Trans-synaptic EphB2–ephrin–B3 interaction regulatesexcitatory synapse density by inhibition ofpostsynaptic MAPK signalingAndrew C. McClellanda,1, Martin Hruskaa,1, Andrew J. Coenena, Mark Henkemeyerb, and Matthew B. Dalvaa,2

aDepartment of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and bDepartment of Developmental Biology and KentWaldrep Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390

Edited by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 25, 2010 (received for review September17, 2009)

Nervous system function requires tight control over the number ofsynapses individual neurons receive, but the underlying cellular andmolecular mechanisms that regulate synapse number remain ob-scure. Herewe present evidence that a trans-synaptic interaction be-tween EphB2 in the presynaptic compartment and ephrin-B3 in thepostsynaptic compartment regulates synapse density and the forma-tion of dendritic spines. Observations in cultured cortical neuronsdemonstrate that synapse density scales with ephrin-B3 expressionlevel and is controlled by ephrin-B3–dependent competitive cell–cellinteractions. RNA interference and biochemical experiments supportthemodel that ephrin-B3 regulates synapse density by directly bind-ing to Erk1/2 to inhibit postsynaptic Ras/mitogen-activated proteinkinase signaling. Together these findings define a mechanism thatcontributes to synapse maturation and controls the number of excit-atory synaptic inputs received by individual neurons.

cell signaling | development | Ras/MAPK | synaptogenesis | competition

Neuronal activity is a key determinant of maintaining andcontrolling the number of synaptic connections (1, 2), but the

molecular mechanisms likely to establish the normal density ofsynaptic contacts are still not well defined (3). Synapse densitycould be controlled by either secreted or cell surface molecules,but trans-cellular interactions are attractive because of their abilityto coordinate events between cells. One family of synaptic adhe-sion molecules that is well suited to control synapse density isephrin-Bs (eBs), a family of three (eB1–3) ligands for the EphBfamily of receptor tyrosine kinases (4). Recent work suggests thatephrins may negatively regulate Ras/MAPK activation in non-vertebrate systems during morphogenesis (5), and that MAPKsignaling can negatively regulate presynaptic terminal maturation(6, 7). Here we show that eB3 controls synapse density and theformation of dendritic spines through a competitive postsynapticmechanism relying on inhibition of MAPK signaling.

ResultsEphrin-B3 Expression Level Controls Synapse Density. Early in de-velopment [5–10 days in vitro (DIV 5–10)], there are few dendriticspines and synapses that are rapidly added on the dendritic shaft.During this time, eB3 is enriched at excitatory synapses but is alsolocalized to many extrasynaptic locations (Fig. 1A). As neuronsmature (DIV 14–21), dendritic spines are formed and become theprincipal site of excitatory synapses (8); eB3 becomes largely re-stricted to spine and shaft excitatory synaptic contacts (Fig. 1A andFig.S1A).The synapticpatternof eB3expression suggests that itmaybe involved in the formation and maturation of excitatory synapses.We asked whether the expression level of eB3 is related to the

density of excitatory synapses on individual neurons by plotting thedensity of endogenous PSD-95 puncta vs. two different measuresof eB3 expression: the density of eB3 puncta and the overall in-tensity of eB3 immunofluorescence.We found that bothmeasuresof eB3 expression vary from neuron to neuron and are correlatedwith PSD-95 puncta density (Fig. 1 F–H). We next examined the

relationship between expression level and synapse density of threedifferent protein families (neuroligins, synCAMs, and EphB2)known to control synapse formation (4), but none of these threefactors had any significant correlation between their intensity ofexpression and PSD-95 puncta density (SI Text and Fig. S2). Thus,unlike other known synaptogenic factors, the level of eB3 ex-pression is positively correlated with the density of PSD-95 puncta.To test whether the amount of eB3 expression in an individual

neuron determines the number of synapses it receives, we artificiallyreduced the expression of eB3 using previously characterized shorthairpinRNA(shRNA) (9) thathadnoeffect on synapsedensitywhenexpressed in neurons from ephrin-B3−/− (eB3−/−) mice (SI Text andFig. S3 A and B). Cortical neurons were cotransfected with eB3shRNAconstructsandGFPatDIV0andstained forendogenouseB3and PSD-95 at DIV 9. Because neurons normally express differentamounts of eB3, knockdown (kd) results in a range of expressionlevels (Fig. 1G).We found that kd of eB3, but not eB1 or eB2, causesa significant decrease in synapse number (Fig. 1 C–E and Figs. S4Band S5 A–C). The effects of eB3 kd did not require neuronalactivity (SI Text), and we found an overall decrease in both eB3 andPSD-95 puncta density relative to control (Fig. 1B). Yet, the linearcorrelation between eB3 expression and PSD-95 puncta density ineB3 kd neurons (Fig. 1 F–H) was statistically indistinguishable fromwild-type (wt)neurons (P=0.4659,eB3staining intensity;P=0.1241,eB3 puncta density; ANCOVA). These results demonstrate that eB3expression and thedensity of synaptic specializations are correlated inboth wt and kd neurons, and that the linear relationship in these twogroups is the same. Therefore, the variability in synapse density inneurons is likely a result of differences in eB3 expression level.To test whether eB3 expression affects the number of functional

