Assessing Neuron-Astrocyte Spatial Interactions Using the Neuron-Astrocyte Proximity Assay.

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Forster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Forster resonance energy transfer imaging in cultured cells Basic Protocol 2: Forster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.