地塞米松（dexamethasone, DEX）是一種合成的糖皮質激素，通常用作消炎藥物。它亦被廣泛用於早產兒身上（prematurely born infant），以治療早產嬰兒的慢性肺部疾病（chronic lung disease)。新生期投予地塞米松治療（neonatal dexamethasone treatment, NDT）已被廣泛研究並證明對中樞神經系統具有長期的不良反應。我們先前的研究結果顯示，新生期投予地塞米松治療（neonatal dexamethasone treatment, NDT）對於成年雄性和青春期雌性大鼠均引起類憂鬱行為（depression-like behavior）的顯著增加。然而，新生期投予地塞米松治療，對成年雌性大鼠的長期影響仍有待探討。除了傳統的alpha （ER）和beta（ER） 型雌激素受體外，G蛋白偶合雌激素受體（G protein-coupled estrogen receptor, GPER舊稱為GPR30）亦廣泛分佈於生殖系統、心血管系統和腦組織中，尤其是在海馬迴（hippocampus）中有大量表現。前人的研究發現GPER是一種位於細胞膜上的雌激素受體，在乳腺、血管內皮細胞及生殖系統相關癌症中呈現異常的表現及作用。然而，對於GPER的神經作用，特別是對行為的影響及作用機制仍待探究。GPER是否會影響海馬迴長期增益效應（long-term potentiation, LTP）的形成？以及杏仁核（amygdala）是否會受到NDT影響？還有待進一步研究。本研究之主旨在為探討GPER是否參與NDT對雌性大鼠產生的長期不良影響。
在此研究中，出生第1天至第3天（postnatal day 1 to 3）對雌性幼鼠以皮下注射方式（subcutaneous injection）投予遞減劑量（0.5, 0.3, & 0.1 mg/kg）的DEX。爾後於10週齡時進行一系列的行為、生化以及電生理實驗。例如以強迫游泳實驗（forced swim test, FST）和蔗糖糖水偏好實驗 （sucrose preference test, SPT） 來評估NDT所誘發的類憂鬱行為。此外使用開放空間測試（open field test, OFT）來評估DEX的非專一效應 (non-specific effect)。為了防止行為實驗對生化分析所造成之干擾，我們以相同的方式準備了另外一批動物，於十週齡時斷頭犧牲，取出其海馬迴進行即時定量聚合酶連鎖反應（real-time polymerase chain reaction, qPCR）分析ERα、ERβ和GPER的表現。部分動物則用於離體胞外電生理記錄實驗 (in vitro extracellular recording)，以檢測海馬迴腦切片中，由高頻電刺激（high-frequency stimulation, HFS）所誘發的長期增益效應（long-term potentiation, LTP），並以直接灌流方式（suprafusion）投予GPER的選擇性致效劑 （selective agonist） G1，評估G1是否能挽回海馬迴中的LTP。再以直接灌流方式（suprafusion）投予GPER的選擇性拮抗劑 （selective antagonist） G15，評估G15是否能阻斷G1所挽回海馬迴中的LTP。
實驗結果顯示，NDT雌鼠在FST中的不動的時間百分比(percent time of immobilization) 明顯增加，而SPT的結果亦顯示其蔗糖偏好指數(sucrose preference index)明顯降低，但NDT與控制組在OPT中的水平運動距離(total horizontal moving distance)並無明顯差異，此項結果顯示NDT成年雌性大鼠的類憂鬱行為增加，應與DEX的非專一效應對活動能力之干擾無關。在qPCR的結果顯示，青少年期時雌性NDT大鼠海馬迴中ER、ER和GPER的表現量，與控制組相比並無明顯差異。然而，在成年期NDT雌性大鼠的海馬迴中GPER的表現量則明顯減少。離體胞外電生理記錄的結果顯示，成年期NDT雌性大鼠海馬迴中，HFS-LTP的強度顯著降低，而在表面灌流GPER的選擇性致效劑G1後，NDT雌鼠海馬迴之LTP可恢復至正常水平，並且在表面灌流GPER的選擇性拮抗劑G15後G1的拯救效果被完全阻斷。
總結各項實驗結果，GPER在NDT對海馬迴中LTP之異常表現中，扮演了十分重要的角色。後續的研究可針對G1是否亦能對NDT所引發的行為異常，具有治療效果進行進一步的探討。利用藥第投予恢復海馬迴中GPER的表現量，或是利用GPER的選擇性致效劑，極可能對NDT所造成之行為異常，具有治療的效果，未來可朝此二個方向進行藥物研發。

關鍵詞: 地塞米松、海馬迴、G 蛋白偶合雌激素受體、長期增益效應

ABSTRACT
Dexamethasone (DEX) is a synthetic glucocorticoid, commonly used as an anti-inflammatory agent. It is also widely used for premature infants to cure the onset of chronic lung disease occurred to them. Neonatal DEX therapy (NDT) has been shown to have long-term adverse effects on the central nervous system. Our previous studies revealed a fact that both early-adult male and adolescent female rats with NDT were found to exhibit a significant increase in a depression-like behaviour. However, little is known about the long-term effect of NDT on adult female subjects. G protein-coupled estrogen receptor 1 (GEPR, also known as GPR30) is widely produced in reproductive system, cardiovascular system, and brain, especially with high expression in