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Tag Archives: GEVI
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
In a recent paper published in the Journal of Neurophysiology, the Knöpfel lab (Riken, Japan) introduced a new design variant of FRET-based voltage sensitive fluorescent proteins, termed VSFP-Butterflies, along with an extensive series of application examples in brain slices and living mice. VSFP-Butterfly 1.2 uses red shifted FP, allowing excitation at 488 to 510 nm with acceptor emission > 600 nm. While the sensitivity of VSFP-Butterfly 1.2 is similar to that of VSFP2.3 (around 22% ΔF/F at half maximal activation, V1/2), its fluorescence to voltage relationship is left shifted, resulting in a more sensitive detection of subthreshold potentials as well as of action potentials (fig. 1). VSFP-Butterfly imaging of voltage signals over the cortex of living mice revealed traveling waves generated by the activity of layer 2/3 pyramidal cells (fig 2). The application examples in this report demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterflies. The authors discuss how VSFP-based voltage imaging will opening new avenues towards a better understanding of the neuronal computations reflected in the dynamics of cortical circuits.