When researchers want to probe the electrical behavior of neurons and neural circuits, they typically attach electrodes to the cell and record what happens. This method, called patch clamping, is exquisitely sensitive but also technically challenging, laborious, and difficult to apply to large numbers of cells.
An alternate approach uses so-called genetically encoded voltage indicators (GEVIs), fluorescent proteins whose photon output changes with the cellular membrane potential. A number of such proteins have been described over the years, but few, if any, are robust enough to work outside of neuronal cell cultures.
In 2012, Vincent Pieribone and Lawrence Cohen at the Yale University School of Medicine described a new GEVI design called ArcLight, built by fusing a voltage-sensitive phosphatase from a sea squirt with a fluorescent protein, and tested it in culture (1). Now Pieribone, in collaboration with Michael Nitabach, Associate Professor of Cellular and Molecular Physiology and of Genetics at Yale, demonstrate that ArcLight has the high signal-to-noise ratio and responsiveness necessary for capturing electrical activity in the Drosophila brain (2).
“We showed for the first time that such a [GEVI] probe could actually be used for effective optical electrophysiology in an intact nervous system,” Nitabach says.
Nitabach and his team used ArcLight to probe neural circuitry both ex vivo and in intact Drosophila brains. They expressed the protein in lateral ventral circadian clock neurons, which control the sleep-wake cycle, and recorded ArcLight activity—depolarization induces a loss in fluorescence, while hyperpolarization causes the cells to glow brighter—by imaging the cells 500 times per second under a microscope. The researchers then compared their results with simultaneously recorded patch clamp data.
Each activity spike recorded by patch clamp was also captured by ArcLight fluorescence, albeit with a slight delay and slower kinetics. But unlike patch clamping, the team was able to capture the activity of multiple cells, and even different cell parts, simultaneously, demonstrating that neurites and cell bodies were largely in sync, as were multiple independent cells in the same circuit.
The team also recorded neural activity in the olfactory center, showing among other things that they could distinguish the response of individual neurons to different odorants.
According to Nitabach, GEVIs offer several advantages over traditional patch-clamping, including the ability to observe entire circuits simultaneously and record deeper in the brain than is possible using electrodes. In addition, unlike traditional organic dye indicators, which feature fast kinetics and linearity, GEVIs can be genetically targeted to specific cells.
Brian Salzberg, Professor of Neuroscience and Physiology at the University of Pennsylvania’s Perelman School of Medicine, says GEVIs also have advantages over another “optogenetic” tool, the genetically encoded calcium indicator.
“Calcium indicators … are expressed throughout the volume of the cell and they respond to calcium changes,” Salzberg explains. “GEVIs are expressed only in the membrane, which is where the voltage changes. And I would argue, as Nitabach et al. do, that if you’re measuring the electrical activity of a circuit, you want to measure electrical activity and not a secondary indicator [such as calcium].”
Furthermore, he notes that GEVIs can capture small membrane potential changes that calcium indicators sometimes miss.
Salzberg, who was not involved in the current study but did participate in the development of ArcLight, says the Yale team’s work “represents incremental, but real, progress in the continuing improvement in optical measurement of membrane potential.”
He notes, for instance, that this study provides the first real application of a GEVI in vivo, but that the molecule is still far slower than patch-clamping, and at least 10-times too slow to accurately match the kinetics of mammalian action potentials.
Salzberg is already using ArcLight in his own lab, in collaboration with his colleague at Penn, Chris Fang-Yen, to study electrical control of locomotion in C. elegans.
Nitabach, who says he plans to use ArcLight to study circadian rhythms in the fly, is also working to improve the GEVI itself. Specifically, he wants to improve its signal-to-noise and response kinetics and develop a second color for multiplexed experiments.
Says Salzberg, “There’s plenty of room for improvement in GEVIs.”
1. L. Jin et al., “Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe,” Neuron, 75:779–85, 2012.
2. G. Cao et al., “Genetically targeted optical electrophysiology in intact neural circuits,” Cell, Aug. 6, 2013. [http:// dx.doi.org/10.1016/j.cell.2013.07.027]