okabe, shigeo. 2013. fluorescence imaging of synapse formation and remodeling.pdf

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Review

Fluorescence imaging of synapse formation and remodeling

Shigeo Okabe*Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 HongoBunkyo-ku, Tokyo 113-0033, Japan*To whom correspondence should be addressed. E-mail: [email protected]

Abstract Brain function is based on proper connectivity between neuronal cells. Inthe developing brain, neurons extend axons and form synaptic connec-tions with appropriate postsynaptic neurons. Molecular mechanismsunderlying establishment of proper synaptic connections are one of themost important topics in the field of developmental neurobiology.Dynamics of synaptic structure and local recruitment of synaptic mole-cules can be studied by live-cell imaging of neurons expressing fluores-cent probes of synaptic molecules. In this review, examples of live-cellfluorescence imaging are presented and their contributions to our under-standing about the molecular mechanisms of synapse formation andremodeling are discussed. Imaging of synaptic proteins in living neuronsrevealed rapid formation of individual synapses within hours and exten-sive remodeling of synaptic connections. Different types of neuronsexpress unique protrusions from dendrites and axons, which play import-ant roles in synapse formation and maturation. Rapid formation of synap-tic structure is associated with continual assembly and disassembly ofsynaptic scaffolding proteins, which are essential building blocks of thepresynaptic active zone and the postsynaptic density (PSD). Quantitativeanalyses of PSD scaffolding proteins further confirmed their essentialroles in maintenance of the synaptic structure. These examples clearlyindicate that fluorescence-based live-cell imaging is an indispensabletechnique in the research on synapse development and its impact willfurther increase in combination with development of new light micro-scopic techniques in the future.

Keywords live-cell imaging, fluorescent proteins, synapse, postsynaptic density, glu-tamate receptors, two-photon microscopy, super-resolution microscopy

Received 26 September 2012, accepted 9 November 2012; online 14 December 2012

Introduction

In the central nervous system (CNS) of vertebrates,neuronal precursors are differentiated from theneuroepithelial cells, migrate within the nervoustissue and settle within specific cortical layers ornuclei. After migration, neurons extend long axonalprocesses and start to connect with their targetneurons. The contact sites between presynapticaxons and postsynaptic dendrites start to differentiateinto synapses, which are the structures important ininformation processing and memory storage.

Electrophysiological and structural properties ofmature synapses were studied extensively in thelate 20th century. However, appropriate techniquesfor the reliable detection and measurement ofnascent synapses during development had not beenavailable until recently. Therefore, researchers ini-tially focused on several types of model synapses inthe peripheral nervous system, such as the neuro-muscular junctions (NMJs), which were larger thansynapses in the CNS and easier to manipulate ex-perimentally [1]. Nascent NMJs could be identified

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Microscopy 62(1): 51–62 (2013)doi: 10.1093/jmicro/dfs083

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© The Author 2012. Published by Oxford University Press [on behalf of The Japanese Society of Microscopy]. All rights reserved.For permissions, please e-mail: [email protected]

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using specific fluorescent probes, and the mechan-isms of synaptic competition between multiple pre-synaptic axons targeted to the same postsynapticmuscle could be analyzed in detail. In the CNS,however, several factors prevented direct detectionand analyses of developing synapses. First, synapticstructures in the CNS are much smaller than NMJsand the resolution of light microscopy is not suffi-cient for the detection of their detailed morphology.Second, the mammalian brain is covered by thecranium and direct access to the brain parenchymarequires invasive surgery. Because the brain tissueis fragile and easy to develop edema, highly sophis-ticated surgical procedures should be invented andapplied to small animals. To circumvent this diffi-culty, isolated preparations of the nervous system,such as dissociated neuronal cultures and slice cul-tures, have been developed and widely used for theanalyses of synapse development within the CNS[2,3]. Imaging experiments in these reduced pre-parations in combination with the development ofgreen fluorescent protein (GFP)-based fluorescentprobes provided essential information about the be-havior of nascent synapses and the time course oftheir differentiation. Another possible approachtoward the understanding of synapse developmentin the CNS is the histological analyses of brain sec-tions taken from different stages of brain develop-ment. Analyses of synapse development in vivo byelectron microscopy revealed structural details ofnascent synapses quantitatively [4]. Fluorescenceimaging of living neurons and ultrastructuralstudies of the fixed brain tissue provide us withcomplementary information and their integrationhas facilitated our understanding about CNSsynapse development.

