呂國棟Lu, Kwok-Tung吳宗訓Wu, Zong-Syun2023-12-082028-08-142023-12-082023https://etds.lib.ntnu.edu.tw/thesis/detail/6662a81992692c80194946a33df96ea9/http://rportal.lib.ntnu.edu.tw/handle/20.500.12235/121344重鬱症 (major depression disorder) 為高盛行率的精神疾病，抗抑鬱藥 (antidepressants) 雖已被廣泛使用於控制及防止重鬱症的惡化，但此類藥物仍存在諸如：從開始服藥至症狀改善之間耗時甚鉅或強烈不良副作用等缺點，因此現存抗抑鬱藥於臨床應用上對於實際改善病患症狀的效果仍十分有限。綜合上述，故現今對於開發新型抗抑鬱藥物的需求是刻不容緩。目前已知早期壓力曝露 (early life stress exposure) 與不良創傷經驗 (adverse traumatic experience) 均為誘發重鬱症的重要風險因子，本研究使用新生期地塞米松投藥處理 (neonatal dexamethasone treatment, NDT) 來模擬早年壓力曝露並成功於小鼠中建立類憂鬱行為模式，並以此模式來探討腦源性神經滋養因子 (brain-derived neurotrophic factor, BDNF)、哺乳動物雷帕黴素靶蛋白 (mammalian target of rapamycin, mTOR) 訊息傳遞路徑與神經發炎反應 (neuroinflammation)、神經可塑性異常 (abnormality on the neuroplasticity) 等如何參與調控NDT所誘發之不良影響。海馬迴 (hippocampus) 為對壓力曝露十分敏感的腦區之一，並與重鬱症的發病機轉高度相關，本研究將著重於探討NDT對於背側海馬迴 (dorsal hippocampus) 內BDNF-mTOR訊息傳遞路徑所造成之影響。於此研究中，實驗動物將於出生後之初三日，逐日接受遞減劑量 (0.5、0.3、0.1 mg/kg) 之地塞米松 (dexamethasone) 皮下注射，並持續飼養直至六週齡時犧牲，並以即時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction, qPCR) 分析相關訊息傳遞分子與目標基因的表現量變異，再以胞外電生理技術 (extracellular recording) 紀錄神經訊號傳遞情形。實驗結果顯示，經NDT處理後實驗動物產生長期之不良影響。首先，NDT組之老鼠體重自出生後7至42日與SAL組相比均明顯較輕。NDT組背側海馬迴內之BDNF、TrkB基因表現量下降，顯示其BDNF訊息傳遞路徑受到影響。胞外電生理紀錄發現，NDT組老鼠其海馬迴突觸前、突觸後傳遞與長期增益效應均顯著劣於SAL組動物，上述結果顯示NDT處理將導致動物海馬迴之神經可塑性異常。此外，Nr3C1與Sgk1的表現量上升與Fkbp5的表現量下降顯示其HPA反應軸失調。ELISA結果顯示NDT組之動物血清內相較SAL組具有較高之糖皮質素表現量，然而此差異並未達統計檢定顯著水準。隨後，經檢驗背側海馬迴組織內相關突觸上離子通道表現量，其結果顯示：於NDT組別中Grin2a、Grin2b、Gria1、Cacna1c與Cacna1e表現量下降；然而，Slc1a6、Slc12a5、Gabra3、Gabra4、Gabbr1及Gabrd表現量顯著上升。近一步檢驗突觸蛋白 (synaptic protein) 與細胞骨架相關基因顯示Syn1、Syn2、Arc、Dlg4、Sptbn2與Khdrbs表現量顯著下降。為近一步了解上述變異之上下游訊息傳遞路徑，實驗結果顯示Raptor、Rictor與Ctnnb1基因表現量於NDT 組中顯著下降。除此之外，於NDT組中轉錄因子Nfkb1 基因表現量顯著增加，而Creb1基因表現量顯著下降，但發炎因子Il6與模式識別受體 (pattern recognition receptor, PRR) Nlrp6卻於NDT組中顯著下降。最後，經由連續兩天給予MHY1485，NDT組於懸尾試驗中之不動時間百分比顯著下降，於開放空間試驗中並未發現明顯之行為異常，雖於投藥過程中動物之體重產生些微下降，惟其可於投藥完成後兩天內回覆至基礎水平。綜合上述，實驗結果顯示mTOR訊息傳遞路徑於NDT所誘發之長期不良影響中扮演關鍵角色。據我等所能了解，本實驗為首次利用MHY1485作為抗憂鬱藥進行壓力所誘發之動物類憂鬱行為進行治療，其優良之抗憂鬱效果可作為未來開發新穎抗抑鬱藥物之參考。Major depression disorder (MDD) is a high-incidence psychiatric disorder worldwide. Antidepressants are widely used to control the symptoms and the deterioration of MDD. Unfortunately, the long latency of the drug effect onset and unpleasant side effects limited its usage and compliance of patients. It is an urgent need to develop new antidepressants. It is well-known that early-life stress is a risk factor for developing MDD in later life. Our previous study successfully used a neonatal dexamethasone treatment (NDT) to mimic early-life stress exposure and induced depression-like behaviors in mice. Here, we used the NDT model to study the involvement of the brain-derived neurotrophic factor& mammalian target of rapamycin (BDNF-mTOR) signaling pathway, neuroinflammation, and neuroplasticity in the long-term adverse impact of NDT. The hippocampus (Hip) is vulnerable to stress exposure and is highly correlated to the pathogenesis of MDD. In the present study, we focused on the NDT effects on the hippocampal BDNF-mTOR