Analysis of Molecular Localization and Synaptic Structure Using ASEM
Abstract: Synapse formation is one of the key steps in the development of neural networks. Precise synaptic connections between nerve cells are crucial for brain development and functions. Recently, several synapse organizers including cerebellar GluRδ2-Cbln1-neurexin triad have been identified. However, their intracellular downstream signals and process of synapse formation are still largely unknown. Atmospheric scanning electron microscope (ASEM) is an electron microscope which was developed recently to directly observe subcellular structures and organelles through a silicon nitride (SiN) film under atmospheric pressure. ASEM dish with the SiN film can be used for incubation of cultured primary neurons. Cultured primary neurons were analyzed by integrated fluoresence microscope and ASEM. Presynaptic differentiation by GluRδ2-NTD-Fc-coated magnetic beads was analyzed by fluoresence microscope and ASEM. Fluorescence signals for presynaptic proteins were accumulated on the beads particularly concentrated at the site of contact with axon. Subsequent ASEM analysis revealed that presynaptic protein, presumably of an indistinguishable neurite branch, surrounded each beads. Thus, ASEM is a potentially useful tool for analysis of molecular localization and fine structure of synapse.
Key words: Atmospheric scanning electron microscope, synapse formation, synapse organizer, glutamate receptor GluRδ2