- 學位論文
壓力如何撩起癮頭:食慾素誘發內源性大麻素促使成癮腦區多巴胺神經元失控
How stress provokes drug craving: Orexin-initiated endocannabinoid-mediated dopaminergic disinhibition in the ventral tagmental area
摘要
已有諸多的文獻指出食慾素系統包含食慾素A和B及其受體，OX1R和OX2R，已知與飲食、回饋、醒覺的生理機能調控有關。而食慾素神經元的在大腦中的分布主要集中於Lateral hypothalamus (LH)、Perifonical area (PFA)以及Dorsomedial hypothalamus (DMH)並透過其軸突向其他腦區廣泛延伸，其中包括腹側被蓋區（VTA），該核區主要與獎勵回饋的生理功能有關，具有相當多的多巴胺型神經元容易受成癮性物質影響。然而，食慾素對腹側被蓋區的調控作用乃至於藥物成癮可說是知之甚少。 從本實驗室先前的文獻已知，食慾素在中腦導水管周圍灰質具有新型鎮痛機制：在急性壓力下LH中的食慾素神經元活化導致下游中腦導水管周圍灰質的食慾素大量上升，透過激活突觸後OX1R，Gq蛋白偶聯受體，進而活化磷脂酶C（PLC），甘油二酯脂肪酶（DAGL）產生內生性大麻酯（2-AG）達到抑制突觸前抑制性神經訊息傳導產生止痛的效果。從而推測如果OX 1R-PLC-DAGL-2-AG這樣的調控機制也存在於腹側被蓋區多巴胺型神經元，或許有相當高的可能性透過抑制多巴胺型神經元上游的抑制性神經元突觸訊息傳導而導致壓力誘發藥物成癮復發的現象，因此本篇論文將透過電生理、行為模式以及生物化學與病理染色檢測來驗證該假說。 首先，從大鼠腦片的電生理數據證實，高濃度的食慾素A（100 nM）可以有效的抑制腹側被蓋區中多巴胺型神經元上游的抑制型神經元突觸訊息而非興奮性神經元傳導。並從Paired pulse ratio (PPR)與mIPSC記錄中觀察到高濃度的食慾素處理下PPR顯著性的上升以及mIPSC中頻率的增加來推斷這樣的抑制性現象係透過突觸前而非突觸後，而這樣的調控透過一系列上述假設的訊息傳導路徑的拮抗劑SB 334867（3μM）(OX1R 拮抗劑) ; Edelfosine（5μM）(PLC 抑製劑) ; THL (Tetrahydrolipstatin, 10μM, 2-AG合成酶（DAGL）抑製劑) ; AM251（3μM）(CB1R 拮抗劑)所逆轉。此外，為求實驗完整性透過直接量測並觀察到在實際生理狀態下食慾素A確實透過上述機轉強化對多巴胺型神經元的活性，而這一項關鍵性的證據搭配上述這些結果初步推論，食慾素A能藉由OX1R-PLC-DAGL-2-AG，這樣的調控機制達到抑制多巴胺型神經元上游的抑制型神經元突觸訊息傳導，進而解釋食慾素系統在藥物成癮行為的貢獻。 已有文獻證實給予實驗動物急性束縛性壓力可活化大腦外側下視丘中食慾素系統，因此論文的第二部分將藉由急性束縛性壓力引發藥物成癮復發動物行為模式(場地制約性偏好測試:實驗動物經過3天的腹腔注射古柯鹼(20 mg/kg)導致與實驗設備發生場地制約化的現象後再經過3天的強制戒斷後，最後一天給予30分鐘的急性束縛壓力)搭配藥理學方法驗證我們上述在體外所發現的新機轉在實驗動物上能夠確實發生。我們觀察到急性束縛性壓力造成位於LH的食欲素神經元大量活化而PFA或DMH且與復發程度息息相關進而使下游腹側被蓋區的內生性食慾素與相對應生成的大麻酯的釋放量上升導致藥物成癮性復發的現象。這樣的現象也分別以系統性或是針對腹側被蓋區的局部給予上述於電生理實驗所用的OX1R拮抗劑、DAGL抑製劑、CB1R 拮抗劑來證實假說確實存在。從結果得知，不論是全身性或是局部性給予拮抗劑皆可以顯著性預防壓力所導致的藥物成癮性復發行為，為求實驗設計完善亦透過CB1R基因踢除的小鼠與局部腹側被蓋區給予食慾素A以及CB1R 拮抗劑來模擬急性束縛性壓力引發藥物成癮復發的機轉，此外，已有部分文獻指出不同的壓力條件所牽涉到的機轉將有所不同，其中研究最為透徹的促腎上腺皮質激素釋放因子(Cortical releasing factor, CRF)系統在某些壓力(如電刺激或是危及生命形式的壓力)所誘發藥物成癮復發的機轉扮演重要角色並非本篇論文所提及的食慾素系統，因此，在行為試驗的最後一部分將驗證食慾素系統以及CRF系統在急性束縛性壓力所引起藥物成癮復發中的角色比重，從結果得知，在腹側被蓋區中食慾素系統相較於CRF系統在急性束縛性壓力下所引起的藥物成癮復發中發揮至關重要的影響力。 從這些結果再次證明這項嶄新的神經生理機轉能在急性束縛性壓力下主要導致LH食慾素神經元的活化使下游腹側被蓋區的食慾素大量上升而非CRF系統，進而透過多巴胺型神經元上OX 1R-PLC-DAGL的訊息傳導路徑產生內生性大麻酯2-AG導致突觸前抑制性訊息傳導受到抑制，最終引發藥物成癮復發現象。
並列摘要
The orexin system, consisting of orexin A and B and their receptors, OX1R and OX2R, is implicated in the regulation of several neuronal functions, such as motivation, feeding, reward and pain. Among these, the role of orexins in reward regulation is especially noteworthy. Orexin neurons, expressed exclusively in the perifornical area (PFA), dorsomedial (DMH) and lateral hypothealamus (LH), project widely throughout the brain including the ventral tegmental area (VTA), an important reward processing site where dopamine neuron plasticity can be altered by addictive substances. Orexins have been known to play an important role in the reward system. However, how orexins regulate the VTA dopaminergic activity to provoke drug craving, especially under a stressful condition, remains unclear. We have previously revealed a novel analgesic mechanism of orexins in the periaqueductal gray (PAG) that mediates the stress-induced analgesia phenomenon. That is, under stress, orexin neurons are activated to release orexins in the PAG. Then, orexins activate postsynaptic OX1Rs, a family of Gq-protein coupled receptors, resulting in synthesis of 2-arachydonoylglycerol (2-AG) via phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic pathway. 2-AG, an endocannabinoid, then produces retrograde inhibition of GABA release (disinhibition) by activating presynaptic cannabinoid 1 receptors (CB1Rs). Here, we examined whether this OX1R-PLC-DAGL-2-AG retrograde disinhibition signaling also exists in VTA dopamine neurons and can contribute to stress-induced extinguished cocaine seeking. We used electrophysiological and behavioral approaches to examine effects of orexin A on inhibitory postsynaptic currents (IPSCs) in dopamine neurons of rat VTA slices and on cocaine-conditioned place preference (CPP) in mice, respectively,. In electrophysiological studies, we found that orexin A (100 nM) depressed GABAergic IPSCs, but not excitatory PSCs, in VTA dopamine neurons and increased paired pulse ratio of paired IPSCs, suggesting a presynaptic effect. Orexin A also decreased the freqquency, but not amplitude, of miniature IPSCs, suggesting orexin A decreases GABA release. The IPSC depressant effect of orexin A was reversed or prevented by SB334867 (3