Structural components of synapses

and molecules involved in the assembly

of presynaptic and postsynaptic

specializations

Detailed morphological characteristics of synapseshave been studied by using transmission electron mi-croscopy of chemically fixed or quickly frozensamples of the nervous tissue (Fig. 1). Synapses arethe sites of membrane adhesion between presynapticaxons and postsynaptic dendrites or cell bodies.

Axonal boutons are the cytoplasmic swellings formedat the contact sites between the axonal plasma mem-brane and the target neurons along the course ofaxonal trajectories. Axonal boutons contain synapticvesicles, which are the storage sites of neurotransmit-ters, such as glutamate and γ-Aminobutyric acid(GABA). Arrival of action potentials to the presynap-tic terminal activates voltage-gated calcium channelsand triggers exocytosis of synaptic vesicles.Neurotransmitters released from the presynapticmembrane diffuse across the synaptic cleft and acti-vate neurotransmitter receptors on the postsynapticmembrane.Most excitatory synapses in the forebrain pyram-

idal neurons are formed onto dendritic spines,small protrusions containing densely packed actinfilaments [5]. Another morphological feature of exci-tatory synapses is the presence of the postsynapticdensity (PSD), located at the plasma membrane ofthe dendritic spine and apposed to the presynapticactive zone [6]. The PSD is composed of a varietyof proteins, including membrane proteins, such asglutamate receptors and cell adhesion molecules,together with PSD scaffolding proteins andsignaling molecules. In addition to the PSD, thepostsynaptic cytoplasm contains several othermicrostructures, such as spine apparatus, endoso-mal membranes and mitochondria.The sizes of both presynaptic axonal boutons and

dendritic spines are in the order of several micro-meters. The PSDs are disk-like structures with dia-meters of 200–500 nm and thicknesses of 30–60 nm[7]. Synapse morphology and distribution of synap-tic proteins are difficult to resolve without theaid of fluorescence microscopy. Under optimizedimaging conditions, structural distinction betweendifferent types of spines, such as thin, stubby andmushroom spines, can be reliably achieved andcould be confirmed by retrospective electron mi-croscopy [8,9]. In order to identify nascent synapticstructures, it is essential to develop techniquesthat enable expression of fluorescent protein (FP)-tagged synaptic molecules and subsequent detec-tion of fluorescence signals in living neurons. Anumber of FP-based probes for presynaptic andpostsynaptic components have been developed andutilized for live-cell imaging. Formation and remod-eling of presynaptic structures can be monitored

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using GFP-tagged synaptophysin or synaptobrevin2/VAMP2 [10,11]. Both of these probes show selectiveaccumulation in axonal boutons. Synaptophysin isone of the most abundant proteins of synaptic vesi-cles. Synaptophysin contains four transmembranedomains with cytoplasmic N- and C-termini.Synaptophysin tagged with GFP at its C-terminuscan be properly recruited to the synaptic vesiclesand is utilized as a reliable marker of local synapticvesicle accumulation [11]. Synaptobrevin2/VAMP2 isalso an integral synaptic vesicle protein with asingle transmembrane domain and a cytoplasmicN-terminal domain. In the process of exocytosis, theassembly of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein [SNAP] receptor)complex from SNAP-25, syntaxin and synaptobre-vin2/VAMP2 is proposed to drive the formation offusion pore between vesicles and the plasma mem-brane. Synaptobrevin2/VAMP2 tagged with GFP atits N-terminus is functional and does not perturbsynaptic vesicle exocytosis. GFP-tagged synaptobre-vin2/VAMP2 is localized at the sites of synapticvesicle accumulation and can be utilized as a reli-able presynaptic marker [10].Detection of the postsynaptic specialization can

be achieved using FP-tagged PSD molecules.2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic

