guidance molecules in synapse formation and plasticity

Guidance Molecules in Synapse Formation andPlasticity

Kang Shen1 and Christopher W. Cowan2

1Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, California 943052Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9127

Correspondence: [email protected]

A major goal of modern neuroscience research is to understand the cellular and molecularprocesses that control the formation, function, and remodeling of chemical synapses. Inthis article, we discuss the numerous studies that implicate molecules initially discoveredfor their functions in axon guidance as critical regulators of synapse formation and plasticity.Insights from these studies have helped elucidate basic principles of synaptogenesis, den-dritic spine formation, and structural and functional synapse plasticity. In addition, theyhave revealed interesting dual roles for proteins and cellular mechanisms involved in bothaxon guidance and synaptogenesis. Much like the dual involvement of morphogens inearly cell fate induction and axon guidance, many guidance-related molecules continueto play active roles in controlling the location, number, shape, and strength of neuronal syn-apses during development and throughout the lifetime of the organism. This article summ-arizes key findings that link axon guidance molecules to specific aspects of synapseformation and plasticity and discusses the emerging relationship between the molecularand cellular mechanisms that control both axon guidance and synaptogenesis.

SYNAPTOGENESIS AND NEURONALSYNAPTIC PLASTICITY

During neuronal development, extendingaxons navigate to stereotyped target re-

gions in the brain and body with the ultimategoal of making a functional synaptic connec-tion. The chemical synapse represents a special-ized functional and morphological cell struc-ture where a presynaptic neuron communicateswith the postsynaptic cell. For most neurons,an action potential in the presynaptic axonstimulates the controlled release of chemical

neurotransmitters that bind and activate spe-cific receptor proteins that reside on the post-synaptic and presynaptic membranes. This trig-gers the opening of ion channels and activationof signaling cascades. There are numerous typesof synapses, often characterized by the type ofneurotransmitter released from the presynapticterminal and the specific cell types involved. Asa central aspect of neurobiology, there are vastnumbers of studies focused on synapse forma-tion, synaptic physiology, and circuit organiza-tion and function. The purpose of this article is

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to focus on recent findings that implicate axonguidance-related proteins in the processes req-uired for synapse formation and plasticity.

Much like axon guidance, the process offorming a new synapse (Fig. 1), or synaptogen-esis, involves coordinated cell morphologicaland structural changes that are instructedthrough ligand-receptor interactions, intracel-lular signaling cascades, and complex fila-mentous actin (F-actin) remodeling. There arealso cell intrinsic processes that contribute tothe pre-establishment of synaptic componentslong before the dendrite or axon arrives atfuture synaptic sites, or that prohibit formationof inappropriate synapses. The processes thatgovern how, when, and where a synapse willform, and specifically, the cell and molecularmechanisms that control synaptogenesis, arestill poorly understood. During the past twodecades, technological advances in microscopyand the use of modern molecular and geneticapproaches have allowed for basic characteriza-tion of key steps in synapse formation andplasticity. Much of our knowledge of synapseformation is based on analysis of either the neu-romuscular junction (NMJ) or the glutamater-gic axo-dendritic synapse formed between twoneurons. However, it is likely that each subtypeof synapse, such as an inhibitory (e.g., GABAer-gic), neuromodulatory (e.g., dopaminergic),or excitatory (e.g., glutamatergic), has distinctmechanisms controlling its formation and plas-ticity. In fact, there are substantial differences inthe processes and molecules involved in the twomost studied synapses, the cholinergic NMJ andthe glutamatergic central synapse.

For the purposes of this article, we havechosen to focus in large part on the axo-dendritic synapse as many guidance-relatedmolecules play a role in these types of synapses.For simplicity, we have also organized the basicsteps of synapse formation into the followingcategories: (1) synaptic prepatterning, (2) den-dritic filopodial motility, (3) contact stabiliza-tion, and (4) synaptic maturation. In addition,functionally mature synapses are not static,but instead alter their strength and number inresponse to experience to facilitate complexbehavioral plasticity.

In this article, we discuss a wealth of litera-ture that indicates an important role for manyaxon guidance-related proteins in synapse for-mation and plasticity. Although dual functionsfor axon guidance molecules in synapse forma-tion may be an efficient use of existing axonaland dendritic proteins, it is interesting howthe same proteins can regulate such diversecell biological processes. Exploring this issuewill be an important area for future research.

AXON GUIDANCE AND SYNAPSEFORMATION

There are numerous similarities between axonguidance and synapse formation/plasticity.Obviously, the targeting of axons to the appro-priate target field is an essential process forproper neural circuit formation, and the axonis an obligate partner of the functional synapse.Axon guidance involves the interaction ofreceptor proteins found on the surface of navi-gating growth cones with secreted or cell-surface-bound ligands encountered en routeto, or in, the target field. Binding of ligand toreceptor often stimulates intracellular signalingcascades that coordinate complex cell morpho-logical changes in the axon growth cone to movetoward (attraction) or away from (repulsion)the guidance cue source. Similarly, the interac-tion between an axon and a postsynaptic cellappears to trigger signaling events necessaryfor synapse formation, and in some cases,involves complex morphological changes thatare largely mediated by regulated assemblyand disassembly of the F-actin cytoskeleton.Regulation of F-actin dynamics is a major proc-ess controlling the morphological changes inboth navigating growth cones and in dendriticspines. Spines represent the site of �90% ofexcitatory glutamatergic synapses in the maturevertebrate brain, and whereas the precise role ofdendritic spines is still debated, it is likely thatthey play an important role in proper synaptictransmission. As such, there are numerous linesof research seeking to understand the mecha-nisms that control dendritic spine formationand remodeling.

