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研究生：	黃郁婷 Yu-ting Huang
論文名稱：	A Study of Synchronized Burst Mechanisms in Neuronal Cultures
指導教授：	陳志強 Chi-keung Chan
口試委員:
學位類別：	博士 Doctor
系所名稱：	理學院 - 物理學系 Department of Physics
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	121
中文關鍵詞：	同步爆發、多電極陣列、神經膠細胞、短期神經可塑性
外文關鍵詞：	Synchronized burst, MEA, glia, STSP
相關次數：	點閱：20 下載：0
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神經同步爆發是一種常見的自發性活動，但其運作原理尚未完全理解。爲了瞭解神經同步爆發的原理，我們利用多電極陣列來量測培養型神經細胞的網絡活動。我們發現同步爆發的形態與網絡發展的天數緊密相關，而不同發展天數的特徵可由藥理學的實驗重現。我們亦利用超高解析度的多電極陣列來研究同步爆發現象的時空分佈。實驗結果指出同步爆發並非隨機發生，細胞在網絡同步爆發時有其偏好的傳播路徑。另外，近期的研究指出神經膠細胞在神經發火行為中可被視為重要的影響因子。我們藉由調整不同神經膠細胞與神經細胞的比例來研究神經膠細胞對同步爆發的影響，證實膠細胞對同步爆發的重要性。上述實驗結果可由短期神經可塑性的均場模型來重現，說明了神經同步爆發與神經網絡的正回饋息息相關。

Synchronized bursting (SB) is a general spontaneous activity of neuronal cultures, however, their origin is still unclear. Here, we investigate the properties of these SBs in a culture on multi-electrode array (MEA) system. We find that characteristics of these SBs can be used to represent the different developmental stages of the cultures and these characteristics can be modified by pharmacological treatments. The spatiotemporal property of SBs and its reverberations are studied by a high-resolution MEA containing 4096 electrodes. The result indicates that SB fires with preferred pathways rather than completely random generation. Furthermore, we produce a neuronal cultures with different amounts of glial cells to study the effects of glia on SBs. The finding indicates that glia are interacting with neurons in the network to coordinate the firings. A mean-field model based on short term synaptic plasticity and recurrent connections has been developed to understand these characteristics. A phase diagram obtained from this model shows that networks exhibiting SBs are in an oscillatory state due to large enough positive feedback provided by synaptic facilitation and recurrent connections. Our finding suggests that networks with SBs have excessive recurrent connections and might have very little information processing capabilities.

摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
謝誌. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
圖目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
表目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
1 Objective and Backgrounds . . . . . . . . . . . . . . 1
1.1 Introduction of Synchronized Burst . . . . . . . . . . 1
1.2 Neurophysiology . . . . . . . . . . . . . . . . . . . . 7
1.3 Neuron-Glia interaction . . . . . . . . . . . . . . . . 10
2 Material and Method . . . . . . . . . . . . . . . . . 15
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Neuronal Cultures . . . . . . . . . . . . . . . . . . . 16
2.2.1 Dissection and Cultures . . . . . . . . . . . . . . . . 16
2.2.2 Immunocytochemistry . . . . . . . . . . . . . . . . . 17
2.3 Multi electrode Array . . . . . . . . . . . . . . . . . 19
2.3.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.2 Increasing signal to noise ratio . . . . . . . . . . . . 21
2.3.3 Recording Procedure . . . . . . . . . . . . . . . . . . 23
2.3.4 Signal Analysis . . . . . . . . . . . . . . . . . . . . . 24
2.4 CMOS-based Multi electrode Array . . . . . . . . . . 26
2.4.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4.2 Increasing signal to noise ratio . . . . . . . . . . . . 28
2.4.3 Recording Procedure . . . . . . . . . . . . . . . . . . 28
2.4.4 Signal analysis . . . . . . . . . . . . . . . . . . . . . 29
2.5 Patch Clamp . . . . . . . . . . . . . . . . . . . . . . 30
2.5.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.5.2 Gigaseal recording . . . . . . . . . . . . . . . . . . . 32
2.5.3 Increasing signal to noise ratio . . . . . . . . . . . . 34
2.5.4 signal recording . . . . . . . . . . . . . . . . . . . . 34
2.6 Calcium image . . . . . . . . . . . . . . . . . . . . . 35
2.6.1 Calcium Fluorescence indicator . . . . . . . . . . . . 35
2.6.2 Recording Procedure . . . . . . . . . . . . . . . . . . 35
2.6.3 Signal analysis . . . . . . . . . . . . . . . . . . . . . 35
3 Reverberation Activities within A Synchronized Burst 37
3.1 Synchronized Burst Activities Vary with Time . . . . 37
3.1.1 Synchronized Burst Firing Histograms Changes in
Different Culture Stages . . . . . . . . . . . . . . . . 37
3.1.2 Features of Synchronized Burst in Different Culture
Stages . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2 Effect of Pharmacology Test in Reverberations . . . . 41
3.2.1 Effect of Magnesium . . . . . . . . . . . . . . . . . . 42
3.2.2 Effect of Bicuculline . . . . . . . . . . . . . . . . . . 42
3.2.3 Effect of Glutamate . . . . . . . . . . . . . . . . . . 42
3.3 Spatiotemporal Properties of Reverberations . . . . . 43
3.3.1 Reverberaiotn Activities in a Long-Lasting Synchronized
Burst . . . . . . . . . . . . . . . . . . . . . . . 43
3.3.2 Spatiotemporal Dynamics of Reverberations . . . . . 47
3.4 Glia Support Neuron Synchronization . . . . . . . . 51
3.4.1 Calcium Image Analysis . . . . . . . . . . . . . . . . 51
3.4.2 Electrophysiology Measurement . . . . . . . . . . . . 52
3.4.3 Effect of NMDA on a Glia Free Culture . . . . . . . 56
4 A Mean Firing short-term Synaptic Plasticity Model
for Reverberation Activity . . . . . . . . . . . . . . . 59
4.1 Vesicles Release and short-term Synaptic Plasticity . 59
4.2 Mean-Firing short-term Synaptic Plasticity Model . . 61
4.2.1 Mean Firing Rate . . . . . . . . . . . . . . . . . . . 61
4.2.2 Vesicle Depletion . . . . . . . . . . . . . . . . . . . . 62
4.2.3 Synaptic Facilitation . . . . . . . . . . . . . . . . . . 64
4.2.4 Slow Modulation of Vesicle Recycling . . . . . . . . . 66
4.3 short-term Synaptic Plasticity of Reverberation Activities
. . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3.1 STSP Model Without Slow Depletion Variables :
TM Model . . . . . . . . . . . . . . . . . . . . . . . 69
4.3.2 STSP Model With A Slow Depletion Variable : TMX
Model . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3.3 Simulating Reverberation Activities with The TMX
Model . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.4 Experimental Prediction of Slow Depletion of Neurotransmitter
. . . . . . . . . . . . . . . . . . . . . . 79
5 Conclusion and Discussion . . . . . . . . . . . . . . . 81
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
A.1 Equipments and Software . . . . . . . . . . . . . 89
A.2 Drugs and Chemicals . . . . . . . . . . . . . . . . 90
Appendix B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
B.1 Culture Medium . . . . . . . . . . . . . . . . . . 91
B.2 Neurobasal Medium . . . . . . . . . . . . . . . . 91
B.3 Digestion Solution . . . . . . . . . . . . . . . . . 91
B.4 Internal Cellular Solution (ICS) . . . . . . . . . 92
B.5 Balanced Salt Solutions (BSS-)/(BSS+) . . . . 92
B.6 Borate Buffer Solution . . . . . . . . . . . . . . . 93
B.7 Coating Solution . . . . . . . . . . . . . . . . . . 93
Appendix C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
C.1 MEA60 Coating Protocol . . . . . . . . . . . . . 95
C.2 BioChip Coating Protocol . . . . . . . . . . . . . 95
C.3 Coverslip Coating Protocol . . . . . . . . . . . . 96
C.4 Dissection and plating Protocol . . . . . . . . . 96

                                

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