EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2). Activation of optoEphB2 in LA of EphB2 mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.

Erythropoietin-producing hepatocellular (Eph) receptors regulate a wide array of developmental processes by responding to cell-cell contacts. EphB2 is well-expressed in the brain and known to be important for dendritic spine development, as well as for the maintenance of the synapses, although the mechanisms of these functions have not been fully understood. Here we studied EphB2's functions in hippocampal neurons with an optogenetic approach, which allowed us to specify spatial regions of signal activation and monitor in real-time the consequences of signal activation. We designed and constructed OptoEphB2, a genetically encoded photoactivatable EphB2. Photoactivation of OptoEphB2 in fibroblast cells induced receptor phosphorylation and resulted in cell rounding ­­­­­­– a well-known cellular response to EphB2 activation. In contrast, local activation of OptoEphb2 in dendrites of hippocampal neurons induces rapid actin polymerization, resulting dynamic dendritic filopodial growth. Inhibition of Rac1 and CDC42 did not abolish OptoEphB2-induced actin polymerization. Instead, we identified Abelson tyrosine-protein kinase 2 (Abl2/Arg) as a necessary effector in OptoEphB2-induced filopodia growth in dendrites. These findings provided new mechanistic insight into EphB2's role in neural development and demonstrated the advantage of OptoEphB as a new tool for studying EphB signaling.

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EphrinB receptors (EphBs) are receptor tyrosine kinases that promote dendritic spine morphogenesis by altering local actin polymerization at axo‐dendritic contacts. Soluble ligands have been used with neuronal cultures to activate EphBs, though local stimulation is more desirable to model local contact and study downstream signaling dynamics. Since EphB signaling requires receptor clustering, we designed an optogenetic tool, named optoEphB, based on an optimized mutant of the plant protein cryptochrome 2 (Cry2olig, residues 1‐498 with E490G mutation), which shows blue light‐induced clustering. OptoEphB consists of the EphB2 cytoplasmic domain fused to Cry2olig and mCherry at the C‐terminus with myristoylation at the N‐terminus. Global and focal blue light illumination of HEK293 cells expressing optoEphB showed reversible optoEphB clustering and retraction of membrane protrusions, indicative of EphB2 signaling. In hippocampal neurons, focal illumination of dendrites produced transient, dynamic protrusions that resembled dendritic filopodia. We observed recruitment of the pleckstrin homology domain of Akt in illuminated regions, suggesting phosphoinositide 3‐kinase (PI3K) activation in this process. Our results demonstrate that optoEphB locally activates EphB signaling and thus represents a novel optogenetic method of Eph receptor activation. Further experiments will determine PI3K‐dependence of filopodial protrusions and use biosensors to measure the spatiotemporal dynamics of downstream Rho GTPase activity, implicated in EphB‐driven spine morphogenesis. These results will bring new insights into current models of dendritic spine development.