K. Shen and C.W. Cowan

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Presynapticspecialization

Axon

Axon Axon


B1 B2

A

Dendrite

Dendrite

C1 C2

Dendrite Dendrite

Axon

Axon

Dendrite

Figure 1. Basic steps of axo-dendritic synaptogenesis. Diagrams illustrate key steps involved in forming genericcentral synapses. (A) A guiding axon and nearby dendrite interact via cell–cell contacts mediated by the growthcone, collateral axon branches, or dendritic filopodial extensions. (B1) In some scenarios, pre-establishedpresynaptic specializations mark the location of future synapses, whereas inappropriate axonal regions donot allow for synapse formation. (B2) In another scenario, random physical interactions between axon anddendrite (red boxes) form transient cell–cell adhesions. If the contacts are stable, then presynaptic andpostsynaptic proteins, vesicles, and ion channels are recruited to the contact site. (C1 and C2) Stable contactsare matured into functional and morphological chemical synapses (spine or shaft synapses).

Guidance Molecules in Synapse Formation

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Another similarity between axon guidanceand synapse formation/remodeling is the com-partmentalization of signaling events. In thenavigating axon, guidance cues initiate localizedchanges in the growth cone morphology thatpromote axon outgrowth or turning (attractionor repulsion). Similarly, a dendritic spine pro-vides a morphological subcompartment of thedendrite that may restrict synaptic activity-induced signaling events to the activated spinesynapse. However, it is important to note thatdendritic spines are not absolutely necessaryfor synapse-specific compartmentalization asshown in aspiny neurons (Goldberg et al.2003; Soler-Llavina and Sabatini 2006).

In an axon growth cone, rapidly extendingand retracting filopodial structures are thoughtto search the local environment for guidancecues. Similarly, highly dynamic, dendritic filo-podia extend from the dendritic shaft duringthe robust periods of synaptogenesis, and appearto search the proximal space for a potential pre-synaptic axon in the vicinity. This process likelyincreases the probabilityof making axo-dendriticcontacts and forming functional synapses. How-ever, the physical interaction between dendriticfilopodium and axon is not sufficient to inducea synapse as many contacts are transient. Likegrowth cone navigation, cell-surface proteinsand intracellular signaling pathways likely deter-mine whether an axo-dendritic interaction pro-ceeds to form a synapse or whether the contactis “repelled.” In some cases, the axon “attracts”the dendrite to form synapses in specific regions(see later).

SPECIFIC ROLES OF GUIDANCEMOLECULES IN SYNAPTOGENESIS

Synaptic Prepatterning

Two distinct types of synapses can be catego-rized based on where the synapses locate withinthe axon: terminal synapses and en passant syn-apses. Terminal synapses are formed at the endof the axon projections, whereas en passant syn-apses locate along the axon shaft and can be faraway from the axon terminal. Both types of syn-apses are quite abundant in both the vertebrate

and invertebrate nervous systems. For example,the vertebrate NMJ comprises terminal synap-ses because motor neurons only elaborate syn-apses at their terminal arbors. For the mostpart, invertebrate synapses are formed en pas-sant, and there are many examples of en passantsynapses in the vertebrate CNS.

Although the locations of terminal synapsesare governed by proper axon targeting to thefuture synaptic site, the precise locations of enpassant synapses are determined independentlyfrom growth cone guidance. During synapseformation, it is generally thought that the dyna-mic movements of dendritic filopodia initiatethe contacts between the axon and its postsyn-aptic partners (see Fig. 1B2). However, there isalso evidence that an axon possesses intrinsicabilities to pattern its presynaptic terminals.For example, in dissociated cortical neurons,the initial formation of presynaptic terminalsoccurs preferentially at predefined sites withinthe axons (Fig. 1B1). Transporting synaptic ves-icle precursors pause repeatedly at these sites,even in the absence of neuronal or glial contact,and “attract” dendrite filopodia to form stablesynapses (Sabo et al. 2006).

If axons can independently pattern theirfuture presynaptic terminals, what factors con-trol this process? The Caenorhabditis elegansDA9 motoneuron elaborates around 25 en pas-sant presynaptic boutons in stereotyped loca-tions along its axon. These presynaptic spe-cializations are found exclusively within a shortsegment in the middle of the axon trajectory.Genetic analyses showed that two diffusibleextracellular gradients formed by the axon guid-ance ligands, LIN-44/Wnt and UNC-6/netrin,play instructive roles in preventing formationof inappropriate presynaptic specializationsoutside of the middle axon segment. LIN-44/Wnt functions through a frizzled receptor,LIN-17, whereas UNC-6/netrin acts throughthe UNC-5 receptor (Klassen and Shen 2007;Poon et al. 2008). Interestingly, both UNC-6/netrin and LIN-44/Wnt also participate inDA9 axon guidance through persistent gra-dients. A gradient of UNC-6/netrin expres-sion (high ventral/low dorsal) repels dorsalaxon migration using the repulsive UNC-5





receptor (Hedgecock et al. 1990); whereas, aposterior–anterior gradient of LIN-44/Wntguides the anterior turn of the axon (V. Poonand K. Shen, unpubl.). Therefore, the sameextrinsic cues provide positional informationfor two distinct cell biological events duringdevelopment.

Much like growth cone guidance, localattractive cues appear to promote synapse for-mation. Recent findings suggest the existenceof guidepost cells, which ensure the stereotypedlocation of certain synapses and help coordinatethe proper formation of neural circuits (Fig. 2)(Chao et al. 2009). For example, in nematodeC. elegans, the thermotactic interneuron, AIY,forms stereotyped synaptic connections ontothe interneuron, RIA. These synapses are for-med in dyadic fashion with another set of inter-neurons, RIB, such that the AIY presynapticterminal faces the two dendrites of RIA andRIB in the synaptic complex (White et al.1986). Therefore, the AIY axon needs to findboth of its postsynaptic targets at the same spa-tial coordinate. This synaptogenesis process iscontrolled by glial-like ventral cephalic sheathcells (VCSCs), which serve as guidepost cells.The thin processes of VCSCs use UNC-6/netrinto define the location of synapses and to coordi-nate the formation of the synaptic complex.However, UNC-6/netrin appears to have dis-tinct effects on the presynaptic AIY and thepostsynaptic neuron RIA. For AIY, local secre-tion of UNC-6/netrin stimulates the formationof presynaptic terminals next to the VCSC,whereas UNC-6/netrin attracts RIA dendriticoutgrowth toward the VCSC (Colon-Ramoset al. 2007). Intriguingly, these distinct cellularresponses are mediated by the same receptor,UNC-40/DCC. Taken together, these findingssuggest that axon guidance, presynaptic special-ization and dendrite outgrowth are intrinsi-cally coupled by the common molecular cue,UNC-6/netrin. UNC-6/netrin uses two dis-tinct receptors, UNC-40/DCC and UNC-5, inthese processes to regulate axon guidance andsynaptogenesis. In both events, UNC-40/DCCseems to confer attraction of growth conesor local construction of synapses, whereasUNC-5 is required for repulsion of axons and

exclusion of synaptic material (Colon-Ramoset al. 2007; Poon et al. 2008).

