We have recently identified a novel role for Eph receptor tyrosine kinases and their ephrin ligands (well known guidance molecules during development) as modulators of synaptic strength in the spinal cord. Activation of EphB receptors in rat dorsal horn induced behavioural thermal hyperalgesia; blocking EphB receptor activation prevented or reduced pain-related behaviour in inflammatory pain and in the formalin test. We have now tested the hypothesis that EphB receptor activation induces hyperalgesia through the modulation of NMDA receptor activity, via phosphorylation of the NR2B subunit of the receptor by the non-receptor tyrosine kinase Src. Intrathecal cannulae were implanted in anaesthetized (Medetomidine, 250μg/kg and ketamine, 60mg/kg, i.p.) adult male Wistar rats. After 1 week, rats received 2 10 μl intrathecal injections (each followed by a 10 μl saline flush) at a 10 min interval according to the following protocol: saline (vehicle solution) + saline (Group 1) or + ephrin-B2-Fc solution (to activate EphB receptors; 2μg/rat) (Group2); the Src family kinases inhibitor PP2 (73 nmoles/rat) + saline (Group 3) or + ephrin-B2-Fc (Group 4); PP2 (7.3 nmoles/rat) + ephrin-B2-Fc (Group 5: only for behavioural analysis). For biochemical analysis, the rats were killed by decapitation. Immunoprecipitation of the NMDA receptor was performed on homogenates of the lumbar region of the spinal cord (dorsal horns only), and followed by Western blotting. Membranes were probed with mouse anti-phosphotyrosine (PY490) or rabbit anti-phosphSrc antibodies. The membranes were then stripped, and probed with antibodies to total NR2B. Densitometric analysis was made using Scion Image software. Behavioural testing was performed using a plantar test (Ugo Basile) 30, 60, 90 and 120 mins after injection. In agreement with our hypothesis, the state of tyrosine phosphorylation of NR2B was significantly increased following ephrin-B2 treatment compared to control levels, as was the phosphorylation level of Src bound to NR2B, while levels of total NR2B remained unchanged: these increases were prevented by prior administration of PP2. We correlated these biochemical findings with behavioural analysis. As expected, intrathecal ephrin-B2 induced thermal hyperalgesia (P<0.001, n=5; ANOVA). Prior administration of PP2 reversed the effect of ephrin-B2 in a dose-dependant manner: animals treated with 7.3nmoles PP2 displayed latencies which were not significantly different from those of either control (n=10) or ephrin-B2 treated rats (n=6), while a dose of 73nmoles totally reversed ephrin-B2- induced hyperalgesia (P<0.001, n=8).