海馬迴是大腦中與學習記憶相關的腦區，許多認知及記憶的功 能異常的疾病皆和這個腦區息息相關，研究者已發現在成熟哺乳類大腦的海馬迴區中的齒狀迴(dentate gyrus)的顆粒細胞下區 (subgranular zone, SGZ)持續有神經新生的情形而此區的顆粒 細胞需要腎上腺分泌的皮質酮才可以生存，因此摘除雙側的腎上腺 會使海馬迴齒狀迴區的顆粒細胞因缺乏皮質酮而大量死亡。實驗室使用腎上腺移除(adrenalectomy;ADX)後的老鼠作為海馬迴齒狀迴區顆粒細胞死亡的動物模型，本實驗室先前研究發現給予sonic hedgelog (Shh)並搭配豐富環境的刺激可以使此區顆粒細胞大量生 長。因此我們在ADX後三個月確定手術成功的老鼠分為兩種實驗組別: (1 ) 短時間組: 給予不同濃度的delta s並放於鼠籠環境或豐富性環境一週後犧牲;(2)長時間組: 給予不同濃度的delta s並放於 鼠籠環境或豐富性環境十週在進行物體辨識行為後犧牲。分別比較藥物濃度、空間、時間的變異對海馬迴齒狀回區細胞的影響，我們發現delta s有適合細胞生長的特定濃度，短時間中環境的差異與細胞的數目多寡相關，長時間的組別中我們發現ADX後處理特定的delta s濃度可以促進神經新生，我們使用一旦受到刺激突觸活化的時候便會表現的Arc來做細胞染色計數，發現新生的神經細胞同時參與物體辨識行為中，在討論部分也針對Shh及本篇使用的delta s 進行短時間組的實驗比較，發現delta s在海馬迴齒狀迴區的新生神經前驅細胞的數目比Shh多，並且能在長時間恢復其學習記憶能力，為具發展潛力的促進神經新生藥物。

海馬迴不僅在學習與記憶中扮演重要的角色，在許多神經退化性疾病中亦佔有重要的地位。海馬迴的齒迴內側區是哺乳動物大腦中成體幹細胞主要來源區域之一，其所新生的海馬迴粒細胞會往上遷移至海馬迴粒細胞層並與固有神經細胞形成功能性連結。 過去的研究發現太少或過量的壓力荷爾蒙均會造成海馬迴粒細胞的死亡，而一定量濃度的皮質固醇對於維持海馬迴粒細胞的生存亦扮演非常重要的角色。在摘除兩側的腎上腺後，海馬迴粒細胞在幾週後會逐漸死亡且造成認知功能的缺損。本實驗即利用雙側腎上腺摘除術建立動物模式，企圖了解海馬迴粒細胞在凋亡的過程中所產生的生理層面的改變。 壓力荷爾蒙（包含皮質固醇，在老鼠稱為corticosterone，在人類稱為cortisol）為腎上腺皮質分泌激素，已知會參與並調控BK 離子通道的選擇性剪接。BK離子通道的孔道形成α次單元由單一基因 (Slo) 負責轉錄，含有STREX外顯子的剪接變異體之α次單元藉由加速神經細胞的再極化，增強過極化電位以及促進鈉離子通道自去活化狀態中回復可造成神經細胞重複激發，而先前的研究已發現過度的激發會對神經細胞產生興奮性毒殺作用。本實驗即探討BK 鉀離子通道選擇性剪接在海馬迴粒細胞凋亡的過程中所扮演的角色。 實驗結果發現，與對照組相比，雙側腎上腺摘除的老鼠海馬迴細胞中含有STREX外顯子的剪接變異體在mRNA含量上確實有改變，而BK 鉀離子通道蛋白質含量亦有所變化。由上述結果推測，含有STREX外顯子的剪接變異體含量可能與海馬迴粒細胞的凋亡機制有關。 / The hippocampus is a brain region central to learning and memory and is a key target of many neurological diseases that have dramatic cognitive consequences, including Alzheimer’s and other forms of dementia, stroke, epilepsy, and chronic stress. Hippocampal granule cells are one of the two cell pools that contain newborn neurons continuously generated from the subgranular zone in adult mammalian brains. The newborn neurons will migrate to the granule cell layer and integrate into preexisting neuron network. Previous studies have indicated that both an excessive and insufficient levels of stress hormones can lead to neuron death. Corticosterone, an adrenal stress hormone, is essential for the survival of granule cells. Bilateral removal of adrenal glands leads to extensive granule cell death over a period of several weeks and gradually causes cognitive deficits. To understand the mechanisms underlying the granule cell death in the hippocampal formation, adrenalectomy (ADX, removal of adrenal glands) was used to specifically eliminate granule cells in the hippocampus, and the subsequent physiological changes in the hippocampal neurons including dentate granule cells are investigated. Stress hormones (corticosterone in rats and cortisol in human) , secreted from the adrenal