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[0] Froemke RC, Merzenich MM, Schreiner CE, A synaptic memory trace for cortical receptive field plasticity.Nature 450:7168, 425-9 (2007 Nov 15)

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ref: -0 tags: GEVI review voltage sensor date: 03-18-2020 17:43 gmt revision:22 [21] [20] [19] [18] [17] [16] [head]

Various GEVIs invented and evolved:

Ace-FRET sensors

  • PMID-26586188 Ace-mNeonGreen, an opsin-FRET sensor, might still be better in terms of SNR, but it's green.
    • Negative ΔF/F\Delta F / F with depolarization.
    • Fast enough to resolve spikes.
    • Rational design; little or no screening.
    • Ace is about six times as fast as Mac, and mNeonGreen has a ~50% higher extinction coefficient than mCitrine and nearly threefold better photostability (12)

  • PMID-31685893 A High-speed, red fluorescent voltage sensor to detect neural activity
    • Fusion of Ace2N + short linker + mScarlet, a bright (if not the brightest; highest QY) monomeric red fluorescent protein.
    • Almost as good SNR as Ace2N-mNeonGreen.
    • Also a FRET sensor; negative delta F with depolarization.
    • Ace2N-mNeon is not sensitive under two-photon illumination; presumably this is true of all eFRET sensors?
    • Ace2N drives almost no photocurrent.
    • Sought to maximize SNR: dF/F_0 X sqrt(F_0); screened 'only' 18 linkers to see what worked the best. Yet - it's better than VARNAM.
    • ~ 14% dF/F per 100mV depolarization.

Arch and Mac rhodopsin sensors

  • PMID-22120467 Optical recording of action potentials in mammalian neurons using a microbial rhodopsin Arch 2011
    • Endogenous fluorescence of the retinal (+ environment) of microbial rhodopsin protein Archaerhodopsin 3 (Arch) from Halorubrum sodomense.
    • Proton pump without proton pumping capabilities also showed voltage dependence, but slower kinetics.
      • This required one mutation, D95N.
    • Requires fairly intense illumination, as the QY of the fluorophore is low (9 x 10-4). Still, photobleaching rate was relatively low.
    • Arch is mainly used for neuronal inhibition.

  • PMID-25222271 Archaerhodopsin Variants with Enhanced Voltage Sensitive Fluorescence in Mammalian and Caenorhabditis elegans Neurons Archer1 2014
    • Capable of voltage sensing under red light, and inhibition (via proton pumping) under green light.
    • Note The high laser power used to excite Arch (above) fluorescence causes significant autofluorescence in intact tissue and limits its accessibility for widespread use.
    • Archers have 3-5x the fluorescence of WT Arch -- so, QY of ~3.6e-3. Still very dim.
    • Archer1 dF/F_0 85%; Archer2 dF/F_0 60% @ 100mV depolarization (positive sense).
    • Screened the proton pump of Gloeobacter violaceus rhodopsin; found mutations were then transferred to Arch.
      • Maybe they were planning on using the Geobacter rhodopsin, but it didn't work for some reason, so they transferred to Arch..
    • TS and ER export domains for localization.

  • PMID-24755708 Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors MacQ-mOrange and MacQ-mCitrine.
    • L. maculans (Mac) rhodopsin (faster than Arch) + FP mCitrine, FRET sensor + ER/TS.
    • Four-fold faster kinetics and 2-4x brighter than ArcLight.
      • No directed evolution to optimize sensitivity or brightness. Just kept the linker short & trimmed residues based on crystal structure.
    • ~5% delta F/F, can resolve spikes up to 10Hz.
    • Spectroscopic studies of the proton pumping photocycle in bacteriorhodopsin and Archaerhodopsin (Arch) have revealed that proton translocation through the retinal Schiff base changes chromophore absorption [24-26]
    • Used rational design to abolish the proton current (D139N and D139Q aka MacQ) ; screens to adjust the voltage sensing kinetics.
    • Still has photocurrents.
    • Seems that slice / in vivo is consistently worse than cultured neurons... in purkinje neurons, dF/F 1.2%, even though in vitro response was ~ 15% to a 100mV depolarization.
    • Imaging intensity 30mw/mm^2. (3W/cm^2)

