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。

兒茶酚胺類神經傳遞系統被認為與包括記憶學習等行為功能有很重要的關連，在記憶多元化理論的假設下，該神經系統與其它者對於特定記憶學習行為應有再確認之必要。過去對於空間性記憶的研究，其相關支持證據所依據之實驗操弄泰半集中在海馬迴系統上。但仍有部份研究指出大腦中的其他區域可能同樣與一般記憶的運作有密切的關連。特別是兒茶酚胺系統所在之紋狀總體組織部位（包括尾狀核與阿控博核），vP A僅與感覺接受、運動反應及增強作用等機制有關外，同時可能也扮演影響記憶表現的重要因子。本研究使用慾求性的八角迷津為工具，藉其地點學習與反應學習這兩種不同的迷津作業，及利用兒茶酚胺類的藥物或神經毒素，探討相關的記憶習得與記憶保持歷程所造成的影響。實驗的操弄包括：(1) 迷津作業之地點學習與反應學習以探討這兩種記憶之行為機制。(2) 記憶習得與記憶保持階段以瞭解這兩種迷津作業所引發記憶之全部歷程。(3) 中樞（阿控博核或尾狀核）神經毒素之破壞以及周邊藥物注射以確認兒茶酚胺類藥物對於記憶之神經藥理機制。本研究分為兩大實驗進行，實驗一以地點學習為主，實驗二以反應學習為主。實驗結果可以簡單歸納如下：(1) 兩 種學習作業的記憶策略有不同的習得歷程及需要不同的處理訊息。(2) 在迷津學習前用神經毒素 6-OHDA 破壞尾狀核或阿控博核，皆會影響地點記憶的習得，但對於反應記憶的習得，則需要同時破壞尾狀核及阿控博核才有類似的干擾效果。(3) 相對於神經毒素 6-OHDA的干擾效果，DSP-4皆不影響地點學習與反應學習的習得歷程。(4) 在記憶保持階段中，周邊注射兒茶酚胺類藥物 d-amphetamine、haloperidol 與 propranolol均會干擾地點記憶的提取，但卻不影響反應記憶的提取表現。(5) 於地點記憶與反應記憶習得後，給予尾狀核加阿控博核的雙側 6-OHDA 注射均不影響這兩種記憶的提取表現。實驗結果顯示兒茶酚胺類神經傳導系統對於記憶功能具有明顯的影響，其中紋狀體扮演了相當重要的角色。相對於不影響記憶提取之歷程，紋狀體的破壞對記憶習得歷程有阻滯之效果，其內部之尾狀核與阿控博核分別依不同之迷津作業具有相異之效果，且多巴胺系統較正腎上腺素系統明顯的參與了影響效果，這些結果顯示兒茶酚胺類神經傳導系統與記憶表現有密切的關連。 / Catecholamine (CA) neurotransmission systems are critically involved in the control of many behavioral functions including learning and memory. The role of CA in mediating learning and memory is recently focused on the basis of multiple memory hypothesis. In addition to the previous finding of spatial memory relevant to the hippocampal areas, the striatum containing the caudate nucleus and the nucleus of accumbens is thought to be important for executing the learning and memory function. By the use of radial arm maze (RAM), the present study examined the effects of CA related neurotoxins and drugs on the acquisition and retention stages of both place and response tasks. Two major parts of experiments were designed to reveal the neurobehavioral mechanisms for the place and response tasks of RAM. Food-deprivated rats were trained to enter the arms baited with chocolate in the eight-arm maze. Specific four arms were baited for each rat in the place task, while randomly selected four arms each cued with a piece of sand paper on the arm entrance were baited for the rat in the response task.The results can be summarized as followings. (1) Differen behavioral processes were shown in performing the place and response tasks. (2) The acquisition deficits were significantly produced by 6-hydroxydopamine (6-OHDA) lesion on either caudate or accumbens for the place task, whereas the acquisition of response task was only impaired by 6-OHDA lesions of both caudate and accumbens together. (3) In contrast to 6-OHDA, adrenergic neurotoxin DSP-4 did not significantly affect subjects to acquire either task. (4) During the retention stage, the performance of place task was significantly disrupted by d-amphetamine, haloperidol, or propranolol. However, this was not the case for the retrieval of response task. (5) Once acquired, neither place nor response task performamce could be influenced by 6-OHDA simultaneously administered on the caudate and accumbens areas.Taken together, these data collected from RAM support the idea that the striatal CA is essential for the leraning and memory. Shift of the CA neurotransmission function induced by either 6-OHDA lesions or relevant drugs can disrupt the RAM behavior, which impairment to be detectable is depended on the learning task itself as well as the time of a specific task being leraned.

