創傷後壓力症候群模型引發嚙齒動物異常神經傳遞所導致焦慮行為及睡眠失調之探討

Abstract

壓力廣泛影響社會，其影響層面包含許多人的生理及心理健康。嚴重的創傷可能增加罹患心理疾病的機會，例如創傷後壓力症候群（post-traumatic stress disorder，PTSD）。目前已有許多動物模式被用於釐清PTSD的機制。然而，不同模型所產生的壓力表徵各有不同。因此，本篇論文中，我們採用不可迴避的足部電刺激壓力（inescapable footshock stress，IFS）作為急性壓力模型；同時建立多次延長壓力（multiple prolonged stress，MPS）做為慢性嚙齒類動物壓力模型。本研究旨在探討兩個議題：IFS誘發神經胜肽分泌變化所引起的睡眠干擾，以及MPS引起的延長類PTSD行為症狀。 過去研究已知IFS會減少快速動眼期（rapid eye movement，REM）睡眠，但其機制尚未明朗。研究表明，起源於側下丘腦區（lateral hypothalamic area,，LHA）的神經肽下丘泌素（hypocretin，hcrt）調節壓力反應及影響REM睡眠。因此，此實驗研究評估大鼠經IFS刺激後，LHA中下丘泌素神經元（hypocretinergic，hcrt-ergic）投射至REM抑制（REM-off）核區中的藍斑核（locus coeruleus，LC）和背側縫核（dorsal raphe nucleus，DRN）的情形以及對睡眠的影響。我們首先發現LHA中的下丘泌素神經元活化，會造成神經肽下丘泌素過度釋放，並傳遞至LC、DRN、腦幹、視丘及杏仁核中。在IFS前給予非選擇性下丘泌素受體拮抗劑（dual hcrt receptor antagonist）TCS1102後，阻斷LC和DRN核區因下丘泌素而活化，可以扭轉REM睡眠減少。此外，抑制LC和DRN的下丘泌素受體還能恢復因IFS而造成的非快速動眼期（non-rapid eye movement，NREM）睡眠中delta波強度的下降，此變化顯示此二腦區的下丘泌素受體阻斷，改善IFS後的睡眠品質。然而，清醒時的theta波強度及行為試驗結果顯示下丘泌素投射至LC和DRN對於焦慮的調節能力有限。綜上所述，我們的結果說明了IFS引起的REM睡眠抑制和NREM睡眠品質下降，是由於LC和DRN接收的下丘泌素過度分泌所致。 截至目前，單次急性壓力刺激普遍應用嚙齒類壓力實驗，而複合式威脅的多次壓力刺激則較少被研究與討論。因此，我們建構了誘導時長為七天的MPS壓力模型。藉由改良原先單一長期壓力（single-prolonged stress，SPS）模型，我們增加重複性、提高刺激強度與不可預期性，產生長期慢性刺激與進而誘導類PTSD症狀。在MPS模型中，我們延長了原先的單一次刺激至連續七天，其內容包含：2小時行為限制、20分鐘強迫游泳、麻醉。同時，結合四次安排於奇數實驗日的IFS作為主要壓力源。雖然除了足部電擊外，每日大鼠接收到的壓力源皆為相同，但每個實驗日皆隨機安排壓力源順序，以增加MPS壓力的不可預期性。本篇研究中，我們持續觀察類PTSD症狀六週以評估長期壓力影響狀況，包含凍結行為、皮質醇表現量、神經交互作用、焦慮行為及睡眠變化。我們的結果顯示，MPS造成的恐懼記憶會持續保留至六週。前額葉皮質區（prefrontal cortex，PFC）、腹側海馬迴（ventral hippocampus，vHPC）、基底外側杏仁核（basolateral amygdala，BLA）中的theta波區域強度及交互連貫性皆因MPS而增加，並向以恐懼固化為主導的方向傳遞訊號。焦慮行為也因長期恐懼記憶回溯後而增加延長。雖然MPS及恐懼記憶回溯會導致REM睡眠減少，但結果也同時顯示強化的theta腦波及睡眠紡錘分別在REM及NREM睡眠時出現，藉此鞏固恐懼記憶。概括而言，以上延長的遠程恐懼記憶、腦核區交互作用、焦慮及睡眠變異之結果皆支持MPS大鼠模型的長期影響。 綜合上述兩個研究，我們的實驗結果闡明IFS和MPS在壓力後對大鼠的影響。不可預測的壓力，由於下丘泌素神經元過度活化，因而向REM-off核區投射大量下丘泌素，進而降低REM睡眠。另外，壓力重複性和強度通過的增加造成清醒期間和睡眠期間的核區聯繫提升，進而延長了行為異常。

Stress is a widespread society issue that affects a considerable number of people and causes abnormalities in their physiology and psychology. A severe traumatic incident may increase the possibility of developing a serious syndrome, such as post-traumatic stress disorder (PTSD). Numerous animal models have been established to clarify the mechanism of PTSD; however, the generated symptoms vary due to diverse manipulations. Thus, in this dissertation, the acute and chronic rodent stress models, the inescapable footshock stress (IFS) and multiple prolonged stress (MPS), were determined and assessed, respectively. Two issues have been investigated: the sleep disruption caused by the IFS-induced neuropeptide and the MPS-derived extended PTSD-like symptoms.

IFS stimulation has been found to suppress rapid eye movement (REM) sleep, while the underlying mechanism remains unclear. Studies indicate that the neuropeptide hypocretin (hcrt) released from the lateral hypothalamic area (LHA) mediates stress response and REM sleep. Therefore, the present research elucidated the involvement of hypocretinergic (hcrt-ergic) projection from LHA to REM-off nuclei, locus coeruleus (LC) and dorsal raphe nucleus (DRN), in the IFS-induced sleep alterations. The results first illustrated that the hcrt-ergic neurons were activated in LHA and overexpressed hcrt in LC, DRN, thalamus, and amygdala. Blocking the LC and DRN regions by administrating TCS1102, a dual hcrt-receptor (hcrtR) antagonist, before IFS reversed the REM sleep reduction. Moreover, inhibiting the LC and DRN hcrtRs also improved the sleep quality after IFS by recovering the decreased intensity of non-rapid eye movement (NREM) sleep delta rhythm. However, the hcrt projections to LC and DRN have a limited effect on innate anxiety, as examined by waking theta powers and behavioral tasks. Overall, the results demonstrated that the IFS-induced REM sleep suppression and impairment in NREM sleep quality were governed by the overexpression of hcrt from LHA to the LC and DRN.

Single-shock acute stressors are commonly applied in rodent models, whereas composite life-threatening multiple-shock stressors have rarely been discussed. Therefore, a seven-day MPS protocol was constructed to initiate long-term chronic PTSD-like symptoms by modifying the repetition, intensity, and uncontrollable from the single-prolonged stress (SPS) model. The original single-day manipulation, consisting of 2 hours of restraint, 20 minutes of force swimming, and anesthesia, was extended over seven continuous days. Additionally, four instances of IFS stimulation (paired with contextual and cued tones) were administered on the odd days as the main stressor. Furthermore, to increase the unpredictability of stress, four different kinds of stressors were randomly administrated on each stress acquisition day.

The PTSD-like symptoms were observed and monitored for six weeks to determine the remote stress effects, including freezing behavior, corticosterone levels, neuronal connectivity, anxiety behavior, and sleep alterations. The results showed that the MPS-induced fear memory persisted for six weeks. Increased theta oscillation power and coherency were observed in the prefrontal cortex (PFC), ventral hippocampus (vHPC), and basolateral amygdala (BLA), with the direction of these transitions predominantly favoring fear consolidation after MPS stimulation. Anxiety behavior also extended after remote fear memory retrieval.

Significant REM sleep disruption was observed after MPS and fear memory retrieval, but with strengthened theta oscillation and sleep spindle during REM and NREM sleep, contributing to fear memory consolidation. The results of the prolonged remote fear memory, brain connectivity, anxiety, and sleep variations supported the long-lasting effect of the MPS rat model.

In summary, the above results have demonstrated the post-stress impacts from both the IFS and MPS rat models. Unpredictable stress decreases REM sleep due to the hyper-activation of hcrt-ergic neuron projections from LHA to the REM-off nuclei. Furthermore, enhanced stress repetition and intensity prolonged the behavioral abnormalities by increasing nuclei connectivity during waking and sleep. Stress is a widespread society issue that affects a considerable number of people and causes abnormalities in their physiology and psychology. A severe traumatic incident may increase the possibility of developing a serious syndrome, such as post-traumatic stress disorder (PTSD). Numerous animal models have been established to clarify the mechanism of PTSD; however, the generated symptoms vary due to diverse manipulations. Thus, in this dissertation, the acute and chronic rodent stress models, the inescapable footshock stress (IFS) and multiple prolonged stress (MPS), were determined and assessed, respectively. Two issues have been investigated: the sleep disruption caused by the IFS-induced neuropeptide and the MPS-derived extended PTSD-like symptoms.

IFS stimulation has been found to suppress rapid eye movement (REM) sleep, while the underlying mechanism remains unclear. Studies indicate that the neuropeptide hypocretin (hcrt) released from the lateral hypothalamic area (LHA) mediates stress response and REM sleep. Therefore, the present research elucidated the involvement of hypocretinergic (hcrt-ergic) projection from LHA to REM-off nuclei, locus coeruleus (LC) and dorsal raphe nucleus (DRN), in the IFS-induced sleep alterations. The results first illustrated that the hcrt-ergic neurons were activated in LHA and overexpressed hcrt in LC, DRN, thalamus, and amygdala. Blocking the LC and DRN regions by administrating TCS1102, a dual hcrt-receptor (hcrtR) antagonist, before IFS reversed the REM sleep reduction. Moreover, inhibiting the LC and DRN hcrtRs also improved the sleep quality after IFS by recovering the decreased intensity of non-rapid eye movement (NREM) sleep delta rhythm. However, the hcrt projections to LC and DRN have a limited effect on innate anxiety, as examined by waking theta powers and behavioral tasks. Overall, the results demonstrated that the IFS-induced REM sleep suppression and impairment in NREM sleep quality were governed by the overexpression of hcrt from LHA to the LC and DRN.

Single-shock acute stressors are commonly applied in rodent models, whereas composite life-threatening multiple-shock stressors have rarely been discussed. Therefore, a seven-day MPS protocol was constructed to initiate long-term chronic PTSD-like symptoms by modifying the repetition, intensity, and uncontrollable from the single-prolonged stress (SPS) model. The original single-day manipulation, consisting of 2 hours of restraint, 20 minutes of force swimming, and anesthesia, was extended over seven continuous days. Additionally, four instances of IFS stimulation (paired with contextual and cued tones) were administered on the odd days as the main stressor. Furthermore, to increase the unpredictability of stress, four different kinds of stressors were randomly administrated on each stress acquisition day.

The PTSD-like symptoms were observed and monitored for six weeks to determine the remote stress effects, including freezing behavior, corticosterone levels, neuronal connectivity, anxiety behavior, and sleep alterations. The results showed that the MPS-induced fear memory persisted for six weeks. Increased theta oscillation power and coherency were observed in the prefrontal cortex (PFC), ventral hippocampus (vHPC), and basolateral amygdala (BLA), with the direction of these transitions predominantly favoring fear consolidation after MPS stimulation. Anxiety behavior also extended after remote fear memory retrieval.

Significant REM sleep disruption was observed after MPS and fear memory retrieval, but with strengthened theta oscillation and sleep spindle during REM and NREM sleep, contributing to fear memory consolidation. The results of the prolonged remote fear memory, brain connectivity, anxiety, and sleep variations supported the long-lasting effect of the MPS rat model.

In summary, the above results have demonstrated the post-stress impacts from both the IFS and MPS rat models. Unpredictable stress decreases REM sleep due to the hyper-activation of hcrt-ergic neuron projections from LHA to the REM-off nuclei. Furthermore, enhanced stress repetition and intensity prolonged the behavioral abnormalities by increasing nuclei connectivity during waking and sleep.