How could the same morphogenetic gra-dients play instructive roles for both axonguidance and synapse formation? Both axonguidance and patterning of presynaptic special-izations can be viewed as symmetry breaking

Dendrite

Dendrite

Axon

A

B

C

Axon

Axon



Guidepost Guidepost

Guidepost Guidepost

Dendrite

Figure 2. Prepatterning of synaptic specializations.Diagrams show the time-course of one form of syn-aptogenesis for en passant synapses. (A) An undiffer-entiated axon and dendrite before synapse formation.(B) Extrinsic cues promote (green) and inhibit (red)local presynapse formation, thereby patterning thesubcellular localization of presynaptic terminals. (C)Postsynaptic dendritic filopodia approach presynapticspecializations to form synapses.





processes. During axon guidance, actin poly-merization is favored in subareas of the growthcone facing the attractive guidance molecules,which likely underlies growth cone turning. Incontrast, synapse formation involves the trans-formation of an undifferentiated patch of pre-synaptic plasma membrane into a highly organ-ized membrane-protein complex specialization.Perhaps a polarized gradient of extrinsic cuesspecifies the synaptic location by initiating theformation of a localized protein complex thatrecruits and captures presynaptic machinery(Fig. 2). Consistent with this idea, the interac-tion between certain cell adhesion molecules,such as neuroligin/neurexin and SynCaM,are sufficient to initiate presynaptic formation(Yamagata et al. 2003). Alternatively, graded re-gulation of synaptic vesicle trafficking mightalso result in specific localization of synapses.Future analysis of the downstream signalingmechanisms will likely provide important newinsights.

Filopodial Motility

Dendritic filopodia and spines were first ob-served over a century ago. However, the essentialrole of these morphological structures hasremained unclear. Detailed time-lapse andultrastructural analysis of dendritic filopodialstructures has revealed that they are rapidlyformed, but are very unstable in young neurons(mean lifetime of minutes to hours [Ziv andSmith 1996; Trachtenberg et al. 2002]). Theyare very abundant on most neurons during earlydevelopment, but as neurons mature, dendriticfilopodia numbers decline dramatically, whereasthe number of dendritic spines, which are mor-phologically distinct and more stable structures,and functional synapses increase (Fiala et al.1998). The energy involved in generating dynam-ic filopodia suggests that they must play animportant role in synaptogenesis, but they donot appear to be essential for a synapse toform. Highly motile dendritic filopodia likelysurvey the proximal dendritic space to increasethe probability of interacting with a synapticpartner (axon) and thereby increase the number

of synapses formed. Interestingly, the axonguidance molecules, BDNF/TrkB and EphB2,appear to facilitate axo-dendritic synaptogene-sis, at least in part, by controlling dendritic filo-podia motility.

BDNF/TrkB

The neurotrophic factor, brain-derived neuro-trophic factor (BDNF), and its high-affinityreceptor, tropomycin-related kinase B (TrkB),are key mediators of axon guidance, synapseformation, and plasticity (Huang and Reichardt2003; Luikart and Parada 2006; Lu et al. 2008)(Fig. 3A). Numerous studies have reportedBDNF-induced changes in dendritic spine den-sity and morphology in a variety of neuronpopulations. Consistently, TrkB-deficient micehave significantly fewer dendritic spines andexcitatory synapses on CA1 hippocampal neu-rons, because of a postsynaptic and cell auton-omous role for TrkB during a defined periodof early synaptogenesis (Luikart et al. 2005).In more stable or mature glutamatergic synap-ses, BDNF/TrkB signaling may be restrictedto regulation of functional and morphologicalsynapse/spine plasticity. TrkB may regulate syn-apse number in early development by stimulat-ing filopodial motility (Luikart et al. 2008)—a process requiring TrkB kinase activity andPI3-kinase signaling (but not ERK signaling)(Fig. 3A). Therefore, BDNF/TrkB signaling ap-pears to increase synapse number by increasingthe probability that a dendritic filopodium willencounter a nearby axon. Interestingly, long-term incubation (24–48 h) of young hippo-campal slice cultures with exogenous BDNFalso increases spine synapse number (Tyler andPozzo-Miller 2001), but this did require ERKsignaling (Alonso et al. 2004).

Other than the involvement of PI3-kinase,the precise mechanisms that link the BDNF-activated TrkB receptor to F-actin remodelingin filopodial motility or synapse/spine forma-tion remain unclear. Two Dbl family guaninenucleotide exchange factors (GEFs), which reg-ulate Rho family GTPases and actin cytoskeletaldynamics, Vav2 and Tiam1, are activated by





BDNF/TrkB signaling. Vav2 is activated by andinteracts with TrkB receptors, and is criticalfor BDNF-induced generation of Rac-GTPin cortical and hippocampal neurons (C. Haleand C. Cowan, unpubl.). Tiam1 can also be

activated by BDNF/TrkB signaling (Miyamotoet al. 2006), which suggests that multiple Rhofamily GEFs may link BDNF/TrkB receptorsignaling to actin cytoskeletal dynamics inneurons.

