| From the first formation of the nervous system, through the entire life span of an animal, to the end of its demise, connections between neurons form and disintegrate, at first establishing the gross neural network, and later refining it to store memories and modify behaviors. However, little is known about the molecular mechanism that regulates this event. This dissertation seeks to understand the first step of this process, namely the first stages of synapse formation. Previous studies have identified a pair of synaptic heterophilic adhesion molecules, Neuroligin and Neurexin that, by itself, is sufficient to trigger pre- and post-synaptic differentiation. Here we focused on the first few hours of the pre-synaptic terminal assembly, and asked two questions: (1) Is the differentiation solely triggered by this pair of synaptic adhesion molecules sufficiently fast to match the same process in neurons? (2) If not, are there other molecules that can regulate this process?; We used an assay developed earlier that utilizes HEK cells to expressing specific adhesion molecules and manipulates them into contact with neurons, thus the nature, the time and the location of interaction are defined. In combination with time-lapse fluorescence imaging and immunocytochemistry, we discovered that alternative splicing of Neuroligin-1 is an important regulator for the rate of synapse formation-the deletion of 9 amino acids from splice site B accelerates the differentiation rate to what is observed between neurons, which is likely to be due to an increase of its affinity to alpha-Neurexin.; In collaboration with Mechanical Engineering experts, we extended my research to the later stage of synapse formation by developing a microfluidic device that enable us to monitor synapse formation for hours to days. We have shown that our device is able to sustain proper neuron growth and exogenous genes can be easily introduced into these cells. Fluorescence time-lapse imaging is compatible with our devices, and the culture can be subjected to immunocytochemistry detection. |
