Hypocretin與theta oscillations在壓力及壓力誘發之睡眠變化中所扮演的角色，以及Cannabidiol (CBD)對壓力引發睡眠失調之作用

Abstract

壓力影響生理和心理健康。許多因素(例如：生活環境和藥物治療)讓研究人員難以釐清壓力如何影響生理和心理健康。因此這篇實驗利用控制良好的環境和實驗動物(大鼠)來研究壓力引起的行為以及相關機制。我們研究神經傳遞物質：下丘泌素(hypocretin, hcrt)或稱食慾素(orexin)的壓力調控功能。論文中有三個研究主題：壓力造成腦波θ波的改變、壓力造成睡眠失調、以及大麻二酚(cannabidiol, CBD)減緩焦慮的機制。 Hcrt上升會造成壓力相關反應。腦波θ波是大鼠遭遇壓力時主要的腦波。Hcrt受體(hcrtR-1和hcrt-R-2)在抑制θ波的腦區—內中縫核(median raphe nucleus, MRN)相當豐富。這可能代表hcrt能調控壓力引起的θ波。在第一部分的實驗是釐清hcrt引發θ波之機轉和MRN在其中扮演的角色。我們的實驗結果顯示對大鼠足電刺激會增加θ波而hcrt受體拮抗劑(TCS1102)會抑制足電刺激引發的θ波。微量注射hcrt-1 (1 and 10 μg) 或 hcrt-2 (10 μg)到MRN中模擬了足電刺激升高θ波之作用。同時注射GABAA受體拮抗劑bicuculline至MRN可以阻斷hcrt或足電刺激引發的θ波。100 Hz的MRN電刺激也抑制了足電刺激的θ波。我們的資料顯示θ波和焦慮感正相關，所以我們再假設抑制θ波可以減緩焦慮。我們利用架高十字迷宮(elevated plus maze, EPM)來測量大鼠焦慮程度。θ波被bicuculline或MRN電刺激抑制後，大鼠進入EPM的open arm時間和比率增高；這些結果代表了壓力會引起hcrt的釋放，經過MRN中GABA神經，減緩了MRN抑制θ波的作用，最後造成θ波增加。除此之外，刺激MRN減少θ波後可減少大鼠焦慮。 壓力同時也是造成睡眠問題的主要原因之一。Hcrt可以增加醒覺，同時也是壓力神經傳遞物。另外，MRN同時調控恐懼反應和睡眠。不過壓力是否會造成hcrt在MRN增加而抑制了睡眠目前不是很了解。我們的結果顯示足電刺激抑制了快速動眼睡眠(rapid eye movement, REM)且微量注射TCS1102到MRN減緩了REM睡眠抑制。hcrt-1 (1 and 10 μg) 或 hcrt-2 (10 μg)注射到MRN模擬了足電刺激引起的睡眠改變。足電刺激也會使hcrt-1和hcrt-2在外側下視丘免疫陽性的神經細胞增加。而注射bicuculline抑制了足電刺激或者hcrt引起的REM睡眠減少。這些結果可能代表了hcrt在MRN中經由GABA神經而抑制REM睡眠。 CBD是大麻中活性主成分且有抗焦慮作用。影像學研究顯示CBD活化了杏仁核(amygdala)而有抗焦慮作用。不過CBD是否可以改善壓力造成的睡眠障礙並不清楚。因為焦慮的產生有時是因為有數天持續的壓力造成，所以我們利用連續四天重複合併測驗(repeated combination test, RCT)也就是50分鐘的曠野測試(open field, OF)接著10分鐘EPM。四天測驗中，大鼠在OF中心的時間以及EPM open arm的時間會漸漸越來越少。CBD注射到杏仁核中心核後(central nucleus of amygdale, CeA)增加了大鼠在OF中心和在EPM open arm的時間，代表了CBD有抗焦慮作用。RCT會抑制入眠後的一小時的非快速動眼睡眠(non-REM, NREM)和第4-10小時的REM睡眠。CBD改善了RCT造成的REM睡眠減少但是對NREM效果不顯著。實驗的結果顯示CBD藉由減緩焦慮而改善了REM睡眠。 總體來說，我們的結果顯示：REM睡眠減少是短暫壓力(例如：足電刺激)最主要造成的影響、壓力造成hcrt在MRN的釋放增加所以使θ波升高，且也抑制了REM睡眠、抑制θ波產生可以減緩焦慮、CBD透過CeA減緩焦慮而改善壓力引起的REM睡眠減少。