Axon

Presynapticdifferentiation

BDNFTrkB

P P

PI3K

Synapse formation?Receptortrafficking

SpinegrowthLTP

Rho GTPases

Vav2, Tiam1 miR-134

LimK1 proteinsynthesis

F-actindynamics

F-actindynamics

?

?

TrkBTrkB

SpineFilopodium

A

P P

P P

Axon

Presynapticdifferentiation

PAK

?

F-actindynamics

Enhancedsynapse formation

Spinegrowth

F-actindynamics

SpineFilopodium

EphrinBEphB2

NMDAreceptors

Kalirin, Tiam,Intersectin, Vav2

Syndecan-2

?

B

PP P P

P

Axon

EphB?

EphB?

EphrinB1

C

EphrinB3

Grb4/GIT1

Spinematuration

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GRIP

Postsynapticscaffolding &maturation

Shaftsynapse

Spinesynapse

Figure 3. Guidance molecules involved in synapse formation and axon branching. (A) Model for EphB2 receptorfunctions and signaling in synaptogenesis. (B) Model for BDNF/TrkB receptor functions and signaling insynaptogenesis. (C) Role of ephrinBs in shaft and spine synapse formation.





EphB2

Similar to TrkB, the axon guidance receptor,EphB2, plays a key role in normal hippocampalsynapse formation (Dalva et al. 2000; Henke-meyer et al. 2003; Kayser et al. 2006; Kayseret al. 2008) (Fig. 3B). EphB2 appears to partic-ipate in several aspects of synapse formation,including filopodial motility. EphB2-deficienthippocampal neurons in culture have dramati-cally reduced filopodial motility and synapsedensity. EphB2 forward signaling was re-quired for the filopodial motility, but artificiallyincreasing dendritic filopodial motility alonedid not rescue the deficit in synapse formationin EphB2-deficient neurons, indicating a keyrole for the EphB2 extracellular domain in syn-aptogenesis (Kayser et al. 2008). These observa-tions suggest that EphB2 kinase-dependentfilopodial motility functions to increase the fre-quency of axo-dendritic contacts, whereas theephrinB/EphB interaction at the surface maystabilize the axo-dendritic contact sites througha cell–cell adhesion mechanism. Alternatively,the EphB2 extracellular domain, which hasbeen shown to recruit NMDA receptors to nas-cent synapses in culture, may facilitate synapseformation/maturation via a cis postsynapticmechanism (Dalva et al. 2000). Taken togetherwith the BDNF/TrkB studies, these findingssuggest that filopodial motility is part of a com-plex synaptogenic process that facilitates propersynapse density.

Contact Stabilization

The ability of a transient axo-dendritic contactto mature into a functional synapse requires sta-bilization of the cell–cell interaction (Fig. 1B).For the most part, this process is poorly under-stood. Recent imaging studies of hippocampalpyramidal neurons revealed that dendritic filo-podia make repeated, transient contacts withaxons, but only some of these contacts becomestabilized (Lohmann and Bonhoeffer 2008).The stable contacts were accompanied bydendritic filopodial calcium transients, butthese transients did not require glutamatereceptors. Although molecular mechanisms

that control axo-dendritic contact stabilizationare unknown, several high-affinity interactionsbetween cell-surface expressed proteins, suchas ephrinB/EphB, Cadherins, SynCaM, andneurexin/neuroligin, are known to regulatesynapses. EphB2 represents a good candidatefor a contact stabilizing protein, because itrequires an extracellular domain function forproper synapse density (Kayser et al. 2008).Another group of proteins implicated in axonguidance and synapse formation is the Cad-herin superfamily of proteins. Recently, anRNA interference (RNAi) screen for neuronalgenes involved in synapse formation identifiedtwo Cadherins, Cadherin 11 and Cadherin 13,as proteins required for proper glutamatergicand GABAergic synapse density (Paradis et al.2007). Although the precise role(s) of these pro-teins in synaptogenesis was not determined,they likely stabilize transient axo-dendriticcontacts via cell–cell adhesion or facilitate theclustering of synaptic proteins. Similarly, N-Cadherin regulates the maturation of dendriticspines and excitatory synapses in part throughstabilization of dendrite filopodia and spines(Togashi et al. 2002; Abe et al. 2004). Inter-estingly, Kalirin-7, a Rac-GEF that is highlyexpressed in dendritic spines and is also impli-cated in EphB- and NMDA/AMPA receptor-mediated spine maturation (Penzes et al. 2003;Xie et al. 2007), appears to function downstreamof N-Cadherin to promote spine enlargementand maturation (Xie et al. 2007). However,Kalirin-7 is only expressed during the later stages(.3 wk postnatal) of hippocampal and corticalsynaptogenesis, which explains why Kalirin KOmice have reduced cortical dendritic spine den-sity in mature, but not juvenile, mice (Cahillet al. 2009). These findings also suggest thatKalirin isoforms regulate cortical dendriticspine/synapse maintenance rather than synapseformation, although it is difficult to separatethese processes in the steady-state environmentof the brain.

Synaptic Maturation

Once a stable contact has been formed, synap-tic machinery must be recruited to the site of





the nascent synapse (Yuste and Bonhoeffer2004; Tada and Sheng 2006), including postsy-naptic recruitment of ion channels, scaffoldingproteins, signaling proteins, and componentssuch as protein translational machinery. Onthe presynaptic membrane, recruitment of synap-tic vesicles, ion channels, transmembrane recep-tors, vesicle docking proteins, mitochondria,etc., must occur at the site of the developingfunctional synapse. In some cases, the pre-synaptic specializations form before the axo-dendritic contact and anchor a location whereappropriate synaptogenesis must occur. Axonguidance molecules are clearly involved in thefunctional and morphological maturation ofsynapses.