cortex regulate the alternative splicing of BK channels (big potassium, calcium-voltage activated potassium channels) in adrenal medulla. An inclusion of STREX (stress axis-regulated exon) exon in pore-forming α subunit encoded by Slo gene promotes repetitive firing by speeding action potential repolarization and augmenting the afterhyperpolarization, as well as facilitating sodium channels de-inactivation. In the present study, the role of BK channel alternative splicing in the ADX-induced granule cell death in the hippocampus was explored. The results indicate that BK channel alternative splicing was regulated by stress hormones in the hippocampus including dentate gyrus. The expression patterns of STREX variant in hippocampus were altered after granule cells death induced by ADX, whilst the expression of total slo gene was changes only in translational level. These observations suggest that the alternation in STREX abundance might be involved in the induction of dentate granule cell death.

在成年的哺乳類動物大腦中有兩個區域，可以不斷的有新的神經細胞生成，一個位於大腦側腦室旁內側(Subventricular zone of anterior lateral ventricle ;SVZ)，另一個位於海馬迴(hippocampus)內的齒狀迴(Subgran- ular zone of dentate gyrus ;SGZ) ，其中海馬迴是本論文主要探討的腦區。 神經前驅細胞(Neural progenitor cells :NPC)因具有自我更新(self -renewal）、增殖(proliferative）、多能(multipotent）的能力以及遷移性(Migration)，所以可利用海馬迴內生性的神經前驅細胞(NPC)，促進其增殖以替代因損傷、老化或疾病而損失的神經細胞。神經前驅細胞經由細胞體外培養過程會形成神經球(Neurospheres)，神經球和神經前驅細胞同樣具有自我更新以及可以分化成其他神經細胞的能力。 本研究觀察到，對成年神經新生進行體外藥物的篩選中，化合物Chemical-X，有明顯的促進神經新生的能力。實驗中取健康成年雄性大鼠為實驗動物，分離出成年大鼠之海馬迴神經前驅細胞。用Chemical-X處理後，觀察神經球自我更新能力，以及再把新生成的神經球利用免疫螢光染色處理，瞭解神經前驅細胞經藥物處理後所新生成的細胞，是否仍維持在神經前驅細胞的狀態。進而評估藥物能否達到促進神經新生的目的。

一、研究動機與目的 本研究的目的，在於探討血壓胺在記憶上是扮演抑制或促進的角色，由於腦中的血壓 胺有百分之八十以上含量分佈在背縫核（B ７）和腹縫核（B ８），而投射到前腦， 背縫核大部份投射到紋狀體，腹縫核大部份投射到海馬迴，兩者在功能上有很多不一 樣的地方。因此，本實驗目的，在於分別破壞紋狀體、海馬迴和前腦中血壓胺的含量 ，來探討血壓胺在記憶上所扮演的角色。 二、研究方法 （一）實驗動物 實驗動物為９０隻雄性大白鼠。 （二）實驗程序 首先分成三個實驗進行，分別手術前腦、紋狀體、海馬迴三個區域。手術十天後，開 始進行實驗，分別給予電擊訓練後，馬上注射藥物至手術的區域，以破壞血壓胺的含 量，四十八小時後，測試大白鼠對於電擊的記憶，測試完畢之後，馬上將大白鼠犧牲 掉，分別取前腦、紋狀體、海馬迴、背縮核以及腹縫核的組織，以作組織化學分析。 （三）統計分析 （１）有關記憶的行為反應，以無母數統計方法進行分析。 （２）有關腦部血壓胺含量的分析，以單因子變異量的模式進行分析。 （四）預期實驗結果 （１）行為分析 破壞血壓胺組的大白鼠，其記憶行為反應較未破壞組的大白鼠差。 （２）組織化學分析 血壓胺的含量：背縫核較腹縫核多。未被破壞組較破壞組多。

本實驗室於先前利用莫氏水迷津試驗篩選學習快與學習慢的大白鼠，取出其海馬迴組織並進行聚合酶連鎖反應差異顯示(PCR differential display)，結果顯示學習快與學習慢的大白鼠背側海馬迴之間共有98個cDNA片段有差異表現。把這些cDNA片段進行定序並利用BLAST資料庫比對，其中一個cDNA片段為大白鼠的pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] 基因。為了瞭解pias1基因的表現是否和空間學習有所關聯，隨機把大白鼠分成兩組，一組為有訓練組別(有空間線索與隱藏式平台)，另一組為無訓練的組別(沒有平台，作為游泳的控制組)同時進行莫氏水迷津學習試驗。試驗完畢，取出海馬迴組織進行即時定量聚合酶連鎖反應與西方墨點法來分析PIAS1的mRNA與蛋白質的表現。結果顯示有水迷津訓練的大白鼠，其PIAS1的mRNA與蛋白質表現皆明顯的高於無訓練的組別。為了更進一步確認PIAS1在空間學習中所扮演的角色，我們利用基因轉染的技術，轉染PIAS1 siRNA至大白鼠海馬迴CA1區域抑制PIAS1的表現。