Research ArticleEphrinB/EphB Signaling Contributes to the Synaptic Plasticity of Chronic Migraine Through NR2B Phosphorylation
Introduction
Chronic migraine (CM), referring to headaches that occur 15 days or more per month for more than three months and have migraine features at least 8 days per month (Carod-Artal et al., 2012), is a common and recurrent neurological disease that affects 2% of the general population (Schwedt, 2014). CM brings intense pain to the sufferer and seriously affects quality of life. Therefore, a therapy for CM is critical. Due to its complex and diverse pathogenesis, CM is still not fully treatable.
One of the major mechanisms of migraine chronification is central sensitization (Lovati et al., 2016). Central sensitization is manifested mainly through the plasticity of the nervous system, that is, enhanced neuronal function and increased synaptic efficacy (Latremoliere and Woolf, 2009). Our previous study found that there were changes in synaptic plasticity in CM, and inhibiting the phosphorylation of NR2B, a main subunit of the NMDA receptor, blocked the enhancement of synaptic plasticity and reduced hyperalgesia induced by CM (Wang et al., 2018a). These results suggested that blocking the enhancement of synaptic plasticity might help prevent migraine chronification. However, while there are currently tyrosine kinase inhibitors and nonreceptor tyrosine kinase Src inhibitors, there is no specific inhibitor of NR2B phosphorylation. Therefore, it is important to explore a new molecular target for regulating synaptic plasticity in CM.
Ephs, consisting of EphAs and EphBs (Aasheim et al., 2005, O'Leary and Wilkinson, 1999, Sasaki et al., 2003), are the largest member of receptor tyrosine kinases (RTKs). Ephs play different roles in mammals by combining with various ligands. Generally, EphAs react with ephrinAs, and EphBs react with ephrinBs, but a few Ephs can also cross-combine to exert diverse biological effects. Ephs/ephrins are regarded as key molecules in regulating synaptic plasticity. EphrinA3 expressed in astrocytes regulates synaptic function by interacting with EphA4 receptors on the dendrites of neurons in the hippocampus (Carmona et al., 2009). Retinotectal synaptic maturation has been shown to be promoted by activating ephrinB reverse signaling (Lim et al., 2008).
It is well known that the interactions between EphB receptors and NMDA receptors are essential for synapse development or function (Dalva et al., 2000). The activation of EphB receptors potentiated NMDAR-induced Ca2+ influx and was necessary for the maintenance of clustering of NMDAR at the synapses (Takasu et al., 2002). The stimulation of EphB receptors can also facilitate the recruitment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) to synapses to increase NMDAR activity and promote c-AMP-responsive element binding protein (CREB) phosphorylation (Takasu et al., 2002).
In addition, ephrinBs and EphBs are widely studied in the pain field. Intrathecal injection of EphB1-Fc and EphB2-Fc, inhibitors of the EphB receptor, have been shown to block the thermal and mechanical hyperalgesia induced by nerve injury (Song et al., 2008). The administration of ephrinB2-Fc in adult rats activated EphB receptors and induced thermal hyperalgesia, while the administration of EphB1-Fc prevented behavioral allodynia and hyperalgesia in a model of inflammation (Slack et al., 2008). Therefore, we hypothesized that ephrinB/EphB signaling may regulate the hyperalgesia of CM and may promote changes in synaptic plasticity through NR2B phosphorylation, thereby contributing to migraine chronification.
Based on the above hypothesis, we investigated the role of ephrinB/EphB signaling in CM in this study. A model of CM was built through 7 days of dura infusion of an inflammatory soup (Melo-Carrillo and Lopez-Avila, 2013). Using this model, we discovered the levels of EphB2 and ephrinB2 increased in the trigeminal nucleus caudalis (TNC). Intracerebroventricular injection of an EphB receptor inhibitor relieved hyperalgesia and reduced the increase in synaptic plasticity mediated by NR2B phosphorylation in CM. In addition, the EphB receptor blocker decreased the expression of CaMKII and pCREB.
Section snippets
Animals
Adult male Sprague-Dawley rats (250–300 g) were used in this study. Since the fluctuation of estrogen affects the prevalence and frequency of migraine (Ibrahimi et al., 2014), male rats were chosen. All animals were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Rats were housed at a temperature of 23 °C ± 1 °C and were under a 12/12 h light–dark cycle for at least 1 week prior to surgery, with free access to food and water. All experimental
Reduction of pain threshold and upregulation of CGRP in a model of CM
Rats recovered for 7 days after cannula fixation and were infused with IS (CM) or PBS (sham) starting from postoperative day 8 (Fig. 1). Before daily injection, the mechanical and thermal pain thresholds were measured. There was no significant difference in the basic pain thresholds (Fig. 2A, B). On the 10th day after surgery, the thresholds for the rats given IS were significantly lower than those given PBS (p < 0.05). In the next few days, the pain thresholds were sharply reduced (p < 0.01).
Discussion
In this article, we firstly assessed the role of ephrinB/EphB forward signaling in CM. First of all, we verified that the mRNA and protein level of EphB2 was upregulated after 7 days of IS infusion. The expression of ephrinB2 similarly increased. Secondly, rats with CM were characterized by the rise of CGRP, mechanical allodynia and thermal hyperalgesia, however the blocker of the EphB receptor inhibited the high production of CGRP and prevented the reduction of pain threshold. The above
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No: 81671093) and the District Science and Technology Projects of Yuzhong Chongqing (No: 20160107). Jiang Wang designed, conducted experiments, analysed data and wrote the manuscript. Lixue Chen provided valuable advice on research and guided the completion of this experiment. Zhaoyang Fei, Jie Liang and Xue Zhou assisted in completing the experiment. Guangcheng Qin and Dunke Zhang provided language help and image
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