Glutamate is the major excitatory neurotransmitter in the mammalian brain. In addition to point-to-point transmission across the synaptic cleft, glutamate has been suggested to mediate volume transmission, escaping from the synaptic cleft to generate extrasynaptic glutamate dynamics (glutamate spillover). Extrasynaptic glutamate dynamics regulates neural and glial functions via the activation of extrasynaptic glutamate receptors. Indeed, defects in extrasynaptic glutamate dynamics have been implicated in neurodegenerative diseases and psychiatric disorders. Despite the immense potential importance of extrasynaptic glutamate dynamics, their characteristics remain elusive because of a lack of appropriate technology.
To overcome this problem, we applied novel glutamate indicators named E (glutamate) Optical Sensor (EOS). 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. EOS labeled the extrasynaptic space through the biotin-streptavidin interaction and was imaged with two-photon microscopy.
Using EOS, we succeeded in imaging extrasynaptic glutamate dynamics in brain slices and in vivo with high-spatiotemporal resolution, and clarified the contribution of neurons and glial cells. Physiologically significant levels of extrasynaptic dynamics were generated upon repetitive inputs in the vicinity of active synapses. Estimated glutamate transients reached micromolar concentrations for tens of milliseconds in response to two to five pulses of stimulation, which are sufficient to activate high-affinity glutamate receptors. In vivo glutamate imaging in the brain in response to sensory input from the extremities revealed glutamate dynamics that were mapped within the appropriate sensory area of the cerebral cortex. The present results shed new light on the extrasynaptic glutamate dynamics during physiological synaptic activities. Furthermore, EOS imaging method may be applicable to the quantification of pathophysiological glutamate dynamics in the brain.