Biophysical Studies on the Neurobiological Phenomena Related to Learning and Memory Using the Combined AFM and CLSM

Abstract

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.