摘要:Orexins (含orexin A 與orexin B)是1998 年被新發現的下視丘胜肽,是第二個被去孤兒化的G 蛋白偶合受體(GPCR)的內生性配體,該受體含OX1R 和OX2R。Orexins 的功能除廣受矚目的覺醒與進食之外,也在脊髓和腦部層次產生止痛作用。我們先前研究發現環導水管灰質(periaqueductal gray; PAG)這個啟發下行止痛路徑的中腦部位是orexins 由腦部產生止痛的重要部位。Orexins 可使突觸後細胞膜電位去極化,亦可在突觸前抑制GABA 釋放,大麻受體CB1R 拮抗劑可阻斷該作用,顯示出內生性大麻酯(endocannabinoids)參與其中。在本計畫我們將進一步來闡述endocannabinoids 在orexins止痛作用中之角色。不少實驗已顯示,活化 mGlu1/5R 和mACh M1/M5R 等Gq 蛋白偶合的GqPCR 會啟動PLC 將細胞膜磷酯phosphotidylinositol 水解成DAG,DAG 可透過DAGL 水解成endocannabinoid,2-AG,所生成的2-AG 能逆行性透過突觸前CB1R 抑制神經傳導物質釋放。OX1R 和OX2R 皆屬GqPCR,我們因此假設:活化突觸後OXR 會經由PLC-DAG路徑產生2-AG,在PAG 產生逆行性的抑制突觸前GABA 釋放,因此興奮PAG 啟動下行止痛路徑。本研究擬藉由OXR, CB1R, PLC, DAG inhibitors 阻斷orexins 的作用,透過動物疼痛行為,腦切片電生理與免疫螢光染色實驗來驗證此假說。本計劃乃延伸我們之前研究進一步闡述 orexins 之止痛機制。Endocannabinoids 之產生已被認為在突觸塑性產生扮演重要的逆行性抑制訊息角色。本假設若經證實,將是orexins 止痛作用的嶄新機制,首度揭開endocannabinoids 於其中扮演逆行性抑制訊息角色。
Abstract: Orexins consisting of orexin A and B, also known as hypocretin 1 and 2, are a novelhypothalamic peptide family identified in 1998. They are the endogenous ligands of thesecond de-orphanized G-protein couple receptor, the OX receptor, including OX1R andOX2R. In addition to orexinergic and arousal actions, orexins have antinociceptive activity atboth spinal and supraspinal levels. We have previously shown that the midbrainperiaqueductal gray (PAG), the site initiating descending pain inhibitory pathway, is involvedin orexins-induced supraspinal antinociception. In addition to inducing postsynapticmembrane depolarization, orexin A also inhibited GABAergic inhibitory postsynaptic currents(IPSCs) presynaptically in a manner blocked by AM-251, a CB1 cannabinoid receptor blocker,suggesting the endocannabinoids are involved. In this project, we will further elucidate therole of the endocannabinoid(s) in orexin-induced antinociception.Activation of Gq-protein couple receptors (GqPCRs), including group I metabotropic(mGlu1/5) and muscarinic M1/M5 receptors, can activate phospholipase C (PLC) whichhydrolyses membrane phosphatidylinositol to yield diacylglycerol (DAG) which is, then,converted by DAG lipase (DAGL) to 2-arachydonlyglycerol (2-AG), an endocannabinoid.The 2-AG generated by GqPCR activation produces retrograde inhibition of neurotransmitterrelease through presynaptic CB1 receptors. Both OX1Rs and OX2Rs are also GqPCRs. We,therefore, hypothesize that activation of postsynaptic OXRs, through PLC-DAG pathway, willlead to 2-AG synthesis and release, and producing retrograde presynaptic inhibition ofGABAergic transmission in the vlPAG and, hence, activating the descending pain inhibitorypathway. Both in vivo and in vitro electrophysiological and immunofluorescent studies will beconducted. The in vivo study will be performed by examining if the antinociception inducedby intra-PAG injection of orexin in the mouse hot-plate test will be reversed by the specificantagonist of CB1 (AM-251), OX1 (SB 33-4867) or OX2 (Compound 29) receptors, and theinhibitors of the enzymes involved in 2-AG synthesis, PLC (U 73122) and DAGL(tetrahydrolipstatin), or be enhanced by the inhibitor of 2-AG degradation enzyme,monoacylglycerol lipase (MGL) (URB 602). We will also examine if the CB receptor agonist(WIN 55212-2) will mimic and occlude orexin-induced antinociception. Electrophysiologicalexperiments will use orexin A-induced depression of IPSCs in rat PAG slices as themeasurement endpoint. We will examine if orexin A-induced IPSC depression will be 1)reversed by SB 33-4867, Compound 29, U 73122 and tetrahydrolipstatin; 2) enhanced byURB 602; 3) occluded by WIN 55212-2. Immunofluorescent labeling of OX1R and OX2Rand DAGL will conducted in the recorded slices to validate the postsynaptic effect of orexinsby the co-localization of OXR and DAGL as well as the co-localization of OXR, DAGL andthe recorded neuron which will be filled with Lucifer yellow added in the recording pipette.This project, extending our previous findings, will further elucidate the antinociceptivemechanisms of orexins. Endocannabinoids have been demonstrated to be synthesized ondemand serving as a retrograde messenger involved in several forms of synaptic plasticity. Ifour hypothesis, orexins produce antinociception in the PAG through a retrogradeendocannabinoid signaling, is verified, this will be the first study revealing a novelantinociceptive mechanism of orexins.