synapses that a neuron receives, we transfected DIV 0 corticalneurons with eB3 shRNA constructs and GFP, and recorded miniexcitatory synaptic currents (mEPSCs) at DIV 9–10. Expression ofeB1 shRNA resulted in mEPSC that occurred at normal (∼1-Hz)frequency but had smaller amplitudes (Fig. 2 A, C, and D). Incontrast, kd of eB3 with two different shRNAs resulted in neuronswith fewer and smaller mEPSCs (∼0.2 Hz; Fig. 2 A and C) withoutaffecting mIPSC frequency (Fig. S6). Cotransfection of eB1 or eB3shRNA with constructs encoding kd-insensitive ephrin-B1 or eB3(HAeB1R or HAeB3R, respectively) resulted in rescue of the ob-served changes, indicating that these effects are specific to loss of

Author contributions: A.C.M., M. Hruska, A.J.C., and M.B.D. designed research; A.C.M.,M. Hruska, A.J.C., and M.B.D. performed research; A.C.M., A.C., and M.B.D. contributednew reagents/analytic tools; M. Henkemeyer provided the ephrin-B3−/− mice; A.C.M.,M. Hruska, A.J.C., and M.B.D. analyzed data; and A.C.M., M. Hruska, and M.B.D. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1A.C.M. and M. Hruska contributed equally to this work.2To whom correspondence should be addressed. E-mail: dalva@mail.med.upenn.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0910644107/-/DCSupplemental.

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the targeted eB molecule (Fig. 2 A, C, and D). We confirmed theeffects on synapse density after kd and rescue using immunostainingfor synaptic marker proteins (Fig. 1 C–E and Figs. S4B and S7).These results indicate that kd of eB3, but not eB1, reduces thedensity of functional excitatory synapses a neuron receives.Next, by varying the degree of kd in single neurons (9) (Fig.

S1B), we tested whether different levels of eB3 kd might result ina graded effect on mEPSC frequency. Consistent with a role incontrolling synapse density, transfecting increasing amounts ofeB3 shRNA constructs into single neurons led to a progressivereduction in mEPSC frequency (Fig. 2E). Increasing eB3 expres-sion by cotransfecting eB3 shRNA constructs with differentamounts of the HA-tagged eB3 rescue construct (HAeB3R) or byoverexpressing eB3 without kd led to mEPSC frequencies greaterthan normal (Fig. 2F; eB3 overexpression: 2.2 ± 0.7 Hz, n = 9).Taken together, these results provide evidence that the post-synaptic expression level of eB3 in an individual neuron is a keydeterminant of the density of synapses that neuron receives.

Ephrin-B3−/− Mice Have Fewer Dendritic Spines but Normal Numbersof Synapses. To test whether eB3 is required for normal dendriticspine density, we examined dendritic spines in cortical and hippo-campal cultures following eB3 kd (SIText andFigs. S1C–F and S5Eand F) or in cortical brain slices from wt or eB3−/− mice. In cellculture, we found a decrease in spine density and a significant de-crease in the density of both spine and shaft synapses following eB3kd (SI Text and Fig. S1 C–F). Pyramidal neurons from eB3−/− cor-

tical brain slices had grossly normal arbors. However, the overalldensity of dendritic protrusions was significantly reduced (Fig. 3 Aand B) with a ∼60% reduction in dendritic spine density (Fig. 3C)and no change in the density of filopodia-like protrusions (Fig. 3D).To address whether cortical neurons from eB3−/− mice have fewersynapses, we determined synapse density in layer 5 of cortical cryo-sections from wt and eB3−/−mice by measuring overlap of pre- andpostsynaptic markers (10). Consistent with previous reports (11–13), we found no significant difference in excitatory synaptic densitybetween wt and eB3−/− mice (Fig. 3 F andG).The majority of excitatory synapses on cortical neurons are

found on spines, yet neurons from eB3−/− mice have fewer spinesthan wt littermates but normal numbers of excitatory synapticspecializations; this suggests that loss of eB3 leads to a relocali-zation of synaptic specializations. Although neurons from sliceslacking eB3 had PSD-95-GFP puncta density that was similar towt (Fig. 3 H–J), PSD-95-GFP puncta were found primarily alongthe dendritic shaft with few puncta found in dendritic pro-trusions, indicating that the reduced number of spines in eB3−/−

mice results in a redistribution of synaptic specializations to thedendritic shaft of cortical neurons.

