Visualizing synapse formation and remodeling: recent advances in real-time imaging of CNS synapses

Neuroscience Research 40 (2001) 291– 300 www.elsevier.com/locate/neures

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Visualizing synapse formation and remodeling: recent advances in real-time imaging of CNS synapses Tatsuya Umeda a, Shigeo Okabe a,b,c a

Department of Anatomy and Cell Biology, School of Medicine, Tokyo Medical and Dental Uni6ersity, 1 -5 -45 Yushima, Bunkyo-ku, Tokyo 113 -8519, Japan b Laboratory of Molecular Neurobiology, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305 -8566, Japan c CREST Japan Science and Technology Corporation (JST), Tokyo, Japan Received 15 January 2001; accepted 19 April 2001

Abstract The formation and maintenance of synaptic connections are critical in the development and plasticity of the central nervous system (CNS). Until recently, there have been few studies that followed the molecular sequences of the CNS synapse formation and maintenance. This situation changed dramatically after the introduction of green fluorescent protein (GFP)-based fluorescent probes and the development of lipophilic tracers of endocytotic membranes. These techniques enabled us to visualize presynaptic and postsynaptic structures in living neurons and illustrated active transport and remodeling of synaptic components. Furthermore, recent attempts to identify correlation between presynaptic and postsynaptic morphogenesis suggested very rapid time course of synapse formation at the individual axo-dendritic contact sites. These recent works clearly demonstrated the power of real-time imaging studies. Development of a wide variety of fluorescent probes and advances in the imaging techniques in future will further extend our knowledge on the molecular events that take place in the process of the development and maturation of synaptic junctions. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Green fluorescent protein (GFP); FM-dye; Synapse; Synaptogenesis; Central nervous system (CNS); Postsynaptic density (PSD); Exocytosis

1. Imaging techniques applied to the studies of the neuromuscular junction Synapses are specialized structural organizations for interneuronal signaling in the CNS. Transmission through synapses is carried out with apparatuses in presynaptic and postsynaptic sites and functionally distinct synapses contain different compositions of synaptic molecules. How specific synaptic connections are generated developmentally and how these connections are remodeled and maintained in mature brain are fundamental questions in neuroscience. Traditionally, * Corresponding author. Present address: Department of Anatomy and Cell Biology, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. Tel.: + 81-3-5803-5140; fax: + 81-3-3818-7170. E-mail address: [email protected] (S. Okabe).

there have been two major approaches toward dissecting molecular events associated with synapse formation in the CNS. The first strategy is the ultrastructural analysis of in vivo synapses along the course of development (Schwartz et al., 1968; Cotman et al., 1973; Miller and Peters, 1981; Pokorny and Yamamoto, 1981; Schwartzkroin et al., 1982; Harris et al., 1992; Boyer et al., 1998; Fiala et al., 1998). The second approach is the immunohistochemical analysis, which characterizes distributions of synaptic molecules in neurons at multiple developmental stages both in vivo and in culture (Fletcher et al., 1991; Killisch et al., 1991; Craig et al., 1993). These ultrastructural studies and immunohistochemical studies gave us only static images of either synapse ultrastructure or distribution of synaptic molecules at particular developmental stages. To reveal the molecular mechanism of synapse forma-