Ephrins and Ephs

An important early study found that addition ofsoluble ephrinB to immature neuronal culturesinduced the formation of EphB/NMDA recep-tor coclusters on dendrites, and increased thenumber of functional synapses. The EphB-NMDA receptor interaction required the extrac-ellular domains of EphB2 and NR subunits,suggesting a molecular mechanism by whichephrinB/EphB binding facilitates synapse mat-uration through direct recruitment of NMDAreceptors to the site of a forming synapse (Dalvaet al. 2000). Activation of EphB2 was also foundto induce Src kinase-dependent phosphoryla-tion of NMDA receptors, which in turn in-creased NMDA receptor calcium permeability(Takasu et al. 2002). Triple and double mutantmice lacking EphB1, EphB2, and EphB3 showedsignificant deficits in dendritic spine formationand clustering of AMPA and NMDA receptors,consistent with a cell-autonomous, forward sig-naling role for EphB receptors in dendritic spineformation and synapse maturation (Henkeme-yer et al. 2003). Also, addition of soluble ephrinBligands to mature dissociated hippocampal neu-rons induced a rapid formation of dendriticspines, which was dependent on the Rac GEF,Kalirin-7 (Penzes et al. 2003). Similar roles forthe Rac GEFs, Tiam1 and Intersectin, in EphB2-mediated dendritic spine and synapse matura-tion were also described (Irie and Yamaguchi

2002; Tolias et al. 2007), suggesting that thereare multiple mechanisms by which activatedEphB receptors stimulate F-actin dynamics dur-ing dendritic spine growth and synapse forma-tion. In addition, EphB2 binds and activatesVav2 in cultured neurons (Cowan et al. 2005),and Vav2 is enriched in developing synapses (C.Hale and C. Cowan, unpubl.), indicating thatmultiple Rho GEFs likely contribute to dendriticspine growth and dynamics.

Using a dominant–negative approach incultured hippocampal neurons, ephrinB1 reve-rse signaling in dendrites was shown to contrib-ute to mushroom-type spine formation via asignaling mechanism involving the adaptor pro-tein, Grb4, and the G protein-coupled recep-tor kinase-interacting protein 1 (GIT1) (Seguraet al. 2007). Taken together with findings thatephrinB3 functions postsynaptically to regulateshaft synapse formation (Aoto et al. 2007), thesefindings suggest that in some contexts, ephrinBreverse signaling in the dendrite can regulatepostsynaptic development (Fig. 3C).

In addition to postsynaptic roles in synapsematuration, the ephrinB/EphB interactions ap-pear to contribute to presynaptic development(Fig. 3B). EphrinB1 was shown to be enrichedin presynaptic axon terminals in the developingXenopus retinotectal system, and activation ofephrinB1 reverse signaling by incubation withthe soluble extracellular domain of EphB2 in-creasedsynapsenumber,basal synaptic transmis-sion, the AMPAR/NMDAR ratio, and activity-induced LTP (Lim et al. 2008), likely because ofaccelerated presynaptic maturation.

Taken together, these findings suggest criticalroles forephrinsandEphsduringsynapsematura-tion. EphBs appear to promote synapse formationthroughboth extracellularandintracellularmech-anismsbyregulatingcell–celladhesion,activationof F-actin remodeling, recruiting synaptic pro-teins to the nascent synapse, and activating eph-rinB reverse signaling.

Wnts

Wnt signaling has been shown to be importantfor various aspects of neuronal develop-ment and plasticity in the nervous system. In





mammalian cerebellar development, Wnt-7awas shown to play an important role in presy-naptic maturation (Hall et al. 2000). Wnt-7amutant mice had significant delays in presynap-tic mossy fiber morphological maturation andclustering of the presynaptic protein, synapsinI (Hall et al. 2000). Synapsins are abundant syn-aptic vesicle proteins that play important mod-ulatory roles in synaptogenesis and synaptictransmission. The cerebellar findings are consis-tent with a recent report that Wnt-7a stimulatespresynaptic protein clustering and facilitatespresynaptic neurotransmitter release probabil-ity in CA3-CA1 hippocampal synapses (Cerpaet al. 2008). In addition, Wnts may also playroles in postsynaptic mechanism of synapto-genesis and plasticity. A recent study reportedthat Wnt-5a induced clustering of PSD-95in dendritic spines through a JNK-dependentsignaling pathway, and Wnt-5a also modulatedglutamatergic synaptic transmission through apostsynaptic mechanism (Farias et al. 2009).

Guidance Molecules in GeneralSynaptogenesis

Semaphorins

Semaphorins are a large family of secreted ormembrane molecules that play diverse roles inaxon guidance (Tran et al. 2007). However, sev-eral recent studies indicate an additional rolefor Semaphorins in synapse formation andstability. An RNAi-based screen for proteinsinvolved in hippocampal synapse formationidentified two class-4 Semaphorins as regulatorsof synaptogenesis. RNAi-mediated reduction ofSema4B significantly decreased both glutama-tergic and GABAergic synapse number in cul-tured neurons, whereas reduction of Sema4D, aclose family member, was found to selectively de-crease GABAergic synapse density (Paradis et al.2007). These observations revealed that there areboth common and synapse subtype-specific fac-tors that regulate synaptogenesis. Semaphorin5B also appears to negatively regulate synapsedensity because overexpression of Sema5B oraddition of exogenous Sema5B reduced synapsedensity, whereas RNAi-mediated reduction of

Sema5B increased synapse density (O’Connoret al. 2009). The receptor(s) that mediates theSema5B synapse effect is unknown, but it is inter-esting to note that Sema5B-induced growth conerepulsion is mediated by a Ca2þ-dependent mec-hanism (To et al. 2007).

Class 3 semaphorins bind to the neuropilinand plexin coreceptors to promote axonal repul-sion orattraction (Tran et al. 2007). Interestingly,Neuropilins are found in synaptic compart-ments of hippocampal neurons. Neuropilin-2null mice have an increased dendritic spine den-sity on CA1 neurons, although this phenotypewas accompanied by a reduced dendritic arborcomplexity and length (Gant et al. 2009). Simi-larly, mice with null mutations for Sema3F,or its coreceptors Neuropilin-2 and PlexinA3, have increased spine sizes and densities indentate gyrus and cortical pyramidal neuronsin apical, but not basal, dendrites (Tran et al.2009).