Ephrin-B3 Controls Synapse Density Through Competitive Cell–CellInteractions. Neurons lacking eB3 have normal synapse numbers,but eB3 kd reduces excitatory synapse density. Based on thesefindings, one likelymechanismmediating eB3 dependent control ofsynapse density is competitive cell–cell interactions. In this model,

Fig. 1. Synapse density is proportional to eB3 expression. (A) DIV-8 (Upper) and DIV-21 (Lower) cortical neuron dendrites transfected with GFP at DIV 0 andstained with anti-eB3 (green), anti-PSD-95 (red), and anti-VGlut1 (blue) antibodies. Grayscale images show the pattern of eB3 staining. (B) Quantification ofeB3 puncta density in neurons transfected with vector control (n = 27), eB3 shRNA#1 (n = 32), or eB3 shRNA#2 (n = 15). (C) DIV-9 neurons transfected with GFPplus indicated shRNA construct at DIV 0 and stained with anti-GFP (green), anti–PSD-95 (red), and anti-VGlut1 (blue) antibodies. Arrows indicate synapticpuncta identified by colocalized PSD-95 and VGlut1 immunostaining. (D) Quantification of colocalized PSD-95 and VGlut1 synaptic puncta density for vectorcontrol (n = 29), ephrin-B1 shRNA (n = 19), eB3 shRNA#1 (n = 31), or eB3 shRNA#2 (n = 16). (E) Quantification of colocalized PSD-95 and VGlut1 synaptic punctadensity for vector control (n = 26), eB3 shRNA#1 (n = 23), or ephrin-B2 shRNA (n = 24). (F) DIV-9 cortical neuron dendrites transfected with GFP plus eB3 shRNAconstructs and stained with anti-GFP (green), anti-eB3 (red), and anti-PSD-95 (blue) antibodies. Arrows and arrowheads indicate eB3 or PSD-95 puncta. (G andH) Plot of (G) eB3 puncta density or (H) eB3 staining intensity in arbitrary units vs. PSD-95 puncta density in DIV-9 neurons expressing GFP plus vector control[filled circles: (G) PSD-95 puncta density × eB3 puncta density, R2 = 0.515, P < 0.0001; (H) PSD-95 puncta density × eB3 staining intensity, R2 = 0.481, P < 0.0001]or eB3 shRNA#1 [open circles: (G) PSD-95 puncta density × eB3 puncta density, R2 = 0.272, P < 0.002; (H) PSD-95 puncta density × eB3 staining intensity, R2 =0.354, P = 0.0002) stained with anti-eB3, anti–PSD-95, and anti-GFP antibodies. Plot is linear regression curve for all cells. (Scale bars, 3 μm.) Error bars indicateSEM. *P < 0.05, **P < 0.0001.

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a neuron expressing more eB3 would receive more synapses thananearby neuronexpressing less eB3, butwithouteB3 therewouldbeno signal generated. To test whether the level of eB3 expressedmight enable competition between neurons, we asked whether theability of eB3−/−neurons tomakenormal numbers of synapsesalongtheir dendrites (Fig. S3 A and B) could be disrupted by simplycoculturing them with neurons expressing normal levels of eB3. Todo this, we made heterogenotypic cultures containing mixtures oflabeled and unlabeled cortical neurons from wt and eB3−/− mice.Labeling was carried out in suspension using electroporation ofGFP (Fig. S8). Although synapse density in the GFP expressingneurons at DIV 10 in wt\wt or eB3−/−\ eB3−/− cultures (Fig. 4,A and B) was similar, GFP-positive eB3−/− neurons mixed with wt

neurons had significantly fewer synapses (Fig. 4 A–C and Fig. S4C).Thus, eB3−/− neurons have normal numbers of synapses when sur-rounded by other eB3−/− neurons, but not when they are surroundedby wt neurons. Moreover, GFP-expressing wt mouse neurons hadsignificantly higher synapse density when cultured with unlabeledeB3−/− neurons (Fig. 4 A–C and Fig. S4C). Thus, changes in therelative level of eB3 expression can selectively increase or decreasethe density of synapses a neuron receives. Because the normal syn-apse density that we observe in eB3−/− neurons is not likely due tocompensation by other synaptogenic molecules (SI Text and Fig.S3C) or culture conditions (SI Text and Fig. S3 A and B), our datasuggest that cell-to-cell differences in eB3 expression level enableneuronal competition that controls overall synapse density.

