Neuropharmacology Laboratory
神經藥理學研究室
Research interests
神經藥理研究室主要探討多巴胺酬償系統及神經胜肽 (NPFF)在中樞神經系統上所扮演的角色，此外我們也探討若干中樞神經與精神病變議題，包括成癮與復犯機制（甲基安非他命及K他命）、精神分裂症、帕金森氏症、疼痛與憂鬱共病的中樞作用機轉。本實驗室主要的研究導向為利用藥物或基因轉殖鼠建立病變的動物模式，待病理的外顯行為確定後，再進行活體和離體的腦部神經化學、訊號傳遞與基因表現等的定性與定量分析，最後以神經藥理作驗證或以細胞培養模式探究其作用機制。在這些疾病中，我們著重於數個神經傳導系統：包括多巴胺、興奮性 (glutamate) 和抑制性(GABA)氨基酸以及HPA axis，希望經由多層次的整體性研究，確定特定神經系統在特殊病理時期的行為和生化反應，以利對疾病的瞭解或開發新型藥物。目前本實驗室發現:
（一）在藥物成癮發展過程中，前腦積核(nucleus accumbens)內的多巴胺第三亞型受體對藥物成癮具有剎車的角色，並和Cdk5-DARPP-32-PP2A這一條訊息路徑的活化有關；此外藉由場地制約行為(conditioned place preference)研究藥癮記憶消除(extinction)和復犯(relapse)則發現前腦額業的興奮性glutamate訊號對extinction具有關鍵的角色；目前我們正繼續分析中腦GABA神經元在藥癮記憶上的角色。
（二）在帕金森氏症研究上，我們發現位於側腦室的一群成體神經幹細胞(adult neural stem/progenitor cells)會受到多巴胺第三亞型受體的刺激而增生，並且能夠在小鼠模式上顯著改善因MPTP神經毒所造成帕金森氏症的動作障礙；目前，我們正在研究帕金森氏症小鼠因為長期服用L-dopa藥物所產生的異動症(dyskinesia)的神經病變機制。此外我們亦發現抑制多巴胺第三亞型受體活性，可以改善因腦部多巴胺過度活化(DAT knockout model)所造成的認知障礙。
（三）有關神經胜肽NPFF的研究，我們實驗室製造出NPFFR2 Tg mice，發現這受體過度表現會使小鼠呈現痛覺過敏(hypralgesia)和憂鬱與焦慮的行為表徵；進一步的研究顯示這基因改造鼠下視丘HPA axis過度活化導致海馬迴神經新生下降，並且醣皮質素受體的負回饋作用缺失，使動物呈現憂鬱與焦慮的狀況。此外，位於脊椎背角區的疼痛胜肽CGRP釋放量會增加，引動上行疼痛路徑在fMRI的偵測下整體活性上升，造成這基因改造鼠在各種疼痛測試中均呈現閾值下降，而產生疼痛致敏化現象。目前我們正在製備NPFFR2基因剔除鼠，並探討該受體在下視丘參與的其他生理功能。
本實驗室主要以系統學 (in vivo) 和細胞層級 (in vitro) 相輔研究，探究受體蛋白和基因在離體時的次細胞信息路徑，以及在活體上的生理與病理上的意義，以期獲致從分子-細胞-生理-行為等層次的綜觀瞭解。實驗室常使用的技術包括有：高液相層析儀(HPLC-EC/ fluorescence detection)、西方墨點法(western blot)、免疫螢光及共軛焦顯微影像分析、定量real-time PCR、磷酸酵素kinase assay、腦組織微透析(microdialysis)與局部藥物注射、細胞培養與基因植入、免疫螢光反應、動物行為分析[水平跑步、刻板行為、水迷宮學習與記憶、焦慮感測試與藥物制約行為]與一般性的生化、分生及組織切片螢光染色分析。
最近本實驗室開發先進的光遺傳學技術(optogenetics)，利用病毒AAV5所攜帶對光敏感的離子通道蛋白(ChR2或NpHR)感染具神經傳導物質專一性的Cre mice特定腦區，再利用植入的光纖維(optic fiber)激發或抑制神經活性，藉以在活體動物瞭解特定神經迴路對特定行為的影響。本實驗室每兩週舉辦一次的lab meeting，可讓實驗室的每位成員瞭解彼此所做的實驗內容和理論基礎，並可有充分溝通的機會。本實驗室與林口長庚醫院神經科學中心(NRC)有長期的合作關係探討帕金森氏症，和分析病患檢體；此外並與基隆長庚醫院合作Ketamine的成癮藥物計劃；與德國的合作團隊與國衛院則探討NPFF的中樞與週邊生理作用。
The focus of Neuropharmacology lab is to understand the cellular and molecular of several neurological and psychiatric disorders, such as drug addiction, depression, schizophrenia, pain and Parkinson’s disease. The efforts by lab participants during previous years had generated many interesting research findings and now we are continuously working on the following research topics:
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Drug addiction acquisition, extinction and relapse: via behavioral sensitization and conditioned place preference (CPP) as addiction model conducted on rodents, we intend to explore the neural signals from prefrontal glutamate that project to the midbrain dopamine cell body in the ventral tegmental area (VTA) and discover how stress hormone CRF reinstates the addiction memory and its underlying cellular mechanism.
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Brain neuropeptide FF (NPFF)- NPFFR system: via research on home-made NPFFR2 Tg mice, we already found this Tg mice exhibit hypralgesia and depression/anxiety and the outcome of NPFFR2 over-expression on HPA axis, ascending nociceptive pathway, CGRP release and hippocampal neurogenesis. We are now producing NPFFR2 KO mice and will characterize their behavioral and biochemical phenotypes in order to understand the physiological role of brain NPFF-NPFFR system.
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Parkinson’s disease and L-dopa-induced dyskinesia (LID): previously, we identified that dopamine D3 receptor stimulation would evoke adult stem/progenitor cell proliferation in the SVZ area and further found systemic D3R agonist treatment could restore the motor dysfunction due to MPTP lesion in the striatum. We are now explore the cellular mechanism of LID via median forebrain bundle 6-OHDA-lesioned mice model, as well as Metabolomic profiling of PD and LID patients.
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Psychosis and cognition: via chronic ketamine administration, we intend to explore the cellular mechanism underlying the ketamine addiction and psychosis, focusing on the nucleus accumbens. In addition, via dopamine transporter (DAT) KD mice, we study the role of dopamine D3R on cognitive ability (novel object recognition test) focusing on the neurochemistry of prefrontal cortex.
The expertise in our lab is behavioral manipulation in rodents and in vivo and in vitro biochemical analyses. Experimental techniques routinely performed in our laboratory include western blot, IHC/ICC, brain microdialysis-HPLC, stereotaxic surgery and cannulation, qPCR, cell culture and transfection, behavioral observation (water maze, forced swim test, open-field activity, tail suspension test, NORT, pre-pulse inhibition, CPP, startling, elevated plus maze, von Frey test, etc.). Recently, we established the optogenetic technique and will conduct on GluT-, Gad2-, Pvalb-cre mice for targeting designated neural circuitry. Upon identifying the altered target(s) in in vivo system, a cell culture system would also be adopted for mechanistic study. It is expected that through comprehensive approaches, i.e. from cellular to systemic, we would be able to understand the causes of particular neural disorder and will eventually benefit the translational research.