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tion and maintenance, direct detection of morphological changes in individual synapses in living neurons is essential and initial attempts to observe synaptogenesis in real time were carried out in dissociation culture of neurons and their target cells. Time-lapse microscopy using differential interference contrast (DIC) optics showed protrusive activity of filopodia and lamellipodia in the growth cone (Bray and Chapman, 1985; Forscher and Smith, 1988). When a growth cone arrived at the vicinity of a target cell, protruding filopodia attached to the target cell and this contact formation initiated successive establishment of a synaptic connection. However, once the contact between a growth cone and a target cell was established, details of the morphology of the contact site were difficult to visualize by using the conventional techniques of light microscopy and non-specific cell surface tracers, such as DiI (Honig and Hume, 1986). These techniques were also insufficient for studying how synaptic molecules accumulated at the contact sites and how the contact sites transformed into mature synaptic structures. Thus, it was necessary to develop techniques that enabled direct observations of synaptic molecules and structures in living cells. A motor neuron axon contacts with a myoblast and forms a specialized synaptic structure, the neuromuscular junction (NMJ) (Fig. 1A). Researches on the basic properties of synaptic transmission were carried out by using the NMJ as a model system. Therefore, this system has been widely used for the initial characterization of imaging techniques to visualize both presynaptic and postsynaptic structures (Sanes and Lichtman, 1999). With regard to the observations of presynaptic structures, endocytosis of synaptic vesicles in the presynaptic terminal was visualized by using lipophilic dye FM 1-43 (Betz and Bewick, 1992; Betz et al., 1992). When FM 1-43 is added to culture medium, it uniformly binds to the plasma membrane (Fig. 2). However, excitatory stimuli on a neuron induce exocytosis– endocytosis cycles at the presynaptic terminal and the dye is trapped inside the recycling vesicles. Replacement of the extracellular solution removes the dye weakly bound to the plasma membrane. Selective labeling of recycling vesicles in the presynaptic terminal, namely synaptic vesicles, can be achieved. Visualization of postsynaptic molecules in the NMJ was performed by using a selective antagonist of the nicotinic acetylcholine receptors (nAChRs), a-bungarotoxin (Fig. 1A). The fluorescently labeled a-bungarotoxin binds to the nAChR localized on the cell surface and densely packed nAChR clusters on the surface of a living myotube were visualized. Using this probe, accumulation of nAChRs during the process of the NMJ formation has been studied (Cohen et al., 1979; Kuromi et al., 1985). Immediately after the nerve terminal had come into contact with the myoblast, accumulation of

nAChRs was initiated. Subsequent maturation process and remodeling of the NMJ structure were extensively studied by using this fluorescent probe (Lichtman et al., 1987; Balice-Gordon and Lichtman, 1990). Fluorescent probes, such as FM1-43 and fluorescent a-bungarotoxin, facilitated the progress of research on the development of the NMJ. Development of other types of probes is important for studying different aspects of the NMJ formation. Progress in the studies on the CNS synaptogenesis had not been remarkable, mainly due to the absence of probes to visualize synaptic structures in living neurons. Recent progress in the visualizing techniques enabled us to monitor synaptic molecules and structures in CNS neurons. In the following section, we will describe the applications of these new imaging techniques to the analyses of synaptic structure and function in the CNS.

Fig. 1. Schematic representations of an NMJ and a typical excitatory CNS synapse. (A) For the NMJ, FM-dye and fluorescently labeled a-bungarotoxin are pre- and postsynaptic markers, respectively. (B) For the CNS synapse, presynaptic markers are FM-dye, fluorescently labeled anti-synaptotagmin I antibody and VAMP-GFP. PSD-95GFP is used for a postsynaptic marker. CaMKII, calcium/calmodulin-dependent protein kinase II; CNS, central nervous system; GFP, green fluorescent protein; nAChR, nicotinic acetylcholine receptor; NMJ, neuromuscular junction; PSD, postsynaptic density; VAMP, vesicle-associated membrane protein.

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Fig. 2. Dynamics of fluorescently labeled presynaptic markers during recycling of synaptic vesicles. (A) FM-dye and fluorescently labeled anti-synaptotagmin I antibodies applied to culture medium bind to the neuronal plasma membrane, through the hydrophobic and antigen –antibody interactions, respectively. (B) The first electrical or ionic stimulation of neurons triggers exocytosis of synaptic vesicles, and then the fluorescent probes are trapped in the endocytotic synaptic vesicles. (C) Removal of FM-dye not incorporated in the synaptic vesicles and free anti-synaptotagmin I antibodies by washing with fresh medium visualizes fluorescently-labeled recycled vesicles in the cultured neuron. (D) The second stimulation induces exocytosis of the labeled synaptic vesicles and the fluorescence becomes undetectable.

2. Applications of new imaging techniques to research on the CNS In most of CNS synapses, transmission of signals at the synaptic site is asymmetric. The presynaptic terminal releases neurotransmitters in response to the arrival of an action potential. The released transmitters bind to receptors on the postsynaptic membrane and activation of the postsynaptic receptors triggers subsequent signaling cascades in the postsynaptic cells. Distinct subsets of synaptic molecules have been identified in both presynaptic and postsynaptic structures (Garner et al., 2000a; Kennedy, 2000). Based on the molecules and structures specific to either pre- or postsynaptic sites, several molecular probes have been devised to visualize CNS synapses. Development of presynaptic and postsynaptic markers will be discussed separately in the following sections.

