Multiplexed Imaging of Glutamate Release and Local Intracellular Ca2+ Dynamics with the Halotag System

Information handling and storage in the brain rely in large part on the Ca2+-dependent release of the neurotransmitter glutamate at small excitatory synapses. The relationship between the dynamics of glutamate release and local pre- and postsynaptic Ca2+ activity provides important clues to the basic mechanisms of excitatory transmission and its use-dependent plasticity. Recent advances in genetically encoded optical sensors and multiplexed imaging have enabled the monitoring of these relationships at identified synaptic connections, with high spatial and temporal resolution. However, targeting the two sensors to the same cell process could be a challenging task, particularly in conditions of the intact brain. The Halotag system could help to deal with this challenge, by having one halotagged sensor expressed in the cell processes of interest, and the other targeted to the same process through the simple procedure of bolus-load application. Here, we describe the method in which the Halotag-fused glutamate sensor SF-iGluSnFR.A184S sparsely expressed in the processes of either neurons or astrocytes in brain tissue helps to target the bolus-loaded Ca2+ sensor JF-646-BAPTA HTL to the same cell processes. The resulting co-localization of the sensors in the target cell processes enables multiplexed imaging of the spatially and temporally concurrent, highly localized glutamate release and Ca2+ dynamics.