서울대학교 중앙도서관
S-Space 소개
My S-Space
로그인이 필요합니다.
Login

S-Space

Publications

Detailed Information

Biophysical Studies on the Neurobiological Phenomena Related to Learning and Memory Using the Combined AFM and CLSM : 원자간 힘 현미경-공초점 레이저 주사 현미경 통합기기를 이용한, 학습 및 기억 관련 신경생물학적 현상에 관한 생물리학적 연구

DC Field Value Language
dc.contributor.advisor이성훈-
dc.contributor.author박애영-
dc.date.accessioned2017-07-14T05:51:52Z-
dc.date.available2017-07-14T05:51:52Z-
dc.date.issued2013-08-
dc.identifier.other000000012574-
dc.identifier.urihttps://hdl.handle.net/10371/125234-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학부 물리화학 전공, 2013. 8. 이성훈.-
dc.description.abstractThis 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.		-
dc.description.tableofcontentsChapter 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……………………………………… 96		-
dc.formatapplication/pdf-
dc.format.extent2703631 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectAtomic force microscopy-
dc.subjectconfocal laser scanning microscopy-
dc.subjectlong-term facilitation-
dc.subjectAplysia-
dc.subject.ddc540-
dc.titleBiophysical Studies on the Neurobiological Phenomena Related to Learning and Memory Using the Combined AFM and CLSM-
dc.title.alternative원자간 힘 현미경-공초점 레이저 주사 현미경 통합기기를 이용한, 학습 및 기억 관련 신경생물학적 현상에 관한 생물리학적 연구-
dc.typeThesis-
dc.contributor.AlternativeAuthorPark, Aee-Young-
dc.description.degreeDoctor-
dc.citation.pagesxi, 106-
dc.contributor.affiliation자연과학대학 화학부-
dc.date.awarded2013-08-
Appears in Collections:
Files in This Item:

Altmetrics

Item View & Download Count

Show Simple Item Record
Find it @ SNU

  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Share

qrcode
SNS Share