探討綑綁處理對於青少年期與成年期條件化恐懼與焦慮行為之影響

Comparison of juvenile and adult immobilization treatment on the conditioned fear and anxiety-like behavior

林維星(Lin Wei-Hsing)

指導教授 ： 呂國棟

國立臺灣師範大學/理學院/生命科學系/碩士(2019年)

https://doi.org/10.6345/THE.NTNU.SLS.006.2019.D01

摘要

在青少年或成年期遭受壓力（stress）或創傷經驗（traumatic experience），例如心理或生理的虐待（physiological or psychological abuse），均會造成長期的不良影響（long-term adverse impact），包括引發創傷後壓力症候群（post-traumatic stress disorder, PTSD），及提高罹患焦慮症（anxiety）之風險，但是目前對於探討青少年壓力或創傷長期影響的動物模式十分稀少。而在青少年期時，下視丘-腦垂體-腎上腺軸（hypothalamic-pituitary-adrenal axis, HPA axis）仍處於發育及調整期，因此遭受創傷時，會較成年期有更嚴重的不良影響。本研究之主要目的為探討青少年的壓力及創傷經驗，造成長期不良影響之機轉。
實驗結果顯示，於小鼠青少年期（五週齡）給予每天兩小時連續兩天綑綁處理（immobilization treatment, IMO），會在隔週的條件化恐懼實驗中，造成恐懼記憶無法削減（extinction）的現象。而只進行單一次的IMO處理，則不會對削減作用有明顯影響，表示可能有累加效應（accumulation effect）的存在。之後在開放空間測試（open field test），IMO 組亦出現類焦慮行為（anxiety-like syndrome）增加的現象。另外在即時定量聚合酶連鎖反應（real-time polymerase chain reaction, qPCR）中，發現經兩次IMO處理的動物，其海馬迴（hippocampus）中Fkbp5的表現量顯著增加。爾後透過離體胞外電生理記錄法（in vitro extracellular recording）測試海馬迴的長期增益效應（long-term potentiation，LTP），結果顯示青少年經IMO處理兩次的動物，在其海馬迴的LTP出現增強的現象。此外，我們發現青少年IMO 處理組（五週齡，IMO_2 (J)）與成年IMO處理組（十一週齡，IMO_2 (A)）皆在十二週齡的條件化恐懼實驗中呈現無法削減恐懼記憶的異常反應，牠們也在高架十字迷宮（elevated plus-maze）中出現類焦慮的行為特徵。透過qPCR發現Fkbp5的表現量，只在IMO_2 (A) 的海馬迴與杏仁核（amygdala）中都明顯增加；另外在Bdnf 的表現量，只在IMO_2 (A) 組動物的杏仁核中明顯增加，而在IMO_2 (J)組皆沒有顯著增加，顯示青少年IMO處理對Fkbp5與Bdnf的表現量只有短期影響，效果不會持續到成年期。
本實驗採用青少年或成年期綑綁方式，模擬創傷經驗對於不同時期條件化恐懼及類焦慮反應之影響，發現青少年的IMO 處理可能有累加效應（accumulation effect）與急性影響（acute effect），而本研究提供往後可供研究青少年期壓力或創傷經驗的動物模式。研究成果具有臨床應用之價值，將有助於未來新一代治療策略及藥物之開發。

關鍵字

青少年 ； 不良對待 ； 綑綁處理 ； 條件化恐懼 ； 開放空間測試 ； 舉臂式十字迷宮 ； 削減作用 ； 電生理的長期增益效應

並列摘要

Previous studies have demonstrated that some adverse experiences in juveniles or adults, such as sexual molest and psychological or physiological abuse, would elicit long-term deleterious effects which might increase the susceptibility of exhibiting the post-traumatic disorder (PTSD) and anxiety disorder in adulthood. Up to now, however, the animal model for the study of these adverse experiences still appears to be rather limited on exploring the long-term effect on juveniles. To juvenile, the hypothalamic–pituitary–adrenal (HPA) axis is still under development and fine-tuning, making it vulnerable to the trauma experience and stress. The present study was aimed to study the possible long-term adverse effect of juvenile immobilization treatment and the underlined mechanism.
In this study, we attempted to investigate the mechanism of early life immobilization treatment effects on the behaviors of the consolidation and extinction of conditioned fear. The juvenile mice (5 weeks old) were subjected to immobilization (IMO) treatment and then tested for conditioned fear and anxiety response at the age of 6 weeks (juvenile) and 12 weeks (adult) old. Several behavioral experiments, including conditioned fear, open field test (OFT) and elevated plus-maze (EPM) were applied to investigate the effect of juvenile IMO on the conditioned fear. Also, we also used real-time polymerase chain reaction (qPCR), to elucidate the molecular mechanism underlying the long-term effect of juvenile immobilization treatment. Brain tissues, including the amygdala and dorsal hippocampus, were collected and subjected to biochemical analysis.
Our results showed that mice with two times of immobilization treatment (two hours per day, for two consecutive days, IMO_2) at the age of 5 weeks old (juvenile) displayed interference on the extinction of conditioned fear. And, such the impairment was not significant in the single IMO treated animals. These results indicated that an accumulation effect was observed for the juvenile immobilization treatment. Besides, an anxiety-like behavior phenotype was also found from the OFT of IMO treated animals. The results of the qPCR revealed a significant increase in the hippocampal Fkbp5 expression in the IMO_2 animals. Furthermore, high-frequency stimulation (HFS) could induce long-term potentiation (LTP) elevation in the IMO_2 animals, suggesting a long-term effect of IMO on the function of the hippocampus.
Based on this study, both juvenile (5 weeks old, IMO_2(J)) and adult (11 weeks old, IMO_2(A)) with twice IMO treatment showed impairment on the extinction of conditioned fear on adulthood (12 weeks old), meaning that the impact of juvenile IMO treatment will be sustained into adulthood. According to qPCR, the expression of Fkbp5 was increased significantly in both hippocampus and amygdala of IMO_2 (A). Similarly, the expression of BDNF revealed the same trend in the amygdala of the IMO_2(A). Above results clearly indicated that the impact of juvenile IMO treatment in the expression of Fkbp5 and Bdnf is just an acute effect, and it could not be sustained into adulthood.
In summary, the present study has adopted juvenile or adulthood IMO to simulate the traumatic experience and study its long-term impact on the conditioned fear response and anxiety-like response. Results showed that IMO treatment might reveal some accumulative and acute effects. We demonstrated the current animal model could be applied in studying the influences of juvenile stress and traumatic experience. Results of this study should as well have contributed to the development of novel therapeutic strategies and pharmacological interventions.

並列關鍵字

juvenile ； maltreatment ； immobilization ； conditioned fear ； open field testing ； elevated plus maze ； extinction effects ； long-term potentiation

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