2.1. Visualization of the dynamics of presynaptic structures Exocytosis of neurotransmitter is operated by a complex molecular machinery within the cytoplasm of the presynaptic terminal. Characteristic membranous structures involved in this process are synaptic vesicles,

endosomes and locally recycling vesicles. The presynaptic cytoplasm also contains soluble proteins necessary for the fusion of synaptic vesicles to the plasma membrane and those required for recovery of the fused vesicle membrane by endocytosis (Bennett and Scheller, 1994; Jahn and Su¨ dhof, 1994) (Fig. 1B). Among these specializations, integral membrane proteins of synaptic vesicles are good candidates for the specific markers of the presynaptic structure. Vesicle-associated membrane protein (VAMP), also called synaptobrevin, is a wellcharacterized protein localized to synaptic vesicles (Trimble et al., 1988). A fusion protein of this synaptic vesicle protein with GFP has been developed. A prerequisite of this marker molecule for studying presynaptic localization is that the fusion protein does not inhibit exo–endocytotic cycles of synaptic vesicles. When rat hippocampal neurons in culture expressed a fusion protein between VAMP and pH sensitive mutant of GFP (pHluorin), neurotransmitter release was successfully visualized (Miesenbo¨ ck et al., 1998; Sankaranarayanan and Ryan, 2000; Sankaranarayanan et al., 2000). Ecliptic pHluorin (epHluorin), one of pH sensitive GFP variants, is scarcely fluorescent at pH6. It emits brighter fluorescence as environmental pH increases. When epHluorin is fused with a lumenal domain of VAMP (VAMP-epHluorin), this molecule

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within a synaptic vesicle is non-fluorescent because the inside of a synaptic vesicle is acidic. When VAMPepHluorin is exposed to the extracellular environment, where pH is nearly 7.4, this molecule starts to emit fluorescence. Following stimulation of a neuron expressing VAMP-epHluorin, the fluorescence emitted from epHluorin was observed. These results suggest that VAMP-GFP variant does not inhibit exo– endocytosis of synaptic vesicles. By transfection of cultured hippocampal neurons with VAMP-GFP cDNA, Ahmari et al. showed that VAMPGFP could be used as a marker of both transported synaptic vesicle precursors and immobile presynaptic structures (Ahmari et al., 2000). They identified mobile fractions of VAMP-GFP containing vesicles within an axon. Furthermore, mobile VAMP-GFP puncta accumulated at contact sites between growth cones and postsynaptic targets within 1– 2 h. To determine whether distinct presynaptic proteins are conveyed in discrete transporting vesicles or in a common transporting structure, immunocytochemical staining of VAMPGFP positive transporting vesicles was performed by using antibodies specific to other synaptic vesicle proteins. The experiments revealed colocalization of VAMP-GFP with other synaptic vesicle proteins, such as SV2, synapsin Ia and amphiphysin I (Jahn and Su¨ dhof, 1994). This suggests the presence of common transporting machinery for various presynaptic vesicle proteins. It is possible that this common transporting system is important for rapid establishment of functional presynaptic structures immediately after initial contacts between pre- and postsynaptic cells. Localization of presynaptic structure can be identified by its presynaptic vesicle recycling associated with neuronal activity. This presynaptic membrane recycling can be visualized by either lipophilic membrane tracers or antibodies against lumenal domains of synaptic vesicle proteins. FM-dye has been utilized as a marker of presynaptic vesicle recycling in the NMJ (Betz and Bewick, 1992; Betz et al., 1992). After the initial application of FM 1-43 to the study of NMJ synapses, similar protocols have been applied successfully to CNS synapses, such as excitatory synapses of hippocampal pyramidal neurons in culture (Ryan et al., 1993; Ryan and Smith, 1995; Ziv and Smith, 1996). Two recent publications showed that application of FM-dye could successfully monitor the process of synaptogenesis in cultured CNS neurons (Ahmari et al., 2000; Friedman et al., 2000). Ahmari et al. analyzed the time course of presynaptic protein accumulation and synaptic vesicle endocytosis by using FM-dye. In rat hippocampal primary cultures, the axo-dendritic contact sites containing VAMP-GFP were also stained with FM 4-64. FM 4-64 staining emerged within 1 h from the onset of the VAMP-GFP accumulation. In a similar culture system, Friedman et al. indicated that there was almost com-