EphB2 Is the Presynaptic Ligand for Postsynaptic Ephrin-B3. Ourheterogenotypic culture experiments suggest that eB3 can controlsynapse density through trans-cellular interactions. Consistent withthis hypothesis, eB3 expressed in nonneuronal cells can induce

Fig. 2. Ephrin-B3 knockdown reduces mEPSC frequency. (A) mEPSCs fromwhole-cell patch-clamp recordings of neurons expressing GFP plus indicatedconstructs (calibration, 40 pA, 1 s) and individual events (Inset: calibration, 20pA, 20 ms). (B) Schematic of eB3 mutants used. HAeB3R is HA-tagged full-length eB3 rendered insensitive to eB3 shRNA#1. mCer-eB3/B1 is mCeruleantagged to the extracellular domain of eB3 fused to the intracellular domain ofephrin-B1. HAeB1/B3 is HA tagged to the extracellular domain of ephrin-B1fused to a knockdown-insensitive intracellular domain of eB3. HAeB3R_L293Ais HAeB3R in which alanine has replaced leucine 293 in a juxtamembranedomain (green star). (C) Quantification ofmEPSC frequency after transfectionwith vector control (n = 14), ephrin-B1 shRNA (n = 11), eB3 shRNA#1, 1.5 μg/well (n = 23), eB3 shRNA#2 (n = 6), eB3 shRNA#1 plus HAeB3R (n = 19), eB3shRNA#1plusmCer-eB3/B1 (n=12), eB3 shRNA#1plusHAeB1/B3 (n=6), or eB3shRNA#1 plus HAeB3R_L293A (n = 7). *P < 0.005 from control. (D) Quantifi-cation of mEPSC amplitude after transfection with vector control (n = 686),ephrin-B1 shRNA (n = 566), ephrin-B1 shRNA plus HAeB1R (n = 861), eB3shRNA#1 (n = 424), or eB3 shRNA#1 plus HAeB3R (n = 2979). ***P < 0.001. (E)Quantification of mEPSC frequency after transfection of 0.25 μg (n = 9) or 1.5μg (n = 5) eB3 shRNA#1 per well. (F) Quantification of mEPSC frequency aftertransfection of eB3 shRNA#1 plus 0.25 μg (n = 8) or 1.0 μg (n = 13) HAeB3R perwell. *P < 0.05. Error bars indicate SEM.

Fig. 3. Reduced spine density in eB3−/−mice. (A) Cortical neuron dendrites inslice culture fromP4-6wt or eB3−/−mice transfectedwithGFP. Arrows indicatespines; arrowheads indicatefilopodia-like protrusions. (B–E) Quantification ofprotrusion density for (B) all protrusions, (C) spines, (D) filopodia-like pro-trusions, and (E) protrusions classified as “other” for wt (n = 16) or eB3−/− (n =20) neurons. (F) (Top) Images of layer 5 cortical cryosections from P6 wt oreB3−/− mice immunostained with anti-VGlut1 (green) and anti-SynGAP (red)antibodies. (Scale bar, 10 μm.) (Bottom) Binary mask illustrating colocalizedVGlut1 and SynGAP puncta in above images. (G) Quantification of colocalizedVGlut1 and SynGAP puncta density in wt (n = 59 images) and eB3−/− (n = 60images) sections. (H) DIV-4–6 cortical neurondendrites in slice culture fromP4-6wtor eB3−/−mice transfectedwith tdTomato (red) andPSD-95-GFP (green) atDIV 2. Arrows indicate PSD-95-GFP puncta in dendritic protrusions. Arrow-heads indicate PSD-95-GFP puncta on the dendritic shaft. (I and J) Quantifi-cation of puncta density for (I) PSD-95-GFP puncta found in protrusions and (J)total PSD-95-GFP puncta for wt (n = 12) or eB3−/− (n = 39) neurons. (Scale bars,3 μm.) Error bars indicate SEM. *P < 0.002.

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presynaptic differentiation when cocultured with hippocampalneurons (12).We tested whether this was a unique property of eB3,or whether other eBs or ephrin-As could also induce presynapticdifferentiation. In heterologous cell assays, we find that HEK293Tcells expressing any of the three eB ligands were able to inducepresynaptic marker accumulation to a similar level as EphB2 (10)(Fig. 5A and Fig. S9). However, when cocultured with neurons,ephrin-A1–expressing HEK293T cells failed to induce an increasein VGlut1 staining (Fig. 5A and Fig. S9), suggesting that the effectswe observe are specific to eB family members and likely occurthrough interactions with presynaptic EphBs.EphB2 copurifies with both presynaptic active zones and the

