臺灣博碩士論文加值系統

藥物成癮是長期使用成癮性藥物後造成腦內類似神經可塑性(synaptic plasticity) 的生理變化進而對藥物產生依賴性，其中腦內的中腦邊緣多巴胺系統 (mesolimbic dopamine system) 的神經迴路改變被認為與成癮發生最為相關，此迴路包含了腹側盖區 ( ventral tegmental area ;VTA )、依核 (nucleus accumbens ; NAC ) 及其他相關的邊緣系統相關區域 (limbic structure) 如海馬迴 (hippocampus) 等。神經可塑性其中的機制之一「長期增益效應 (long-term potentiation ;LTP)」是突觸間的神經迴路受到外界的刺激而使突觸間傳導強度改變的現象，為目前最常被用來解釋學習以及記憶形成的機制之一。在近來的研究中發現蛋白激酶MMζ (Protein Kinase Mζ ; PKMζ ) 可藉由增加hippocampus 中突觸後
細胞膜上AMPA (α-amino-3-hydroxyl-5-methyl-4 -isoxazole-propionate)受器的數目來增加突觸間的傳遞強度，進而在記憶形成時扮演重要角色，故在本實驗中以古柯鹼誘導成癮後，希望探討PKMζ 在中腦邊緣多巴胺系統中的VTA 以及其他相關區域中所扮演的角色及可能的
調控路徑。在本實驗中給予不同年齡層的大鼠腹腔注射古柯鹼而誘導成癮的發生後分別在不同時間點取出VTA、NAc、hippocampus 等區域的腦組織並分析其PKMζ 表現量的變化。我們發現年輕組的大鼠(Postnatal day ; P18~P30) 在接觸單一劑古柯鹼注射後的 VTA 其
PKMζ 的確會被誘導而表現，並且在第30 分鐘有顯著增加，而此PKMζ 蛋白質在 1小時以內的增是來自於對既存的 PKMζ mRNA 轉譯的調控，其中包含了 PKMζ 對自身表現正回饋的調控，以及受到MEK1 (mitogen-activated protein kinase kinase 1)、蛋白激酶A
(cAMP-dependent protein kinase ; PKA)、PI3K (Phosphatidylinositol 3-kinases)、多巴胺受器D1- 及D2- 亞型 (dopamine receptor D1- 及D2- subtype) 等的影響，並且不受 CaMK II(Calcium/calmodulindependent
protein kinase II) 的影響。同時於 VTA 的 PKMζ mRNA 表現量也在第1 小時增加，顯示具有轉錄上的調控。此外單一劑古柯鹼注射也會24 小時內於 NAc 與 hippocampus 中誘導出PKMζ 的表現。而重複連續注射古柯鹼 5 天也會在VTA 、NAc、hippocampus等區域誘導PKMζ 持續的表現。在VTA 與 NAc 中，年長組 (P45~P50)的大鼠對古柯鹼所誘導的PKMζ 表現量較年輕組的不顯著，並且年長組對古柯鹼所誘導行為影響 (locomotor sensitization) 也較年輕組
不明顯。胚胎時期即接觸到古柯鹼也會減少於出生後再度接觸到古柯鹼在 VTA 與 NAc 中所誘導出的 PKMζ 表現量，並且對古柯鹼所誘導行為影響 (locomotor sensitization) 也相近於年長組的表現。

The mesolimbic dopamine system, including dopaminergic projections from the ventral tegmental area (VTA) to nucleus accumbens (NAc), is critically involved in the development of addiction to many drugs of abuse, including cocaine (CA). Although there is an attractive hypothesis that the modifications of mesolimbic reward circuit following repeated drug exposure are responsible for cocaine-addicted causes behaviors change, however, our understanding in the underlying molecular mechanisms at the neural circuit level is still in its infancy. It has been suggested PKMzeta, a constitutively active atypical isoform of PKC, plays a critical role in spatial memory formation and long-term synaptic potentiation in hippocampus. To define the relationship among PKMzeta, CA-induced synaptic long-term potentiation and CA addiction, we examined the regulation of PKMzeta after CA administration in Sprague-Dawley rat. We found single CA injection elicits an increase in PKMzeta protein expression in the VTA region. The increase was first observed 10 min after CA administration and lasted for 7 days, the longest sampling time point of our experimental design. The PKMzeta protein expression can also be induced in 10 minutes while incubating the acute isolated brain slice with CA, the expression within 1 hr can be eliminated at the present of Chelerythrine (PKC inhibitor) and ZIP (PKMzeta inhibitor) suggests a positive feedback loop. The PKMzeta mRNA expression can be induced within 1 hr, and Actinomycin d (transcription inhibitor) had no effect on the PKMzeta protein expression indicating CA increases PKMζ translation from preexisting PKMζ mRNA. Furthermore,real time PCR-based analysis showed resembling increase profile ofPKMζ mRNA after single CA injection, suggesting a co-upregulation of transcription and translation of PKMζ after CA administration in VTA.
Eticlopride (dopamine receptor D2-subtype antagonist) 、SCH-23390(dopamine receptor D1-subtype antagonist)、H-89 (PKA inhibitor)、
Wortmannin (PI3K inhibitor)、PD98059 (MEK1 inhibitor) decreasedcocaine-induced PKMζ expression within 1 hr in VTA. On the contrary,
KN-62 (CaMK II inhibitor) has no obvious effect on PKMζ expression.
CA challenge not only induces the PKMζ expression in the VTA region but also in the NAc and hippocampus region. The CA-induced PKMζ
expression is more obvious in elder group (>45 days in age) than younger group (18~30 days in age), similar results also showed in the locomotor
activity assay. Prenatal CA exposure decreased the postnatal CA-induced PKMζ expression and the locomotor sensitivity in younger group.
Overall, results from our current experiments have raised the possibility of PKMζ involvement in CA addiction. How CA regulates PKMζ
expression and the context dependence between PKMζ and CA-induced behavior change and synaptic long-term potentiation remains further elucidation.

頁數
圖表索引 1
縮寫表 2
中文摘要 3
英文摘要 5
緒論 7
實驗材料與方法
實驗動物 15
1.腦組織切片製備 15
2.西方點墨法
2-1.蛋白質萃取 17
2-2.蛋白質定量 18
2-3.硫酸十二酯鈉-聚丙烯醯胺凝膠電泳 18
2-4.蛋白質電泳轉印 19
2-5.免疫染色 19
2-6. 冷光持續曝光拍攝 20
3.即時-反轉錄聚合酶連鎖反應
3-1. RNA 萃取 21
3-2. RNA 定量及反轉錄 21
3-3.聚合酶連鎖反應 23
4.運動活性測量 24
5.實驗數據統計與分析 26
6.實驗試劑與供應商 26
7.實驗試劑的作用 27
結果 28
討論 43
參考文獻 52
圖表 58

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