  • PMID-24952910 All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins QuasAr1 and QuasAr1 2014
    • Directed evolution approach to improve the brightness and speed of Arch D95N.
      • Improved the fluorescence QY by 19 and 10x. (1 and 2, respectively -- Quasar2 has higher sensitivity).
    • also developed a low-intensity channelrhodopsin, Cheriff, which can be activated by blue light (lambda max = 460 nm)dim enough to not affect QuasAr.
    • They call the two of them 'Optopatch 2'.
    • Incident light intensity 1kW / cm^2 (!)

  • PMID-29483642 A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Archon1 2018
    • Started with QuasAr2 (above), which was evolved from Arch. Intrinsic fluorescence of retinal in rhodopsin.
    • Expressed in HEK293T cells; then FACS, robotic cell picking, whole genome amplification, PCR, cloning.
    • Also evolved miRFP, deep red fluorescent protein based on bacteriophytochrome.
    • delta F/F of 80 and 20% with a 100mV depolarization.
    • We investigated the contribution of specific point mutations to changes in localization, brightness, voltage sensitivity and kinetics and found the patterns that emerged to be complex (Supplementary Table 6), with a given mutation often improving one parameter but worsening another.
    • If the original QY of Arch was 9e-4, and Quasar2 improved this by 10, and Archon1 improved this by 2.3x, then the QY of Archon1 is 0.02. Given the molar extinction coefficient is ~ 50000 for retinal, this means the brightness of the fluorescent probe is low, 1. (good fluorescent proteins and synthetic dyes have a brightness of ~90).
    • Big paper, moderate improvement.
    • SomArchon1 and SomCheriff serve as the basis of Optopatch4, e.g. All-optical electrophysiology reveals excitation, inhibition, and neuromodulation in cortical layer 1
    • Slow photobleaching, consistent with other Arch based GEVIs.

VSD - FP sensors

  • PMID-28811673 Improving a genetically encoded voltage indicator by modifying the cytoplasmic charge composition Bongwoori 2017
    • ArcLight derivative.
    • Arginine (positive charge) scanning mutagenesis of the linker region improved the signal size of the GEVI, Bongwoori, yielding fluorescent signals as high as 20% ΔF/F during the firing of action potentials.
    • Used the mutagenesis to shift the threshold for fluorescence change more negative, ~ -30mV.
    • Like ArcLight, it's slow.
    • Strong baseline shift due to the acidification of the neuron during AP firing (!)

  • Attenuation of synaptic potentials in dentritic spines
    • Found that SNR / dF / F_0 is limited by intracellular localization of the sensor.
      • This is true even though ArcLight is supposed to be in a dark state in the lower pH of intracellular organelles.. a problem worth considering.
      • Makes negative-going GEVI's more practical, as those not in the membrane are dark.

  • Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain ASAP3 2018
    • Opsin-based GEVIs have been used in vivo with 1p excitation to report electrical activity of superficial neurons, but their responsivity is attenuated for 2p excitation. (!)
    • Site-directed evolution in HEK cells.
    • Expressed linear PCR products directly in the HEK cells, with no assembly / ligation required! (Saves lots of time: normally need to amplify, assemble into a plasmid, transfect, culture, measure, purify the plasimd, digest, EP PCR, etc).
    • Screened in a motorized 384-well conductive plate, electroporation electrode sequentially activates each on an upright microscope.
    • 46% improvement over ASAP2 R414Q
    • Ace2N-4aa-mNeon is not responsive under 2p illum; nor is Archon1 or Quasar2/3
    • ULOVE = AOD based fast local scanning 2-p random access scope.

