원자간 힘 현미경-공초점 레이저 주사 현미경 통합기기를 이용한, 학습 및 기억 관련 신경생물학적 현상에 관한 생물리학적 연구

학위논문 (박사)-- 서울대학교 대학원 : 화학부 물리화학 전공, 2013. 8. 이성훈.This thesis research focuses on the development of the combined atomic force microscopy and confocal laser scanning microscopy (AFM-CLSM) system and its application to understanding learning and memory. The developed combined AFM-CLSM system is an efficient and suitable method to study the fine 3D structural changes in live cells and the relationship between morphological/structural changes and intracellular functional changes such as the dynamic change of synaptic connections related to neuronal mechanism or phenomena. Brain-derived neurotrophic factor (BDNF) is a major regulating neurotrophin of synaptic transmission and plasticity at adult synapses in many regions of the central nervous system (CNS). The diverse functions of BDNF are mediated by their specific interaction with tyrosin kinase B (TrkB) receptors. We have utilized a quantum dot (QD)-based immunocytochemistry technique to detect TrkB receptor proteins. We applied the combined AFM-CLSM system to detect accurate localization of endogenous TrkB receptor proteins, which are targeted to the surface membrane for the function in mouse hippocampal neurons. We found that endogenous TrkB receptor proteins are distributed in a highly diffuse manner in the central soma, discrete in the neurites. This result shows that the combined AFM-CLSM system is highly efficient for discovering precise correlation between localization of immunocytochemically labeled specific proteins and cellular structures in a three-dimensional manner. Structural and functional plasticity of Aplysia mechanosensory presynaptic neurons has been investigated to understand the mechanism of learning and memory. Long-term facilitation, a well-known cellular model for long-term memory in Aplysia, is known to be accompanied by new synaptic growth. We applied the combined AFM-CLSM system to image accurate and volumetric changes in presynaptic structure (varicosities) of live Aplysia neurons. We found that preexisting varicosities filled with synaptic vesicles showed a volumetric increase following a continuous (massed) application of serotonin (5-hydroxytriptamine, 5-HT) in conjunction with an increase in the total number of varicosities. This volumetric change in synaptic structure improves strength of the synaptic connection and efficiency of the synaptic transmission due to an increase in active zones of synaptic areas and synaptic vesicle pools of synaptic varicosities. This result shows that the combined AFM-CLSM system is highly efficient for measuring accurate and detailed structural changes together with the functional changes in synaptic contacts of live neurons.Chapter 1 Introduction 1.1 Motivation……………………………………………………………………………… 2 1.2 Introduction to Light Microscopy……………………………………………………… 5 1.2.1 Light Microscopy………………………………………………………………… 5 1.2.2 Fluorescence Microscopy……………………………………………………… 6 1.2.3 Confocal Laser Scanning Microscopy (CLSM)…………………………………… 7 1.3 Introduction to Electron Microscopy………………………………………………… 10 1.4 Introduction to Atomic Force Microscopy (AFM)…………………………………… 11 1.4.1 Principle of Operation………………………………………………………… 11 1.4.2 Operation Modes of AFM……………………………………………………………… 14 1.4.3 AFM Applications Specific to Biology…………………………………………………………… 16 1.5 Combined AFM and CLSM…………………………………………………………… 17 1.6 Outline of this Thesis…………………………………………………………… 19 1.7 References……………………………………………… 20 Chapter 2 Development of Combined AFM and CLSM 2.1 Introduction …………………………………………………………………… 27 2.1 Requirements of the Combined AFM and CLSM……………………………………………… 28 2.3 Set-up of the Combined AFM and CLSM……………………………………………… 29 2.3.1 Combination between AFM and CLSM instruments...…………………………………… 29 2.3.2 Fluorescent Filters…………………… 33 2.3.3Preparation of Sample Dishes...……………………………………………………… 33 2.3.4 Alignment of Cantilever Tip to Optical Axis………………………………………………… 35 2.4 Performance of the Combined AFM and CLSM……………………………………………… 35 2.4.1 Performance of the AFM………………………………………………… 35 2.4.2 Performance of the CLSM……………………………………………… 40 2.5 Data Analysis……………………………… 42 2.5.1 Data Processing for the Integration between AFM and CLSM Images: Matlab Routine…………………………………… 42 2.6 Results……………………………………… 42 2.6.1 AFM Imaging of Neuronal Cells under Fixed and Live Conditions..………………………………… 42 2.6.2 Integrated Images Acquired by the Combined AFM and CLSM System……………………………… 46 2.7 Conclusions………………………………… 48 2.8 References………………………………… 49 Chapter 3 Development of TrkB receptors distributed in cultured Hippocampal neurons 3.1 Introduction …………………………………… 54 3.2 Experimental Section……………………… 57 3.2.1 Chemicals……………………………… 57 3.2.2 Single-step Synthesis of Quantum dots (QDs) with Chemical Composition Gradients…………………………………………… 58 3.2.3 Surface Modification of CdSe/ZnS QDs…………………………………………………… 59 3.2.4 Conjugation between MPA-capped QDs and Neutravidin (NTV)………………………………… 59 3.2.5 Cell Culture………………………………… 60 3.2.6 Cell Fixation…………………………… 60 3.2.7 Detection of TrkB Receptors on a Mouse Hippocampus Neuron Cell Surface…………… 61 3.3 Measurements and Analysis……………… 61 3.3.1 Measurement of Quantum Yield (QY) of CdSe/ZnS QDs …………………………………… 61 3.3.2 Transmission Electron Microscopy (TEM)………………………………………………………… 62 3.3.3 Fourier Transform Infrared Spectroscopy (FT-IR)………………………………………………………… 62 3.3.4 Measurement of Hydrodynamic Size of CdSe/ZnS QDs…………………………………………………… 62 3.3.5 Confocal Laser Scanning Microscopy (CLSM) ………………………………………………………… 63 3.3.6 Combined AFM and CLSM…………… 63 3.4 Results and Discussion…………………… 64 3.5 Conclusions………………………………… 76 3.6 References………………………………… 78 Chapter 4 Volumetric changes in presynaptic structure of Aplysia sensory neurons during LTF 4.1 Introduction ………………………………………………………… 83 4.2 Experimental Section…………………………85 4.2.1 Sensory-to-Motor Neuron Coculture… 85 4.2.2 Microinjection of DNA Constructs into Aplysia Neurons……………………………………………… 85 4.2.3 Long-term Facilitation Protocol ……… 86 4.3 Measurements and Analysia……………………………………………… 86 4.3.1 Combined AFM and CLSM………………… 86 4.3.2 Quantification of the Total Varicosity Number and the Volume of Varicosity…………………………… 87 4.4 Results and Discussion …………………… 88 4.4.1 Concurrent Functional and Structural Changes Following 5-HT Treatment ………………………… 88 4.4.2 Presynaptic Structural Changes Accompanying Long-Term Facilitation Are Analyzed by Actual Volumetric Changes ……… 92 4.5 Conclusions……………………………………… 95 4.6 References……………………………………… 96Docto

