PI3K & ephrinBs on Spinal Nociceptive Information Pretreatment of EphB1-Fc (0.5 mg) prevented CCI-induced thermal hyperalgesia in paw-withdrawal latency (PWL) test and mechanical allodynia in paw-withdrawal threshold (PWT)test. * P,0.05, ** P,0.01, compared with EphB1-Fc-CCI, n = 8 mice in each group. (B) The representative immunohistochemical staining and the quantitative data for the decreased CCI-induced spinal Fos expression by pretreatment with EphB1-Fc. ** P,0.01 compared with saline (Sal)-CCI group, n = 6 mice in each group, Scale bar = 100 mm. (C) The representative bands and the quantitative data for the decreased expression of spinal PI3K-p110c and p-AKT by CCI after pretreatment with EphB1-Fc. 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-Sham group were set at 1 for quantifications. * P,0.05 or ** P,0.01 compared with Sal -Sham group, # P,0.05 compared with Sal-CCI group, n = 4 mice in each group. (D)Posttreatment of EphB1-Fc reversed CCI-induced thermal hyperalgesia and mechanical allodynia. * P,0.05, compared with CCI-EphB1-Fc, n = 8 mice in each group. (E) The reversed CCI-induced spinal Fos expression by posttreatment with EphB1-Fc. ** P,0.01 compared with CCI-Sal group, n = 6 mice in each group. (F) The decreased expression of spinal PI3K-p110c and p-AKT by CCI after posttreatment with EphB1-Fc. *P,0.05 or ** P,0.01 compared with Sham -Sal group, # P,0.05 compared with CCI-Sal group, n = 4 mice in each group. doi:10.1371/journal.pone.0040930.g006 used as a maker for neuronal activation in the central nervous system and has been widely used as a tool in functional mapping of neuronal circuits in response to various defined stimuli. There is a positive correlation between the quantity of Fos protein expression and the degree of sensitization induced by nociceptive stimuli in spinal cord neurons. Long-term facilitation of c-fiber-evoked firing of wide dynamic range neurons in the spinal dorsal horn in response to conditioning stimulation of afferent fibers was accompanied with the frequency-dependent increase of c-fos- labeled cells in superficial, intermediate, and deep laminae of the dorsal horn on the stimulated side [35]. Inhibition of tetanically sciatic stimulation-induced LTP of spinal neurons was also accompanied with the suppressed tetanic stimulation-induced spinal Fos expression [36]. In this study, we further confirmed the previous electrophysiologic findings that activation of ephrinBs- EphBs receptor signaling is required for induction of c-fibers- evoked LTP of dorsal horn neurons and lowers stimuli threshold of the LTP [17] by means of Fos protein expression. Spinal application of ephrinB1-Fc, which can activate EphBs receptors, induced a dose-dependent increase in spinal Fos protein expres- sion. Blocking of EphBs receptors with intrathecal injection of EphB1-Fc also suppressed the increased expression of spinal Fos protein expression in CCI and formalin pain models. Although we cannot identify which EphBs subgroup was activated dominantly by ephrinB1-Fc, our study demonstrates clearly that activation of ephrinBs/EphBs system is required for development of the pain- related,long-lasting alterations of excitability and synaptic plastic- ity of spinal neurons, which lead to increased sensitivity to noxious stimuli (hyperalgesia) or nonnoxious stimuli (allodynia). EphBs receptors transduce signals bidirectionally by interacting with membrane-anchored ephrinBs ligands expressed on adjacent cells. Application of ephrinB1-Fc reagent can activate EphBs receptors (forward signaling) and also prevent activation of endogenous ephrinBs ligands (reverse signaling). EphB1-Fc can disturb the signaling by the interaction of EphBs and ephrinBs and activate endogenous ephrinBs ligands. In this study, we could not confirm whether reverse signaling produced by ephrinBs activa- tion was involved in hyperalgesia induced by activation of spinal ephrinBs/EphBs signaling. However, injection of EphB1-Fc could not induce any pain behavior and not affect basal pain threshold. It is possible that reverse signaling has no contribution to this process, or EphB1-Fc induces only weak agonist activity. A recent finding in bone cancer model may support that EphB1 receptor forward signaling is critical to the development and maintenance of pain [37]. When EphB1 is inhibited, pain behaviors and NMDA receptors activation are inhibited even though the ephrinB2 continues to be active. When EphB1 receptor is activated, NMDARs are activated and pain is induced even though ephrinB2 is down regulated. But another recent study, by deleting ephrinB2 in Nav1.8+ nociceptive sensory neurons, indicates that presynaptic ephrinB2 expression may play a role in regulating inflammatory pain and some types of neuropathic Figure 7. PI3K mediated spinal ERK activation induced by i.t. ephrinB1-Fc. PI3K inhibitor wortmannin (0.4 mg) or LY294002 (5 mg) or DMSO was intrathecally injected at 30 min before i.t. ephrinB1-Fc (0.5 mg). The expression of spinal p-ERK was assayed at 30 min after i.t. ephrinB1-Fc injection. Pre-treatment with both wortmannin (A) and LY294002 (B) inhibited spinal ERK activation induced by i.t. ephrinB1-Fc. The representative bands (top) and the quantitative data (bottom) for the expression of spinal p-ERK after i.t. ephrinB1-Fc (0.5 mg). The fold change for the density of each p-ERK normalized to ERK for each sample. The fold change of p-ERK level in DMSO-Sal group was set at 1 for quantifications. ##P,0.01, compared with DMSO-Sal group, *P,0.05, **P,0.01 compared with DMSO-ephrinB1-Fc group, n = 6 mice in each group. doi:10.1371/journal.pone.0040930.g007 PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e40930