postsynaptic density fraction (14). To identify whether a pre-synaptic EphB receptor mediates eB-dependent presynaptic in-duction, we used a modified coculture assay (9) in which axonsexpressing shRNA targeting potential presynaptic receptors arecocultured with eB expressing HEK293T cells (9). We transfectedneurons with constructs expressing shRNA targeting EphB2 (10,15) along with a GFP-tagged presynaptic marker synaptophysin(syn-GFP) to label transfected axons, and at DIV 7 transfectedneurons were cocultured with HEK293T cells expressing eitherHA-tagged eB3 (HAeB3) or RFP. At DIV 8, single axons at sitesof contact with eB3-expressing HEK293T cells resulted in a sig-nificant ∼1.3-fold increase in the density of syn-GFP (Fig. 5 B andC). The expression of either of two independent EphB2 shRNAconstructs in axons (15) blocked the increase in syn-GFP density(Fig. 5 B and C). These effects were rescued by coexpression ofa kd-insensitive EphB2 (Fig. 5 B and C). Thus, EphB2 appears tobe necessary for eB3-dependent presynaptic induction, suggestingthat EphB2 is the presynaptic ligand for eB3.

Erk and Ephrin-B3 Interact to Control Synapse Density. To determinehow eB3 regulates synapse density, we used a whole-cell patchclamp to recordmEPSCs in DIV 9–11 neurons and asked whetherthe intracellular domain of eB3 is required for changes in mEPSCfrequency. Although the known signaling domains of eBs areconserved (12, 16), eB3 also contains a nonconserved,∼60–amino

acid juxtamembrane domain. We constructed two chimeric pro-teins consisting of the extracellular domains of eithermCer-eB3 orHAeB1 fused to the intracellular domains of either eB1 or eB3(Fig. 2B). Both chimeric molecules reached the cell surface whenexpressed in HEK293T cells. However, expression of mCer-eB3/B1 in neurons expressing eB3 shRNA failed to rescue mEPSCfrequency, whereas coexpression of HAeB1/B3 was able to rescuemEPSC frequency (Fig. 2G). These results indicate that the reg-ulation of synapse density by eB3 requires intracellular residuesthat are distinct from those found in eB1.Sequence analysis revealed a putative Erk binding domain (D-

domain) centered at leucine 293 (L293) in the juxtamembrane re-gion of eB3. To test for an eB3-Erk interaction, we asked whethereB3 and Erk physically associate at synapses by immunoprecipita-tion from mouse brain synaptosomes. Consistent with previousreports (11) and our immunostaining data, eB3 was enriched in thesynaptosomal fraction (Fig. 6A). In purified synaptosomes (17), wefound that whereas both Erk1 and Erk2 were present at similarlevels (Fig. 6A), only Erk2 effectively coimmunoprecipitated witheB3 (Fig. 6B). Thus, eB3 andErk2appear to associate at synapses inbrain lysates.To test whether the putative Erk-D domain that we identified

mediates the association between eB3 and Erk, we conducteda GST-pulldown assay from E19 rat brain. GST fusions of eB3cytoplasmic domain effectively pulled down Erk, whereas a GSTfusion with a point mutation in the putative Erk binding domainshowed a significant reduction pull-down (Fig. 6 C and D).Consistent with our in vivo results, quantification of the relativepull-down revealed that Erk2 interacts with the intracellulardomain of eB3 more strongly than Erk1 (Fig. 6 C and D).

Fig. 4. Competitive control of synapse density by ephrin-B3. (A) Heterge-notypic cultures of P0 wt and eB3−/− littermate mice. Unlabeled neurons ofone genotype were plated followed by a second group labeled by electro-poration with GFP. Cultures were stained at DIV 10 with anti-GFP (green),anti-PSD-95 (red), and anti-VGlut1 (blue) antibodies. Arrows indicate syn-aptic puncta identified by colocalized PSD-95 and VGlut1 immunostaining.(B) Quantification of synaptic puncta density for GFP-labeled neurons inheterogenotypic cultures (wt/wt, n = 44, eB3−/−/wt, n = 67, eB3−/−/eB3−/−, n =50, and wt/eB3−/−, n = 70). (C) Model of experimental design and results.(Scale bar, 3 μm.) Error bars indicate SEM. * P < 0.002, ** P < 0.0003.

