PI3K & ephrinBs on Spinal Nociceptive Information Figure 5. Intrathecal (i.t.) pretreatment with EphB1-Fc inhibited formalin-induced inflammatory pain and spinal Fos protein expression, accompanied by the decreased expression of spinal PI3K-p110c and phosphorylated AKT (p-AKT). The amount of time spent licking the injected paw and the number of flinching paw were measured from 0- to 5-min (the first phase) and from 10- to 40-min (the second phase) after formalin injection. EphB1-Fc was injected at 30-min before formalin injection. The expression of spinal Fos protein and spinal PI3K-p110c and p-AKT was measured at 2-h and 30-min after injection of formalin, respectively. (A) Pretreatment of EphBs receptors blocker EphB1-Fc (0.5 mg) decreased the time of licking paw and the number of flinching paw induced by formalin. *P,0.05 or **P,0.01 compared with saline (Sal)-Formalin group, n = 8 mice in each group. (C) The representative immunohistochemical staining and the quantitative data for the decreased formalin-induced Fos expression by pretreatment with EphB1-Fc (0.5 mg), not saline, in the spinal cord of mice. ** P,0.01 compared with Sal-Formalin group, n = 6 mice in each group, Scale bar = 100 mm. (D) The representative bands and the quantitative data for the decreased expression of spinal PI3K-p110c and p-AKT by injection of formalin after pretreatment with EphB1-Fc (0.5 mg). The fold change for the density of PI3K-p110c and p-AKT normalized to GAPDH or AKT for each sample. The fold change of PI3K-p110c and p-AKT levels in Sal-Sal group was set at 1 for quantifications. *P,0.05 compared with Sal-Sal group, # P,0.05 compared with Sal-Formalin group n = 4 mice in each group. doi:10.1371/journal.pone.0040930.g005 by i.pl. or i.t. injection of ephrinB1-Fc [33,34]. Pre- or post- treatment with MEK inhibitor prevented or reversed hyperalgesia induced by i.pl. or i.t. injection of ephrinB1-Fc.Moreover, ERK is a potential downstream target of PI3K and a recent study has shown that PI3K activity is required for ERK activation induced by i.pl. injection of ephrinB1-Fc in periphery [28]. Thus, we asked whether spinal PI3K pathway contributes to ERK activation induced by ephrinB1-Fc as the same as in periphery. To address this question, two kinds of PI3K inhibitor wortmannin (0.4 mg) or LY294002 (5 mg) at 30 min before ephrinB1-Fc injection was intrathecal administered. We measured activation of spnial ERK by western blot using a phosphopeptide specific antibody that recognizes phosphorylated ERK(p-ERK) at 30 min after ephrinB1-Fc injection. We found that both wortmannin and LY294002 prevented spinal ERK activation induced by ephrinB1- Fc(P,0.05 or P,0.01, compared with DMSO-ephrinB1-Fc group; fig. 7). Discussion This study showed the following findings (1) spinal administra- tion of ephrinB1-Fc produced a dose-dependent thermal hyper- algesia and mechanical allodynia, which was accompanied with the increased expression of spinal Fos protein and of spinal PI3K- p110c expression as well as activation of AKT, a downstream target of PI3K.(2) inhibition of spinal PI3K prevented or reversed ephrinB1-Fc-induced hyperalgesia and spinal Fos protein expres- sion;(3) blocking EphBs receptors alleviated formalin and CCI- induced pain behaviors and inhibited activation of spinal AKT; and (4) PI3K activity is required for ERK activation induced by i.t. injection of ephrinB1-Fc. These findings demonstrated PI3K/ AKT signaling as a novel mechanism for ephrinBs/EphBs system involved in spinal nociceptive information modulation. ephrinBs/EphBs participate in central sensitization Central sensitization, an activity-dependent functional plasticity in spinal cord neurons, is one of the main causes of behavior hyperalgesia under pathologic conditions and has been under intensive investigation [1,2,3]. Fos protein expression has been PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e40930