瑞特氏症(Rett syndrome, RTT)為第二型甲基化CPG結合蛋白(2methyl-CpG binding protein 2, MeCP2)基因發生突變所造成的神經發育疾病，其症狀包含了嚴重的運動障礙及自閉傾向等特徵。由於紋狀體(striatum)為運動控制的重要腦區，我們假設RTT的運動障礙主要為紋狀體的功能異常所造成，故利用RTT模式小鼠來研究紋狀體是否為RTT運動障礙的致病原因。利用敞箱試驗(open field test)及加速滾輪測試(accelerating rotarod task)結果發現，Mecp2基因剔除小鼠的活動力明顯下降，並伴隨有運動協調能力的缺失。以免疫組織染色法及西方點墨法分別標定紋狀體中的mu-opioid receptor及calbindin蛋白，發現二者表現量均有明顯下降，然而表現parvalbumin的中間神經元細胞數目卻大量增加。我們發現在紋狀體中多巴胺D2受體的表現量顯著增加，但多巴胺合成酶tyrosine hydroxylase與多巴胺訊號傳遞下游分子DARPP-32蛋白並沒有明顯減少。為了更進一步確認紋狀體的致病角色，我們利用特定在紋狀體中缺少MeCP2的「Mecp2條件缺失小鼠」，觀察其運動行為的改變。結果發現，Mecp2條件缺失小鼠不管是在活動力或是運動學習上都表現出和Mecp2基因剔除小鼠相似的運動障礙，顯示紋狀體所調控的正常活動力及運動學習能力皆需要MeCP2的參與。我們接著進一步探討是否擁有完整MeCP2表現的紋狀體就足以執行正常的運動功能。當Mecp2基因剔除小鼠的紋狀體重新表現MeCP2(即「Mecp2條件回復小鼠」)，MeCP2缺失所造成的運動障礙可被回復到接近野生型小鼠運動能力的正常水準。顯示紋狀體中MeCP2的存在為正常運動控制的充要條件。在以cyclin-dependent kinase-like 5 (Cdkl5)突變小鼠研究MeCP2的磷酸化是否會影響到運動行為，發現Cdkl5突變小鼠在出生早期及成年時期皆存在與Mecp2基因剔除小鼠一致的運動協調能力缺失。免疫組織染色及西方點墨法結果顯示，Cdkl5突變小鼠的紋狀體中mu-opioid receptor表現量有明顯下降，但parvalbumin的中間神經元數目並無改變，而在大腦皮質中多巴胺轉運子DAT1蛋白表現量明顯上升。CDKL5突變造成與RTT相似症狀的原因還須更進一步的探討。綜上所述，本研究為「紋狀體異常可能為RTT運動障礙的主要致病原因」提供動物模式的實驗證據，並提供了一個新的觀點用於未來治療RTT或防止其症狀的惡化。

甲基安非他命是一種被廣泛濫用的非法神經興奮劑，而且使用之後常伴隨著精神疾病的發生，動物研究也顯示，施打甲基安非他命所引起的神經毒性不僅會造成多巴胺神經元及血清素神經元的損傷，也引起認知功能和社交行為的缺失，同時對於產生迷幻作用的5-HT2A受體作用劑的行為反應增強。N,N,N-trimethylglycine (TMG)和N,N-dimethylglycine (DMG)是甘胺酸的甲基化衍生物，由於這兩種藥物具有治療神經系統疾病的潛力，因此本研究的目的為評估TMG及DMG是否可以預防或改善小鼠在甲基安非他命的暴露下所導致的行為缺失包括新位置辨識測試，新物體辨識測試，社交行為互動測試以及使用5-HT2A受體作用劑DOI 誘導小鼠頭部抽搐(head twitch )的行為。實驗方式為腹腔注射給予雄性ICR小鼠甲基安非他命，一天注射四劑（4 × 5mg/kg），每劑間隔兩小時。實驗一，小鼠在暴露甲基安非他命，先確認行為改變後，給予腹腔注射TMG及DMG (10或30 mg/kg)連續七天，評估TMG及DMG的治療效果。實驗二在施打每劑甲基安非他命30分鐘前給予TMG及DMG (100 mg/kg)，七天後進行行為評估，實驗三，評估TMG及DMG個別及混合劑量的治療效果，小鼠給予甲基安非他命之後，先確認行為改變，再給予腹腔注射TMG及DMG (20、5+5或是10+10 mg/kg) 連續七天，七天後進行行為測試。實驗四，檢測TMG及DMG的治療效果是否藉由活化NMDA受體glycine binding site，小鼠給予甲基安非他命七天之後，腹腔注射TMG及DMG (20 mg/kg)並在給予TMG及DMG前30分鐘給予glycine binding site 拮抗劑7-chlorokynurenic acid (7-CK) (1 mg/kg)，連續給藥七天，七天後進行行為評估。實驗結果發現連續給予七天TMG及DMG在個別劑量及混合劑量中都能夠恢復甲基安非他命所造成的認知功能缺損，社交退縮和降低由DOI 誘導小鼠頭部抽搐行為表現，以及在紋狀體中酪氨酸羥化酶的蛋白質表達減少情況。而前給予7-CK則阻斷TMG及DMG對甲基安非他命所造成的認知功能缺損，社交退縮的改善作用，但是對TMG及DMG對DOI 誘導小鼠頭部抽搐的行為的改善作用影響較小，顯示TMG及DMG可能都是經由活化NMDA 受體的glycine binding site改善甲基安非他命所造成的認知功能缺損，社交退縮，這些發現表示，TMG及DMG具有治療甲基安非他命成癮者所造成的精神分裂等異常症狀的潛力。 / Methamphetamine (METH) is a widely abused illicit psychostimulant. METH use is commonly associated with psychosis. A neurotoxic regimen of METH, which damages the dopaminergic and serotonergic neurons, causes cognitive dysfunction, social interaction deficits, and supersensitivity to hallucinogen in mice. N,N,N-trimethylglycine (TMG) and N,N-dimethylglycine (DMG) are methyl derivatives of amino acid glycine and naturally occur as intermediate metabolites in choline-to-glycine metabolism. Growing evidence shows that both compounds have potential to treat some neurological disorders. The aim of this study was to examine the protective and therapeutic effects of TMG and DMG on METH-induced behavioral aberrations. The novel location recognition test (NLRT), the novel objective recognition test (NORT), the social interaction and the hallucinogenic 2, 5-dimethoxy-4-iodoamphetamine (DOI)-induced head twitch response were evaluated. Male ICR mice received one day drug treatment with four injections of METH (4 × 5 mg/kg, i.p.) or saline at 2h interval. First, TMG or DMG (10 or 30 mg/kg, i.p.) were separately administered once daily for seven consecutive days after the behavioral impairment was confirmed in METH-treated mice. Seven days after final injection of TMG and DMG, the behavioral tests were monitored. Secondly, the preveting effects of TMG and DMG were examined by TMG and DMG (100 mg/kg, i.p.) pretreatment, 30 min prior to each dose of METH. Third, the lower dose (20 mg/kg) and combined effects of TMG and DMG (5+5 or 10+10 mg/kg i.p.) were evaluated. Fourth, in order to determine if the improving effects of TMG and DMG are mediated by NMDA receptor glycine binding site, the glycine binding site antagonist 7-CK (1 mg/kg, i.p.) was administered 30 min prior to each dose of TMG and DMG (20 mg/kg, i.p.), TMG and DMG dose-dependently improved, but not prevented the METH-induced cognition deficits, social withdrawal and hypersensitivity to hallucinogen with additional effect. Pretreatment of 7-CK, reversed the improving effects of TMG and DMG on behavioral deficits after METH exposure, yet had minor effect on hypersensitivity to hallucinogen. These results demonstrate that TMG and DMG might activate the glycine binding site of NMDA receptor to improve METH-induced cognition deficits and social withdrawal. TMG and DMG may be the novel therapeutic agents for psychiatric disorders related to METH abuse.

蛋白激酶CK2（Casine kinase 2）為四單體所構成，針對配受質蛋白之絲胺酸或蘇胺酸位置進行磷酸化，先前研究已經發現在紋狀體腦區之CK2的表現量與活性皆高於大腦中其餘腦區，而紋狀體腦區主要神經細胞為-氨基丁酸神經元（GABAergic neurons）的medium spiny neuron（MSN），會受到來自黑質多巴胺神經細胞（dopaminergic neurons）的調控。此外，DARPP-32（dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDA）蛋白亦被發現大量表現於在MSN細胞中，且為CK2之受質蛋白質。雖然CK2已被證實參與多巴胺神經元的神經保護機制，但是否參與MSN細胞對運動行為調控之生理機制仍未清楚。由於已有研究發現施予1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）藥物處理造成黑質-紋狀體腦區受損之老鼠腦內-氨基丁酸（GABA）的生合成酵素─麩胺酸脫羧酵素67（GAD67）表現量與正常老鼠不同，因此本論文研究的主題擬在大鼠實驗模式中利用MPP+造成投射至紋狀體之多巴胺神經細胞受損，探討當多巴胺調控紋狀體神經細胞能力缺失的狀態下，MSN細胞之CK2、DARPP-32和GAD蛋白表現與動物運動行為之相關性。 實驗結果發現，直接於紋狀體給予1-甲基-4-苯基碘化啶 （MPP+ Iodide）皆會造成CK2、DARPP-32以及GAD67之蛋白質含量的減少，多巴胺及其代謝物和GABA等神經化學傳遞物質亦有減少的現象；另外，在MPP+給予前分別操弄CK2或DARPP-32 胺基酸Ser102磷酸化的表現，皆會改變GAD67蛋白質含量與黑質酪胺酸羥化酶（Tyrosine Hydroxylase, TH）蛋白質含量，同時神經化學傳遞物質的含量或代謝亦有改變。由現有之結果推測CK2/DARPP-32/GAD67細胞訊息傳遞機制可能參與巴金森氏症運動行為失常之細胞層面的調控。 / Protein kinase CK2 is a heterotetrameric and serine/threonine protein kinase. Its protein levels and activity are found to be elevated in the striatum when compared to other brain areas. CK2 is known to involve in the neuroprotective effects of dopaminergic neurons, whether it also regulates the neuronal function relative to motor behaviors is still unclear. DARPP-32 protein is known as one of the substrates for CK2 and is highly expressed in the GABAergic medium spiny neurons (MSN) responsible for dopamine stimulation in the striatum. Furthermore, other studies have indicated that the expression of glutamic acid decarboxylase 67 (GAD67) mRNA and protein was different in the striatum of MPTP vs. naïve animals, which is one of the enzymes responsible for the synthesis of neurotransmitter GABA. In the present study, we observed that the parallel changes in protein levels of CK2, DARPP-32 and GAD67 in the striatum and TH in the substantia nigra of MPP+-treated. We also found that manipulation of CK2 or DARPP-32 gene expression aggravated the MPP+-induced neuropathological dificts. The present results suggest that CK2/DARPP-32/GAD67 signaling pathway might involve in the cellular mechanism of motor-deficit in Parkinson’s disease.

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.