Fig. 5. EphB2 is a presynaptic ligand for eB3. (A) Quantification of inductionof VGlut1 puncta area in axons under HEK293T cells transfected with vectorcontrol (n = 78), HAeB1 (n = 70), HAeB2 (n = 63), HAeB3 (n = 68), or eA1 (n = 23).(B) DIV-10 axons from cortical neurons transfected with synaptophysin-GFP(green) and indicated shRNA constructs cocultured with HEK293T cells trans-fected with RFP or HAeB3 (red) for 16–18 h. Arrowheads indicate synapto-physin-GFP puncta colocalized with HEK293T cells. (Scale bar, 3 μm.) (C) Quan-tification of fold increase in synaptophysin–GFP density in axon segmentsunderneath HEK293T cells comparedwith adjacent axon segments for neuronstransfected with indicated shRNA constructs (cocultured with RFP-expressingHEK293T cells, HAeB3-expressing HEK293T cells): vector control (RFP: n = 111,HAeB3: n = 86), EphB2 shRNA#1 (RFP: n = 81, HAeB3: n = 85), EphB2 shRNA#2(RFP: n = 41, HAeB3: n = 45), EphB2 shRNA#2 + EphB2R (RFP: n = 55, HAeB3: n =58). Error bars indicate SEM. *P < 0.006.

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We next asked whether the putative Erk-D domain in eB3 isnecessary for eB3-dependent control of synapse density. Wegenerated a full-length HAeB3R construct in which L293 wasconverted to alanine (HAeB3_L293A, Fig. 2B) that reached thesurface of HEK293T cells and had no effect on mEPSC frequencywhen expressed inneurons alone.However, unlike coexpressionofa full-length eB3 construct insensitive to the shRNA, coexpressionofHAeB3R_L293Awith eB3 shRNAfailed to rescue the decreasein mEPSC frequency or synapse density measured by colocaliza-

tion of synaptic marker proteins (Fig. 2B and Fig. S7). Thesefindings suggest that the eB3–Erk interaction is required for eB3regulation of synapse density.To test whether eB3 dependent regulation of synapse number

relies on activation or inhibition of theMAPKpathway in culturedcortical neurons, we activated or inhibited Erk1/2 signaling byexpressing a dominant negative (DN) or constitutively active (CA)MEK (DN-MEK or CA-MEK) (18). Expression of either CA-MEK or DN-MEK alone in neurons had no significant effect on

Fig. 6. Postsynaptic eB3 acts through Erk1/2 to control synapse density. (A) Various purification fractions from three P30 mouse brains. Blots are probed withantibodies recognizing eB3, NR1, GluR2, PSD-95, and Erk1/2 (n = 3). (B) EB3 immunoprecipitated from synaptosomes and resultingWestern blots were probedwithanti-Erk1/2andanti-eB3antibodies (n=3). Lysates fromthe samepreparationsare shown in lowerblots. (C)GST fusionproteinsofwt (eB3intra)or L293Amutant (eB3L293A) intracellulardomainsofeB3wereexpressed inbacteria andused inGSTpull-downassaywith E19 ratbrain lysates. Resultingelutates and input to the columnswereanalyzedbyWesternblottingwithanti-Erk1/2antibody. FusionproteinexpressionwasanalyzedonaWesternblotwithanti-GSTantibody. (D)Quantificationofrelative Erk1 and Erk2 pull down as comparedwith total Erk1 and Erk2 input (n = 3). (E–G) Density of excitatory synapses in rat cortical neurons transfectedwith DN-MEKand either control or eB3shRNA constructs at DIV 10. (E) Quantification of the frequency ofmEPSCs vector control as in Fig. 2, DN-MEK (n = 11), CA-MEK (n = 7),eB3 shRNA#1 plus DN-MEK (n = 12), eB3 shRNA#1 plus CA-MEK (n = 6). (F) Examples of staining formarkers PSD-95 (red) and VGlut (blue) in transfected neurons. (G)Quantificationof the synapse density. Vector control (n=27), eB3 shRNA#1 (n= 39, P= 0.1212). (H) EB3−/−andwt neurons transfectedwithGFP aloneorGFP andDN-MEK cultured on wt mouse cortical neurons. GFP (green), PSD-95 (red), and vGlut1 (blue). Arrowheads indicate colocalized PSD-95 (red) and vGlut (blue) puncta intransfected neurons. (I) Quantification of synaptic density in heterogenotypic cultures (wt/wt, n = 18; eB3−/−\wt, n = 30; eB3−/−\wt+DN-MEK, n = 34). (J) Neuronstransfected with GFP and vector control or eB3 shRNA at DIV 0 [anti-GFP (green), anti-Erk1/2 (red), and anti-VGlut1 (blue)]. (Scale bar, 5 μm.) Arrowheads indicateVGlut1puncta. Straight arrows indicatecytoplasmicErk1/2; curvedarrows indicatenuclear Erk1/2. (K)Quantificationofpercentnuclear Erk1/2after transfectionwithvector control (n= 6 transfections) or eB3 shRNA (n = 6 transfections). A total of 50–300 cellswere scored for each experiment. (L) Dendrites of control or eB3 shRNA–expressing neurons stained for GFP (green), Erk1/2 (red), and vGlut1 (blue). Arrowheads indicate VGlut puncta in transfected neurons. (M) Quantification of ratio ofsynaptic Erk todendritic Erk staining in control andeB3 shRNA-expressingneurons. Vector control (n=22), eB3 shRNA#1 (n=24). (J–M) Experimentswere conductedfollowing a 1-h treatment with blockers of neuronal activity (1 μM TTX), NMDA receptors (50 μMAPV), and L-type calcium channels (50 μMnifedipine). (Scale bars,3 μm except in J.) *P < 0.03, **P < 0.01, ***P < 0.0001. Error bars indicate SEM.

