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There has been almost two decades of research in the field of genetically-encoded voltage indicators (GEVIs). Early probes carried channel-based voltage sensors (e.g. derived from potassium and sodium channels) but showed poor membrane localization. Membrane localization of GEVIs was greatly improved by replacing channel-based sensors with the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSD). Despite this major improvement, the goal to record single action potentials and subthreshold electrical events in mammalian neurons with adequate temporal and spatial resolution still presents a challenge. The latest step toward this goal was taken by the Pieribone lab which came up with two Ci-VSD-based monochromatic GEVIs.
The first one, called ArcLight and published in Neuron, was obtained by combining the Ci-VSD and a super ecliptic pHluorin that carries a critical point mutation (A227D). Based on ArcLight, five probes were engineered with super ecliptic pHluorin A227D relocated closer to the S4 domain of the Ci-VSD, after amino acids Q239, M240, K241, A242, or S243. Out of these 5 variants, ArcLight A242 emerged as the best, exhibiting -35% change in ΔF/F in response to 100mV depolarizing steps in HEK293 cells and up to -5% change in ΔF/F for single action potentials in neurons. ArcLight A242 … Continue reading
Genetically encoded optical voltage sensors expand the optogenetic toolkit to enable the imaging of electrical activity from genetically defined populations of neurons. In a recent paper that appeared in the Journal of Neurophysiology, Wang et al reported the imaging of electrical activity in hippocampal slices from transgenic mice expressing hybrid voltage sensors (hVoS). hVoS probes are membrane targeted fluorescent proteins that have been optimized for a FRET interaction with dipicrylamine, a lipophilic molecule that partitions into lipid bilayers. A change in voltage alters the FRET interaction between the fluorescent protein and dipicrylamine to produce an optical signal that can be imaged.
Among the various genetically encoded voltage sensors currently under development in various labs, hVOS probes have a signal amplitude comparable to other probes (20-30% for 100 mV), but a very rapid response time (~0.5 msec). Thus, these probes are rapid enough to detect action potentials. Wang et al generated transgenic mice with two different high-performance hVoS probes under control of a neuron-specific thy-1 promoter. Hippocampal slices from these animals present distinct spatial patterns of expression, and electrical stimulation evoked fluorescence changes as high as 3%.
In some instances, clear responses were recorded in a single trial without averaging. One … Continue reading