acid (AMPA)-type and N-methyl-D-aspartic acid(NMDA)-type glutamate receptors are enriched inthe biochemically purified PSD fraction and theirpostsynaptic localization was confirmed by immu-noelectron microscopy. AMPA receptors andNMDA receptors are essential components ofglutamate-mediated synaptic transmission and theirpresence at the postsynaptic sites can be detectedby expression of FP-tagged receptors. In the case ofAMPA receptors, their distribution on dendriticmembrane is diffuse and local density of AMPAreceptors on the postsynaptic membrane is not ex-tremely high compared with extrasynaptic recep-tors [12]. Furthermore, it is proposed that nascentsynapses may lack AMPA receptors (silent synap-ses) and local activation of NMDA receptors trig-gers recruitment of AMPA receptors to silentsynapses [13]. From these considerations, FP-taggedAMPA receptor subunits, such as GFP-GluA1 andGluA2, have not been utilized frequently to detectnascent synapses. However, these GFP-taggedAMPA receptors are useful in the detection ofactivity-dependent modifications of postsynapticfunctions [14]. Recruitment of NMDA receptors tothe postsynaptic sites takes place in the early stageof synaptogenesis. NMDA receptors are tetramerscomposed of two GluN1 subunits and two GluN2

Fig. 1. Ultrastructure of the excitatory synapse. (a) Transmission electron micrograph of an excitatory synapse in the mouse hippocampus.Scale bar, 200 nm. (b) Major cytoplasmic components of the presynaptic and postsynaptic cytoplasm of the excitatory synapses.

S. Okabe Fluorescence imaging of synapse formation 53



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subunits. NMDA receptors tagged with GFP at theirextracellular N-termini have been utilized for thedetection of postsynaptic sites in cultured corticalneurons [15].PSD scaffolding molecules are highly concen-

trated in the postsynaptic sites and are good candi-dates for postsynaptic markers. PSD-95 is one of themost abundant scaffolding molecules in the PSD[16]. PSD-95 can interact directly with NMDA recep-tors and also indirectly with AMPA receptorsthrough its binding to transmembrane AMPA recep-tor regulatory proteins. PSD-95 interaction partnersare not restricted to glutamate receptors, butinclude cell adhesion molecules, such as neuroliginsand synCAMs, scaffolding molecules, such as guany-late kinase-associated protein (GKAP), and signalingmolecules, such as synaptic GTPase-activatingprotein for Rac (SynGAP), neuronal nitric oxide syn-thase (nNOS) and spine-associated Rap-Gap (SPAR)[17]. Multiple binding partners of PSD-95 indicate itsessential role in PSD organization. PSD-95 with itsC-terminal tagged with GFP shows postsynaptic

distribution indistinguishable from that of endogen-ous PSD-95 and has been utilized as a reliable fluor-escent marker of the postsynaptic specialization(Fig. 2a) [18,19]. In addition to PSD-95, otherGFP-tagged PSD scaffolding proteins, such asGFP-Homer and GFP-Shank, have also been utilizedas probes for the PSD detection (Fig. 2b) [20,21].The basic properties of these scaffolding proteinsare similar, with quantitative difference in their localturnover rate and response to neuronal activity.Electron microscopic studies reported ultrastruc-

tural difference between asymmetrical (Gray type 1;correspond to excitatory glutamatergic synapses)and symmetrical (Gray type 2; correspond to inhibi-tory GABAergic synapses) synapses [22]. Thesestructural differences are derived from the differ-ence in thickness of the postsynaptic membranespecialization. Postsynaptic membranes of excita-tory synapses develop thicker meshwork of proteinassembly and are detected as prominent electron-dense structures [6]. Typical PSD proteins, such asPSD-95, Homer and Shank, are specifically

Fig. 2. Clustering of PSD scaffolding proteins tagged with GFP at the postsynaptic sites and their turnover in living hippocampal neurons inculture. (a) A cultured hippocampal neuron expressing PSD-95-GFP (green) stained with a lipophilic dye DiI (red). Arrows indicatecolocalization of PSD-95-GFP puncta (green) and dendritic spines (red). Arrowheads indicate a single straight axon devoid of PSD-95 puncta.Scale bar, 10 μm for the main panel and 4 μm for the lower small images. (b) Formation and elimination of the PSD detected by expression ofGFP-tagged Homer1c in cultured hippocampal neurons. Imaging of the same dendritic segments with an interval of 24 h for 6 days revealedcontinual remodeling of the PSDs, with gradual increase in the total number of GFP-Homer1c clusters. Scale bar, 4 μm. Reprinted fromOkabe et al. [19].




