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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 閔明源(Ming-Yuan Min) | |
| dc.contributor.author | Jung-Chien Hsieh | en |
| dc.contributor.author | 謝戎建 | zh_TW |
| dc.date.accessioned | 2021-06-17T06:04:11Z | - |
| dc.date.available | 2019-02-12 | |
| dc.date.copyright | 2019-02-12 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-01-24 | |
| dc.identifier.citation | Aston-Jones, G., & Cohen, J. D. (2005). An integrative theory of locus coeruleus-norepinephrine function: adaptive gain and optimal performance. Annu. Rev. Neurosci., 28, 403-450.
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Takeuchi, T., Duszkiewicz, A. J., Sonneborn, A., Spooner, P. A., Yamasaki, M., Watanabe, M., . . . Greene, R. W. (2016). Locus coeruleus and dopaminergic consolidation of everyday memory. Nature, 537(7620), 357. Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J. I., Obata, K., & Kaneko, T. (2003). Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67‐GFP knock‐in mouse. Journal of Comparative Neurology, 467(1), 60-79. Usher, M., Cohen, J. D., Servan-Schreiber, D., Rajkowski, J., & Aston-Jones, G. (1999). The role of locus coeruleus in the regulation of cognitive performance. Science, 283(5401), 549-554. Verstegen, A. M., Vanderhorst, V., Gray, P. A., Zeidel, M. L., & Geerling, J. C. (2017). Barrington's nucleus: Neuroanatomic landscape of the mouse “pontine micturition center”. Journal of Comparative Neurology, 525(10), 2287-2309. Walling, S. G., Brown, R. A., Miyasaka, N., Yoshihara, Y., & Harley, C. W. (2012). Selective wheat germ agglutinin (WGA) uptake in the hippocampus from the locus coeruleus of dopamine-β-hydroxylase-WGA transgenic mice. Frontiers in behavioral neuroscience, 6, 23. Yoshihara, Y., Mizuno, T., Nakahira, M., Kawasaki, M., Watanabe, Y., Kagamiyama, H., . . . Tabuchi, K. (1999). A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene. Neuron, 22(1), 33-41. Yuste, R. (2008). Circuit neuroscience: the road ahead. Frontiers in neuroscience, 2, 17. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71597 | - |
| dc.description.abstract | 跨突觸追蹤(Transneuronal tracing)可以提供主要神經元和中間神經元連結的直接證據，但現今仍沒有適合的追蹤劑能夠在微迴路尺度下標定特定的細胞群。為了解決這個問題，我們設計了cre重組酶活化小麥凝集素血清二型類腺病毒(AAV2-CMV-DIO-WGA)，此病毒可以使帶有cre重組酶的細胞表現小麥凝集素(wheat germ agglutinin)(WGA)，並在細胞內累積進行跨突觸運輸，達到追蹤的效果。搭配免疫組織化學染色法，我們發現由這隻病毒所製造的小麥凝集素在海馬迴(CA3)、網狀視丘(Reticular thalamus,RT)以及下視丘室旁核(paraventricular nucleus, PVN)中具有逆向追蹤(retrograde tracing)的特性。在轉基因小鼠的實驗裡，我們將cre重組酶活化小麥凝集素血清二型類腺病毒打入TH-cre小鼠的藍斑核(locus coeruleus)，的確僅具有cre重組酶的主要神經元會表現小麥凝集素。除此之外，我們也發現一些位於peri-LC區域的非主要神經元也有小麥凝集素免疫反應，我們推論這些細胞就是和主要神經元有連結的中間神經元，因為在橋腦區域除了藍斑核以外沒有其他的兒茶酚安類家族的神經元，且TH-cre這隻基轉小鼠在藍斑核中並沒有cre重組酶表現外漏的問題。利用TH-cre和GAD-GFP轉基因小鼠的雜交後代，我們發現這些藍斑核的中間神經元有16%是丙胺基丁酸神經元(gamma-Aminobutyric acid -ergic neuron)(GABAergic)、27%具有FoP2這個轉錄因子。在形態學上，我們的實驗結果提供了藍斑核中間神經元存在的直接證據，這些中間神經元可能在功能上，彙整來自不同腦區的訊息到藍斑核主要神經元，進而調節其電生理性質，但至於這些中間神經元的生理功能仍需近一步的實驗分析。 | zh_TW |
| dc.description.abstract | In morphology, trans-synaptic tracing can provide direct evidence of the connectivity between nucleus. In modern neuroscience, there are lots of trans-synaptic tracers to use, such as fluoro-gold, chlorea toxin B, pseudo-rabies virus. But none of them can perform cell-type specific tracing under the scale of microcircuit due to non-cell type specificity and leak out expression. Yoshihara Yoshihara et al. have validated the usefulness of wheat germ agglutinin, a commonly used transneuronal tracer, in several genetic approaches (Yoshihara et al., 1999). We chose the AAV-mediated WGA method described by Yoshihiro Yoshihara, and designed AAV2-CMV-DIO-WGA, a serotype 2 adeno-associative virus carrying double floxed inverted sequence of wheat germ agglutinin(WGA). Using this virus, we found the WGA expressed by this virus had retrograde trans-synaptic activity (CA3, RT, PVN) and had no leak-out-expression. We are currently interested in the microcircuit of locus coeruleus. We then used TH-cre mice to perform the cell-type specific microcircuit tracing. This resulted in the observation of interneuron pool in peri-LC region, which have not been reported in mice. Among these interneurons, there were about 16% GABAergic interneurons and 27% FoxP2 posotive interneurons. The result here provided a direct evidence for the existence of LC interneurons in peri-LC region, and these interneurons may play a role in integrating information from other brain regions, such as amygdala and pre-frontal cortex. We need more experiments to analyze the physiological function of these interneurons. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T06:04:11Z (GMT). No. of bitstreams: 1 ntu-108-R05b21008-1.pdf: 2936977 bytes, checksum: 8b984d5a5bba8c86331dffdb5337f399 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii Abstract iv Content v Chapter 1 Introduction 1 Chapter 2 Materials and Methods 4 2.1 Animals 4 2.2 Virus preparation and injection 4 2.3 Perfusion and section 5 2.4 Immunohistochemistry 6 2.5 Confocal imaging 9 2.6 Electrophysiology 9 2.7 Opto-stimulation 10 Chapter 3 Results 11 3.1 AAV2-CMV-floxed stop-WGA construction and experimental flow chart. 11 3.2 Validation of transneuronal tracing efficiency of AAV2-CMV-DIO-WGA in CA3. 11 3.3 Cell-type specific WGA tracing of RT using Vgat-cre mouse. 12 3.4 Cell-type specific WGA tracing of PVN using AVP-cre mouse 12 3.5 Cell-type specific WGA tracing of LC using TH-cre mouse 13 3.6 Leakage control of cre expression in LC of TH-cre mouse using AAV-eF1a-DIO-eYFP 13 3.7 Interneurons targeted by WGA were 16.7% GABAergic in peri-LC region. 14 3.8 Interneurons targeted by WGA were 26.7% FoxP2 positive in peri-LC region. 14 3.9 GABAergic interneurons targeted by WGA in peri-LC receiving input from CeA. 15 3.10 Non-LC neurons in peri-LC region receiving pure GABAa input from CeA in ex-vivo recording. 15 3.11 Validation of the effect of the input from CeA on LC using 40 hz opto-stimulation. 16 Chapter 4 Discussion 18 4.1 AAV2-CMV-floxed stop-WGA is a good material to perform cell-type specific tracing in the scale of microcircuit. 