Sho Iinuma

Optical glutamate sensor for spatiotemporal analysis of synaptic transmission.

Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand‐binding domain of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high‐resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS‐based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.

Glutamatergic neurotransmission in the procerebrum (Olfactory Center) of a terrestrial mollusk

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real‐time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum.

Imaging glutamate spillover from synaptic clefts using the fluorescent glutamate indicator

The serine/threonine kinase SAD regulates several synaptic functions such as synapse development, axon/dendrite polarization, and neurotransmitter release. In mammalian central nervous system (CNS), SAD is localized on synaptic vesicles and at the active zone in nerve terminals where SAD appears to phosphorylate the active zone protein RIM1 implicated in neurotransmitter release. However, expression and localization of SAD in peripheral nervous system (PNS) is currently unknown. Here we have attempted to examine its expression and localization at synapses of neuromuscular junctions (NMJs). In the mouse diaphragm, quadriceps femoris muscle, and lumbrical muscle, the immunoreactivity for SAD is colocalized with that for -bungarotoxin, a major marker for NMJs, which binds to acetylcholine receptors. These results suggest that SAD is localized at NMJs and a presynaptic component at the PNS as well as the CNS.