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研究生:王靖雅
研究生(外文):Jin-Ya Wang
論文名稱:神經胜肽NPFF對於前腦多巴胺活性之細胞機制探討
論文名稱(外文):Cellular Mechanism of Neuropeptide FF on Forebrain Dopamine Activity
指導教授:陳景宗
指導教授(外文):Jin-Chung Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:66
中文關鍵詞:神經胜肽NPFF
外文關鍵詞:NPFF
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NPFF是屬於FMRM-NH2類且具有拮抗鴉片類物質的神經胜肽,參與鴉片類止痛、學習記憶、內分泌調控以及藥物依賴性等生理功能。由於目前對於大腦中NPFF接受器與中腦皮質邊緣多巴胺系統的活性關聯尚缺乏直接證據,因此本論文的主要目的為探討NPFF 接受器在中腦皮質邊緣多巴胺系統所扮演之角色,並探討其細胞作用機轉。
首先,在腹側蓋區或依核區給予大白鼠NPFF,觀察對於多巴胺D1接受器致效劑SKF81297 (皮下注射, 2 mg/kg) 及多巴胺D2接受器致效劑quinpirole (腹腔注射, 2 mg/kg) 所引起的動物行為活動力之影響。腹側蓋區給予NPFF能夠強而有效的抑制quinpirole所誘發之高行為活動力,然而對於SKF81297所引起的動物行為則沒有影響。且此抑制作用只發生在腹側蓋區,因為依核區給予NPFF對於quinpirole所誘發之高行為活動力並沒有影響。若在前腦額葉區給予多巴胺D1接受器致效劑SKF81297,而非D2接受器致效劑,則可以部份抑制quinpirole所誘發的行為活動力。免疫組織螢光染色的解剖學證據顯示,在腹側蓋區與黑質體緻密區中,NPFF接受器多表現於多巴胺神經元上,極少分佈於依核區。此外,在微透析動物實驗中,腹側蓋區給予NPFF對於前額葉區的多巴胺代謝產物DOPAC有些微但持續的增加,對於依核區則沒顯著影響,顯示NPFF對中腦多巴胺系統的活化,是透過前額葉皮質D1接受器的作用,來抑制周邊注射quinpirole 對依核活化產生高活動力的影響。欲探究NPFF接受器的細胞生理機制,我們也利用新生大白鼠腹側中腦組織細胞培養,進一步研究NPFF接受器所調控之下游訊號路徑。初步結果顯示給予1 μM NPFF能夠隨著時間增加而活化ERK1/2訊息。根據目前的研究進度,我們推測活化腹側蓋區的NPFF接受器,能透過活化中腦皮質多巴胺活性並增加此區多巴胺的釋放,進而抑制中腦邊緣多巴胺的活性,而NPFF接受器的活化可能是透過MAPK的作用進行。
Neuropeptide FF (NPFF) is a FMRF-NH2-like peptides which functions as an anti-opioid peptide that involves in opioid analgesia, learning/memory, endocrine regulation and drug dependence. Due to a lack of direct evidence linking brain NPFF receptors with mesocorticolimbic dopamine activity, the aim of this study was to identify the impact of NPFF receptors on mesocorticolimbic dopamine system, attempting to provide a mechanistic explanation. First, NPFF was locally infused into the VTA (ventral tegmental area) or NAcc (nucleus accumbens) while animals (male SD rats, 300-350 gm) were systemically injected with dopamine D1 (SKF81297) or D2 (quinpirole) agonists. Intra-VTA injection of NPFF resulted in a potent inhibition on quinpirole (2 mg/kg, i.p.)-induced hyperlocomotion, but it could not modify SKF81297 (2 mg/kg, s.c.)-induced behavioral activation. This effect was also specific for VTA since NAcc-injected NPFF could not modify quinpirole-induced behavioral activation. Intra-mPFC injection of D1 receptor agonist, but not D2 ligand, partially inhibited the systemic quinpirole-evoked behavioral activation which suggests that the behavioral effect of VTA-injected NPFF might be mediated through the mesocortical dopamine D1 receptor activation. Anatomical evidence illustrated that NPFF receptors expressed on dopamine neurons at both VTA and SNc (pars compacta), rarely expressed in the NAcc. In addition, via in vivo microdialysis, we detected a two-fold but sustained increase in the level of DOPAC in the mPFC, but not in the NAcc after intra-VTA NPFF administration. In order to delineate the NPFF receptor-mediated signaling, primary neural cultures of rat postnatal ventral mesencephalon were prepared. The preliminary results indicate that NPFF (1 μM) induced pERK1/2 in a time-dependent manner. Overall, we hypothesize that activation of NPFF receptors in the VTA could activate the mesocortical dopamine activity. The enhanced dopamine release in the mPFC would, possibly through D1 receptor activation, suppress the mesolimbic dopamine activity, via either pre-synaptic (dopamine terminals) or post-synaptic (on D1 or D2 receptor-expressing MSNs) event.