hippocampus. Previous studies have indicated that GPER is a functional estrogen receptor that is involved in some reproductive related cancers, such as breast cancer. It also acts in vascular endothelial cells and causes vasodilation, thereby reducing the antihypertensive effect of estrogen. However, the detailed physiological role of GPER is still unclear. Whether GPER can affect the formation of long-term potentiation (LTP) in the hippocampus and whether the amygdala will be affected by neonatal DEX treatment remains to be further elucidated.
In this study, the female pups were subject to a tapering dosage (0.5, 0.3, & 0.1 mg/kg, subcutaneously) of DEX from postnatal day 1 to day 3. The associated behavioural, biochemical, as well as electrophysiological recordings, were performed at 10 weeks old of (adult female rats). Forced swim test (FST) and sucrose preference test (SPT) were used to eveluate the NDT-induce depression-like behavior. An open field test (OFT) was applied used to analyze the non-specific effects of NDT. Adult female rat’s hippocampus was later dissected for real-time polymerase chain reaction (qPCR) to quantify the expression of estrogen receptor alpha (ERα) and beta (ERβ) and GPER. Finally, hippocampus brain slices were further examined by using an in vitro extracellular recording to reveal LTP induced by high-frequency stimulation (HFS). In addition, a hippocampal suprafusion of GPER selective agonist G1 and GPER selective antagonist G15 was also used in extracellular recording to verify the role of the receptor.
Our results showed an increase in depression-like behavior in adult NDT female rats. The FST showed an increase in the percent time of immobility. The SPT also showed a decrease in the sucrose preference index. However, there was no difference in the total distance travel distance between the NDT and SAL groups during the behavior test. From the results of RT-PCR, we observed no significant difference in hippocampal ERα, ERβ and GPER expression in adolescent female rats. However, the hippocampal GPER expression exhibited a significant decline in adult female rats. The extracellular recording showed a decrease in HFS-LTP in hippocampal CA3-CA1, and G1 could restore LTP formation and the rescue effect of G1 can be blocked by GPER selective antagonist G15 completely.
Conclusively, the present study has demonstrated that GPER is critical to the NDT-induced impairment on the hippocampal LTP formation. Further studies should be worthwhile to more clearly understand the functional role of GPER in the NDT-induced behavioral abnormalities. We suggest either restore the GPER expression or directly activate the GPER might be an effective and promising strategy for the development of novel therapeutic approaches for curing the sequel of NDT.