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accumulated at excitatory asymmetrical synapsesand their GFP-tagged probes can be utilized as spe-cific markers of this type of synapses. In turn,GFP-tagged scaffolding proteins showing specificlocalization at inhibitory postsynaptic sites, such asgephyrin-GFP, can be utilized for the detection ofsymmetrical synapses in living neurons [23,24].By using GFP-tagged presynaptic and postsynap-

tic molecules, time-lapse imaging of living neuronsrevealed the process of synapse formation andtiming of recruitment of synaptic molecules. Theseanalyses revealed basic principles of synapse for-mation and mechanisms of molecular assembly atthe synaptic junctions [11,25–27].

Live-cell fluorescence imaging of synaptic

molecules and structure

Before GFP technology enabled us to visualizedynamics of synaptic molecules in living neurons,discussions on the time-course of synapse develop-ment had been based on the comparison of im-munocytochemical and electron microscopic dataof fixed preparations. Because the density of synap-ses and dendritic spines increases gradually in bothculture preparations and in vivo, it was widelyaccepted that the differentiation of individualsynapses is also a slow process which may take afew weeks. However, time-lapse imaging of PSD-95tagged with GFP in hippocampal pyramidalneurons in culture revealed rapid establishment ofPSD-95-GFP fluorescent clusters on time scales ofseveral hours [19]. These PSD-95-GFP clusters wereapposed to the presynaptic boutons and were se-lectively localized within dendritic spines, suggest-ing their identity as synaptic PSD structures [11]. Asimilar time course of postsynaptic molecular as-sembly was confirmed by several independentgroups and has been taken as convincing evidencefor rapid establishment of synaptic specialization[28,29]. If synapse assembly is a rapid process,how is relatively slow increase of overall synapticdensity achieved? A possible explanation is thatsynapse addition and elimination take place simul-taneously and the rate of addition is maintained tobe moderately higher than the rate of elimination.Indeed, this difference in addition and eliminationof PSD-95-GFP clusters existed in culture

preparations and could quantitatively explain theoverall rate of synapse density (Fig. 2b) [18,19].It is possible to determine the time course of

molecular assembly at presynaptic and postsynapticsites simultaneously by using multicolor imaging ofFP-tagged synaptic proteins. We expressed PSD-95tagged with yellow fluorescent protein (YFP) to-gether with synaptophysin tagged with cyan fluores-cent protein (CFP) to determine whetherpresynaptic and postsynaptic molecules appear atsynaptic sites with different time courses [11]. Wefound a strong temporal correlation between cluster-ing of PSD-95-YFP and that of synaptophysin-CFP atthe synaptic contact sites (Fig. 3d and e).

Fig. 3. Simultaneous detection of multiple synaptic components bydual-color time-lapse imaging. (a–c) Imaging of the PSDs byexpression of PSD-95-yellow fluorescent protein (YFP) (b) togetherwith detection of spine structure by cyan fluorescent protein (CFP)as a volume marker. (a) Overlay of PSD-95-YFP (red) and CFP(green) was also presented. (c) Formation of a PSD-95-YFP cluster(arrows) is coordinated with the enlargement of the spine head.Scale bar, 3 μm. (d–e) Simultaneous imaging of synaptophysin-CFP(d) and PSD-95-YFP. (e) Accumulation of synaptic vesicles detectedby synaptophysin-CFP is synchronized with clustering ofPSD-95-YFP (arrows). Scale bar, 3 μm. Reprinted from Okabe et al.

[11,21].




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Interestingly, appearance of synaptophysin-CFP clus-ters tends to precede clustering of postsynapticPSD-95-YFP with time intervals of <1 h, indicatingthe existence of temporal orders in synapsedifferentiation.Immature hippocampal pyramidal neurons express

dendritic filopodia, which are highly motile and havelifetimes shorter than those of spines. It has been pro-posed that dendritic filopodia serve as sensors of thesurrounding tissue environment and are transformedinto spines after physical interaction with nearbyaxons. To determine structural changes of immaturedendritic protrusions and their relationship to cluster-ing of PSD-95, we expressed both PSD-95-YFP andCFP in dissociated hippocampal neurons [11]. CFPwas utilized as a volume label for morphological iden-tification of dendritic protrusions. Imaging ofPSD-95-YFP and CFP revealed the presence offilopodia-like protrusions on pyramidal neuron den-drites and appearance of PSD-95-YFP clusters in asmall subset of these protrusions (Fig. 3a–c). After es-tablishment of PSD-95-YFP clusters, these protru-sions were stabilized and increased their volume,indicating their morphological transformation intospine-like structures. Importantly, none of thePSD-95-YFP clusters within dendritic shafts inducedthe formation of dendritic protrusions. These obser-vations indicate that filopodia-like protrusions fromdendritic shafts play an important role in initialcontact with nearby axons and subsequent differenti-ation of spines with synaptic contacts.

