piccolo, syntaxin, SNAP-25 and N-cadherin. Because two components of these vesicles, Bassoon and Piccolo, are nearly always found at new functional presynaptic sites [29•,49••], these findings were interpreted to suggest that these 80-nm vesicles constitute active-zone precursor vesicles, and that their fusion with the presynaptic plasma membrane might result in the rapid formation of new active zones. Presumably, the formation of new SV release sites is followed by the recruitment of SVs to these sites. Interestingly, SVs seem to be transported along axons [50,51] and recruited to new axodendritic sites [28••] in the
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Figure 2 legend Synaptic differentiation by insertion of pre-assembled precursor vesicles versus sequential in situ recruitment of synaptic components. In this very simplistic simulated ‘time-lapse sequence’, presynaptic differentiation is shown to occur by the insertion of precursor vesicles containing full complements of active zone cytoskeleton complexes, which leads to the rapid formation of functional active zones. Postsynaptic differentiation is shown to occur by the sequential, in situ, recruitment of PSD scaffolding molecules followed by glutamate receptors and PSD signaling molecules. The differentiation processes are presumed to be initiated by interactions between the external aspects of axonal and dendritic membranal molecules. The time points represent the approximate time course of these processes in minutes from the point of axodendritic contact.
form of multivesicular packets, recapitulating the motif of assembly through ‘modular’ units at the presynaptic site (Figure 2).
Postsynaptic assembly: component by component In contrast to the relatively coherent picture that is emerging on presynaptic differentiation, the phenomenology of postsynaptic differentiation remains unclear. This may seem surprising considering how much has been learned on the molecular composition of the glutamatergic PSD. In spite of much uncertainty, some principles are emerging that we will discuss here. Contemporary methods such as yeast two-hybrid assays, colocalization analysis and, recently, large-scale protein complex analysis methods, such as mass spectrometry [52,53], have revealed that the glutamatergic PSD is composed of several large multimolecular complexes, each composed of dozens of molecules. These molecules fall into several categories, including glutamate receptors, scaffolding and adapter molecules, cell-adhesion molecules, cytoskeleton molecules, protein kinases/phosphatases, and other signaling molecules. One group of molecules that has received much attention is the group of scaffolding/adapter molecules that includes SAP90/PSD-95, PSD-93, SAP 102, ProSAP/Shank, SAP 97, PICK, Stargazin [54••] and many others [4,32]. Owing to their multiple binding sites for glutamate receptors on the one hand and other scaffolding/cytoskeletal molecules (and various signaling molecules) on the other, it has been postulated that these molecules might serve to build macromolecular receptor-associated signaling complexes, as well as to cluster these complexes specifically at postsynaptic sites.
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These findings might suggest that, in common with the active zone, the PSD is assembled from ‘prefabricated’ complexes; however, current data are not entirely consistent with this possibility and indicate that PSD components may be recruited sequentially to postsynaptic sites. NMDA-type and AMPA-type glutamate receptors do not seem to be part of the same core multimolecular postsynaptic complexes [53]. NMDA-type receptor 2B
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subunits are shuttled to synaptic sites on discrete transport vesicles, which contain some molecules, but not others, belonging to the multimolecular complex associated with
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NMDA receptors [55], whereas AMPA receptors seem to be recruited from diffuse pools [56,57] by rapid exocytosis and endocytosis mechanisms (reviewed in [2,58,59]). Furthermore, the recruitment of glutamate receptors to nascent synaptic sites seems to lag behind the recruitment of the scaffolding molecules SAP90/PSD-95 [29•] and ProSAP/Shank (H Vardinon-Friedman, T Boeckers, E Gundelfinger, NE Ziv, unpublished data). Finally, studies based on GFP-tagged SAP90/PSD-95 suggest that this molecule is recruited to nascent synaptic sites in a gradual manner, and not in discrete packets ([26]; and see Update). Thus, PSD assembly seems to be a multistep sequential process that occurs directly at the postsynaptic site (Figure 2).
Activity: important but not essential A key step in postsynaptic differentiation is the insertion of glutamate receptors into the postsynaptic membrane. In hippocampi of postnatal rats, as well as in other developing systems, some synapses seem to contain primarily NMDA receptors, and few or no AMPA receptors. As development proceeds, the fraction of synapses lacking AMPA receptors is reduced, but some such synapses remain, even in adult animals. These synapses may constitute the so-called ‘silent synapses’, that is, glutamatergic synapses that exhibit NMDA-, but not AMPA-receptor-mediated synaptic currents. Some researchers have proposed that excitatory synapses start out as ‘silent’ synapses and evolve into fully functional synapses by activity-dependent, NMDA-receptor-mediated, recruitment of AMPA receptors into the postsynaptic membrane (reviewed in [60,61]). Is electric activity or neurotransmitter release absolutely essential for glutamatergic synapse formation per se? The answer seems to be no. Synapses form in cultured hippocampal neurons in the presence of pharmacological agents that block spontaneous spiking activity [62] or synaptic transmission [29•,31,63]. Furthermore, mice deficient in either one of the presynaptic molecules Munc 13-1 or Munc 18-1 develop morphologically normal synapses in spite of partial [64] or complete [65••] loss of neurotransmitter release. Are electric activity or NMDA receptor activation essential for AMPA receptor recruitment to new synapses? Here, too, the answer seems to be no. AMPA receptors accumulate at new synaptic junctions when spontaneous activity and/or glutamate receptors (NMDA receptors included) are blocked [29•,31,56,62,66,67]. The recruitment of one AMPA receptor subunit, GluR4, to synapses formed in the developing rat hippocampus does seem to depend on activity and NMDA receptor activation [68•]. The expression of this subunit, however, is largely restricted to the first 4–5 postnatal days, and is reduced to less than 3% of the total AMPA receptor subunit pool by postnatal day 6 (see supplementary data for [68•]). As vigorous synaptogenesis continues beyond postnatal day 6, the general significance of these findings is currently unclear.
