Molecular Mechanism of Neurotransmission Studied by Ultra-High Power Light Microscopy

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Physiological activities in nerve terminals are vital for humoral control and brain function. Therefore, analyses of these activities are very important for understanding biological functions of the higher order. In this study, we aim at elucidation of molecular mechanisms of nerve terminals by observing them under a video-enhanced DIC light microscope. In cultured bovine chromaffin cells, terminals of the processes made synapselike contacts with neighboring cells. Electrical stimulation of the presynaptic terminal induced many popping responses of granules in the terminal. These responses were [Ca^<2+>]o dependent and suppressed by Cd^<2+> or La^<3+>. The smallest granule responded was 0.1 mum in diameter. The frequency of such responses were very high especially at areas which were in contact with the neighboring cell. Repetitive stimulation induced facilitation. Stimulation with many pulses induced fatigue characterized by a lack of granules in the terminals. These finding indicated that popping responses bear properties common to synaptic responses. Therefore, we concluded that these popping responses are exocytosis of granules in the terminal. Simultaneous Ca-imaging and DIC-imaging demonstrated a signal transmission from one cell to the other in the synapse-like contact, providing evidence for direct visualization of exocytosis in the synapse. Neither the swelling hypothesis nor the collision hypothesis is confirmed for initiation of exocytosis. Probably, membrane fusion at the final step of exocytosis is initiated by some Ca-dependent key molecules in the plasma membrane. In addition to this, we also found rapid sprouting of filopodia induced by electrical stimulation in nerve terminals. This sprouting may be deeply involved in the synaptic plasticity.