Guidance Molecules in Synapse Plasticity

Functional synapse plasticity is generally de-fined as a process by which the strength of a syn-apse is changed, and as a likely mechanism forlong-term memory and behavioral plasticity, itrepresents a major area of current neuroscienceresearch. Recent findings suggest a potentialcausal relationship between morphological den-dritic spine plasticity and functional synapseplasticity. In the mammalian brain, establisheddendritic spines were once thought be fairlystatic and stable structures. However, the devel-opment of time-lapse in vitro and in vivo imag-ing techniques has allowed for a more carefulanalysis of CNS dendritic spine dynamics. Ingeneral, established dendritic spines continueto change shape and size over time (Dunaevskyet al. 1999; Trachtenberg et al. 2002; Honkuraet al. 2008); however, the magnitude of thechanges appears to be smaller in mature neu-rons than younger ones, and larger dendriticspines are less dynamic than smaller spines(Yasumatsu et al. 2008).

As dendritic spines are rich in F-actin, it isnot surprising that intrinsic and extrinsic regu-lation of F-actin assembly, branching, and





disassembly control spine dynamics. Fluctua-tions in spine size/shape in slice cultures appearto be mediated by both synaptic activity-de-pendent and -independent mechanisms (Yasu-matsu et al. 2008). The activity-independentfluctuations are likely because of cell intrinsicproperties and may reflect the “noise” of basalactin cytoskeleton remodeling and basal activityof actin remodeling proteins in the spine, per-haps similar to the dynamic changes observedin the constantly changing growth cone mor-phology during basal axon outgrowth. Theimportance of basal dendritic spine “morph-ing” to synaptic and neural circuit function isunclear. In contrast, synaptic activity-inducedspine enlargement is correlated with long-termsynaptic potentiation (LTP), and vice versa forspine head shrinking and induction of long-term depression (LTD) (Matsuzaki et al. 2004;Okamoto et al. 2004; Honkura et al. 2008; Tan-aka et al. 2008). Indeed, pharmacological in-hibition of F-actin polymerization blocks thelate phase of LTP, but not the early phase (Kimand Lisman 1999; Krucker et al. 2000; Fukazawaet al. 2003; Rex et al. 2007), suggesting thatF-actin remodeling is critical for stabilizingthe activity-induced changes that underlie

potentiation (e.g., insertion of surface AMPAreceptors at the synapse and expansion of thePSD). The molecular and cellular mechanismsby which F-actin polymerization stabilizes func-tional synapse plasticity are not clear, but genesinvolved in growth cone F-actin dynamics aregood candidates for mediating dendritic spineplasticity (Fig. 4).

BDNF/TrkB

BDNF/TrkB signaling has been linked to func-tional synapse plasticity in numerous studies(Fig. 4). Recent findings indicate that F-actinremodeling and dendritic spine growth mayunderlie the BDNF/TrkB effects on long-last-ing LTP. Genetic or functional interferencewith BDNF or TrkB results in LTP deficits(Korte et al. 1995; Patterson et al. 1996; Mini-chiello et al. 1999; Pozzo-Miller et al. 1999;Kossel et al. 2001; Rex et al. 2007). Consistentwith these observations, addition of exogenousBDNF to wild-type hippocampal neuronsincreases tetanus-induced LTP in a Trk kinase-dependent manner (Figurov et al. 1996; Rexet al. 2007), and this effect correlates with anincrease in spine F-actin content (Rex et al.

Axon

PresynapticLTP

Presynapticplasticity

EphB2

EphB?TrkB

BDNF

Proteinsynthesis

Spinegrowth

?

CA3-CA1MF-CA3CA3-CA1

GRIPGRIP

AMPAreceptor

clustering

LTP LTD

Glutamate

NMDAreceptors

EphrinB3 EphrinB2

EphrinB3

EnhancedLTP

PP

P PP

Figure 4. Model for ephrinB, EphB, and BDNF/TrkB signaling in structural and functional synapse plasticity inthe hippocampus.





2007). Also, a long-lasting form of dendriticspine enlargement, which was induced by localglutamate uncaging combined with postsynap-tic spikes (spike-timing protocol), was depend-ent on BDNF/TrkB signaling (Tanaka et al.2008). Addition of glutamate to a dendritic spineis sufficient to induce transient spine headenlargement (Matsuzaki et al. 2004; Tanakaet al. 2008) in a NMDA receptor-dependentmanner, but endogenous BDNF/TrkB signalingappears to stabilize the spine enlargement.Together, these findings suggest that stimulus-induced BDNF release and TrkB signaling maystabilize dendritic spine growth and LTP via anF-actin-dependent process.

Although the effects of BDNF on dendriticspine morphology/density and LTP are wellestablished, the downstream signaling mecha-nisms by which TrkB mediates these effects areless clear. Several studies have reported that theBDNF-induced effects on spine and synapseplasticity require new protein synthesis. A recentstudy (Schratt et al. 2006) revealed that a micro-RNA, miR-134, is located in dendrites and spinesand suppresses local translation of Lim kinase(LimK), a F-actin-regulating protein that is acti-vated by Rac/Cdc42 family GTPases (via PAK).BDNF appears to block miR-134’s translationalrepressor function, which allows for local synap-tic translation of LimK to facilitate dendritespine growth. Consistent with this idea, LimK-deficient mice have smaller synapses and spineslacking actin (Meng et al. 2002).

Rho-family GTPases have been implicated inspine growth and maturation (Tashiro and Yuste2004). As such, BDNF likely controls spine plas-ticity by regulating Rho family GTPases. Werecently found that BDNF/TrkB activates Vav2,a Rho/Rac family GEF that is highly expressedin the developing brain and synapses. Vav GEFsare required for BDNF-induced Rac-GTPactiva-tion and CA1 dendritic spine growth (C. Hale,K. Dietz, and C. Cowan, unpubl.), revealing animportant role for Vav GEFs in BDNF-dependentspine growth and synapse plasticity. Similarly,Tiam1mediatesBDNF-inducedRac-GTPforma-tion and BDNF-induced chemotaxis in the devel-oping cerebellum (Zhou et al. 2007b), and Tiam1also appears to mediate BDNF-induced neurite

outgrowth in cultured cortical neurons (Miya-moto et al. 2006), suggesting that BDNF-inducedF-actin dynamics may be mediated by different ormultiple Rho GEFs, depending on the neuronalpopulation, development stage, and subcellularstructure.