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mEPSC frequency (Fig. 6E). In neurons expressing shRNAs tar-geting eB3, expression of CA-MEK had no effect on mEPSCfrequency, but expression of DN-MEK rescued the decrease inmEPSC frequency and reduction in synapse density (Fig. 6 E–Gand Fig. S4D). To test whether Erk has a similar function in ourheterogenotypic neuronal cell culture system,we coexpressedDN-MEK and GFP in neurons cultured from eB3−/− mice and mixedthese cells with neurons from wt littermates. Remarkably, ex-pression of DN-MEK in neurons from eB3−/− mice was sufficientto rescue defects in synapse density normally found in eB3−/−/wtheterogenotypic cultures (Fig. 6H and I), suggesting that the Ras/MAPK pathway is a negative regulator of synapse density and thateB3 negatively regulates this pathway.Extracellular signaling can cause the translocation of Erk to the

nucleus and activation of downstream effector molecules. There-fore, we tested whether eB3 kd would alter Erk localization. Al-though, in control-transfected neurons, Erk1/2 was excluded fromthe nucleus, kd of eB3 resulted in a significant increase in thepercentage of neurons with nuclear Erk1/2 localization (Fig. 6 Jand L). In addition, eB3 kd reduced the amount of Erk stainingcolocalized with synaptic markers (Fig. 6 K andM). These resultsindicate that the eB3–Erk interaction is important for the sub-cellular localization of Erk1/2 and suggest that eB3 may regulatesynapse number in part by preventing translocation of Erk to thenucleus or by retaining Erk at synapses.

DiscussionOur data suggest that control of synapse density and perhaps neu-ronal excitability may not be left only to activity-dependent ho-meostatic mechanisms, but are enabled by activity-independentcompetitive mechanisms as well. Analogous to morphogen proteingradients in embryonic development, eB3 regulation of synapsedensity depends on the amount of eB3 expressed rather than simplywhether or not it is expressed. Howmight eB3 expression influencesynapticdensity?Theheterogenotypic cultures provideevidence fora model in which the relative levels of eB3 expressed betweenneighboring neurons mediate competition for a presynaptic ligand,EphB2, enabling neurons expressing higher levels of eB3 to formmore synapses. Future studies will be needed to determine how eB3expression is controlled and whether mismatches in EphB2–eB3interactions between two neurons determine synapse density.EB3 kd reduces both spine and synapse density, whereas neurons

from eB3−/− mice only have fewer spines. The lack of an effect onsynapses in eB3−/− mice is consistent with our in vitro findings andsuggests that eB3 has two distinct functions: noncompetitive controlof spine formation and competitive control of excitatory synapsedensity. Notably, expression of DN-MEK rescues defects in heter-

ogenotypic cultures of eB3−/− and wt neurons, indicating eB3-de-pendent control of synapse density relies on Erk signaling. In eB3−/−

mice, the loss of spines and the corresponding increase in excitatoryshaft synapses in eB3 mice is intriguing and should enable elucida-tion of the in vivo role of dendritic spines.Our experiments establish eB3 as a member of a small group of

transmembrane proteins that can interact with Erk (19, 20) andprovide evidence for a biological function of the interaction. TheErk2–eB3 interaction appears to negatively regulate Erk signalingpossibly by retaining Erk in the dendrites, or by virtue of eB3’sselective interaction with Erk2. One model suggested by ourfindings is that Erk1 and Erk2 act to oppose each other. However,more work is needed to explore Erk-dependent control of synapsedensity. In conclusion, we provide evidence that eB3 controlssynapse density through a competitive cell–cell interaction withEphB and by the negative regulation of MAPK signaling.

Materials and MethodsCortical Cell Culture and Transfection. Primary dissociated rat cortical neuronsand transfections were conducted as described previously (10, 15). Primarydissociated cortical neurons from eB3−/− mice and wt littermate controlswere prepared from postnatal day 1 (P1) mice. Organotypic slice cultureswere prepared and transfected as previously described (10).

