Contribution Of SIP30 In The Anterior Cingulated Cortex To The Pain-related Negative Emotion In Rats

According to the definition of pain by the International Association for the Study of Pain (IASP), pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, which including a sensory-discriminative and an emotional-affective component. The neuropathic pain, which is induced by the damage of peripheral or central nerve system associated with illness or injury, is a common kind of chronic pain. Accumulating clinical observations indicate that the patients with chronic pain suffer from as much emotional disturbance as pain sensation. The quanlity of life of the patients suffering from chronic pain is much damaged by persistent pain as well as pain related negative emotion such as fear, anxiety, loneliness and aversion. In view of neuropathic pain and pain-related aversion are still short of effective treatment, to seek and find new targets for treatment become more urgent.Application of cDNA array, Northern blot and In situ techniques, neuropathic pain related genes were screened. Differential gene expression results indicate that sip30 is one of the genes which are highly related with neuropathic pain. SIP30 (SNAP25 interacting protein of 30 kD) consisting of 266 amino acids was first identified in cochlear hair cells in 2002. SIP30 is highly expressed in the central nervous system, especially in the brain. So far, little is known about the function of the SIP30. Previous study indicated that SIP30 was up-regulated in the spinal cord by ERK through the recruitment of CREB to the promoter of the sip30 gene, which contributed to the initiation and maintenance of CCI-induced neuropathic pain in rats.As an important component of pain, pain related negative emotion and memory have got more and more attention in recent years. Lots of results show that the activation of anterior cingulate cortex (ACC) contributes to pain-related emotional responses as well as pain perception, memory and expectations. Our previous results indicated that the activation of NMDA receptor-dependent cAMP/PKA-ERK-CREB signaling pathway in the ACC was necessary for the formation and expression of pain-related aversion. So, is SIP30 in the ACC involved in the induction of pain-related aversion? If participates, does SIP30 act as the downstream target of cAMP/PKA-ERK-CREB signaling pathway? What is the possible mechanism? By using behavioral training, molecular biology, immunohistochemistry and whole-cell patch clamp recording on ACC slices, we firstly investigated the contribution of SIP30 in the ACC to the pain-related aversion and its possible cellular and molecular mechanisms.Firstly, we observed the relationship between the upregulated SIP30 and pain-related aversion by immunohistochemistry, Western Blot and behavioral training. The results showed that the expression level of SIP30 was significantly increased in bilateral rACC (the rostral anterior cingulate cortex) induced by peripheral nerve injury (unilateral sciatic nerve chronic constriction injury, CCI). The upregulated SIP30 expressed mainly in the pyramidal neurons in layers II-III in ACC, but not in astrocytes or microglia. Intra-ACC microinjection of SIP30 shRNA could significantly reduce the time spent in the light side of the chamber, without affecting the CCI induced allodynia and thermal hyperalgesia and the formation of non-painful stimuli induced conditioned place avoidance, suggesting that the upregulation of SIP30 in the rACC specifically involved in the induction of pain-related aversion.To study the upstream regulatory pathways of SIP30 expression, we observed the relationship between SIP30 and PKA-ERK-CREB signaling pathway. Immunohistochemistry results indicated that pPKA, pERK and pCREB upregulated in the bilateral ACC induced by peripheral nerve injury. Double immunohistochemistry results showed that SIP30 and pERK, SIP30 and pCREB, pPKA and pERK, pPKA and pCREB could both co-localized with each others. Bilateral intra-ACC microinjection of pPKA inhibitor Rp-cAMP, or MEK (ERK kinase) inhibitor PD98059 could both inhibit the upregulation of SIP30 in the rACC and decrease the time spent in the light side of the chamber, without affecting the CCI induced allodynia and thermal hyperalgesia. These results suggested that the PKA-ERK-CREB signaling pathway-dependent the upregulation of SIP30 in the rACC was necessary for the induction of pain related negative emotion.Previous co-immunoprecipitation and yeast two-hybrid results showed that SIP30 could directly interact with SNAP25, which is an important component of SNARE complex playing an important role in neurotransmitter release. To further investigate the downstream molecular mechanisms of SIP30, whole-cell patch clamp recording in ACC slice, in vivo microdialysis and high performance liquid chromatography (HPLC) were used. Compared with negative control (negative control, NC) group, the excitatory postsynaptic current (EPSC) recorded in SIP30 shRNA group decreased significantly, suggesting that silencing the expression of SIP30 in the rACC inhibited the excitatory synaptic transmission. Both the amplitude and the frequence of spontaneous excitatory postsynaptic current (sEPSC) in the SIP30 shRNA group significantly decreased. Compared with NC group, paired pulse ratio (PPR) in the SIP30 shRNA group dramtically increased at different intervals. All these electrophysiological results indicated that silencing the expression of SIP30 could both inhibit the excitatory synaptic transmission and the presynaptic release of excitatory neurotransmitter. In vivo microdialysis and HPLC results showed that the concentration of extracellular glutamate significantly decreased after intra-ACC microinjection of SIP30 shRNA, suggesting that silencing the SIP30 expression inhibited the glutamate release.Long-term potentiation could be induced by theta burst stimulation (TBS) in rat ACC slice. Downregulation of SIP30 in the ACC completely blocked the induction of LTP, suggesting that the silent expression of SIP30 in the ACC destroyed the formation of synaptic plasticity.Taken together, the activation of PKA-ERK-CREB signaling pathway upregulated the expression of SIP30 in the ACC, in turn affected the presynaptic glutamate release and synaptic plasticity, leading to the formation of pain-related aversion.