Measurement of astrocytic glutamate release using genetically encoded probe combined with TIRF microscopy

Abstract

Astrocytes modulate brain function such as memory and cognition by processing neuronal activity through the release of transmitters such as glutamate, ATP and d-serine. Various release mechanisms have been proposed depending on pathophysiological conditions. Among them, inverted transport, ion channels mediated release, and vesicular release. To better understand how cellular interactions alter glutamate release, we used live imaging techniques to monitor the dynamics of glutamate release in various cell culture conditions. For that purpose we combined total internal reflection fluorescence (TIRF) microscopy with a genetically encoded fluorescent glutamate sensor (iGluSnFR) which is an intensity-based glutamate-sensing fluorescent reporter that has a high signal-to-noise ratio. This system allows a direct measurement of glutamate in real time. Our goal is to provide guidelines on how to use this approach and to highlight its pros and cons.