Roles of dendritic and axonal

protrusions in synaptogenesis

Neuronal networks in the mammalian cortex arecomposed of two types of neurons, excitatorypyramidal-shaped neurons and inhibitory neurons.Although glutamatergic excitatory synapses areformed on dendrites of both pyramidal neurons andinterneurons, their postsynaptic morphology andmolecular composition are distinct. The mostobvious difference is the absence of dendriticspines in excitatory synapses on interneuron den-drites [30,31]. Dendritic filopodia from pyramidalneurons are thought to be the precursors of spinesand important in searching nearby axons. It has notyet been clarified whether interneuron dendrites

have any searching systems to contact nearbyaxons. Without such a mechanism to enhance thechance of contacting nearby axons, the ability ofinterneurons to increase synaptic density should bequite limited. However, previous electron micro-scopic studies reported that dendritic shafts ofmature interneurons are densely covered with gluta-matergic synapses, suggesting the presence ofinterneuron-specific strategy to increase synapticcontacts [32]. To solve this problem, time-lapseimaging of synapse formation on interneuron den-drites was performed and two important observa-tions were made [33]. First, dendritic protrusiveactivity of interneurons was developmentally regu-lated. Although dendrites of mature interneuronshad few protrusions, immature interneuronsexpressed numerous dendritic protrusions, whichwere longer than typical filopodia of pyramidalneurons. Second, PSD-95 clusters were frequentlyobserved on these dendritic protrusions andshowed slow translocation toward the base of pro-trusions (Fig. 4). These observations indicated thatdendritic protrusions of interneurons serve as con-duits for retrograde translocation of synaptic struc-ture to the parental dendrites. This translocationwas dependent on microtubules present within den-dritic protrusions and was driven by dynein motorsystem. These experimental data indicate that thebehavior of synaptic structure may differ betweendifferent types of synapses even within the samebrain area. In the case of pyramidal neurons, thepositions of individual synapses along dendriticshafts are precisely determined by the position ofinitial protrusive activity of dendritic filopodia. Onthe other hand, the positions of excitatory synapsesalong interneuron dendrites may be more flexibleand the synaptic connectivity can be increasedeffectively by using long protrusions as synapticconduits.Assembly of synaptic structure is a stochastic

process and the behavior of a large number ofsynapses with different stages of maturation is diffi-cult to be classified and characterized. If synapsematuration can be synchronized, the time course ofsynapse development may be determined moreprecisely and molecular markers and structuralfeatures for each developmental stage may be char-acterized more easily. In the cerebellum,




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synaptogenesis between the axons of granule cells(parallel fibers) and the dendrites of Purkinje cellsis regulated by a soluble factor Cbln1, a C1q familyprotein produced and secreted from granule cells.Cbln1 released from parallel fibers binds to bothpresynaptic receptor neurexin (Nrx) and postsynap-tic receptor glutamate receptor delta 2 (GluD2).It is likely that the tripartite Nrx-Cbln1-GluD2complex affects synaptogenesis of both presynapticand postsynaptic components. We took advantageof slice cultures derived from Cbln1 knockout (KO)mice and induced synaptogenesis in slices by ex-ogenous application of Cbln1 [34]. Without Cbln1,parallel fibers showed little morphological

remodeling and subsequently generated feweraxonal boutons. However, application of Cbln1increased both motility of synapses and their subse-quent transformation into axonal boutons. On thebasis of the data obtained by using this artificialsynchronization system of synaptogenesis, we pro-posed ‘bidirectional interaction model’ of synapsematuration between parallel fibers and Purkinje celldendrites. This model is based on our observationthat a specific type of axonal protrusions withcomplex morphology appears during the intermedi-ate stage of synapse formation and increase in thecontact area between axons and dendritic spinesenhances both Nrx-dependent presynaptic matur-ation and GluD2-dependent spine maturation.We learned from this study that time-lapse imagingof the synchronized formation of neural circuits ishighly informative in defining precise stages ofsynaptogenesis.

