Project Summary / Abstract (30-line maximum) In this R01 Renewal project, we will examine contribution of the EphB2 receptor-NMDA receptor (NMDAR) interaction to excitatory synaptic neurotransmission in dorsal horn (DH) pain circuitry and neuropathic pain (NP) after spinal cord injury (SCI). NP is a highly debilitating and often chronic outcome that occurs in a major portion of SCI cases. Importantly, this NP is particularly refractory to treatment, urgently calling for identification of targets that both robustly regulate pathological pain and avoid the devastating effects of opioid-based interventions. NMDAR-mediated hyperexcitability of DH circuitry is a major substrate for pathological pain. NP has been linked to EphB receptor tyrosine kinases via potentiation of NMDAR function, as EphB signaling regulates multiple facets of NMDAR biology, including NMDAR-dependent Ca2+ influx and synaptic plasticity. We discovered that EphB2 modulation of NMDARs is mediated by a direct, extracellular interaction between EphB2 and the NMDAR. Importantly, we elucidated the mechanism regulating this interaction: phosphorylation of a single extracellular amino acid of EphB2 (Y504) interacts with a positive surface charge on the N-terminal domain of the NMDAR GluN1 subunit. We find in vitro that Y504 phosphorylation is required in spinal cord neurons for EphB2-NMDAR interaction, NMDAR synaptic localization, and excitatory synapse function. In our cervical contusion SCI model that results in forepaw hyperalgesia and allodynia, and ongoing pain, this NP phenotype is accompanied by long-lasting increases in EphB2 expression and signaling, Y504 phosphorylation, and EphB2- NMDAR co-localization at excitatory synapses in cervical DH, in particular in the highly-important population of DH pain projection neurons. Excitingly, inducible DH neuron-specific EphB2 knockout or inducible chemogenetic inhibition of EphB tyrosine kinase signaling robustly reverses already-established NP-related behavior after SCI. We therefore hypothesize that modulating EphB2-NMDAR interaction in DH pain projection neurons after cervical contusion SCI will impact: (1) NMDAR synaptic localization, (2) excitatory synaptic transmission in DH pain circuitry, and (3) NP. In Aim 1, we will determine whether inducible EphB2 knockout selectively in DH projection neurons reduces NMDAR synaptic localization and NP-related behaviors using a novel floxed-EphB2 mouse. In Aim 2, we will determine if preventing EphB2-NMDAR interaction selectively in DH projection neurons impacts NMDAR synaptic localization, excitatory synaptic transmission and NP behaviors using our innovative knockin mouse that allows for inducible expression of EphB2-Y504F (EphB2 with single amino acid switch that blocks interaction with the NMDAR). In Aim 3, we will determine the role of EphB2 intracellular tyrosine kinase activity in NP using a chemogenetic mouse that allows for specific, inducible and reversible inhibition of EphB2 kin...