plete overlap between newly developed sites of FM 4-64 incorporation and immunoreactivity of a presynaptic protein Bassoon (tom Dieck et al., 1998). These observations indicate that the accumulation of presynaptic proteins precedes the functional recycling of synaptic vesicles. Utilization of FM-dye during the process of synaptogenesis accompanies some problems. First, electrical or ionic stimulation is required to induce incorporation of FM-dye to the presynaptic vesicles. In addition, researchers have introduced sodium channel blockers in the labeling process of FM-dye to prevent overexcitation of neurons (Friedman et al., 2000). Both stimulation and suppression of neuronal activity themselves can potentially influence the normal process of synaptogenesis (Rao and Craig, 1997; O’Brien et al., 1998; MaleticSavatic et al., 1999; Gomperts et al., 2000). Therefore, loading procedures of FM-dye should be carefully controlled. Second, in a strict sense, recycling vesicles are not specific to the presynaptic sites. It has been reported that endocytotic activities at dendritic shafts could be visualized by FM-dye with a different loading protocol of the dye (Maletic-Savatic and Malinow, 1998; Maletic-Savatic et al., 1998). This suggests that FM-dye can potentially mark other neuronal components. Finally, in certain culture systems, staining with FM-dye yields high background. To overcome this problem, methods to reduce the background of FM-dye have been developed recently (Pyle et al., 1999; Kay et al., 1999). FM-dye is a unique marker for the visualization of presynaptic structure, but combination of this imaging technique with other synaptic markers to verify the reliability of staining is an important consideration. Another marker of presynaptic vesicle recycling is a fluorescently labeled anti-synaptotagmin I antibody. Synaptotagmin I is one of synaptic vesicle proteins and serves as a calcium ion sensor for exocytosis (Brose et al., 1992). Antibodies that recognized lumenal domains of synaptotagmin I were generated (Matteoli et al., 1992; Malgaroli et al., 1995). These antibodies can bind to synaptotagmin I, which has been exposed to the cell surface after fusion of synaptic vesicles (Fig. 2). The bound antibodies are trapped within synaptic vesicles and subsequent washout of unbound antibodies reveals localization of recycling vesicles. This technique has been applied to the study of localization and turnover of synaptic vesicles in hippocampal neurons and also to the study of identifying new presynaptic sites after induction of long-term potentiation (Kraszewski et al., 1995; Malgaroli et al., 1995).

2.2. Visualization of the dynamics of postsynaptic structures Postsynaptic sites of CNS neurons contain several specialized structures (Fig. 1B). Both excitatory and

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inhibitory postsynaptic sites are enriched with neurotransmitter receptors. Ionotropic types of neurotransmitter receptors are localized to the synaptic junctions, where the concentration of presynaptically released transmitter molecules should be highest (Petralia and Wenthold, 1992; Petralia et al., 1994; Takumi et al., 1999). Some of metabotropic types of receptors are distributed outside of the synaptic junction (Lujan et al., 1996). It is generally assumed that the distribution of different receptor types is related to their critical functions in the synaptic transmission. In glutamatergic excitatory synapses in the CNS, plasma membranes of postsynaptic sites are thickened. This structure, postsynaptic density (PSD), was initially identified by electron microscopic observation of excitatory synapses (Cotman et al., 1974; Blomberg et al., 1977; Matus and Taff-Jones, 1978). Subsequent biochemical analyses have identified a number of proteins localized to this structure (Ziff, 1997; Garner et al., 2000b). It is likely that these proteins function in clustering neurotransmitter receptors (Kornau et al., 1995). Thus, glutamate receptors themselves and PSD proteins are good candidates for generating specific markers of postsynaptic sites. Another characteristic structure associated with glutamatergic synapses in the CNS is the dendritic spines (Harris and Kater, 1994). They are small protrusions on the surface of dendritic shafts, with an average length of 0.2–2 mm in the case of hippocampal pyramidal neurons. Exact roles of dendritic spines are still speculative, but researchers postulate their roles in the coupling of electrical and chemical signaling to dendritic shafts (Harris and Kater, 1994). It will be also possible to utilize proteins that are concentrated in the spine structure, such as cytoplasmic actin, to visualize postsynaptic sites (Fischer et al., 1998, 2000). For the visualization of postsynaptic structures in CNS neurons, there have been few suitable chemicals that have sufficient selectivity and affinity to faithfully visualize the distribution of glutamate receptors and GABA receptors, such as the fluorescent a-bungarotoxin in the NMJ (however, see Benke et al., 1993). From this reason, observations of postsynaptic molecules in the CNS neurons have mostly relied on immunocytochemistry of fixed cells (Craig et al., 1993; Rao et al., 1998; Petralia et al., 1999; Liao et al., 1999). However, several recent studies opened the possibility of visualizing postsynaptic structures by using GFPbased reporter molecules. Calcium/calmodulin-dependent protein kinase II (CaMKII) is an abundant protein in the PSD fraction (Kennedy et al., 1983; Kelly et al., 1984). Possible involvement of this protein in the plasticity of synapses has been studied extensively (Malinow et al., 1988; Silva et al., 1992; Pettit et al., 1994; Mayford et al., 1995; Lledo et al., 1995). A fusion protein of CaMKII

