Abstract
In mammalian central nervous system, upon arrival of an action potential, glutamate is released from presynaptic terminals by exocytosis. Direct imaging of neurotransmission should greatly contribute to analyze an exocytosis dynamics at synapses and improve our understanding of the mechanisms in synaptic transmission. Aiming at imaging glutamate, we developed a novel optical glutamate probe named EOS, which consists of a ligand binding domain of AMPA receptor GluR2 subunit and a small molecule fluorescent dye. Glutamate binding to EOS can be reported as a change of fluorescence intensity. By immobilizing EOS on the cell surface of the hippocampal neuronal culture, we successfully visualized released glutamate following presynaptic firing with a single synapse resolution. Furthermore, we succeeded in continuous monitoring of the changes in presynaptic activity induced by phorbol ester. These results indicate that EOS can be used in evaluation of presynaptic modulation and plasticity. To clarify the mechanism of exocytosis in an excitatory synapse, we tried to quantitatively analyze released glutamate at individual synapses. We found that released glutamate varied in amount among synapses. Also, changes in release probability by manipulating extracellular calcium concentration were associated with changes in the amount of glutamate released at individual synapses. Our results suggest that single hippocampal synapses contain several release units. In summary, EOS-based glutamate imaging method is useful to address numerous fundamental issues in exocytosis process in the central nervous system. [J Physiol Sci. 2008;58 Suppl:S24]