我們發現轉染PIAS1 siRNA至CA1區域會抑制大白鼠在水迷津的行為表現，然而轉染野生型的PIAS1質體基因至CA1區域卻會增進水迷津試驗的學習能力，同時我們也以西方墨點法發現，當轉染PIAS1 siRNA會增加STAT1 Tyr701的磷酸化，而轉染PIAS1 WT則會抑制STAT1 Tyr701的磷酸化。為了探討PIAS1促進記憶形成的分子機制，我們發現當轉染突變型的STAT1 Y701F質體基因至CA1區域，會抑制PIAS1 siRNA所造成記憶的損害。這些實驗結果代表著PIAS1會抑制STAT1 Tyr701的磷酸化，而PIAS1促進記憶的形成可能是藉由抑制STAT1 Tyr701的磷酸化而達成。另外，我們也單獨轉染突變型的STAT1 Y701F質體基因至CA1區域，水迷津實驗結果顯示會促進空間記憶的形成。目前PIAS1在免疫的角色已有許多研究證實，但是本篇研究是第一個提出PIAS1會參與哺乳類動物學習與記憶形成探討。 / Our laboratory has previously identified 98 cDNA fragments by using PCR differential display from rat dorsal hippocampus that are differentially expressed between fast learners and slow learners from the water maze learning task. After sequencing and BLAST analysis, one of these cDNA fragments encodes the rat pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] gene. In order to determine whether pias1 gene expression is associated with spatial learning, naïve rats were randomly assigned to the trained group (with visual cues and platform been present) and the non-trained group (without the platform as the swimming control). The dorsal hippocampus from these animals was dissected out at the end of the training and was subjected to RNA and protein extraction for real-time PCR and Western blot analysis of PIAS1 expression, respectively. Results revealed that the pias1 mRNA level and protein level was both higher in the hippocampus of trained rats than non-trained rats. To further examine the role of PIAS1 involved in spatial learning and memory, the specific PIAS1 siRNA was used to knockdown the expression of PIAS1 in rat hippocampal CA1 region. We found that transfection of PIAS1 siRNA to the CA1 area impaired water maze performance, whereas transfection of the wild-type PIAS1 DNA plasmid to the CA1 area facilitated water maze performance in rats. Meanwhile, PIAS1 siRNA increased STAT1 phosphorylation at Tyr701 whereas PIAS1 WT decreased STAT1 phosphorylation at this residue. In the examination of molecular mechanism underlying PIAS1-mediated memory facilitation, we have found that transfection of the STAT1 Y701F mutant plasmid antagonized the memory-impairing effect of PIAS1 siRNA, whereas transfection of STAT1 Y701F alone facilitated spatial memory formation. These results together suggest that one of the molecular mechanisms underlying PIAS1-mediated memory facilitation is through decreased STAT1 phosphorylation at Tyr701. All these manipulations did not affect visible platform learning in rats. In addition to the well documented role of PIAS1 in the immune system, here we have been the first to demonstrate a novel role of PIAS1 involved in spatial memory formation in rats.