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with GFP (CaMKII-GFP) is a possible candidate for postsynapse specific probes. Recent studies of Shen et al. indicate that CaMKII-GFP is not only localized to PSDs, but also associated with actin cytoskeleton (Shen and Meyer, 1999; Shen et al., 2000). Interestingly, highfrequency stimulations of cultured neurons expressing CaMKII-GFP induced translocation of CaMKII-GFP to PSD in a time scale of several minutes. This dynamic property of CaMKII-GFP will be useful in monitoring the activity-dependent process within dendrites. However, the dynamic property of CaMKII-GFP hinders its application to the analyses of long-term changes of synaptic structure. In this sense, scaffold proteins stably associated with the structure of PSD are good candidates for postsynapse specific markers. Among them, PSD-95 is a dominant protein in the biochemically isolated PSD fraction (Cho et al., 1992; Kistner et al., 1993; Kornau et al., 1995) and has been shown to be localized to the PSDs in vivo by immunoelectron microscopy (Hunt et al., 1996). A fusion protein of PSD95 with GFP (PSD-95-GFP) has been generated and the role of palmitoylation at the N-terminus of PSD-95 has been characterized by using GFP-tagged mutant proteins of PSD-95 (Craven et al., 1999; Arnold and Clapham, 1999). Turnover of postsynaptic structures was analyzed by using PSD-95-GFP as a postsynaptic marker (Okabe et al., 1999) (Fig. 3). Until recently, it was not clear whether CNS synapses are stable over lifetime. In the NMJ, synaptic connections do not disappear, even though extensive remodeling of the junctional morphology takes place postnatally (Lichtman et al., 1987; Balice-Gordon and Lichtman, 1990). A time-lapse study of PSD-95-GFP clusters in hippocampal pyramidal neurons revealed that 20% of total PSD-95-GFP clusters newly appeared and a similar proportion of total PSD-95-GFP clusters disappeared in 24 h. This suggests that the exchange of excitatory synapses is extensive even in mature neurons in culture, where the total number of synaptic connections remains stable over time. Whether CNS synapses in vivo also show a similar level of extensive remodeling after maturation is a question to be answered. Shi et al. expressed GluR1, a subunit of AMPA-type glutamate receptors, tagged with GFP (GFP-GluR1) in hippocampal pyramidal neurons in a slice culture using virus-mediated gene transfer (Shi et al., 1999). This N-terminal fusion construct of GFP to GluR1 retained its basic properties as a ligand-gated ion channel. They observed the translocation of GFP-GluR1 to dendritic spines after application of electrical stimuli, which produced long-lasting potentiation of the synaptic transmission in this system. This report confirmed the postulated mechanisms of the expression of long-term potentiation in hippocampus by changing distribution of glutamate receptors themselves. Long-term change

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of GluR1 distribution along the time course of synaptogenesis will be an intriguing question. Combined with the development of other GFP-based probes, such as GFP-tagged NMDA receptors, temporal orders of the accumulation of glutamate receptors at the developing synapses will be studied in living neurons in future. The translocation of GFP-GluR1 in a slice culture was observed by using a two-photon laser scanning microscope. This method can detect fluorescence from not only the surface but also a deeper layer in a tissue, such as a slice culture, because less light is scattered as wavelength increased. This imaging technique has been also applied to the visualization of synapse morphology in vivo. Lendvai et al. showed motility of dendritic spines in the developing barrel cortex in vivo (Lendvai et al., 2000). They indicated that the sensory experiences affected the dynamics of dendritic protrusions. By combination of synapse-specific markers with this new imaging technique, more detailed structural analyses of neurons in vivo will be possible.

