Orexins在大鼠中腦環導水管灰質經由內生性大麻訊息傳遞抑制GABA媒介的神經傳遞-一個嶄新的orexin止痛機制

Abstract

Orexins，又稱做hypocretins，是一個嶄新的下視丘神經胜肽，由prepro-orexin來的。orexins包含了兩個成員，orexin A (hypocretin 1)和orexin B (hypocretin 2)。orexin A和orexin B分別是由33和28個胺基酸所組成的胜肽，胺基酸序列的同源相似性為46%。它們在1998年被確定是孤兒G蛋白偶合受體(orphan GqPCR)，OX1和OX2受體的內生性致效劑。OX1受體對於orexin A有較高的親和力；而OX2受體則對orexin A和orexin B有相似的親和力。含有orexin的神經元主要只位在外側下視丘和穹窿區，但它的軸突會廣泛地投射至其他腦區，包括啟動中腦疼痛下行路徑的核區，腹外側環導水管灰質區 (ventrolateral periaqueductal; PAG)。orexins與許多中樞神經功能有關，包括進食、覺醒、酬賞、疼痛調控、運動功能和自主神經功能。 大麻，包含植物成分的Marijuana，△9-tetrahydrocannabinol和許多的大麻合成物，已在動物及臨床研究中證實大麻具有中樞及周邊作用的止痛機制。大麻受體有兩中亞型，其中中樞作用主要是透過大麻CB1受體來止痛。在PAG中有許多的大麻CB1受體和內生性大麻酯anandamide和2-arachidonoylglycerol (2AG)，可能可以說明在PAG中大麻在疼痛控制扮演重要的角色。 我們實驗室之前研究發現，在vlPAG微量注射orexin A在小鼠的疼痛模式中可以觀察到有止痛的反應，可能可以說明orexin A在vlPAG有止痛的作用。在本篇實驗，我們使用盲補綴全細胞電生理記錄更進一步探討orexin A在中腦vlPAG止痛作用的細胞機轉。orexins和內生性大麻酯在PAG都參與了疼痛的調控，我們將研究在vlPAG的腦片中這兩種內生性止痛胜肽系統是否具有交互作用。 本實驗結果發現，給與orexin A 10-300 nM會抑制突觸後膜抑制性電流(IPSC)，且依劑量增加對IPSC的抑制情形有增加的情形。orexin A (100 nM)的抑制作用可以被OX1受體拮抗劑SB 334867 (3 μM)所對抗，但是無法被OX2的拮抗劑Compound 29 (30 μM)所對抗。orexin A會增加paired-pulse facilitation (PPF)的比值，同時降低微小電流(miniature IPSC; mIPSC)的頻率而不影響振幅，可能可以說明是一個突觸前的作用。有趣地，orexin A抑制IPSC的作用可以被大麻CB1受體拮抗劑AM 251 (3 μM)所對抗。且orexin A抑制IPSC的作用可以用大麻CB1受體致效劑WIN55, 212-2 (3 μM)所模擬。此外，orexin A抑制IPSC的作用可以分別被phospholipase C (PLC)，U 73122 (5 μM)，和diacylglycerol (DAG) lipase的抑制劑，tetrahydrolipstatin (THL) (10 μM)，所阻斷。這些結果指出orexin A抑制突觸前神經末梢GABA的釋放是透過內生性大麻酯作用在突觸前GABAergic神經末梢上的大麻CB1受體。orexin A抑制IPSC的效果是透過突觸後OX1受體偶合到Gq蛋白而活化PLC，產生DAG,，再透過DAG lipase轉換成內生性大麻酯2-AG。這個研究是第一個說明了在vlPAG中，活化突觸後的OX1受體可以經由逆行性內生性大麻酯訊息來抑制GABAergic的傳遞，進而活化了下行的止痛路徑。

Orexins, also known as hypocretins, are a novel family of hypothalamic neuropeptides derived from prepeo-orexin and consist of two members, orexin A (hypocretin 1) and orexin B (hypocretin 2). Orexin A and orexin B consist of 33 and 28 amino acids, respectively, and share 46 % sequence homology. They were identified in 1998 to be the endogenous agonists of an orphan Gq-protein coupled receptor (GqPCR) family., OX1R and OX2R. OX1R has a higher affinity for orexin A, while OX2R displays equal affinity to both orexin A and orexin B. Orexin-containing neurons are limited localized in the lateral and perifonical area of hypothalamus, but project widely to numerous brain regions, including the ventrolateral periaqueductal gray (vlPAG), a crucial midbrain region for initiating the descending pain inhibitory pathway. Orexins have been implicated in several central neuronal functions, including feeding, arousal, rewarding, pain regulation, locomotion and autonomic functions. Cannabinoids, including the plant constituent of Marijuana, 9-tetrahydrocannabinol and a number of synthetic cannabinoids, have been known to have analgesic effects from animal and clinical studies with both central and peripheral sites of action. The central effect is mainly mediated by the cannabinoid 1 (CB1) subtype, one of two types of CB receptors. The abundance of CB1 receptors and endogenous cannabinoids, anandamide and 2-arachidonoylglycerol (2AG), in the PAG suggests that endocannabinoids might play a role in the pain control within the PAG. Our laboratory have previously found that intra-vlPAG microinjection of orexin A redcued the nociceptive response in a pain animal model, suggesting that the vlPAG is the site of antinociceptive action of orexin A. In this study, we further investigated the underlying cellular meachanisms of orexin A-induced antinociception in the vlPAG using the blind-patch whole cell recording technique in rat midbrain slices containing the vlPAG. Given that both orexins and endocannabinoids are involved in the pain regulation in the PAG, we also investigated if there is interaction between these two endogenous analgesic peptide systems in vlPAG slices. Orexin A (10-300 nM) concentration-dependently depressed evoked inhibitory post-synaptic currents (IPSCs). The effect of orexin A (100 nM) was antagonized by SB 334867 (3 μM), an OX1R antagonist, but not Compound 29 (30 μM), an OX2R antagonist. Orexin A increased the paired-pulse facilitation ratio of IPSCs and reduced the frequency, but not amplitude, of miniature IPSC, suggesting a presynaptic site of action. Interestingly, orexin A-induced depression of IPSCs was blocked by AM 251 (3 μM), a CB1 receptor antagonist, and was mimicked by WIN55,212-2 (3 μM), a CB1 receptor agonist. The effect of orexin A was blocked by U73122 (5 μM), a phospholipase C (PLC) inhibitor, and tetrahydrolipstatin (THL) (10 μM), a diacylglycerol (DAG) lipase inhibitor. These results suggest orexin A inhibits GABA release from presynaptic terminals through a retrograde endocannabinoid acting on the presynaptic CB1 receptors of GABAergic terminals. This effect was mediated by OX1R, coupled by Gq protein-PLC pathway, yielding DAG, which could be converted to 2-AG, an endocannabinoid. For the first time, we proved that activation of the postsynaptic OX1R can inhibit GABAergic transmission via retrograde endocannabinoid signaling in the vlPAG, and hence activating the descending pain inhibitory pathway.