![\"Confocal](\"\/\/media.nature.com\/w800\/magazine-assets\/d41586-019-03064-8\/d41586-019-03064-8_17234850.jpg\")
<\/div>\n<\/div>Fluorescent proteins that react to voltage changes show signalling between cells in the brain of a zebrafish (Danio rerio<\/i>).<\/span>Credit: A.S Abdelfattah\u00a0et al.\/Science<\/i><\/p>\n<\/figcaption><\/figure>\n <\/p>\n <\/p>\n Douglas Storace still has the dollar bill that he triumphantly taped above his laboratory bench seven years ago, a trophy from a successful wager. His postdoctoral mentor, Larry Cohen at Yale University in New Haven, Connecticut, bet that Storace couldn\u2019t express a protein sensor of voltage changes in mice back in September 2012. Storace won.<\/p>\n The bill is a handy reminder that the experiments he aims to try in his new lab can work. And it\u2019s a testament to just how tricky it is to correctly express these sensors and track their signals. Storace, now an assistant professor at Florida State University in Tallahassee, plans to use these sensors, known as genetically encoded voltage indicators (GEVIs), to study how neurons in the olfactory bulb sense and react to smells.<\/p>\n GEVIs are voltage-sensitive, fluorescent proteins that change colour when a neuron fires or receives a signal. Because GEVIs can be targeted to individual cells and directly indicate a cell\u2019s electrical signals, researchers consider them to be the ideal probes for studying neurons. But they have proved frustratingly difficult to use. \u201cBeing able to visualize voltage changes in a cell has always been the dream,\u201d says neuroscientist Bradley Baker at the Korea Institute of Science and Technology in Seoul. \u201cBut probes that looked great often didn\u2019t behave in ways that were useful.\u201d<\/p>\n Early GEVIs disappointed on several levels. They were bright when a cell was resting and dimmed when the cell fired an action potential, producing signals that were tough to distinguish from the background. And they failed to concentrate in the nerve-cell membranes, where they function. But researchers are beginning to solve these issues. Some are turning to advanced fluorescent proteins or chemical dyes for better signals; others are using directed evolution and high-throughput screens to make GEVIs more sensitive to voltage changes. Meanwhile, biologists are putting these molecules through their paces. GEVIs, says neuroscientist Katalin Toth at Laval University in Quebec City, Canada, are not yet widely used, but they\u2019re getting there. \u201cThey are becoming brighter and faster \u2014 and growing in popularity,\u201d she says. \u201cI think this is the dawn of GEVIs.\u201d<\/p>\n <\/p>\n Glimmer of promise<\/strong><\/p>\n When a mouse smells a banana and races towards the treat, it is the inevitable result of a well-organized orchestra of neural circuits. Researchers can tap into these pathways using patch-clamping (in which electrodes and pipettes are placed on cells to track electrical activity in a given brain region) and voltage-sensitive dyes (which can reveal overarching electrical changes).<\/p>\n Genetic probes are another option. In a similar way to dyes, these molecules fluoresce in response to electrical signals. But researchers can use genetic tricks to limit the probes\u2019 expression to specific cells. Genetically encoded calcium indicators (GECIs), such as GCaMP proteins, are made by fusing a fluorescent protein to one that can bind to calcium. Calcium floods a nerve cell after it has fired an electrical signal, causing a change in the binding protein\u2019s shape that triggers a change in fluorescence.<\/p>\n But GECIs are only proxies for neural electrical activity. Although they are sensitive to action potentials, which are the basic units of neural communication, they cannot capture the smaller, sub-excitatory cues that help nerve cells to compute and integrate different kinds of information.<\/p>\n In 1997, Ehud Isacoff at the University of California, Berkeley, developed the first GEVI, named FlaSh, by fusing green fluorescent protein with a voltage-sensitive potassium channel1<\/a><\/sup>. Imperial College London neuroscientist Thomas Kn\u00f6pfel, then at RIKEN in Wako, Japan, followed suit in 2010 by fusing a voltage-sensing phosphatase enzyme derived from\u00a0Ciona intestinalis<\/i>, a marine invertebrate, to a fluorescent protein2<\/a><\/sup>. Other designs followed, including the 2012 discovery that a random mutation in one protein made it 14-fold more sensitive to voltage changes, leading to one of the biggest early GEVI successes3<\/a><\/sup>, ArcLight.<\/p>\n Today, there are three major classes of GEVI (see \u2018Flavours of fluorescence\u2019). Probes such as ArcLight fuse voltage-sensitive protein domains (VSDs) to fluorescent proteins, whereas others such as Archer and QuasAr2 rely on fluorescent membrane-spanning bacterial proteins known as rhodopsins. Ace\u2013mNeon represents a third group known as opsin\u2013FRET (fluorescence resonance energy transfer) probes. These molecules combine light-sensitive opsins that are similar to rhodopsin with a second fluorescent protein to create an energy transfer \u2014 detectable as a change in fluorescent colour \u2014 when the proteins are excited. \u201cUnlike GCaMP, where everyone was focused on one scaffold, each GEVI has its own developmental path,\u201d says neuroscientist Michael Lin at Stanford University in California.<\/p>\n <\/p>\n Ideally, a GEVI will yield a bright, stable signal that consistently follows a change in voltage or action potential, and produce minimal background fluorescence. But this doesn\u2019t always happen. Unlike GCaMPs, which can fill a cell\u2019s volume, GEVIs must be localized to the cell membrane to be effective. They cannot be tested in bacteria, because it is difficult to maintain a membrane potential in these cells. And the change in fluorescence when a GEVI fires is much smaller than that seen with GECIs.<\/p>\n <\/p>\n <\/p>\n The millisecond pace of neural electrical activity is also a problem, both for GEVIs and the cameras that image them. And generating a sufficiently bright signal requires intense excitation light, which can overheat cells and cause the GEVI to bleach within minutes.<\/p>\n As a result, most biologists still look to GCaMPs to study fine-scale neuronal activities. \u201cWhat the field would love to have is a solution like GCaMP. Calcium imaging works consistently in anyone\u2019s hands,\u201d says neuroscientist Eric Schreiter at the Howard Hughes Medical Institute\u2019s Janelia Research Campus in Ashburn, Virginia. \u201cThere are very few reports of existing GEVIs being used\u00a0in vivo<\/i>, and they\u2019re quite limited in their scope.\u201d<\/p>\n <\/p>\n Brighter, faster \u2026 better?<\/strong><\/p>\n But that is beginning to change, thanks to directed-evolution approaches, high-throughput screening strategies and more-stable fluorescent molecules.<\/p>\n Schreiter and his team, for instance, removed the fluorescent portions of rhodopsin-based sensors and replaced them with a protein that binds to a synthetic dye molecule in response to voltage changes. Synthetic dyes are significantly brighter and more photo-stable than fluorescent proteins. One such probe, dubbed Voltron, produced a signal that was several-fold brighter than its parent GEVIs and lasted upwards of 15 minutes without bleaching4<\/a><\/sup>.<\/p>\n![\"\"](\"\/\/media.nature.com\/w800\/magazine-assets\/d41586-019-03064-8\/d41586-019-03064-8_17263768.jpg\")
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