18 4.2 Cell-type specific tracing using AAV2-CMV-DIO-WGA revealed an interneuron pool in peri-LC region 19 4.3 WGA expression level in first order neurons is different in CA3, RT, PVN and LC tracing experiment 20 4.4 WGA expressed by AAV2-CMV-floxed stop-WGA exhibit multi-synaptic tracing 21 4.5 Physiological effect of CeA to LC was not robust in opto-stimulation experiments 21 Chapter 5 Summary 23 Chapter 6 Reference 24 Chapter 7 Figures 26 Figure 1. WGA tracing method and hypothetical model of interaction between principle neuron and interneuron in Locus coeruleus. 26 Figure 2. Plasmid structure. 27 Figure 3. Cre-dependent expression in HEK293 cell line 28 Figure 4. Virus package and injection 29 Figure 5. Transneuronal evaluation of AAV2-CMV-DIO-WGA in CA3 of wild type mouse. 30 Figure 6. Transneuronal evaluation of AAV2-CMV-DIO-WGA in reticular thalamus (RT) of vesicular GABA transporter-cre (Vgat-cre) mouse. 31 Figure 7. Cell type specific WGA tracing in paraventricular nucleus (PVN) of vasopressin (AVP)-cre mouse using AAV2-CMV-DIO-WGA. 33 Figure 8. DAB staining of cell type specific WGA tracing in PVN of AVP-cre mouse using AAV2-CMV-DIO-WGA. 34 Figure 9. Cell type specific WGA in LC of TH-cre mouse using AAV2-CMV-floxed stop-WGA. 35 Figure 10. DAB staining of cell type specific WGA tracing in LC of TH-cre mouse using AAV2-CMV-DIO-WGA. 36 Figure 11. Leakage control of cre expression in LC of TH-cre mouse using AAV-eF1a-DIO-eYFP. 37 Figure 11. Population of GABAergic interneurons in LC of TH-cre X GAD-GFP mouse using AAV2-CMV-DIO -WGA. 38 Figure 12. Population of GABAergic interneurons and FoxP2 interneurons in LC of TH-cre X GAD-GFP mouse using AAV2-CMV-DIO -WGA. 40 Figure 13. WGA tracing infection rate, transneuronal rate and interneurons population 41 Figure 14. Input from CeA targeting GABAergic interneurons in peri-LC. 42 Figure 15. Non-LC neurons in peri-LC region receiving pure GABAa input from CeA in ex-vivo recording. 43 Figure 16. Delayed activation of phasic response of LC principle neurons using 40hz 10s blue light stimulation. 45 Chapter 8 Supplementary data 47 Supplementary data 1. Another example of Interneuron in peri-LC region receiving pure GABAa input from CeA in ex-vivo recording. 47 Supplementary data 2. Whole predicted sequence of Plasmid structure of AAV2-CMV-DIO-WGA 49 | |
| dc.language.iso | en | |
| dc.subject | 微迴路追蹤 | zh_TW |
| dc.subject | 藍斑核 | zh_TW |
| dc.subject | 抑制性中間神經元 | zh_TW |
| dc.subject | 小麥血清素 | zh_TW |
| dc.subject | Wheat germ agglutinin | en |
| dc.subject | microcircuit tracing | en |
| dc.subject | Locus coeruleus | en |
| dc.subject | GABAergic interneuron | en |
| dc.subject | FoxP2 | en |
| dc.title | 利用小麥凝集素進行跨突觸追蹤探討藍斑核中間神經元之分布 | zh_TW |
| dc.title | Identification of locus coeruleus interneuron using trans-synaptic tracer wheat germ agglutinin | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳志成(Chih-Cheng Chen),陳示國(Shih-Kuo Chen),楊琇雯(Hsiu-Wen Yang) | |
| dc.subject.keyword | 小麥血清素,微迴路追蹤,藍斑核,抑制性中間神經元, | zh_TW |
| dc.subject.keyword | Wheat germ agglutinin,microcircuit tracing,Locus coeruleus,GABAergic interneuron,FoxP2, | en |
| dc.relation.page | 52 | |
| dc.identifier.doi | 10.6342/NTU201900180 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-01-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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