臺灣博碩士論文加值系統

摘要

我們研究大白鼠皮質層神經元網路在低鎂離子濃度下之同步發放行為。利用共軛焦雷射掃瞄顯微技術，同步發放的鈣離子動力學得以量測。我們發現上述同步發放動力與網路型態有密切相關。神經元網路在高密度下形成三維巨大團簇，以粗大絲狀神經元捆束相互連結。低密度神經元網路則形成二維小團簇，以單神經突觸相互連結。我們發現高密度神經元網路比低密度神經元網路更早達到同步發放，並且在成長後期展現有趣的發放動力型態。我們在其中並發現有一長時間的休歇期間與高頻發放動力共存，形成一緩慢之調幅效應。我們利用非線性動力學分析手法，量測其中發放時間間隔分佈並以此重建其奇異吸子。我們發現其中休歇時期與高頻發放期間具有有趣的關聯性，並以空間中離子濃度匱乏理解上述現象。我們並利用高速雷射掃瞄量測在同步發放中之領先與落後神經元的時空相關性，佐證在神經元網路系統中的時空異質性。神經元網路同步發放行為可經由以化學蝕刻之微米鎢針作局部調控。神經元團簇之間的連接可被鎢針切割，造成非同步發放。我們發現神經元網路的集體動力行為展現小世界網路之拓樸特性。

Abstract

We study the synchronous firing activities in cortical neuronal network culture. Under depletion of extracellular Mg2+, synchronous firing activity is observed using confocal fluorescence microscopy. The firing patterns of the synchronous firing activities are found to closely correlated to the network morphology. It is found that the high density culture develops into synchronous firing prior to the low density one at early stage. The firing frequency of synchronous firing is generally an increasing function of day in vitro (DIV). It is further found that the high density culture develops into unique firing pattern with high rate bunched firing peaks separated by long quiescence periods. Using non-linear dynamics analysis, we analyze the phase diagram of the inter-event interval (IEI) of synchronous firing and reconstruct the strange attractor structure. It is found that the strange attractor exhibits period two characteristics and the histograms of IEI show double peak and broader distribution for the unique firing pattern. The mechanism behind the emergence of the long quiescence period is investigated by measuring the correlation between the long quiescence and long bursting period, and explained in term of extended ion depletion. Using fast line laser scanning, we identify master and slave during synchronous firing, showing spatiotemporal heterogeneity in the synchronous firing activity. Micro-dissection is performed to perturb the synchrony of the network firing activity as a preliminary est for the small world network architecture.

Contents


1. Introduction 1
2. Background 7
2.1 Integrate-fire systems………………………………………………………7
2.2 Neurobiology……………………………………………………………….9
2.21 Basic blue print for living organisms…………………………………...9
2.2.2 Biology of neuron at cellular level……………………………………..11
2.2.3 Developmental evolution of neuronal subtypes in cerebral cortex……..20
2.2.4 Synchronization in cerebral cortex: epilepsy due to inhibition failure vs. synchronous firing under Mg2+ depletion………………………………21
2.3 Synchronization in complex network: Small world architecture………….22
2.4 Non-linear dynamical model for Neuronal Network………………………27
3. Experiment and data analysis 27
3.1 Primary cortical neuronal culture………………………………………… 28
3.2 Fluorescence staining: calcium indicator……………………………… 29
3.3 Observation platform: confocal microscopy and in-situ incubation chamber…………………………………………………………………….30
3.4 Electrophysiological measurement and local cell manipulation techniques………………………………………………………………….33
4. Results and discussions 36
4.1 Cell density dependence of cortical neuronal network morphology… ……37
4.2 Synchronous firing in rat cortical neuronal network in vitro: Electrical field potential and fluorescence measurement…………………………………..40
4.3 Developmental dynamics of firing pattern of cortical neuronal network during maturation………………………………………………………….44
4.3.1 Data analysis: histogram of inter-event interval and strange attractor reconstruction………………………………………………………...46
4.3.2 Long quiescence period during synchronous firing at maturation…………………………………………………………….48
4.4 Emergence of synchronization: identifying the master and slave in synchronous firing…………...……………………………………………..58
4.5 Manipulating the firing dynamics: local micro-dissection ………………………………………………………...64
5. Conclusion and future work 67
6. Appendix 69
A1. Protocol for primary cortical neuronal culture…………………………..68
A2. Protocol for chemical preparation……………………………………….77
A3. Protocol for fluorescence staining……………………………………….83
A4. Protocol for micropipette preparation…………………………………...85

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