  • Bright and tunable far-red chemigenetic indicators
    • GgVSD (same as ASAP above) + cp HaloTag + Si-Rhodamine JF635
    • ~ 4% dF/F_0 during APs.
    • Found one mutation, R476G in the linker between cp Halotag and S4 of the VSD, which doubled the sensitivity of HASAP.
    • Also tested a ArcLight type structure, CiVSD fused to Halotag.
      • HarcLght had negative dF/F_0 and ~ 3% change in response to APs.
    • No voltage sensitivity when the synthetic dye was largely in the zwitterionic form, eg. tetramethylrodamine.

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ref: -0 tags: VARNUM GEVI genetically encoded voltage indicators FRET Ace date: 03-18-2020 17:12 gmt revision:5 [4] [3] [2] [1] [0] [head]

PMID-30420685 Fast in-vivo voltage imaging using a red fluorescent indicator

  • Kannan M, Vasan G, Huang C, Haziza S, Li JZ, Inan H, Schnitzer MJ, Pieribone VA.
  • Other genetically encoded voltage indicators (GEVI):
    • PMID-22958819 ArcLight (Peribone also last author) ; sign of ΔF/F\Delta F / F negative, but large, 35%! Slow tho? improvement in speed
    • ASAP3 ΔF/F\Delta F / F large, τ=3ms.\tau = 3 ms.
    • PMID-26586188 Ace-mNeon FRET based, Acetabularia opsin, fast kinetics + brightness of mNeonGreen.
    • Archon1 -- fast and sensitive, found (like VARNUM) using a robotic directed evolution or direct search strategy.
  • VARNAM is based on Acetabularia (Ace) + mRuby3, also FRET based, found via high-throughput voltage screen.
  • Archaerhodopsin require 1-12 W/mm^2 of illumination, vs. 50 mw/mm^2 for GFP based probes. Lots of light!
  • Systematic optimization of voltage sensor function: both the linker region (288 mutants), which affects FRET efficiency, as well as the opsin fluorophore region (768 mutants), which affects the wavelength of absorption / emission.
  • Some intracellular clumping (which will negatively affect sensitivity), but mostly localized to the membrane.
  • Sensitivity is still imperfect -- 4% in-vivo cortical neurons, though it’s fast enough to resolve 100 Hz spiking.
  • Can resolve post-synaptic EPSCs, but < 1 % ΔF/F\Delta F/F .
  • Tested all-optical ephys using VARNAM + blueshifted channelrhodopsin, CheRiff, both sparsely, and in PV targeted transgenetic model. Both work, but this is a technique paper; no real results.
  • Tested TEMPO fiber-optic recording in freely behaving mice (ish) -- induced ketamine waves, 0.5-4Hz.
  • And odor-induced activity in flies, using split-Gal4 expression tools. So many experiments.

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ref: -0 tags: surface plasmon resonance voltage sensing antennas PEDOT imaging spectroscopy date: 12-05-2019 16:47 gmt revision:1 [0] [head]

Electro-plasmonic nanoantenna: A nonfluorescent optical probe for ultrasensitive label-free detection of electrophysiological signals

  • Use spectroscopy to measure extracellular voltage, via plasmon concentrated electrochromic effects in doped PEDOT.

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ref: -0 tags: voltage sensitive dyes fluorescent protein date: 01-02-2013 05:08 gmt revision:0 [head]

PMID-20622860 Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins.

  • Interesting: Most fluorescent fusion proteins form intracellular aggregates during long-term expression in mammalian neurons, although this effect appears to be minimal in Aequorea victoria–derived fluorescent proteins.
  • See also {1185}

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ref: -0 tags: optical recording voltage sensitive dyes redshirt date: 01-02-2013 03:17 gmt revision:3 [2] [1] [0] [head]

PMID-16050036 Imaging brain activity with voltage- and calcium-sensitive dyes.