In addition to a postsynaptic role for BDNF/TrkB signaling during induction/maintenanceof LTP or dendritic spine growth, BDNF hasalso been reported to alter presynaptic func-tions, such as regulation of synaptic vesicle re-lease probability (Tyler and Pozzo-Miller 2001)(Fig. 4). Similarly, mice lacking presynapticTrkB in the CA3 hippocampal neurons havestructural and electrophysiological presynapticdefects consistent with TrkB playing an essentialpresynaptic role in development and plasticity(Luikart et al. 2005).

Ephrins and Ephs

In addition to a clear role in modulating synapseformation during early hippocampal develop-ment, ephrins and Ephs have also been reportedto regulate morphological spine plasticity andLTP (Fig. 4). EphrinB2 knockout mice havesevere deficits in both LTP and LTD (Grunwaldet al. 2004; Bouzioukh et al. 2007). Phosphory-lation of five ephrinB2 intracellular domaintyrosines is required for normal CA3-CA1 hip-pocampal LTP, but not for LTD, whereas thePDZ-binding site on the carboxyl terminus ofephrinB2 is required for both LTP and LTD(Bouzioukh et al. 2007). The molecular mecha-nisms are not clear; however, ephrinB2 appearsto regulate AMPA receptor trafficking and sta-bilization of surface AMPAR expression throughrecruitment of the glutamate receptor-inter-acting proteins (GRIP1 and GRIP2) (Essmannet al. 2008).

Similar to ephrinB2, mice lacking ephrin-B3 have deficits in CA3-CA1 LTP (Grunwaldet al. 2004; Rodenas-Ruano et al. 2006); how-ever, no deficit was observed in another study(Armstrong et al. 2006). Interestingly, ephrin-B3-mediated LTP does not require its intra-cellular domain (i.e., reverse signaling) for CA3-CA1 LTP (Rodenas-Ruano et al. 2006). In con-trast, mossy fiber-CA3 LTP, which involves a





presynaptic ephrinB3 function, does requireephrinB3 reverse signaling (Armstrong et al.2006). Interestingly, ephrinB1 in the developingXenopus retinotectal synapses appears to regu-late neurotransmitter release and postsynapticdevelopment and function via a presynapticmechanism (Lim et al. 2008).

Ephs also contribute to functional and mor-phological synapse plasticity. In EphB2 nullmice, CA3-CA1 LTP is reduced (Hendersonet al. 2001), but no LTP defects are observed inEphB2 knock-in mice lacking its intracellularregion (Henkemeyer et al. 1996). This suggeststhat EphB2 functions presynaptically as theligand for ephrinBs (postsynaptic), which arerequired for normal LTP (see previous), or thatEphB2 might function on the postsynaptic neu-rons through an extracellular domain-dependentprocess, or both. The former idea is the moststraightforward considering the phenotypes ofephrinB2/3 null mice, but the extracellular do-main of EphB2 mediates an interaction withNMDA receptors (Dalva et al. 2000) andNMDA receptors are required for LTP. Therefore,future studies withselective knockout ofEphB2 inCA3versus CA1neurons will be helpful toaddressthis issue. Note that the kinase-independent roleof EphB2 for LTP is in contrast to its kinase-dependent role in spine formation, filopodialmotility, and synapse formation, suggesting thatonce synapses are formed, EphB2 functions in anon-cell autonomous fashion to regulate synapticplasticity.

The situation in the mossy fiber-CA3 LTP ismore complex than CA3-CA1 LTP. In this case,postsynaptic EphB receptors appear to facilitateLTP via a PDZ domain-dependent interactionwith GRIPs, which cluster AMPA receptors,and by acting as a ligand for presynaptic eph-rinBs (Contractor et al. 2002). Activation ofephrinB reverse signaling with soluble EphB2-Fc increased basal synaptic transmission, andthis occluded tetanus-induced LTP, suggest-ing that enhancements of presynaptic functionfacilitate mossy fiber-CA3 LTP. Unfortunately,developmental defects in CA3 region in theEphB2 null mice precluded a more defini-tive analysis of the CA3 EphBs involved in thisprocess.

EphA receptors and ephrinAs have also beenimplicated in synaptic plasticity. Hippocampalperfusion of a soluble EphA5-Fc fusion proteinreduced tetanus-induced LTP, possibly by bloc-king EphA forward signaling, whereas incuba-tion with ephrinA5-Fc partially mimicked LTP(Gao et al. 1998). In addition, mice lackingEphA4 show kinase-independent deficits inCA3-CA1 LTP (Grunwald et al. 2004), andCA1-specific EphA4 knockout mice have CA3-CA1 LTP deficits (Filosa et al. 2009). In thiscase, CA1-expressed EphA4 appears to activateephrinA3 reverse signaling in astrocytes, whichin turn regulates glial glutamate transporterlevels. Consistently, ephrinA3 null mice alsohave CA3-CA1 LTP deficits (Filosa et al. 2009).In contrast, EphA4 appears to contribute to de-ndritic spine retraction in an Eph kinase-dependent manner (Murai et al. 2003; Bourginet al. 2007; Richter et al. 2007; Zhou et al. 2007a;Carmona et al. 2009). This process may be me-diated by a Cdk5-dependent phosphorylationof Ephexin1 and activation of RhoA (Fu et al.2007) and/or by inhibition of b1-Integrin sig-naling that control spine stability through inter-actions with the extracellular matrix (Bourginet al. 2007). EphA4 in dendritic spines appearsto be activated by glial cell-expressed ephrinA3,and mouse knockouts of either of these geneshave dendritic spine abnormalities (Muraiet al. 2003; Carmona et al. 2009). However, therelevance of the ephrinA3/EphA4 regulationof spine morphology for functional synapseplasticity is not yet clear.

