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Synaptic Elasticity Synaptic Elasticity Ju Yang Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2018 © 2018 Ju Yang All Rights Reserved Abstract Synaptic Elasticity Ju Yang Synapses play a critical role in neural circuits, and their highly specialized structures and biochemical characteristics have been widely studied in learning and memory. Along with their role in signal transmission, synapses also serve as adhesion structures, yet their mechanical characteristics have not received much attention. Given the important role of mechanics in cell adhesion, mechanical studies of synapses could offer insights into synaptic development, maintenance, and function. Here, I investigated synaptic elasticity in cultured rat hippocampal neurons and suggest that mechanical elasticity may be related to synaptic plasticity. I used torsional harmonic atomic force microscopy (TH- AFM) to measure the nanomechanical properties of functional mature excitatory synapses, whose identity and activity was verified by fluorescence microscopy. I combined TH-AFM with transmission electron microscopy and found that high stiffness of synapses originated from postsynaptic spines, not presynaptic boutons. I observed that spines at functional mature excitatory synapses were on average 10 times stiffer than dendritic shafts and that the distribution of spine stiffness exhibited a lognormal-like pattern. Importantly, I found that spine stiffness was correlated with spine size, and it is well established that spine size is correlated with synaptic strength. Based on the stiffness measurements and theoretical modelling of cell adhesion stability, I suggest that stiffness not only helps maintain spine morphology in the presence of synapse adhesion, but also helps stabilize synaptic adhesion. I propose a mechanical synaptic plasticity model. According to this model, mechanical strength leads to functional strength, which could provide a potential causal link between structural plasticity and functional plasticity of synapses. Table of contents List of charts, graphs, illustrations ................................................................................................................... ii Acknowledgements ............................................................................................................................................ v Dedication ......................................................................................................................................................... vii Chapter 1 Introduction ........................................................................................................................... 1 Chapter 2 TH-AFM: a tool to study cell mechanics .......................................................................... 9 Chapter 3 Live nanomechanical imaging with TH-AFM reveals stiff synapse-like structures .. 18 Chapter 4 Correlative TH-AFM/fluorescence imaging reveals stiff and functional mature excitatory synapses ........................................................................................................................................... 28 Chapter 5 Correlative TH-AFM/TEM imaging reveals ultrastructure of stiff synapses ........... 39 Chapter 6 Spines are substantially stiffer than shafts ....................................................................... 49 Chapter 7 Spine stiffness and actin networks ................................................................................... 65 Chapter 8 Mechanical synaptic plasticity model ............................................................................... 76 Chapter 9 Conclusion ........................................................................................................................... 87 References ......................................................................................................................................................... 96 i List of charts, graphs, illustrations Figure 1-1 Neurons communicate through synapses. .................................................................................. 2 Figure 1-2 Synapses are mechanically interesting structures. ...................................................................... 4 Figure 2-1 AFM principles. ............................................................................................................................ 10 Figure 2-2 AFM applications in biological samples. ................................................................................... 12 Figure 2-3 Force-distance curves and force-volume imaging with AFM. ............................................... 14 Figure 2-4 Torsional harmonic AFM. ........................................................................................................... 17 Figure 3-1 T-shaped cantilever. ..................................................................................................................... 20 Figure 3-2 Nanomechanical imaging platform. ........................................................................................... 21 Figure 3-3 Nanomechanical imaging of live cultured neurons. ................................................................ 23 Figure 3-4 Force-distance curves during TH-AFM imaging. .................................................................... 25 Figure 3-5 Three-dimensional AFM image of a stiff synapse-like structure in live neurons. ............... 25 Figure 3-6 Stiffness of a synapse-like structure does not vary significantly during imaging. ................ 27 Figure 4-1 Optical and TH-AFM imaging reveals stiff synapse-like structures. .................................... 31 Figure 4-2 Μolecular organization at synapse. ............................................................................................ 32 Figure 4-3 Functional labeling of synaptic terminals with FM dyes. ........................................................ 33 Figure 4-4 Fluorescence imaging of neurons after TH-AFM. .................................................................. 35 Figure 4-5 Correlative TH-AFM/fluorescence imaging shows stiff synapse-like structures are functional mature excitatory synapses. ......................................................................................................... 36 Figure 4-6 Stiff synapse-like structures are labeled with synaptic markers. ............................................. 37 Figure 5-1 Workflow of correlative TH-AFM/TEM imaging. ................................................................. 40 Figure 5-2 Applications of TEM in the study of synaptic ultrastructure. ............................................... 42 Figure 5-3 A homemade glass bottom dish with a gridded coverslip. ..................................................... 44 ii Figure 5-4 Correlative TH-AFM/TEM imaging. ........................................................................................ 44 Figure 5-5 Correlative TH-AFM/TEM imaging of stiff synapses. .......................................................... 45 Figure 5-6 Examples of correlative TH-AFM/TEM images of synapses. .............................................. 46 Figure 6-1 Contact mechanics models. ......................................................................................................... 52 Figure 6-2 Spine morphological heterogeneity. ........................................................................................... 54 Figure 6-3 Distribution of spine stiffness and shaft stiffness.................................................................... 55 Figure 6-4 Distribution of apparent spine size. ........................................................................................... 57 Figure 6-5 Spine stiffness is correlated with spine size. ............................................................................. 57 Figure 6-6 A subgroup of synapses identified by immunofluorescence microscopy do not show high stiffness. ............................................................................................................................................................. 60 Figure 6-7 Colocalization detection with Caltracer. .................................................................................... 61 Figure 6-8 A shaft synapse does not display high stiffness. ...................................................................... 62 Figure 6-9 Immature protrusions are not stiff. ............................................................................................ 63 Figure 7-1 Spines contain dense actin networks regulated by actin binding proteins. .......................... 67 Figure 7-2 Elasticity of actin networks comes from cross-linking density or tension. .......................... 68 Figure 7-3 F-actin is enriched in a stiff spine head. .................................................................................... 69 Figure 7-4 Latrunculin A reduces F-actin level in neurons. ...................................................................... 70 Figure 7-5 Spine stiffness is not affected by acute Latrunculin A treatment. ......................................... 71 Figure 7-6 Actin branching and elongation in structural persistence. ...................................................... 72 Figure 7-7 Spine stiffness is not affected by acute Blebbistatin treatment. ............................................. 73 Figure 8-1 Stiffness helps maintain
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