  • Voltage-sensitive dyes are well suited for measuring synaptic integration, as:
    • Electrodes are too blunt to effectively record these fine, < 1um diameter structures.
    • The surface area to volume ratio is highest in the dendrites
    • Voltage-sensitive dyes also permeate internal membranes not subject to voltage gradients, hence this does not contribute to the signal, leading to a decreased ΔF/F\Delta F / F .
  • Dominant experimental noise is shot noise, statistical -- see {1181}.
  • modern dyes and imagers can reliably record single action potentials; spatial averaging yields similar resolution as electrical recording.
  • They performed optical recording of Aplysia sensory ganglia, and discovered following light tail touch: "It is almost as if the Aplysia nervous system is designed such that every cell in the abdominal ganglion cares about this (and perhaps every) sensory stimulus. In addition, more than 1000 neurons in other ganglia are activated by this touch..."
      • These results force a more pessimistic view of the present understanding of the neuronal basis of apparently simple behaviors in relatively simple nervous systems.
  • Used calcium imaging on olfactory glomeruli of mice and turtles; measurements were limited by either shot-noise or heart/breathing artifacts.
  • Confocal and two-photon microscopes, due to their exchange of spatial resolution for sensitivity, are not useful with voltage-sensitive dyes.
    • The generation of fluorescent photons in the 2-photon confocal microscope is not efficient. We compared the signals from Calcium Green-1 in the mouse olfactory bulb using 2-photon and ordinary microscopy. In this comparison the number of photons contributing to the intensity measurement in the 2-photon confocal microscope was about 1000 times smaller than the number measured with the conventional microscope and a CCD camera.
  • By the numbers, quote: Because only a small fraction of the 10e16 photons/ms emitted by a tungsten filament source will be measured, a signal-to-noise ratio of 10e8 (see above) cannot be achieved. A partial listing of the light losses follows. A 0.9-NA lamp collector lens would collect 0.1 of the light emitted by the source. Only 0.2 of that light is in the visible wavelength range; the remainder is infrared (heat). Limiting the incident wavelengths to those, which have the signal means, that only 0.1 of the visible light is used. Thus, the light reaching the
preparation might typically be reduced to 1013 photons/ms. If the light-collecting system that forms the image has high efficiency e.g., in an absorption measurement, about 1013 photons/ms will reach the image plane. (In a fluorescence measurement there will be much less light measured because 1. only a fraction of the incident photons are absorbed by the fluorophores, 2. only a fraction of the absorbed photons appear as emitted photons, and 3. only a fraction of the emitted photons are collected by the objective.) If the camera has a quantum efficiency of 1.0, then, in absorption, a total of 10e13 photoelectrons/ms will be measured. With a camera of 1000 pixels, there will be 10e10 photoelectrons/ms/pixel. The shot noise will be 10e5 photoelectrons/ms/pixel; thus the very best that can be expected is a noise that is 10e−5 of the resting light (a signal-to-noise ratio of 100 db). The extra light losses in a fluorescence measurement will further reduce the maximum obtainable signal-to-noise ratio.

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ref: -0 tags: optical coherence tomography neural recording squid voltage sensitive dyes review date: 12-23-2012 21:00 gmt revision:4 [3] [2] [1] [0] [head]

PMID-20844600 Detection of Neural Action Potentials Using Optical Coherence Tomography: Intensity and Phase Measurements with and without Dyes.