Nogo Recepter

Nogo ligand and Nogo Receptors were origi-nally described for their function in blockingaxon outgrowth after spinal cord injury; how-ever, recent reports indicate that Nogo Receptor1 (NgR1) is also involved in hippocampal syn-apse plasticity (Lee et al. 2008). Specifically,mice lacking NgR1 had attenuated CA3-CA1LTD, while expressing normal LTP. However,the molecular mechanism by which NgR1 regu-lates LTD is unknown, but may involve modula-tion of co-receptors, like the FGF receptors (Leeet al. 2008).





Semaphorins and Plexins

Class 3 semaphorins (Sema3A-3F) function assoluble, secreted ligands that bind and activatethe co-receptor complexes that contain plexinsand neuropilins (de Wit and Verhaagen 2003).These ligands and receptors are not only ex-pressed in the developing brain, but are alsoexpressed in the adult brain. Neuropilin1 and2 are both found in the synapse (Sahay et al.2005; Bouzioukh et al. 2006), and recombinantSema3A and Sema3F bind throughout the juve-nile and adult hippocampus. Addition of solu-ble Sema3F increased synaptic responses fromboth CA1 pyramidal and dentate gyrus granuleneurons through a postsynaptic mechanism(s)(Sahay et al. 2005). Sema3F null mice werefound to be prone to seizures, but it is difficultto know if this phenotype is related to theobserved effects on synaptic transmission. Incontrast to the effects of Sema3F, acute applica-tion of soluble Sema3A induced rapid, synapticdepression in adult CA1 hippocampal neuronsthat was reversed on Sema3A washout (Bou-zioukh et al. 2006). Interestingly, mRNA levelsof some semaphorins and neuropilins are regu-lated by experience and activity (Shimakawaet al. 2002; Barnes et al. 2003; Holtmaat et al.2003; O’Donnell et al. 2003), suggesting that re-gulation of these ligand/receptor genes mayplay a role in synapse plasticity. A key down-stream target of Sema3A/Npn1/PlexinA signal-ing in axon guidance is the collapsin responsemediator protein (CRMP), which binds to tu-bulin dimers and regulates microtubule assem-bly (Fukata et al. 2002). Interestingly, CRMP1null mice had reduced CA3-CA1 LTP anddeficits in spatial memory (Su et al. 2007), sug-gesting the interesting possibility that CRMP-dependent regulation of microtubules may becritical for synapse plasticity.

Wnts

Wnt signaling has been shown to modulate bothpresynaptic and postsynaptic functions, suchthat Wnt-7a can increase presynaptic CA3-CA1hippocampal vesicle release probability (Cerpaet al. 2008) and Wnt-5a appears to modulate

synaptic transmission through a postsynapticmechanism possibly involving PSD-95 clus-tering in dendritic spines (Farias et al. 2009).Wnt signaling appears to play an importantrole in experience-dependent changes in synap-tic connectivity in the hippocampus in vivo.Mice exposed to an enriched environmentshowed increased MF-CA3 spine and synapsenumbers, which involved postsynaptic expres-sion of Wnt-7a/b in CA3 hippocampal neurons(Gogolla et al. 2009). Taken together, thesestudies suggest that Wnt signaling will emergeas a critical regulator of synapse function invertebrates.

Synaptic Scaling

The TGF-b family of secreted proteins serves asmorphogens that play diverse functions in de-velopmental biology. The TGF-b family mem-bers, BMP7 (Augsburger et al. 1999; Butlerand Dodd 2003) and UNC-129 (Colavita et al.1998), act as instructive cues for axon guidance.However, TGF-b family genes also appear tomediate synaptic scaling during development,a process whereby the nervous system expandsto accommodate the growth of body size. AtDrosophila neuromuscular junctions, loss-of-function mutants of the BMP7 homolog, gbb,or its receptor wit, Thv, and Sax, or downstreamsignaling molecules Mad and Medea, displaydecreased number of synapses, ultrastructuraldefects, and severely compromised synaptictransmission during the larval growth stage(Marques 2005). Gbb is expressed in the postsy-naptic muscles, whereas the receptors and sig-naling components are required in the axon(McCabe et al. 2003). Hence, Gbb can poten-tially act as a retrograde signal to stimulate thegrowth of NMJs. One study indicates thatdiminished presynaptic glutamate release orinsufficient Ca2þ entry in the muscle cells leadsto reduced activity of CaMKII in the muscle,which then triggers the release of the Gbb. Inturn, Gbb acts through the presynaptic Witreceptor to activate the compensatory growthprogram (Haghighi et al. 2003). Therefore, Gbbis the output of a feedback loop to coordinatethe growth of the muscle and the presynaptic





terminals to ensure sufficient depolarization dur-ing development.

SUMMARY AND CONCLUSIONS

Overwhelming evidence suggest that many mol-ecules that were initially discovered for their func-tions in axon guidance also play importantfunctions in synapse formation. The reuse ofmolecules in different developmental processesis not a new concept. For example, many morph-ogens such as Wnts and Hedgehogs are criticalfor cell fate induction at early developmentalstages. However, they also play important rolesin later events including axon guidance. Whereasthere are many examples of molecules involved inboth axon guidance and synapse formation, theexisting literature offers very little insight regard-ing how diverse cellular events can be regulated bythe same membrane receptors. Future researchon the downstream components of these receptorpathways in both axon guidance and synapse for-mation will shed light on how they are mechanis-tically linked.

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March 10, 20102010; doi: 10.1101/cshperspect.a001842 originally published onlineCold Spring Harb Perspect Biol

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David A. Feldheim and Dennis D. M. O'Leary

Human Genetic Disorders of Axon GuidanceElizabeth C. Engle

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