Turnover of postsynaptic scaffolding

molecules and their structural roles

We proposed that synapse addition and eliminationtake place simultaneously and the slight excess ofadded synapse over eliminated synapse determinesthe gradual increase of overall synapse density. Thismodel indicates the importance of a regulatorymechanism that controls the rates of synapse forma-tion and elimination through the assembly and disas-sembly of synaptic molecules. To determine theturnover rate of a given synaptic molecule at localsynaptic sites precisely, fluorescence recovery afterphotobleaching (FRAP) and fluorescence activationwere performed. With both techniques, the turnoverrate of fluorescent molecules in a local environmentcan be directly measured. Interestingly, FRAP ana-lyses of four different PSD scaffolding moleculesrevealed distinct kinetic properties (Fig. 5) [20].PSD-95, a scaffolding molecule directly interactingwith the plasma membrane via its S-palmitoylation,showed low mobility by FRAP analysis. In contrast,Homer1c, a scaffolding molecule containing bindingsites for actin, showed a much higher turnover rate.Because actin molecules in spines show very rapidexchange between soluble and polymerized pools[35], it is likely that dynamics of PSD scaffolding pro-teins is heterogeneous and scaffolding proteins

Fig. 4. PDS mobility along dendritic protrusions of interneurons.(a) Time-lapse images of an interneuron dendrite expressingPSD-95-GFP (green) and RFP (magenta) in a dissociatedhippocampal culture. A PSD-95-GFP cluster (arrows) showsretrograde translocation. Scale bar, 3 μm. (b) Time-lapse images ofan interneuron expressing GFP-Homer1c and RFP in a cortical sliceculture. A GFP-Homer1c cluster showed movement in adistal-to-proximal direction (arrows). Scale bar, 5 μm. Reprintedfrom Kawabata et al. [33].




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enriched at the cytoplasmic surface of PSDs are de-pendent on actin cytoskeleton for their localizationand stability.To further clarify the roles of PSD scaffolding

proteins in the molecular architecture of PSDs, in-formation about the molecular content of individualscaffolding proteins per single PSD is essential.Even if functional studies suggest importance of agiven molecule by gene knockout and knockdownapproaches, its importance as a structural compo-nent can be proved only by measuring its molecularcontent per synapses. To this end, we developed atechnique of quantitative measurement of proteincontent per synapses by using fluorescent micro-spheres as a calibration standard of GFP molecules

(Fig. 6) [36]. We first determined the fluorescenceintensity of single GFP molecules and comparedthe fluorescence of single microspheres with that ofsingle GFP proteins. Our measurements indicatedthat the fluorescence microspheres are equivalentto the fluorescence intensity of about 4000 GFPproteins. By using these calibrated microspheres,

Fig. 6. Determination of absolute numbers of PSD scaffoldingproteins in single synapses. (a) A confocal microscopic image of adendritic segment expressing PSD-95-GFP. The neuron waschemically fixed, reacted with anti-VGLUT1 antibody (red) andanti-MAP2 (blue) and sprinkled with fluorescent microspheres(arrow). The fluorescence signal from PSD-95-GFP clusters(arrowheads) was compared with the signal from fluorescentmicrospheres. Scale bar, 2 μm. (b) Calculation of the endogenousPSD-95 protein content by comparison of the immunofluorescenceintensity of control neurons and neurons infected with recombinantadenoviruses for the expression of PSD-95-GFP. After determinationof the number of functional GFP fusions in a single postsynapticsite (363 molecules), the number of endogenous PSD-95 moleculesper synapse was calculated to be 308, based on the ratio of totalPSD-95 content between control and infected neurons (1:2.47). (c)Cumulative distribution of scaffolding protein numbers in singlepostsynaptic sites of hippocampal neurons on day 25 in culture. Thesynaptic contents of PSD-95 and total membrane-associatedguanylate kinase proteins were 208 ± 86.6 and 308 ± 128(mean ± standard deviation). Reprinted from Sugiyama et al. [36].

