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EphrinB-EphB receptor tyrosine kinases have been demonstrated to play important roles in pain processing after peripheral nerve injury. We have previously reported that ephrinB-EphB receptor signaling can regulate excitability and plasticity of neurons in spinal dorsal horn, and thus contribute to spinal central sensitization in neuropathic pain. How EphB receptor activation influences excitability of primary neurons in dorsal root ganglion (DRG), however, remains unknown. Here, we report that EphB receptor activation facilitates calcium influx through N-methyl-D-aspartate receptor (NMDAR) dependent and independent manners. In cultured DRG cells from adult rats, EphB1 and EphB2 receptors were expressed in neurons, but not the glial cells. Bath application of EphB receptor agonist ephrinB2-Fc induced NMDAR-independent Ca2+ influx, which was from the extracellular space rather than endoplasmic reticulum. EphB receptor activation also greatly enhanced NMDAR-dependent Ca2+ influx and NR2B phosphorylation, which was prevented by pretreatment of Src kinase inhibitor PP2. In nerve-injured DRG neurons, elevated expression and activation of EphB1 and EphB2 receptors contributed to the increased intracellular Ca2+ concentration and NMDA-induced Ca2+ influx. Repetitive intrathecal administration of EphB2-Fc inhibited the increased phosphorylation of NR2B and Ca2+-dependent subsequent signals Src, ERK, and CaMKII as well as behaviorally expressed pain after nerve injury. These findings demonstrate that activation of EphB receptors can modulate DRG neuron excitability by facilitating Ca2+ influx directly or through Src kinase activation-mediated NMDA receptor phosphorylation and that EphB receptor activation is critical to DRG neuron hyperexcitability, which has been considered critical to the subsequent spinal central sensitization and neuropathic pain.

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trans-10b- 3b and 3c, were obtained from the valence isomerization of their corresponding [2.2]cyclophanedienes 28a and 28b. The long internal alkyl chain in each case was previously introduced via a Wittig reaction followed by reduction. The cyclophanedienes were synthesized from their corresponding dithia-[3.3]cyclophanes via a Wittig rearrangement-Hofmann elimination sequence. Proton chemical shifts of H1′ to H9′ of the alkyl chains in 3b and 3c, respectively, are identical, suggesting that the C1′-C9′ section of the long alkyl chain in 3b or 3c has similar averaged conformational movement. Application of the Johnson-Bovey (J-B) model to 3b does not give a satisfactory correlation between the proton ring current shift and the out-of-plane distance. A direct empirical plot of the out-ofplane distance, D, against the proton ring current shifts, ∆δ, however, gives a curve similar to that obtained from the J-B model of benzene. This curve in fact corresponds to a linear relationship between D and log |∆δ|. Calculated ring current shifts of several derivatives of 10b,10c-dihydropyrene based on the above empirical correlation agree well with the reported experimental values. Our model shows that the experimental detectable limit for the diatropicity of 10b,10c-dihydropyrene is about 14 Å from the mean molecular plane. A similar empirical treatment based on the carbon chemical shifts of the n-undecyl chain in 3b gives unsatisfactory results. Our model does not support the prediction that the magnitude of ring current effect on proton and carbon chemical shifts is essentially the same at the sample position in space relative to the mean plane of delocalization of an aromatic π-electron cloud.