Immunocytochemistry. Cells and tissue were fixed and stained as describedpreviously (10, 15). Antibodies used were: chicken anti-GFP (1:2,500; Upstate),rabbit anti-Erk1/2 (1:500; Promega), rabbit anti-eB3 (1:50; specificity confirmedbyobserving lossof stainingafter incubationwithacetone-precipitatedproteinsfromwt but not eB3−/− brain tissue; Zymed), mouse anti–PSD-95 (1:250; AffinityBioReagents), rabbit anti-SynGAP (1:1,000; Affinity BioReagents), and guineapig anti-VGlut1 (1:5,000; Chemicon).

Electrophysiology.Whole-cell recordings weremade from rat cortical neuronstransfected in suspension as described previously (10).

Heterologous cell coculture and shRNA constructs were previously de-scribed (9, 10, 15). All data were collected from a minimum of three in-dependent experiments, and data from genetically engineered mice werecollected from a minimum of three animals per condition.

Additional details are provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank G. Bashaw and members of the Dalvalaboratory for helpful advice; M. Kayser for help and advice throughout theproject; A. Markowitz for conducting some pilot experiments that led to ourinitial findings; A. Burnet for the CA-MEK, DN-MEK, and Erk1/2 expressionconstructs; and T. Biederer and P. Scheiffele for their gifts of antibodies tosynCAM and Neuroligin. This work was supported by the Training Programin Developmental Biology (A.C.M., M.H.), and the Whitehall Foundation,Dana Foundation, National Institute of Mental Health, and National In-stitute of Drug Addiction (M.B.D).

1. Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB (1998) Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391:892–896.

2. Rao A, Craig AM (1997) Activity regulates the synaptic localization of the NMDAreceptor in hippocampal neurons. Neuron 19:801–812.

3. Verhage M, et al. (2000) Synaptic assembly of the brain in the absence ofneurotransmitter secretion. Science 287:864–869.

4. Dalva MB, McClelland AC, Kayser MS (2007) Cell adhesion molecules: Signallingfunctions at the synapse. Nat Rev Neurosci 8:206–220.

5. Shi W, Levine M (2008) Ephrin signaling establishes asymmetric cell fates in anendomesoderm lineage of the Ciona embryo. Development 135:931–940.

6. Yan D,Wu Z, Chisholm AD, Jin Y (2009) The DLK-1 kinase promotes mRNA stability andlocal translation in C. elegans synapses and axon regeneration. Cell 138:1005–1018.

7. Wairkar YP, et al. (2009) Unc-51 controls active zone density and protein compositionby downregulating ERK signaling. J Neurosci 29:517–528.

8. Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and function of dendriticspines. Annu Rev Physiol 64:313–353.

9. McClelland AC, Sheffler-Collins SI, Kayser MS, Dalva MB (2009) Ephrin-B1 and ephrin-B2 mediate EphB-dependent presynaptic development via syntenin-1. Proc Natl AcadSci USA 106:20487–20492.

10. Kayser MS, McClelland AC, Hughes EG, Dalva MB (2006) Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors. J Neurosci 26:12152–12164.

11. Grunwald IC, et al. (2004) Hippocampal plasticity requires postsynaptic ephrinBs. NatNeurosci 7:33–40.

12. Aoto J, et al. (2007) Postsynaptic ephrinB3 promotes shaft glutamatergic synapse

formation. J Neurosci 27:7508–7519.13. Rodenas-Ruano A, et al. (2006) Distinct roles for ephrinB3 in the formation and function

of hippocampal synapses. Dev Biol 292:34–45.14. Bouvier D (2008) Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in

adult mouse forebrain. J Neurochem 106:682–695.15. Kayser MS, Nolt MJ, Dalva MB (2008) EphB receptors couple dendritic filopodia

motility to synapse formation. Neuron 59:56–69.16. Segura I, Essmann CL, Weinges S, Acker-Palmer A (2007) Grb4 and GIT1 transduce

ephrinB reverse signals modulating spine morphogenesis and synapse formation. Nat

Neurosci 10:301–310.17. Lau CO, Ng FH, Khoo HE, Yuen R, Tan CH (1996) Inhibition of sodium-dependent

uptake processes in purified rat brain synaptosomes by Lophozozymus pictor toxin

and palytoxin. Neurochem Int 28:385–390.18. Pagès G, Brunet A, L’Allemain G, Pouysségur J (1994) Constitutive mutant and

putative regulatory serine phosphorylation site of mammalian MAP kinase kinase

(MEK1). EMBO J 13:3003–3010.19. Zhou J, et al. (2005) A novel six-transmembrane protein hhole functions as a suppressor

in MAPK signaling pathways. Biochem Biophys Res Commun 333:344–352.20. Ishihara K, Tsutsumi K, Kawane S, Nakajima M, Kasaoka T (2003) The receptor for

advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like

docking site. FEBS Lett 550:107–113.

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