Nervous system function requires tight control over the number of synapses individual neurons receive, but the underlying cellular and molecular mechanisms that regulate synapse number remain obscure. Here we present evidence that a trans-synaptic interaction between EphB2 in the presynaptic compartment and ephrin-B3 in the postsynaptic compartment regulates synapse density and the formation of dendritic spines. Observations in cultured cortical neurons demonstrate that synapse density scales with ephrin-B3 expression level and is controlled by ephrin-B3-dependent competitive cell-cell interactions. RNA interference and biochemical experiments support the model that ephrin-B3 regulates synapse density by directly binding to Erk1/2 to inhibit postsynaptic Ras/mitogen-activated protein kinase signaling. Together these findings define a mechanism that contributes to synapse maturation and controls the number of excitatory synaptic inputs received by individual neurons.N euronal activity is a key determinant of maintaining and controlling the number of synaptic connections (1, 2), but the molecular mechanisms likely to establish the normal density of synaptic contacts are still not well defined (3). Synapse density could be controlled by either secreted or cell surface molecules, but trans-cellular interactions are attractive because of their ability to coordinate events between cells. One family of synaptic adhesion molecules that is well suited to control synapse density is ephrin-Bs (eBs), a family of three (eB1-3) ligands for the EphB family of receptor tyrosine kinases (4). Recent work suggests that ephrins may negatively regulate Ras/MAPK activation in nonvertebrate systems during morphogenesis (5), and that MAPK signaling can negatively regulate presynaptic terminal maturation (6, 7). Here we show that eB3 controls synapse density and the formation of dendritic spines through a competitive postsynaptic mechanism relying on inhibition of MAPK signaling. ResultsEphrin-B3 Expression Level Controls Synapse Density. Early in development [5-10 days in vitro (DIV 5-10)], there are few dendritic spines and synapses that are rapidly added on the dendritic shaft. During this time, eB3 is enriched at excitatory synapses but is also localized to many extrasynaptic locations (Fig. 1A). As neurons mature (DIV 14-21), dendritic spines are formed and become the principal site of excitatory synapses (8); eB3 becomes largely restricted to spine and shaft excitatory synaptic contacts (Fig. 1A and Fig. S1A). The synaptic pattern of eB3 expression suggests that it may be 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 the density of endogenous PSD-95 puncta vs. two different measures of eB3 expression: the density of eB3 puncta and the overall intensity of eB3 immunofluorescence. We found that both measures of eB3 expression vary from neuron to neuron and are correlated with PSD-95 punct...