Fig. 5. Differential stability of PSD scaffolding proteins revealed byFRAP. (a) Organization of the PSD by layered distribution ofmultiple types of PSD scaffolding proteins. (b) Differentialstabilization of four PSD scaffolding proteins measured by FRAP.Fluorescence recovery within 5 min was measured and quantitated.Scale bar, 1 μm. Reprinted from Kuriu et al. [20].




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we next determined the number of GFP-tagged PSDscaffolding proteins present in single postsynapticsites. We could successfully estimate the absolutenumbers of four key scaffolding proteins, PSD-95,GKAP, Shank and Homer, per synapses.Interestingly, the synaptic contents of the fourscaffolding proteins were quite similar and were inthe range 100–450 molecules per synapses. Becausethese four scaffolding molecules were knownto interact with each other, we proposed that theirbinding stoichiometry is relatively simple.Furthermore, the total protein mass of four scaf-folding proteins was calculated to be 100 MDa,which corresponded to about 10% of the total massof single PSD (1.1 GDa). This suggests that onlyfour types of PSD scaffolds occupy a substantialfraction of the total PSD mass. This conclusion isconsistent with the idea that a small subset of PSDscaffolding proteins are responsible for the con-struction of the main PSD structure, which mayfunction for anchoring and stabilization of otherfunctional molecules, such as neurotransmitterreceptors and cell adhesion molecules [16].Several hundreds of PSD-95 molecules in a single

synapse are sufficient to anchor glutamate recep-tors, which are estimated to be accumulated tosingle synapses in the range 10–200 tetramers persynapse [37,38]. It is important to note that thenumber of PSD-95 per synapse and the number ofglutamate receptor complexes per synapse are setto be in a similar range. This relationship mayenable efficient regulation of glutamate receptornumbers through the synaptic content of PSD-95.This idea was supported by electrophysiologicalrecordings of slices from PSD-95 knockout mice.In the absence of PSD-95 and related membrane-associated guanylate kinase proteins, synaptictransmission, which was mediated by AMPA-typeglutamate receptors, could be detected, but its amp-litude was significantly down-regulated, suggestingPSD-95-dependent regulation of AMPA receptorcontent per synapse [39].

Future directions of imaging research

on synapse dynamics

Recent advancement of imaging technologies pro-vided a wide range of opportunities for the

applications of new optical principles, light sourcesand sample preparations techniques to the analysesof synapse formation and remodeling. Especially,two important technologies, in vivo two-photonimaging and super-resolution microscopy, maygreatly accelerate our understanding of synapsedynamics.Two-photon excitation laser scanning microscopy

is a technique appropriate for high-resolutionimaging of neuronal microstructure in vivo [40]. Aninfrared pulsed laser beam can penetrate the braintissue more effectively than the shorter wavelengthlight and can be focused to the volume smallenough to resolve submicron structures within thenervous tissue [41,42]. By using two-photon micros-copy, activation of fluorescence molecules isrestricted to a small volume without unnecessaryexcitation above and below the focal plane, whichmay induce phototoxic effects after repeatedimaging of a large volume of samples. Two-photonmicroscopy enabled researchers to resolve dendrit-ic spines filled with GFP in the neocortex of livingmice with time intervals of several days to months(Fig. 7) [43,44]. By comparing the images of dendrit-ic segments and attached spines at different timepoints, the rate of formation and elimination ofspines could be successfully measured in vivo.Initial studies revealed the highly stable nature ofdendritic spines in the mature neocortex: more

Fig. 7. In vivo imaging of layer 5 pyramidal neurons expressingGFP in the somatosensory cortex. (a) Two-photon imaging of theapical dendrites in the layer 1 of an adult transgenic mouseexpressing GFP under the control of Thy1 promoter. (b) Dendriticspines visualized in vivo with an interval of 14 days. Arrowindicates an eliminated spine. Most of the imaged spines werestable over 14 days in vivo. Scale bar, 5 μm.