STAT1是一個轉錄因子，在細胞生理功能中是非常重要的訊息傳遞者，在免疫系統具有抗病毒的角色，但是目前為止對於STAT1在中樞神經系統所扮演的角色仍不清楚。爲證實STAT1的表現與空間記憶的形成有關聯，我們將大白鼠分成兩組，一組為有訓練的組別，另一組則為無訓練的組別分別進行水迷津試驗，試驗完畢後取出大鼠的海馬迴CA1區域組織進行即時定量聚合酶連鎖反應與西方墨點法分析。結果顯示，經過水迷津訓練的刺激下，STAT1 mRNA與蛋白質分別減少約34 %及40 %，而STAT2 mRNA及蛋白質的表現則不受空間學習的影響。爲了進ㄧ步探討STAT1在空間學習記憶過程中所扮演的角色，實驗利用STAT1 siRNA轉染至海馬迴CA1區域抑制STAT1的表現，發現降低STAT1表現會促進大白鼠在水迷津試驗的學習能力，實驗同時也轉染STAT2 siRNA至CA1區域，結果顯示STAT2不參與大白鼠空間記憶的形成。本實驗室先前發現降低laminin β1表現量會促進大白鼠的空間學習記憶 (unpublished observation, 附錄二)，此外laminin β1基因啟動子上具有STAT1結合序列：interferon-γ activated site (GAS)。因此，實驗利用PC12細胞進行laminin β1報導基因分析，結果顯示STAT1會促進 laminin β1啟動子的轉錄活性。而爲了進一步探討在STAT1影響空間學習與記憶歷程中與laminin β1的關聯性，實驗利用STAT1 siRNA抑制大白鼠海馬迴CA1區STAT1的表現並促進空間學習與記憶的同時，發現laminin β1 mRNA及蛋白質表現量都受到STAT siRNA的抑制，而轉染野生型STAT1-Flag質體則會增加laminin β1 mRNA及蛋白質的表現量，顯示STAT1正向調控laminin β1的表現。本篇論文提出海馬迴CA1區域的STAT1參與動物空間學習與記憶的形成，其中可能與STAT1正向調控laminin β1的表現有關。 / STAT1 is a signal transducer and transcription factor in the cell. Several reports have indicated that STAT1 plays a critical role in immune response against virus infection in animals. However, the role of STAT1 in the central nervous system is still unclear. In the present study, we aimed to examine the role of STAT1 involved in spatial memory formation in rat and the possible downstream gene that STAT1 regulates. Rats were randomly divided into the trained group and the non-trained group. Animals were subjected to water maze learning according to the previous behavioral paradigm. Their hippocampus CA1 tissues were dissected out for STAT1 mRNA level and protein level determination. Results indicated that spatial training markedly decreased STAT1 mRNA level and protein level in the CA1 area, but this change was not found for STAT2 mRNA and protein expression. To further confirm the role of STAT1 involved in spatial learning and memory, animals were transfected with STAT1 siRNA in the CA1 area. Results showed that STAT1 siRNA transfection significantly facilitated water maze performance, whereas their water maze performance under STAT2 siRNA transfection was not altered. Previous studies from our laboratory have demonstrated that laminin β1 impairs spatial memory formation in rat (unpublished observation). In addition, promoter analysis indicates that the laminin β1 promoter region contains two GAS elements, which is the STAT1/STAT1 and STAT1/STAT3 binding site. Results from luciferase reporter assay revealed that transfection of STAT1 siRNA decreased laminin β1 promoter activity, whereas transfection of STAT1 wild-type plasmid increased laminin β1 promoter activity. To further study the relationship between STAT1 and laminin β1 in spatial memory formation, we used STAT1 siRNA to knockdown STAT1 expression and these animals were subjected to spatial training. We then determined their laminin β1 expression. Results showed that the laminin β1 mRNA level and protein level were both significantly decreased by STAT1 siRNA transfection. Besides, STAT1 wild-type plasmid transfection increased laminin β1 mRNA level and protein level in the CA1 area associated with spatial memory impairment. These results together suggest that STAT1 negatively regulates spatial memory formation. Further, STAT1 may impair spatial memory formation through increased laminin β1 expression.

