Optical glutamate sensor for spatiotemporal analysis of synaptic transmission

Abstract

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 alpha-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.

Imaging extrasynaptic glutamate dynamics in the brain

“…K716A-EOS, in which lysine residue at position 716 was replaced by alanine, has a high affinity [dissociation constant (K d ) = 174 nM; Fig. 1B] similar to that of the original EOS but has a greater amplitude of the maximal change in fluorescence intensity (29.1 ± 1.7%, n = 9; measured after labeling on the cell surface, mean ± SEM; SI Text) (21). This improvement allowed for glutamate imaging with a higher signal-to-noise ratio.…”

“…EOS sister indicators were produced and evaluated as described in ref. 21. Cerebellar, neocortical and hippocampal slices were prepared from postnatal day 20-24 C57/B6 mice (44).…”

“…EOS is a hybrid-type fluorescent indicator consisting of the glutamate-binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluR2 and a fluorescent small molecule conjugated near the glutamate-binding pocket. EOS changes its fluorescence intensity upon binding of glutamate, for which it has both high affinity and high selectivity (21). We successfully used EOS in cultured hippocampal neurons to observe spontaneous and evoked synaptic activities (21).…”

“…EOS changes its fluorescence intensity upon binding of glutamate, for which it has both high affinity and high selectivity (21). We successfully used EOS in cultured hippocampal neurons to observe spontaneous and evoked synaptic activities (21). Because EOS has a high affinity to glutamate, we considered whether the indicator could be used to detect extrasynaptic glutamate activities in acute slice preparations.…”

“…We developed glutamate indicators derived from the E (glutamate) optical sensor (EOS) (21). EOS is a hybrid-type fluorescent indicator consisting of the glutamate-binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluR2 and a fluorescent small molecule conjugated near the glutamate-binding pocket.…”