— Last updated on December 19, 2023 —
Neurons use the voltage across the membrane to rapidly transmit information through action and synaptic potentials (post). Patch-clamp is the gold-standard method to record this electrical activity from individual neurons using electrodes (post). However, there is no such reference method for recording multiple action potentials at once because microelectrode arrays (post) or calcium indicators are very effective but indirect techniques. Therefore, the new Genetically Encoded Voltage Indicators (GEVIs) are seen as very promising.
GEVIs are light-sensitive proteins genetically introduced into neuron membranes that emit fluorescence in response to changes in voltage with millisecond resolution (top image from Xu et al, 2017). These proteins are generally either microbial opsins (light-sensitive proteins) or voltage-sensitive proteins coupled to another fluorescent protein.
GEVIs have allowed measuring both action potentials and subthreshold events as shown in the video below. These voltage indicators have also been applied in vivo to record defined neuronal populations during the behavior of many animals. For example, hippocampal place cells of mice during navigations tasks, subcellular responses during visual processing in Drosophila, and olfactory sensory neurons in zebrafish.
Voltage imaging with GEVIs is not yet widespread due to technical limitations of GEVIs such as low expression levels, modest voltage sensitivity (often less than 1-fold fluorescence changes), relative and non-linear responses, as well as significant photobleaching. Thus, the biophysical properties of each GEVI need to be considered when choosing the correct probe for each experiment. For these reasons, studies that compare GEVIs under the same experimental conditions serve as helpful resources for the community (Bando et al., 2019, Milosevic et al., 2020, Davis et al., 2023). In short, there is still no reference GEVI such as GCaMP for calcium imaging.
However, improved versions of GEVIs have been steadily released (see table below). For instance, more photostable GEVIs like JEDI-2P and ASAP4 have enabled deep in vivo imaging using two-photon microscopy. Another way to improve the photostability and the dynamic range is by using indicators that increase the fluorescence in response to neuronal depolarizations and action potentials. Whereas most initial GEVIs were negative-going, there are now (2023) positive-going indicators with high sensitivity such as ASAP4 and SpikeyGi. As a result, dual voltage imaging of two neuronal populations can be also achieved by combining indicators with different polarities (e.g., Ace-mNeon and VARNAM). In addition, most GEVIs can be combined with other optical tools such as optogenetics (post) for all-optical physiology experiments.
Despite the technical challenges, the goal of developing a technology capable of imaging action potentials from large populations of identified neurons would be a significant breakthrough in neuroscience and well worth the effort.
| GEVI | Fluorophore | polarity | ΔF/F (1 Ap) | Tau on (fast/slow) | TAU on (fast/Slow) | LINEARITY | photobleaching (5 min) | Reference |
|---|---|---|---|---|---|---|---|---|
| Archon2 | Archaerhodopsin 3 | Positive | 20% | 0.06 / 6.7 ms | 0.17 / 7 ms | Yes | 10% | Piatkevich et al., 2018 |
| ASAP4 | GFP | Positive | 20-40% | 1.5 / 6.1 ms | 1.5 / 7.8 ms | No | 25% | Evans et al., 2023 |
| Quasar6 | Archaerhodopsin 3 | Positive | 10% | 0.8 / 6.6 ms | 0.8 / 6.0 ms | High | 20% | Tian et al., 2023 |
| SpikeyGi | GFP | Positive | 20% | ? | ? | High | 10% | Platisa et al., 2023 |
| Voltron 2 | Janelia Fluor dyes | Negative | -5% | 0.7 / 3.2 ms | 1.1 / 6.2 ms | No | 25% (two-photon) | Abdelfattah et al., 2023 |
More resources
List of available GEVIs (search for Target: V+) by Fluorescent Biosensor Database.
List of current GEVIs by Piatkevich lab. Up-to-date with the characteristics of each GEVI.
Spikes and bursts 