摘要:我們最近發現在中腦環導水管灰質腹側區(ventrolateral periaqueductal gray, vlPAG)中注射食慾素(orexin A)產生的止痛是經由一個全新的止痛機制: 外給食慾素(Orexin A)活化vlPAG神經細胞突觸後OX1受體(OX1R),繼而活化phospholipase C (PLC)以水解膜磷酯產生diacylglycerol (DAG),接著由突觸後胞內DAG lipase (DAGL)將DAG轉變為一種內生性的大麻酯2-arachidonoylglycerol (2-AG),2-AG再從突觸後逆行到突觸前活化大麻CB1受器(CB1R)進而抑制GABA釋放。PAG的GABA釋放減少可以活化下行疼痛抑制路徑,進一步產生止痛反應。這個全新的止痛機制已被J. Neurosci.雜誌認同而刊登,我們擬研究內生性食慾素是否也可經由該新機制產生止痛,進一步驗證該止痛新機制的生理意義。緊張所致的止痛效果正是一種由內生性食慾素所媒介的止痛,因此在本計畫中我們將驗證緊張所致的止痛效果與PAG中該止痛新機制(OX1R-PLC-DAGL-2-AG-CB1R)有關。致痛素(nociceptin) (我們實驗室長期研究的主題)之如此被命名乃因其具有痛覺過敏(hyperalgesia)作用。一般認為致痛素產生痛覺過敏是因為抑制了進行腦室內(i.c.v.)注射操作過程所產生的緊張所致止痛 (stress-induced analgesia, SIA)。致痛素也被證實會直接抑制食慾素神經細胞,此作用被認為與致痛素抑制緊張止痛的現象有關,但是內生性大麻素是否參與此過程卻未被探討。因此在本研究中,我們也會驗證致痛素產生的痛覺過敏現象是否經由其對該食慾素受體引發的內生性大麻素訊息,因而抑制緊張所致止痛效果。總言之,本研究將驗證兩個假說: 1) 緊張所致的止痛效果是因緊張時,下視丘食慾素神經細胞活化,釋出的食慾素在PAG藉由OX1R-PLC-DAGL-2-AG-CB1R-媒介的止痛新機制所致;2) 致痛素產生的痛覺過敏現象是因為抑制了上述食慾素受體活化所引發內生性大麻素訊息媒介的緊張止痛效果。我們計畫透過行為、神經化學、免疫組織化學和電生理的實驗來探討五大目標:1) 建立小鼠緊張止痛模式,並確認在該模式中參與的PAG食慾素受器亞型;2) 證實在食慾素調控的緊張止痛中參與的內生性大麻素是否為2-AG;3) 確認2-AG所媒介的緊張止痛效果是源自食慾素受體 (OX1R) 活化後的下游反應;4) 在緊張止痛模式時,下視丘食慾素神經細胞是否被激活; 5) 研究活化致痛素系統是否能抑制內生性食慾素引發的內生性大麻訊息路徑來抑制緊張所致止痛。假若我們的假設得到證實,則不只可以證明我們發現的食慾素引發內生性大麻訊息這個新止痛機轉具有生理上的重要意義,而且可以對緊張止痛的機制與致痛素所致痛覺過敏作用提出新銓釋;也可證實食慾素系統於生物體面對壓力時的反應扮演重要角色。後者的發現,可以延伸研究食慾素在其他壓力相關失調疾病的貢獻,如創傷後壓力症候群、憂鬱症、壓力誘發成癮者的癮頭發作或是壓力引起的動作失調症。
Abstract: Recently, we found that microinjection of orexin A into the rat ventrolateral periaqueductal gray (vlPAG) induced analgesia by a novel mechanism: Orexin A activates the postsynaptic orexin 1 receptor (OX1R) in the vlPAG, leading to phospholipase C (PLC) activation and generating diacylglycerol (DAG). DAG is then converted by DAG lipase (DAGL) into 2-arachydonoylglycerol (2-AG), an endocannabinoid which produces retrograde inhibition of GABA release (disinhibition) by activating presynaptic cannabinoid 1 (CB1) receptors in the vlPAG, leading to analgesia via activating the descending pain inhibitory pathway. This is a novel analgesic mechanism and was appreciated by J. Neurosci. In this proposal, we will further explore the physiological significance of this analgesic mechanism by examining if endogenous orexins also produce analgesia through this mechanism. Stress-induced analgesia (SIA) is a phenomenon involving endogenous orexins. Therefore, we will validate a hypothesis that this novel analgesic mechanism, OX1R-PLC-DAGL-2-AG-CB1-mediated disinhibition in the vlPAG, contributes to SIA. In addition, we will also examine if the hyperalgesic effect of nociceptin/orphanin FQ (N/OFQ) is attributed to its inhibition of this OX1R-initiated and endocannabinoid-dependent SIA. N/OFQ, one of our long-term studied themes, is so named because it induces hyperalgesia when given by intraventricular (i.c.v.) injection. The hyperalgesic effect of N/OFQ was suggested to be due to its inhibition of SIA generated during the i.c.v. procedure. A direct inhibitory effect of N/OFQ on orexin neurons was suggested to contribute to its inhibition of SIA. However, it remains to be elucidated if endocannabinoid is involved in N/OFQ inhibition of SIA.Together, two hypotheses will be validated: 1) Endogenous orexins released due to hypothalamic orexin neuron activation during stress contributes to SIA through a novel analgesic mechanism, OX1R-PLC-DAGL-2-AG-CB1-mediated disinhibition in the vlPAG. 2) Nociceptin induces hyperalgesia through inhibition of this novel analgesic mechanism-mediated SIA. Five specific aims will be pursued via behavioral, neuronchemical, immunohistochemical and electrophysiological approaches: 1) To establish restraint SIA and examine if it is mediated by endogenous orexins in the PAG; 2) To elucidate if 2-AG in the PAG is involved in endogenous orexins-mediated SIA; 3) To elucidate if 2-AG-mediated SIA is down stream to OX1 receptor activation; 4) To examine if hypothalamic orexin neurons are activated during restraint stress; 5) To verify if activation of the N/OFQ-NOR system inhibits endogenous orexin-mediated and 2-AG-dependent SIA.If our hypotheses are proven, it will not only substantiate the physiological significance of the novel analgesic mechanism (orexin-initiated endocannabinoid analgesia) we revealed, but also disclose a novel mechanism for SIA as well as for N/OFQ-induced hyperalgesia. In addition, this study will also substantiate the contribution of the orexin system in living organisms upon coping with stress, and the findings in the interactions among N/OFQ, orexin and endocannabinoid systems in the phenomenon of SIA may be extended to other stress-related disorders, such as post-traumatic stress disorder, depression, stress-induced cue reinstatement of abusing substances or stress-elicited movement disorders.