Contents
指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
論文著作授權書 ………………………………………………………iii
致謝 ……………………………………………………………………iv
中文摘要 ……………………………………………………………… v
Abstract …………………………………………………………………vi
Abbreviations……………………………………………………………ix
І. Introduction …………………………………………………………… 1
1. Neuropeptide FF …………………………………………………… 1
2. Physiological functions of Neuropeptide FF ……………………… 2
2.1 Pain and Analgesia …………………………………………… 2
2.2 Learning and Memory ………………………………………… 3
2.3 Feeding behavior ……………………………………………… 4
3. Neuropeptide FF receptors ………………………………………… 4
3.1 Distribution of NPFF receptors………………………………… 5
3.2 Physiological Functions………………………………………… 6
3.3 Signaling Pathways …………………………………………… 8
4. Dopamine and Dopamine receptor classification ………………… 9
5. Mesocorticolimbic Dopamine System …………………………… 10
6. NPFF receptors and Dopamine…………………………………… 12
II. Specific Aims ……………………………………………………… 13
III. Materials and Methods …………………………………………… 14
1. Subjects……………………………………………………………14
2. Surgical procedures……………………………………………… 14 3. Drug treatments……………………………………………………15
4. Locomotion and Stereotypy assessment………………………… 16
5. Brain slice immunocytochemistry ……………………………… 16
6. Mesencephalon primary neuronal cultures……………………… 17
7. Western immunoblot………………………………………………18
8. In vivo Brain Microdialysis……………………………………… 18
9. HPLC-ECD analysis………………………………………………19
10. Data analysis and statistics………………………………………20
IV. Experimental Designs and Results …………………………………20
1. The effect of local intra-VTA or NAcc NPFF on behavioral activation induced by dopamine D1 or D2 receptor agonist………20
1.1 The effect of intra-VTA NPFF on dopamine D1 or D2 receptor agonist-evoked behavioral activation…………………………… 21
1.2 Effect of intra-NAcc NPFF on dopamine D2 receptor agonist induced behavioral activation ……………………………………22
2. Effect of co-administration of intra-mPFC D2R antagonist raclopride and intra-VTA NPFF on systemic quinpirole-induced behaviors…23
3. Effect of intra-mPFC D1R agonist SKF 81297 on the behavioral activation evoked by systemic quinpirole…………………………24
4. Localization of NPFF receptors in TH-enriched VTA as well as NAcc………………………………………………………………26
5. Effect of intra-VTA NPFF on in vivo efflux of monoamines and their metabolites from mPFC and NAcc ………………………………27
6. NPFF receptor-mediated cellular signaling in primary ventral mesencephalon neuronal/glial cultures……………………………28
6.1 Establish the rat postnatal ventral mesencephalon neuronal/glial cultures……………………………………………………………28
6.2 NPFF receptor-mediated signaling in the primary mesencephalic
cultures ………………………………………………………… 29
V. Discussion……………………………………………………………30
VI. Reference……………………………………………………………38
Figures ………………………………………………………………… 48
VI. Reference
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