Keyword: Dexamethasone, Hippocampus, G-protein coupled estrogen receptor, GPER, Long-term potentiation

Table of Contents
中文摘要 VI
ABSTRACT IX
INTRODUCTION 1
The long-term impact of early life adversities and traumatic experiences 1
The hypothalamic–pituitary–adrenal axis- 2
Estrogen Receptors and mental disorders 4
METERIALS AND METHODS 7
Animals 7
Neonatal Dexamethasone Administration Protocol 7
Somatic Growth Monitoring 8
Administration of G1, a Selective Agonist of G-protein Coupled Estrogen Receptor 8
Administration of G15, a Selective Antagonist of G-protein Coupled Estrogen Receptor 8
RESULTS 9
Neonatal Dexamethasone Treatment Lessen the Somatic Growth of Female Rats Temporarily 9
Neonatal Dexamethasone Treatment Promotes the Depression-like Behavior in the Adult Female Rats Demonstrated by Forced Swim Test 11
The NDT Induced Depression-like Behavior Demonstrated by Sucrose Preference Test 12
The Gene Expression of Hippocampal Gper was Decreased in NDT Adult Female Rats Analysed by Real-time Polymerase Chain Reaction 14
The Hippocampal LTP Formation was Decreased in NDT Adult Female Rats and Restored by Perfusion of GPER Agonist G1 and Blocked by Perfusion of GPER Antagonist G15 in NDT Adult Female Rats Analysed Using in vitro Electrophysiological Recording 16
DISCUSSION 19
FIGURES 26
Figure 1: The hypothalamic-pituitary-adrenal (HPA) axis response to the stress(Adapted from Raabe & Spengler, 2013). 26
Figure 2: The HPA axis plays a key role in the regulation homeostasis and the response to stress (Adapted from McGowan & Matthews, 2018). 27
Figure 3: The negative feedback control pathways of the glucocorticoids release (Adapted from Andrew J et al., Autopsy Pathology: A Manual and Atlas, 3th edition. Elsevier, 2016, ch.9). 28
Figure 4: Glucocorticoid signalling pathways (Adapted from Schoneveld et al., 2011). 29
Figure 5: Role of glucocorticoids in health and disease (Modified from Kadmiel & Cidlowski, 2013). 30
Figure 6: G-protein-coupled estrogen receptor (GPER) involved in regulation physiological responses and disease. (Modified from Prossnitz & Barton 2011). 31
Figure 7: The mechanisms of E2 in object recognition memory in ovariectomized mice. Adapted from (Adapted from Frick et al., 2018a). 32
Figure 8: Classical (genomic) and non-classical (non-genomic) mechanisms of E2 action (Adapted from Frick, 2015). 33
Figure 9: The Caveolin protein is responsible for the separation of functionally distinct signalling pathways in neurons (Adapted from Luoma et al., 2008). 34
Figure 10: Schematic representation of G-protein coupled estrogen receptor mediated signalling (Adapted from Liu et al., 2012). 35
Figure 11: Neonatal dexamethasone treatment impaired somatic growth of the female rats in a temporary manner. 36
Figure 12: Forced swim test for the depression-like behavior of NDT adult female rats. 37
Figure 13: Sucrose preference ratio for the depression-like behavior of NDT adult female rats. 38
Figure 14: Sucrose preference index for the depression-like behavior of NDT adult female rats. 40
Figure 15: qPCR Analysis on the Neonatal Dexamethasone Treatment Effect on the Hippocampal ERs Gene Expression in the Female Rats 41
Figure 16: GPER Agonist G1 Could Restore the High Frequency Stimulation- induced Hippocampal Long-term Potentiation in the Adult Female Rats. 42
Figure 16: GPER Agonist G1 Could Restore the High Frequency Stimulation- induced Hippocampal Long-term Potentiation in the Adult Female Rats. 43
Figure 17: GPER Antagonist G15 Could Blocked the GPER Agonist Effect on the High Frequency Stimulation-induced Hippocampal Long-term Potentiation in the Adult Female Rats. 45
Abbreviation table 46
References 50