周邊注射安非它命能夠影響動物受試在表現與時間知覺有關的操作式制約行為作業，歷來被研究者認為是大腦多巴胺神經系統與動物時間知覺系統有關的主要證據之一。本研究所共同採用的研究方法為先注射多巴胺受體專屬拮抗劑再於大鼠受試周邊腹腔注射安非它命的方式探討安非它命影響大鼠時間知覺的大腦機制為何。實驗一利用區辨性增強低頻反應作業觀察周邊注射多巴胺受體專屬拮抗劑何者可以反制周邊安非它命對此作業的影響效果，結果發現多巴胺D1受體拮抗劑SCH23390與D2受體拮抗劑raclopride均可反制周邊安非它命的效果。實驗二同樣利用區辨性增強低頻反應作業，但是將SCH23390與raclopride分別注入海馬迴、背側中區紋狀體、腹側側邊紋狀體、依核、內側前額葉皮質以及腹側頂蓋區等六個部位，觀察何種多巴胺受體拮抗劑可在那些大腦部位產生反制周邊安非它命的效果。結果發現SCH23390可在海馬迴、依核、內側前額葉皮質以及腹側頂蓋區等四個部位產生反制周邊安非它命的效果，而raclopride可在腹側側邊紋狀體與內側前額葉皮質兩個部位產生同樣的反制效果。實驗三利用高峰時距作業觀察SCH23390在海馬迴與內側前額葉皮質是否能反制周邊安非它命對此作業的影響效果，結果發現SCH23390僅在海馬迴會影響大鼠受試的正常表現，特別是在與周邊安非它命同時注射的時候。綜合以上結果顯示，周邊注射安非它命能夠使大鼠受試在區辨性增強低頻反應作業當中表現出時間知覺變快的傾向，這個效果需要同時透過大腦內的海馬迴、依核、內側前額葉皮質以及腹側頂蓋區的多巴胺D1類受體和腹側側邊紋狀體與內側前額葉皮質的多巴胺D2類受體。 / The central dopaminergic system has been hypothesized to play a role in time perception based on the results that peripheral injections of d-amphetamine alter the responses in time-related operant conditioned behavioral tasks. The present study investigated the effect by injecting specific dopamine receptor antagonists before peripheral d-amphetamine injections in rats. Data from Experiment I showed that both peripheral the dopamine receptor D1 antagonist SCH23390 and D2 antagonist raclopride could attenuate the response alteration on differential reinforcement of low-rates responding task induced by peripheral d-amphetamine. By using the DRL task, Experiment 2 employed the microjeciton technique to determine the neural substrates for the DA receptor antagonist to attenuate the effect of peripheral d-amphetamine. The infusion sites for DA receptor antagonist were the hippocampus, the dorsomedial striatum, the ventrolateral striatum, the nucleus accumbens, the medial prefrontal cortex, and the ventral tegme ntal area. The results showed that SCH23390 infused into the hippocampus, the nucleus accumbens, the medial prefrontal cortex, the ventral tegmental area could attenuate the effect induced by peripheral d-amphetamine, and such attenuation effects were also observed for raclopride infused into the ventrolateral striatum, the medial prefrontal cortex. Experiment 3 tried to confirm the results of Experiment 2 by microinjecting SCH23390 in hippocampus and medial prefrontal cortex under peak-interval task. Only SCH23390 in the hippocampus altered the subject's normal performance in this task especially when combined with peripheral injection of d-amphetamine. In conclusion, that the response alteration on the DRL task induced by peripheral injection ofd-amphetamine suggests the subject's timing perception being accelerated. These effects of d-amphetamine were mediated by simultaneous activation of multiple dopamine receptor subtypes including D1 receptors located in the hippocampus, nucleus accumbens, medial pref rontal cortex, ventral tegmental area, as well as D2 receptors located in the ventrolateral striatum, medial prefrontal cortex.