층상이중수산화물 (Zn-Cr-Ds 체계)의 유기실란에 의한 공유결합적 표면개질에 관한 연구

본 연구에서 중점적으로 시도한 연구는 LDHs 표면을 여러 가지 화합물들이 결합할 수 있는 표면으로 개질(modification) 하는 것이었다. 표면을 개질하기 위해서 bridge 역할을 할 수 있는 coupling reagent인 3-aminopropyltriethoxysilane (H_(2)N(CH_(2))_(3)Si(OC_(2)H_(5))_(3): 이하 APTES로 표기)와 LDHs표면을 유기 용매 상에서 반응시켰다. LDHs 층 사이의 DS 이온은 첨가한 cationic surfactant인 cetyltrimethylammonium bromide ([C_(16)H_(33)N(CH_(3))_(3)]^(+) : 이하 CTAB로 표기)와 반응하여 친유성 염을 이루게 되고 유기 용매로 용출되어 나오게 된다. 동시에 APTES가 그 자리에 치환되어 들어가면서 수산 화기 사이에 공유 결합인 oxane bond (M-O-Si (M=Zn, and Cr))를 만들게 된다. 반응에 쓰인 유기 용매의 극성이 표면 개질하는 반응에 영향을 주게 된다. 무극성 유기 용매에서 반응시키면 공유 결합된 APS만 층 사이에 남게 되고, 극성 유기 용매에서 반응시켰을 때는 공유결합한 APS와 층간 삽입된 DS 이온이 함께 LDHs의 층 사이에 존재하게 된다. 층 간격은 Powder X-ray를 통해서 확인하였고, LDHs의 표면 위의 APS 이온의 Si 원자 결합 형태는 고체 상태 Si^(29) NMR로 확인하였다. APS가 존재함은 FT-IR과 Ninhydrin test의 Ruhemann's purple을 UV-Vis spectra 로 확인하였다.;Interlayer surface modified Zn/Cr layered double hydroxides (LDHs) were obtained by reacting (3-aminopropyl) triethoxysilane (APTES) with hydroxyl groups of LDHs in organic solvent. The procedure of reaction was that the salts formed between dodecylsulfate (DS^(-)) anionic and cetyltrimethylammonium (CTA^(+)) cationic surfactant in interlayers of LDHs were extracted into organic phase. And then APTES were simultaneously replaced and covalent bonds with surface of LDHs were formed. Polarity of the organic solvents used affects the reaction between host and guest species. When the reaction was occurred in nonpolar solvent, only covalently bonded APS moieties were observed between layers. However, the reaction was occurred in polar solvent, the covalently bonded APS moieties together with intercalated DS ions were observed between layers. The basal spacing conditions were characterized by powder X-ray diffraction. The chemical environments of silicon atoms of the APS on the LDHs’ surface are characterized by ^(9)Si CP/MAS NMR experiments. Presence of the APS is confirmed by FT-IR and UV-vis spectrometric detection of Ruhemann’s Purple in ninhydrin test. TEM is used to analyse the layered morphology of LDHs.Contents = Ⅲ List of Figures = Ⅴ List of Tables = Ⅶ Abstract = Ⅷ Brief Review of Layered Double Hydroxides = 1 A. Introduction = 12 B. Experiments = 15 1. Materials = 15 2. Synthesis = 15 a. Synthesis of Zn/Cr-DS LDHs = 15 b. Surface modification of Zn/Cr-DS LDHs = 16 3. Analysis = 17 a. Powder X-ray Diffraction Analysis (PXRD) = 17 b. Ninhydrin test / UV-Vis Spectroscopy = 17 c. Infrared spectroscopy (FT-IR) = 18 d. Solid-state ^(29)Si Cross Polarization/Magic Angle Spinning (CP/MAS) NMR spectroscopy = 19 e. High-Resolution Transmission Electron Microscopes (HRTEM) = 19 C. Results and Discussions = 21 Powder X-ray Diffraction = 21 Ninhydrin Test/UV-Vis Spectroscopy = 23 Infrared Spectroscopy (FT-IR) = 26 Solid-state ^(29)Si Cross Polarization/Magic Angle Spinning (CP/MAS) NMR spectroscopy = 27 High-Resolution Transmission Electron Microscopes (HRTEM) = 29 D. Conclusions = 35 E. References = 36 국문초록 = 4