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研究生:張欽賀
研究生(外文):Chin-He Chang
論文名稱:神經膠細胞在神經同步活動及鈣離子波傳遞中之角色
論文名稱(外文):Investigation of the role of glial cells in the synchronous firing and calcium wave propagations in rat cortical neural cultures
指導教授:陳志強陳志強引用關係洪蘭洪蘭引用關係
指導教授(外文):Chi-Keung ChanDaisy L. Hung
學位類別:碩士
校院名稱:國立中央大學
系所名稱:認知與神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:79
中文關鍵詞:神經膠細胞鈣離子波同步活動
外文關鍵詞:calcium wavephotolysissynchronous firingastrocytesglial cell
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「自發性神經同步活動」在哺乳類神經系統中是個普遍的現象,而這樣的現象對於神經細胞的發育、突觸(synapse)的形成,扮演著極重要的角色。事實上,哺乳類動物的大腦中除了神經細胞之外,佔大多數的卻是神經膠細胞(glial cell)。隨著近年來大量的研究結果顯示,神經膠細胞除了傳統上認為支持、提供養分的角色之外,還參與了神經細胞的許多活動,同時亦可以擔任調節突觸活動的功能。甚至還有證據認為神經膠細胞在突觸的連結與形成的機制上,扮演著舉足經重的角色,這樣的結果的確隱含著神經膠細胞有著直接影響神經可塑性(plasticity)與高等認知功能的緊密關係。由於神經細胞要達到同步化,必要的條件是細胞與細胞之間的連結數目要夠多,意即細胞之間的突觸要達到某一程度的連結。由於最近的研究顯示,神經膠細胞和神經細胞的突觸之間有著緊密的連結,並且參與了活動的調節,因此我們推論神經膠細胞不僅能提供神經細胞養分,同時在自發性神經同步活動中,也可能扮演著重要的作用。
有別於傳統電生理(electrophysiology)技術上的限制,無法同時記錄整個神經網絡的活動變化,本實驗利用鈣離子螢光造影技術,藉由螢光亮度的整體變化來記錄神經細胞的同步活動。同時在6∼21的培養天數內操弄神經膠細胞的有無、數量多寡以及出現的時間,希望能找出神經與神經膠細胞間的交互關係。
實驗的結果顯示,即使在沒有含膠細胞的培養中,神經的同步現象仍然可以產生,然而不同的是,神經膠細胞的有無亦造成了不同程度的同步現象。
此外,我們也使用光解作用(photolysis)的方法,利用UV雷射破壞單一細胞而引發鈣離子波,同時紀錄影像分析,發現在有大量的膠細胞情況下,鈣離子波的傳遞速率較沒有膠細胞的情形下來得慢。
Glial cells are traditionally thought to play the peripheral and supportive role in the CNS (Central Nervous System). However, recent studies are shown that glia have a variety of functions in the synaptic transmission. Therefore, recognition of the importance of glial cells in nervous system functions is increasing, especially regarding the modulation of neural activity.
Synchronized spontaneous Ca2+ spikes in neuronal network cultures represent periodic bursting, which are believed to play a major role during the development of the CNS. It is known that glia, especial astrocytes, are the most abundant cell-type in the CNS, which will also release neurotransmitters. How the neuronal network are shaped and modulated by the glia remain to be studied. In this work, we study the role of glial cells in spontaneous activities by monitoring changes in [Ca2+]i with fluorescence dye. The result displays that the neuronal synchronized firing (SF) remains to be observed under the glia-suppressed cultures (GSCs). However, it can be seen that there are the different patterns in the cultures with the different number of glial cells. These studies demonstrate that glia play an important role in the neuronal network activity during development.
Furthermore, photolysis by using a UV laser is used to create intercellular Ca2+ waves to investigate the effects of glia on synaptic transmission. It is shown that the velocity of Ca2+ wave propagation in the glia-enhanced cultures (GECs) is slower than that of in the GSCs.
Abstract Ⅰ
Contents Ⅱ
List of figures Ⅲ
List of tables Ⅴ

1 Chapter 1 introduction 1

1.1 Synchronization phenomenon in the natural world 1
1.2 Synchronized activities of neural network in the central nervous system 1
1.3 The relationship between synchronous firing 4
1.4 Basic neurobiology of neuron-glia interactions 5
1.5 Do glial cells participate in synchronous firing and epilepsy? 8
1.6 The role of astrocytic calcium waves 11
1.7 The underlying effect of glial cells on the cognitive functions 13
1.8 The approaches and the purposes 15

2 Chapter 2 Experimental setup and procedures 16

Overview 16

2.1 Sample preparations 16
2.1.1 Neuron-glia cocultures (NGCs) 17
2.1.2 Pure astrocytic cultures and preparation of glia-conditioned medium (GCM) 17
2.1.3 Glia-suppressed cultures (GSCs) 18
2.1.3.1 GSCs with the GCM 18
2.1.3.2 GSCs in NB/B27 medium 18
2.1.4 Glia-enhanced cultures (GECs) 19
2.1.4.1 Pre-plated astrocytes 19
2.1.4.2 Post-plated astrocytes 19

2.2 Imaging system 20
2.2.1 Microscope 20
2.2.2 Optics set-up 21
2.2.3 Temperature controller 22
2.2.4 Intensified Charge-Coupled Device (ICCD) video camera 22
2.2.5 Automation 23

2.3 The UV laser photolysis 23
2.3.1 Fluorescence calcium indicator 23
2.3.2 Pulsed UV laser machaine 24
2.3.3 Flash photolysis-induced calcium waves 24

2.4 Immunocytochemistry 26

3 Chapter 3 Results 27

3.1 Identification of neurons and astroglia in the primary cultures 27
3.2 The different SF patterns of three types of cultures during development 38
3.3 Calcium waves measurements in the three cultured conditions 46
3.4 UV laser-induced Ca2+ propagations induce the neuronal rapid Ca2+ oscillations in the glia-free cultures 47
3.5 Laser-induced the astrocytes wavy-like Ca2+ oscillations 48

4 Chapter 4 discussion 50
4.1 The manipulation of glial cells on the cultures during development 50
4.2 The amount of glial cells influences the neuronal Sf (SF frequency) 53
4.3 The role of astroglia in the Ca2+ waves propagation 54

5 Chapter 5 summary and conclusions 59

6 Reference 60

7 Appendix A 65

8 Appendix B 67
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