We are interested in understanding the molecular mechanisms underlying the formation and function of excitatory synapses in the CNS. To reach this goal, we are currently undertaking two approaches: (1) analyzing the molecular events during the formation of synapses in the brain and under in vitro conditions and (2) studying the function of the protein constituents of excitatory synapses. We are also participating in the development of three technologies: multielectrode array recording systems, nanodepth tagging methodology and patterned neuronal cultures.
To study the molecular events leading to the formation of synapses, we are using thin brain slices containing intact, developing thalamocortic connections from rat embryos as a model. The function and molecular constitution of the newly formed synapses in the cortical plate layer are studied by electrophysiological and immunocytochemical means, respectively. As supported by the Research Program for Genomic Medicine (National Science Council, Taiwan), we are employing MEMS (MicroElectroMechanical System) technologies to make micropatterns on the surface of glass coverslips. These patterned coverslips are used to culture neurons dissociated from embryonic rats. Different subcellular components of neurons, including axons, growth cones and presynaptic terminals, will be collected for systematic proteomic analysis.
To study the neural circuits in embryonic brains, we are currently moving into the field of neural engineering. In collaboration with Professors Yeh and Chiao of our Institute and Professors Fang, Liu and Yao of the Institute of Microelectromechanical System, we are fabricating multi-electrode arrays and making electronics for data acquisition which will allow us to monitor the electric activities of many neurons, between 16 to 100 neurons, of a neural circuit.
To study the function of the protein constituents of the synapses, we are presently analyzing the proteins in the postsynaptic density (PSD), a unique protein complex associated with excitatory synapses, isolated from the cerebral cortex of adult porcine brain. The function of individual PSD proteins is studied by monitoring their distributions in normal neurons and also by analyzing neurons in which the expression of a particular PSD protein has been compromised or increased. The experimental tools used for this part of study include Mass spectrometry, 2-D gel electrophoresis, confocal microscopy, molecular biology methodologies and voltage clamp recording facilities.
To probe the 3-dimensional organization of various proteins in the PSD, our laboratory is supported by the Research Program for Genomic Medicine (National Science Council, Taiwan) to develop a methodology that allows us to selectively isolate proteins residing at different depths from the surface of a supramolecule. By using the methodology developed thus far, we have already partially resolved the laminar organization of various proteins in the PSD.
2012.11.15 更新版: