Targeted AAV Transduction for Fluorescent Imaging of the Tripartite Synapse

Central synapses are typically ensheathed by nanoscopic protrusions of the astrocytic cell body known as perisynaptic astrocytic processes. While advances in in vivo laser-scanning microscopy and bioengineering have provided ample tools to measure intracellular activity of both neurons and astrocytes through fluorescent biosensors, there remain significant challenges when assessing specific responses at single synapses that incorporate activity from both astrocytic and neuronal compartments. In this chapter, we describe a simple strategy to label the astrocytic population present at axonal terminations using viral vectors, in combination with sparse labeling of the axonal projections themselves, such that individual axonal boutons en passant within the astrocytic arbor can be optically assessed using two-photonTwo-photon light microscopy. We discuss some ascending and descending pathways that are amenable to this strategy, describe the various protocols needed to set up such experiments, and consider the arousal state of the animal in order to account for constitutive or cryptic activity. With more recent advances in optogenetic actuators and genetically encoded indicators, including those for a wide variety of neurotransmitters, this strategy can be applied to more precisely investigate how astrocytesAstrocytes impact synaptic efficacy and plasticity at individual synapsesSynapses in the central nervous system.