signaling pathway. To achieve this goal, NDT animals were subjected to a tapering dose (0.5, 0.3, and 0.1 mg/kg) of dexamethasone subcutaneous injection from postnataldays 1 to 3. The qPCR was applied to examine the possible gene expression alteration. The hippocampal neural activities were examined using a brain-slice extracellular recording.The result demonstrated the long-term adverse impact of NDT. First, the body weight measurement showed a continuous decrease from PND 7 to 42 in the NDT group. (Experiment 1) The disturbance of the BDNF signaling pathway was verified via the evidence of the downregulation in both Bdnf and Ntrk2 gene expression in the NDT group (Experiment 2). The extracellular recording of the Schaffer collateral pathway in the dorsal hippocampus (dHip) displayed a defect in HFS-LTP formation, I/O curve, and PPF recording. (Experiment 3) The upregulation of Nr3C1, Sgk1, and the downregulation of Fkbp5 gene expression in the NDT group revealed the dysregulation of the HPA axis. (Experiment 4-1) Results of ELISA showed a tendency of higher averages in corticosterone secretion in the NDT group. However, the difference was not achieved in statistical significance. (Experiment 4-2) Results showed decreasing gene expression in excitatory synaptic transmission-related channels in the NDT group, including Grin2a, Grin2b, Gria1, Cacna1c, and Cacna1e. Interestingly, the Slc1a6 gene expression was increased in the NDT group. (Experiment 4-3) Examination of synaptic protein showed decreased gene expression of Syn1, Syn2, Arc, and Dlg4. (Experiment 4-4) Data of the GABA transmission-related gene showed an upregulation in Slc12a5, Gabra3, Gabra4, Gabbr1, and Gabrd. (Experiment 4-5) Moreover, results showed downregulation in Raptor, Rictor, and Ctnnb1 gene expression in the NDT group. (Experiment 5) Further study about actin skeleton showed no alternation in Actb but downregulation in Sptbn2 and Khdrbs1 gene expression in the NDT group. (Experiment 6) As for neuroinflammation, the pro-inflammation transcription factor Nfkb1 gene expression was upregulated, and Creb1 gene expression was downregulated in the NDT group. Unexpectedly, Il6 and Nlrp6 had a decreased expression in the NDT group. (Experiment 7) Finally, with two days of MHY1485 injection, the immobility duration of the NDT group in the tail suspension test (TST) was significantly lower. No notable deviations were detected in the open field test (OFT), but a slight body weight drop was recorded, which would soon recover in two days. (Experiment 8)In summary, we highlighted that mTOR signaling was essential in NDT-induced long-term adverse impact. To our knowledge, we are the first who administrated MHY1485 as an antidepressant to cure stress-related depression-like behavior, which might provide hints for developing new pharmacological strategies for MDD patients.新生期地塞米松投藥處理背測海馬迴BDNF-mTOR 訊息傳遞路徑神經可塑性類憂鬱行為MHY1485neonatal dexamethasone treatmentdorsal hippocampusBDNF-mTOR signalingneuroplasticitydepression-like behaviorMHY1485etd