  • Optical methods of recording have been investigated since the 1940's:
    • During action potential (AP) propagation in neural tissue light scattering, absorption, birefringence, fluorescence, and volume changes have been reported (Cohen, 1973).
  • OCT is reflection-based, not transmission: illuminate and measure from the same side.
    • Here they use spectral domain OCT, where the mirror is not scanned; rather SD-OCT uses a spectrometer to record interference of back-scattered light from all depth points simultaneously (Fercher et al., 1995).
    • Use of a spectrometer allows imaging of an axial line within 10-50us, sufficient for imaging action potentials.
    • SD-OCT, due to some underlying mathematics which I can't quite grok atm, can resolve/annul common-mode phase noise for high temporal and Δphase\Delta phase measurement (high sensitivity).
      • This equates to "microsecond temporal resolution and sub-nanometer optical path length resolution".
  • OCT is generally (intially?) used for in-vivo imaging of retinas, in humans and other animals.
  • They present new data for depth-localization of neural activity in squid giant axons (SGA) stained with a voltage-sensitive near-infrared dye.
    • Note: averaged over 250 sweeps.
  • ΔPhase>>ΔIntensity\Delta Phase &gt;&gt; \Delta Intensity -- figure 4 in the paper.
  • Use of voltage-sensitive dyes improves the resolution of ΔI\Delta I , but not dramatically --
    • And Δphase\Delta phase is still a bit delayed.
    • Electrical recording is the control.
      • It will take significant technology development before optical methods exceed electrical methods...
  • Looks pretty preliminary. However, OCT can image 1-2mm deep in transparent tissue, which is exceptional.
  • Will have to read their explanation of OCT.
  • Used in a squid giant axon prep. 2010, wonder if anything new has been done (in vivo?).
  • Claim that progress is hampered by limited understanding of how these Δphase\Delta phase signals arise.

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ref: Sakai-2001.06 tags: voltage scensitive fluorescent protein flourophore VSFP1 endoscope date: 01-24-2012 06:07 gmt revision:5 [4] [3] [2] [1] [0] [head]

http://www.blackwell-synergy.com/doi/full/10.1046/j.0953-816x.2001.01617.x PMID-11454036[0]

____References____

[0] Sakai R, Repunte-Canonigo V, Raj CD, Knöpfel T, Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein.Eur J Neurosci 13:12, 2314-8 (2001 Jun)
[1] van Roessel P, Brand AH, Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins.Nat Cell Biol 4:1, E15-20 (2002 Jan)
[2] Guerrero G, Siegel MS, Roska B, Loots E, Isacoff EY, Tuning FlaSh: redesign of the dynamics, voltage range, and color of the genetically encoded optical sensor of membrane potential.Biophys J 83:6, 3607-18 (2002 Dec)
[3] Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ, In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy.J Neurophysiol 92:5, 3121-33 (2004 Nov)
[4] Sjulson L, Miesenböck G, Optical recording of action potentials and other discrete physiological events: a perspective from signal detection theory.Physiology (Bethesda) 22no Issue 47-55 (2007 Feb)

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ref: Froemke-2007.11 tags: nucleus basalis basal forebrain acetylcholine auditory cortex potentiation voltage clamp date: 10-08-2008 22:44 gmt revision:2 [1] [0] [head]

PMID-18004384[0] A synaptic memory trace for cortical receptive field plasticity.

  • nucleus basalis = basal forebrain!
  • stimulation of the nucleus basalis caused a reorganization of the auditory cortex tuning curves hours after the few minutes of training.
  • used whole-cell current-clamp recording to reveal tone-evoked excitatory and inhibitory postsynaptyic currents.
  • pairing of nucleus basalis and auditory tone presentation (2-5 minutes) increased excitatory currents and decreased inhibitory currents as compared to other (control) frequencies.
  • tuning changes required simultaneous tone presentation and nucleus basalis stimulation. (Could they indiscriminately stimulate the NB? did they have to target a certain region of it? Seems like it.)
    • did not require postsynaptic spiking!
  • Pairing caused a dramatic (>7-fold) increase in the probability of firing bursts of 2+ spikes
  • Cortical application of atropine, an acetylcholine receptor antagonist, prevented the effects of nucleus basalis pairing.
  • the net effects of nucleus basalis pairing are suppression of inhibition (20 sec) followed by enhancement of excitation (60 sec)
  • also tested microstimulation of the thalamus and cortex; NB pairing increased EPSC response from intracortical microstim, but not from thalamic stimulation. Both cortical and thalamic stimulation elicited an effect in the voltage-clamped recorded neuron.
  • by recording from the same site (but different cells), they showed that while exitation persisted hours after pairing, inhibition gradually increased commensurate with the excitation.
  • Thus, NB stimulation leaves a tag of reduced inhibition (at the circuit level!), specifically for neurons that are active at the time of pairing.

____References____