Abstract
Voltage imaging in living cells offers the tantalizing possibility of combining the temporal resolution of electrode-based methods with the spatial resolution of imaging techniques. Our lab has been developing voltage-sensitive fluorophores, or VoltageFluors, that respond to changes in cellular and neuronal membrane potential via a photoinduced electron transfer (PeT)-based mechanism. This unique mechanism enables both the fast response kinetics and high sensitivity required to record action potentials in single trials, across multiple cells without the need for stimuli-triggered averaging. In this chapter, we present a methodology for imaging membrane potential dynamics from dozens of neurons simultaneously in vitro. Using simple, commercially available cameras, illumination sources, and microscope optics in combination with the far-red synthetic voltage-sensitive fluorophore BeRST-1 (Berkeley Red Sensor of Transmembrane potential) provides a readily applied method for monitoring neuronal activity in cultured neurons. We discuss different types of voltage-sensitive dyes, considerations for selecting imaging modalities, and outline procedures for the culture of rat hippocampal neurons and performing voltage imaging experiments with these samples. Finally, we provide an example of how changes to the metabolic input to cultured hippocampal neurons can alter their activity profile.