2.3. Correlati6e obser6ation of the dynamics of presynaptic and postsynaptic structures Synapse formation is not a cell autonomous phenomenon. Communication between pre- and postsynaptic sites should be a complex process, which involves a variety of cell surface receptors, their ligands (Torres et al., 1998; Buchert et al., 1999; Garcia et al.,

2000; Huang et al., 2000; Dalva et al., 2000) and cell adhesion molecules (Ichtchenko et al., 1995; Fannon and Colman, 1996; Uchida et al., 1996; Tanaka et al., 2000; Scheiffele et al., 2000). Spatial and temporal correlation between pre- and postsynaptic maturation is the first step toward understanding the interaction between these structures. To reveal functional relationship between presynaptic and postsynaptic sites, techniques that visualize the activity of synaptic molecules selectively in either pre- or postsynaptic sites will be important. As described above, in the process of the NMJ formation, accumulation of nAChRs in the postsynaptic sites occurs immediately after initial contacts between approaching nerve growth cones and postsynaptic myoblasts (Kuromi et al., 1985). In contrast, maturation of NMJ structure is a slow process, which takes more than 2 weeks in rodent muscles (Sanes and Lichtman, 1999). These two distinct observations in the NMJ formation have influenced interpretations of data on synapse development in the CNS. Analyses of fixed preparations of the CNS provided evidence for the gradual transition of the synapse structures with time. In the case of hippocampal neurons, structural maturation of excitatory synapses follows a stereotyped pattern and is completed in the postnatal 3 weeks (Schwartzkroin et al., 1982; Boyer et al., 1998; Fiala et al., 1998). On the other hand, electrophysiological analyses revealed rapid changes of synaptic func-

Fig. 3. Clustering of PSD-95-YFP in cultured hippocampal neurons. (A) DIC image of a neuron expressing PSD-95-YFP (green). PSD-95-YFP forms clusters and some are localized at dendritic spines. (B) Time-lapse imaging shows a newly-formed PSD-95-YFP cluster (arrow). Time stamps are shown in minutes in the lower left corners. Scale bar, 3 mm.

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tions and it has been claimed that some of these functional modifications are based on the rapid morphological changes of synapses (Buchs and Muller, 1996; Toni et al., 1999). Friedman et al. examined the time scale of assembly of presynaptic and postsynaptic proteins at the site of synaptic contacts, which were identified by FM 4-64 incorporation in cultured hippocampal neurons (Friedman et al., 2000). Their retrospective immunocytochemistry of a presynaptic protein, Bassoon, suggests that the establishment of presynaptic specializations precedes initiation of vesicle recycling at the contact sites. In the case of postsynaptic specializations, it took :1 h for major postsynaptic proteins, PSD-95, AMPA receptor subunit GluR1, and NMDA receptor subunit NR1, to accumulate at the sites of FM 4-64 staining after the initial appearance of vesicle recycling. These observations indicate two important features of CNS synaptogenesis. First, presynaptic differentiation precedes accumulation of postsynaptic molecules. Second, the differentiation of presynaptic and postsynaptic structures takes place in a time scale of 1–2 h. This time course is significantly shorter than the time course of the gradual shift of receptor distribution in developing hippocampal neurons in culture (Craig et al., 1993; Rao et al., 1998). Whether a single model of synapse development can reconcile the rapid development of individual synapses with a gradual change of population properties of synapses is an intriguing question. In future, simultaneous detection of presynaptic and postsynaptic proteins with different wave-length variants of GFP will be possible and this type of experiments will provide further insights into the temporal pattern of presynaptic and postsynaptic differentiation. In the early stage of excitatory synapse formation in hippocampus, only NMDA receptor response is detected electrophysiologically. As development proceeds, AMPA receptor response increases (Liao et al., 1995; Petralia et al., 1999). These observations led to the silent synapse hypothesis, which states that developing synapses initially express only NMDA receptors and AMPA receptors become functional only after coupling of postsynaptic membrane depolarization with glutamate release from a presynaptic terminal (Durand et al., 1996). By using techniques to monitor generation of new presynaptic sites combined with retrospective immunocytochemistry of postsynaptic receptors, Friedman et al. attempted to detect silent synapses in cultured hippocampal neurons (Friedman et al., 2000). They examined whether newly formed FM 4-64 positive recycling sites were immunopositive with a GluR1 subunit of AMPA receptors and an NR1 subunit of NMDA receptors. They reported that the kinetics of the increase of GluR1 clusters was similar to that of NR1 clusters. This observation indicates that newly formed synapses start to accumulate both AMPA and

