Visualizing the activity of neurons in living and behaving animals can shape our understanding of neuronal function in the context of an intact network. Genetically encoded voltage indicators report fluctuations in cellular membrane potential and therefore sense neuronal activity more directly than calcium indicators. In contrast to traditional electrophysiological techniques, these sensors allow for analysis of neuronal activity in many defined neurons at the same time. Despite recent progress in their development, fluorescent voltage indicators that work well in in vivo applications, especially in mammals, are still missing.

Imaging membrane potential in living animals. Credit: Katie Vicari/Nature Publishing Group

Ideally, these sensors would combine fast kinetics with high sensitivity while requiring low laser excitation power to minimize phototoxicity in living tissue. Sensors with these properties would enable the recording of single action potentials in vivo without the need for averaging signals from many trials. Furthermore, a good signal-to-noise ratio would be useful for monitoring subthreshold events such as excitatory or inhibitory postsynaptic potentials, which are less prominent than action potentials.

In recent years, notable improvements to voltage sensors have made them suitable for many ex vivo applications. Reports of using voltage sensors in living animals, on the other hand, are few: these include ArcLight in Drosophila melanogaster (Cell 154, 904–913, 2013) and Archer1 in Caenorhabditis elegans (Nat. Commun. 5, 4894, 2014). In Drosophila, ArcLight could report individual action potentials even in single trials. Although the recently developed sensor MacQ-mCitrine has improved characteristics compared to ArcLight, sensing single action potentials in live mice is still at the edge of its capabilities (Nat. Commun. 5, 3674, 2014).

We expect to see further improvements in the near future that will make voltage sensors suitable for robust in vivo imaging, in challenging scenarios similar to those for which calcium sensors are being used. These developments might include tweaks to existing sensor classes or involve completely new designs with improved kinetics, sensitivity and signal-to-noise ratio. Advances in image-based voltage sensing will bring within reach the answers to many important questions about neuronal activity in living animals.