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than 90% of spines imaged in the adult mouse neo-cortex could survive when they were re-examinedseveral months later [43,45]. This stability is in con-trast with rapid turnover of synapses in culture con-ditions. There are two possibilities that can explainthis discrepancy. First, synapse dynamics in cultureconditions may be artificially upregulated by thepresence of factors specific to the culture conditionor the absence of factors present in the nativetissue environment. For example, culture mediumand serum supplement may upregulate synapseelimination in vitro. It is also possible that matureastrocytes may protect newly formed synapses andthe lack of glial support may explain the higherturnover rate of synapses in culture [41]. Thesecond possibility is that rapid turnover of synapsesin vitro reflects the situation of synapses in theearly phase of synapse development in vivo.Indeed, quantitative analyses of spine density in theearly postnatal period of rat visual cortex revealedthat significant increase in spine density occursbetween postnatal days 6 and 21 [46]. Applicationof in vivo two-photon imaging in the early postnatalperiod may clarify this point. However, thisexperiment has not yet been accomplished, mainlybecause of the technical difficulty of imagingnascent synapses in vivo. At present, most of thein vivo synapse analyses rely on dendritic spines asa structural marker of synapses. In the future [47],application of GFP-tagged postsynaptic proteins,such as PSD-95-GFP, will increase the precision ofidentifying nascent synapses in vivo and will revealthe degree of synapse turnover in the early post-natal period.Another important advancement in the field of

synapse imaging is application of super-resolutionimaging to molecular distribution and dynamicsof synaptic proteins [48]. Recently, two papersreported significant improvement in spatial reso-lution of postsynaptic molecular movement andPSD structure by using photoactivated localizationmicroscopy (PALM) and stochastic optical recon-struction microscopy (STORM) techniques [49,50].PALM is the imaging technique based on photoacti-vation of FPs and STORM is based on stochasticactivation of organic fluorescent dyes attached toappropriate antibodies. Recent application of PALMto the behavior of single actin molecules revealed

the presence of actin flow in spines and confirmedthe results of previous photoactivation studies ofactin tagged with photoactivatable GFP [50].Furthermore, these PALM studies revealed hetero-geneity in the speed of actin movement withinsingle spines. In the cytoplasm close to the PSD,actin velocity was specifically higher than in theother area. These PALM experiments of actin mobil-ity in spines suggested that overall organization ofactin filaments in spines is based on short filamentswith less aligned orientation and heterogeneous vel-ocity. It is also possible to evaluate molecular distri-bution of multiple synaptic proteins at the synapticjunctions by using super-resolution imaging techni-ques. Dani et al. reported distribution of PSD scaf-folding proteins and glutamate receptors withinthe postsynaptic membrane of fixed brain sectionslabeled with antibodies against presynaptic andpostsynaptic molecules [49]. They could successful-ly detect differential distribution of AMPA andNMDA receptors in individual synapses by usingthe STORM technique. In the future, precise organ-ization of glutamate receptors and scaffolding pro-teins can be determined by using the PALM/STORMtechniques and their intrasynaptic redistributionassociated with synaptic plasticity may be directlyvisualized.

Conclusions

Before the introduction of live imaging techniquesof synaptic structure and function, electrophysio-logical recording was the only possible techniqueto analyze functional modification of synapses.Although electrophysiological recording is an indis-pensable technique to record activity of synapticreceptors and ion channels, it usually lacks theinformation of single synapses and is not suitablefor long-term monitoring of synapse behaviors.Introduction of GFP technology and its applicationto the research on synapse formation led to directmonitoring of single synapse behavior for a longtime with minimal perturbation. These imagingstudies in the last decade provided a clear pictureof synapse formation in the mammalian neocortexand hippocampus. Two-photon excitation micros-copy, combined with various gene expression tech-nologies, such as generation of transgenic mice and




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infection of viral vectors, further extended thepossibility of imaging synapse formation andremodeling within tissue environment. Especially,two-photon imaging of neocortical neurons throughoptical windows on the rodent cranium enabledresearchers to monitor the lifetime and the behav-ior of single synapses in vivo. In the near future,using in vivo imaging technologies combined withtechniques of manipulating neuronal activity, wecan expect that the basic principles of neural con-nectivity in living animals can be extracted withfirm experimental evidences.

Funding

This work was supported by Grants-in-Aid forScientific Research (18200025, 20019013, 21220008,and 22650070 to S.O.) from the Ministry ofEducation, Culture, Sports, Science and Technologyof Japan. A part of this study was the result of‘Development of biomarker candidates for social be-havior’ carried out under the Strategic ResearchProgram for Brain Sciences by the Ministry ofEducation, Culture, Sports, Science and Technologyof Japan (S.O.), and also by Takeda ScienceFoundation (S.O.).

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