To confound matters further, Renger et al. [30••] have shown recently that individual immature synapses that display ‘silent-synapse’-like postsynaptic currents when stimulated presynaptically do have functional AMPA receptors — the presence of which is revealed by glutamate microiontophoresis. If this finding has general applicability, it might suggest that AMPA receptors appear at synapses much earlier during development than has been indicated previously by electrophysiological methods. Can activity regulate the AMPA receptor content (both receptor number and subunit composition) of new synapses? Apparently it can (reviewed in [61]); however, in addition to activity-dependent mechanisms, there seem to be constitutive pathways for AMPA receptor insertion into postsynaptic membranes that are insensitive to treatments that block activity-regulated AMPA receptor insertion [59,69]. It thus seems that activity or NMDA receptor activation are not indispensable for the formation of functional glutamatergic synapses. This conclusion is actually quite logical: if all synapses are initially ‘silent’, and if NMDA receptor activation is an absolute requisite for synaptic gain of function, how would neurons be excited sufficiently to activate these NMDA receptors? Although excitability in developing networks may be driven by additional mechanisms, such as γ-amino benzoic acid A receptor (GABAA)-based synaptic transmission [70], we feel that the findings described above are more congruent with the concept that AMPA receptor recruitment involves both activity-independent and activity-dependent routes, providing a satisfactory mechanism for setting up initial connections and refining them later on in a use-dependent manner.
Conclusions We have attempted to highlight general principles that seem to be emerging from the expanding body of literature concerning the formation of glutamatergic synapses. In the process, we have purposely glossed over many molecular details, aiming to focus on the ‘forest’, and not on the ‘trees’. Even so, important issues were not discussed, including among others those concerning the capacity of activity to induce new synaptic connections, the roles of activity in maintaining existing glutamatergic synapses, the roles of scaffolding molecules in PSD differentiation, the roles of active zone cytoskeleton molecules in activezone differentiation, and the mechanisms of glutamate receptor targeting to nascent synapses. Nevertheless, it is apparent that considerable progress has been made in our understanding of the cellular physiology of glutamatergic synapse formation, and we have no doubt that with time, our view of the forest will become even clearer.
Update During the time between the submission of this review and the correction of the proofs, several noteworthy studies have been published that lend further credibility to ideas
Principles of glutamatergic synapse formation Ziv and Garner
summarized above. Of particular interest is the study by Okabe et al. [71•], on the formation of new dendritic spines and the recruitment of synaptic molecules to these new synaptic structures. By expressing molecules tagged with spectrally separable variants of GFP, the authors demonstrate that clusters of SAP90/PSD-95 can form in new dendritic filopodia/spines within ~45 min of their extension. Retrospective immunohistochemistry revealed that most new SAP90/PSD-95 clusters are associated with clusters of AMPA receptors within ~90 minutes of their appearance. Furthermore, all new SAP90/PSD-95 puncta were observed to be associated with clusters of SVs within ~60 min of their appearance. Finally, clustering of SVs (labeled by cyan fluorescent protein-tagged synaptophysin) was observed to precede the accumulation of SAP90/PSD95 at new synaptic sites. These findings provide strong support to the notion that dendritic filopodia can initiate synaptogenic contacts and thereafter evolve into dendritic spines [9–13,14••,15•,16–18], and are in good agreement with previous studies concerning the time course and temporal order of glutamatergic synapse formation [28••,29•]. A second report we wish to mention here is a study by Bresler et al. [72•], showing that GFP-tagged SAP90/PSD95 is recruited to new synaptic sites gradually from diffuse cytoplasmic pools, with kinetics fitting a single exponential with a mean time constant of ~23 min, supporting the proposition that PSD assembly occurs sequentially in situ. In agreement with previous studies [28••,29•] and the study mentioned above [71•], the authors show that SAP90/PSD-95 clusters at new axodendritic contact sites within 20–60 minutes of contact establishment, and that such sites rapidly acquire a capacity for evoked SV recycling.
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Acknowledgements
17.
Work in the lab of NE Ziv was supported by a grant from the Israel Science Foundation (139/98). Work in the lab of CC Garner was supported by grants from the National Institutes of Health (RO1 NS39471, PO1 AG06569). NE Ziv is a member of the Bernard Katz Minerva Center for Cell Biophysics.
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