pain through the regulation of synaptic plasticity in the DH [38].In conclusion, to date, we could confirm that the forword signaling produced by EphBs activation was involved in hyperalgesia induced by activation of ephrinBs/EphBs signaling. However, whether the reverse signaling produced by EphrinBs activation was involved in hyperalgesia induced by activation of ephrinBs/ EphBs signaling still unclear. PI3K mediated the role of ephrinBs/EphBs in central sensitization PI3K is widely expressed in spinal, particularly in the laminae I– II of the dorsal horn, the important sites for modulation of nociceptive information [24,26,39]. Ample and growing evidence showed that PI3K is involved in central sensitization induced by various stimuli [21,22,23,24,25,26,27]. Such as, Granulocyte- Colony Stimulating Factor (G-CSF) induces mechanical hyperal- gesia via spinal activation of PI3K in mice [22]. Intradermal injection of capsaicin results in activation of PKB/Akt in the lumbar spinal cord [27]. Intrathecal wortmannin, a PI3K inhibitor, dose dependently attenuated carrageenan-induced thermal and tactile hyperalgesia and reversed an established thermal hyperalgesia when given as a posttreatment [40]. In Painful Inflammatory Conditions, Phosphatidylinositol 3-Kinase Is a Key Mediator of Central Sensitization, PI3K is required for the full expression of spinal neuronal wind-up, intrathecal adminis- tration of LY294002, a potent PI3K inhibitor, dose-dependently inhibited pain-related behavior and spinal application of LY294002 reduces the ‘‘wind-up’’ of deep dorsal WDR neurons and inhibits electrically evoked responses [24]. There also some evidence supports the notion that activation of central receptor tyrosine kinase (RTK) system and the related downstream signaling pathway contribute to the development of central sensitization [23,25,41]. For example, activation of TrkA, TrkB or G-CSF receptor tyrosine kinases by central administration of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or Granulocyte-Colony Stimulating Factor (G-CSF) produces hypersensitivity and pain in humans and reduces nociceptive threshold in animal models of pain. Blockage of NGF/TrkA, BDNF/TrkB system also attenuates excitability of neurons and prevents mechanical and thermal hyperalgesia [22,42,43,44].Furthermore, PI3K activation mediates NFG, BDNF or C-GSF induced central sensitization and hyperalgesia [21,22,45].Moreover, several lines of evidence have shown that regulation of PI3K pathway is associated with EphBs receptors activation [28,29,30]. Some previous studies have indicated that ephrinBs acted as a sensitizer, which coupled with second- messenger systems through their EphBs receptors, to participate in central sensitization relevant to hyperalgesia [16,17,28,33,34]. In the present study, we found that ephrinB1-Fc-induced hyperalgesia was companied with the time- and dose-dependently increase of spinal PI3K-p110c expression and activation of AKT. Increased phosphorylation of p-AKT was prevented by pre- treatment with PI3K inhibitors wortmannin and LY294002. Inhibition of spinal PI3K prevented and reversed pain behaviors and spinal Fos protein expression induced by i.t. injection of ephrinB1-Fc. blocking EphBs receptors alleviated formalin and CCI-induced pain behaviors and inhibited activation of spinal AKT. These results indicated that, like other RTK systems, pain behaviors induced by activation of spinal ephrinBs/EphBs signaling were mediated by activation of spinal PI3K. PI3K & ephrinBs on Spinal Nociceptive Information The crosstalk between PI3K and ERK signaling in ephrinBs/EphBs system Some recent studies [28,33,34] and our results suggested that both peripheral and central ERK or PI3K activation are involved in pain behaviors induced by activation of peripheral and central ephrinBs/EphBs system. ephrinB1-Fc-induced hyperalgesia was companied with the time- and dose-dependently increase of p- ERK or PI3K expression. Inhibition of ERK or PI3K prevented and reversed pain behaviors and spinal Fos protein expression induced by i.pl. or i.t. injection of ephrinB1-Fc. pre-treatment with EphB1-Fc, which inhibited EphBs receptors, suppressed the pain behaviors and the increase of p-ERK or PI3K expression induced by inflammation and neuropathic pain. Furthermore, PI3K inhibitors blocked ERK activation induced by i.pl. or i.t. injection of ephrinB1-Fc. Our and other results support the crosstalk between PI3K and ERK signaling is involved in ephrinB1-Fc- induced pain model. Two recent studies have showed that peripheral and Spinal NMDA receptor contributed to activation of ERK by ephrinB1-Fc [33,34]. Previous results indicated that NMDA receptor-mediated [Ca2+]i increase can activate ERK pathways in a PI3K dependent manner [46,47]. Therefore, we thought that exogenous ephrinBs- Fc or increased expression of ephrinBs by tissue or inflammation directly activates EphBs receptors in nociceptors or neuronal cells. Activated EphBs receptors can modulate the NMDA receptor function via a Src-dependent mechanism and then enhance NMDAR-mediated Ca2+. This [Ca2+]i elevation in turn directly facilitates ERK and AKT signaling via PI3K pathways. The activated ERK and AKT signaling further phosphorylated NMDA receptor [48], which increases membrane excitability and induces nociceptor sensitization or neuron exciting. On the other hand, the activated ERK and AKT signaling could modulate the expression of transcriptional factors, such as CREB and Fos, and promotes the transcription of downstream genes. The increased expression of pain-related genes may contribute to the maintenance of ephrinBs/EphBs signaling-induced hyperal- gesia. Conclusion This study demonstrates that activation of spinal ephrinBs/ EphBs system induces hyperalgesia through a PI3K-mediated mechanism. These findings may have important implications for exploring the roles and mechanisms of ephrinBs/EphBs system underlying central nervous system diseases and for understanding the molecular basis for underlying physiologic and pathologic pain. In addition, it suggests that ephrinBs/EphBs pathway would be a new potential target for treatment of pathologic pain and other CNS diseases. Materials and Methods Animals Adult, male Kunming mice (20–25 g) were employed in the present study. Mice were housed under a 12-h/12-h light–dark cycle regime, with free access to food and water. The animals were provided by Experimental Animal Center of Zhejiang University. All experimental protocols were approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, Zhejiang Province, China) and were in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 80-23, revised 1996). PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e40930