Abstract Stress impairs physical and mental health. Multi-factors, such as living environments, make investigators difficult to clarify how stress affects health. Thus this study utilized well-controlled environments and experimental animals (rats) to elucidate stress-induced behaviors and the related underlying mechanisms. We investigated the stress-related functions of a novel neurotransmitter, hypocretin (hcrt). Three main issues have been investigated in current dissertation, including: the stress-induced alteration of theta frequency in the electroencephalograms (EEGs), stress-induced sleep disturbances, and the mechanism of a potential anxiolytic, cannabidiol (CBD). Increased hcrt mediates stress-related responses. Theta frequency of EEGs is predominant during stress in rats. Hcrt receptors are abundant in the median raphe nucleus (MRN) which is a brain region desynchronizing hippocampal theta oscillation, suggesting a possible role of hcrt in modulating theta rhythm. First part of experiments clarified the involvement of hcrt in the stress-induced theta waves and the role of the MRN. Our results indicated that the intensity of theta waves was enhanced by the footshock and that TCS1102 (hcrt receptor antagonist) suppressed the footshock-induced theta waves. Administration of hcrt-1 (1 and 10 μg) and hcrt-2 (10 μg) directly into the MRN simulated the effect of footshock and increased theta waves. Co-administration of GABAA receptor antagonist, bicuculline, into the MRN blocked the increase of theta waves induced by hcrt or footshocks. Electrical stimulation of MRN also suppressed footshock-induced theta waves. We hypothesized that suppression of theta waves reduces anxiety. We exploited the elevated plus maze (EPM) to measure the anxiety level of subjects. After suppression of theta waves by either bicuculline or electrical stimulation, the duration in open arms increased. These results suggested that stress enhances the release of hcrts, activates GABAergic neurons in the MRN, blocks the ability of MRN to desynchronize theta waves, and subsequently increases the intensity of theta rhythm. Furthermore, stimulation of MRN blocked theta waves, which in turn reduced anxiety levels. The MRN modulates fear responses and also regulates sleep, but it is unclear whether stress-induced hcrts in the MRN disrupt sleep or not. Our data demonstrated that the footshock reduced rapid eye movement (REM) sleep and microinjection of TCS1102 into the MRN blocked the decrease of REM sleep in rats. Administration of hcrt-1 or hcrt-2 to the MRN mimicked the footshock-induced sleep alterations. Co-administration of bicuculline suppressed the decrease of REM sleep, induced either by footshock stimuli or administration of hcrts. These observations suggest that hcrt in the MRN is involved in the stress-induced reduction of REM sleep and this action is mediated by the GABAergic neurons in the MRN. CBD, an active component of marijuana, is reported to have the anxiolytic effect. Image studies showed that CBD decreases anxiety by activation of the amygdala. However, the ability of CBD to improve stress-induced sleep disturbances is unclear. We employed the repeated combination tests (RCT), consisting of a 50-minute open field (OF) and a subsequent 10-minute EPM, for four consecutive days to simulate the development of anxiety. Time spent in the centre arena of OF and during open arms of the EPM was substantially decreased in latter days of RCT, whereas microinjection of CBD into the amygdala blocked the reduction, further confirming its anxiolytic effect. The suppression of REM sleep during hours 4-10 were observed after the RCT. CBD efficiently blocked anxiety-induced suppression of REM sleep. In summary, our results have shown that REM sleep reduction is the main sleep disturbance induced by the acute stress, such as the inescapable footshock. Stress increases hcrt in the MRN which results in increase of theta rhythms and subsequently decreases REM sleep. Desynchronization of theta waves reduces the anxiety level of rats. Moreover, CBD in the amygdala reduces anxiety and reverses the REM sleep reduction induced by stressors.