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NMDA type of glutamate receptors in a similar time course and is seemingly contradictory to the silent synapse hypothesis. One possible explanation is that GluR1 clusters do not represent cell surface receptors. It is necessary to detect synaptic currents from individual synaptic sites to obtain a definitive answer to this problem. One possible approach is to utilize cagedcompound of glutamate to locally activate the cell surface receptors (Denk, 1994). Combination of GFPbased receptor molecules with caged glutamate molecules will be a powerful approach to characterize morphological and functional properties of individual synaptic sites.

2.4. Future applications of the new imaging techniques to research on the synaptogenesis To further characterize the relationship between preand postsynaptic development, information on the functional state of individual synapses should be combined with morphological data. Challenging questions are whether synaptic activity itself can be measured by GFP-based technology and whether synaptic activity can be dissected into several different components by using imaging techniques. As stated previously, VAMPepHluorin was reported to serve as a marker of presynaptic exocytosis (Miesenbo¨ ck et al., 1998; Sankaranarayanan and Ryan, 2000; Sankaranarayanan et al., 2000). One problem associated with this probe is its high background. The development of epHluorinbased probes with low background will facilitate the imaging of synaptic functions. How can we detect synaptic activity at the postsynaptic sites? One possibility is to use calcium-sensitive GFP-based reporters, such as cameleons (Miyawaki et al., 1997, 1999). By attaching an appropriate synapse-targeting sequence to cameleon, local increase of calcium concentrations can be measured. Combination of cameleon with a recently identified fluorescent protein from sea anemone (Matz et al., 1999) would be a possible method to detect synaptic activity and morphology simultaneously. Another possibility is the development of probes using the translocating property of CaMKII-GFP (Shen and Meyer, 1999; Shen et al., 2000). It is not yet determined quantitatively whether stimulus–response relationship of CaMKII-GFP translocation is suitable for experiments to monitor the synaptic activation. It is also possible to utilize other molecules that have been reported to change their localization in response to the activity of cells, such as PKCs and pleckstrin homology domain of PLC-d1 (Hirose et al., 1999; Wang et al., 1999; Maasch et al., 2000). To monitor the translocation of GFP-labeled proteins within neurons, GFP fluorescence should be stable within the range of pH shift associated with stimulation. However, pH-dependent change of fluorescence is not negligible in the case

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of glutamate-stimulated neurons expressing a longer wavelength variant of GFP (YFP) (Miyawaki et al., 1999). Improvement of pH-resistance of GFP will be important to monitor local synaptic activity by using GFP-based imaging techniques in future.

3. Conclusion DIC imaging and non-selective membrane staining methods showed us the dynamic changes of presynaptic and postsynaptic structures before their contact formation. Non-selective markers for endocytosis and antibodies to lumenal domains of synaptic vesicle proteins can be utilized to identify presynaptic structure. Fusion between synaptic molecules and GFP allows us to observe dynamic movements of synaptic molecules in presynaptic and postsynaptic sites. Therefore, theoretical bases for the techniques to visualize structural details of the synapse formation have been well established. Future development of various fluorescent probes for synaptic functions, combined with technical advances in the fluorescence detection methods, will lead to the extension of our knowledge on how the complex molecular machinery within the synapse develops and acquires its function and how this process is regulated.

Acknowledgements We thank Drs Akihiro Inoue and Hidehito Kuroyanagi for critical reading of the manuscript.

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