Abstract
Glutamate is an essential excitatory neurotransmitter in the central nervous systems. For the understanding of mechanisms underlying synaptic transmission, we developed a novel optical glutamate probe called S403C-OG which consists of a recombinant glutamate binding domain derived from GluR2 subunit of AMPA receptor and a fluorescent dye. To visualize synaptically released glutamate, we immobilized S403C-OG on the cell surface of the cultured hippocampal neurons and captured fluorescence images with CCD camera. With this maneuver, we successfully detected the glutamate release along active synapses in response to electrical stimuli. The amount of glutamate release considerably varied among locations within the same neuron, suggesting the spatial heterogeneity among the release sites. We also observed spontaneous and transient glutamate release events without electrical stimuli, which was stochastic, variable in amplitude and spatially confined in small regions. Neither application of tetrodotoxin nor removal of extracellular calcium blocked the spontaneous glutamate release. The frequency of the release increased upon application of high concentrations of sucrose which is known to increase the frequency of miniature EPSC. Furthermore, we succeeded in continuous monitoring of the changes in presynaptic activity induced by phorbol ester, indicating that our probe enable to directly visualize the presynaptic activity. In summary, S403C-OG is useful to address many fundamental issues related to glutamatergic synaptic transmission in the central nervous system. [J Physiol Sci. 2006;56 Suppl:S166]
