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Tag Archives: Indicators
The simultaneous optogenetic sensing of the intracellular concentrations of Cl− and H+ is a challenging task, which requires probes with high sensitivity allowing reliable quantitative measurements without perturbation of cell functioning. The recent paper in Frontiers of Molecular Neurosciences by Mukhtarov et al. (2013) describes the intracellular calibration and functional characterization of three genetically encoded probes developed for these purposes. The first is recently proposed combined Cl−/pH sensor (ClopHensor), which was obtained by fusion of a red fluorescent protein (RFP) with a GFP variant, E2GFP, which contains a specific Cl−-binding site (Arosio et al., 2010). The second is PalmPalm-ClopHensor, a variant that is preferentially expressed at the plasma membrane thanks to the addition of two palmitoylation sites at the N-terminus. The third ClopHensor variant contains a two additional mutations (H148G/V224L) in the GFP moiety conferring improved Cl− affinity and reduced pH dependence.
For functional analysis, constructs were expressed in CHO cells and neurons. In CHO cells ClopHensor and the H148G/V224L mutant exhibits cytoplasmic intracellular distribution while the PalmPalm-ClopHensor construct, as expected, is preferentially localized in the vicinity of membranes (FIG.1).
In order to evaluate the dynamic range and sensitivity of constructs to ions, CHO cells were co-transfected with Cl−-selective glycine … Continue reading
One of the primary goal of neuroscience research is to dissect the role of different cell-types in neuronal circuits. Thanks to the development of optogenetic reporters and control tools in recent years, researchers can now optically control and monitor the activity of genetically defined neuronal populations. Going deeper into the understanding of neuronal circuit requires to follow and control the activity of individual cells within these genetically-defined populations. Imaging the activity of individual neurons in vitro and in vivo can be achieved with classical microscopy techniques but labelling and following the activity of arbitrarily defined subsets of cells in a given population remains hard to implement. Optical highlighters which can be photoactivated or photoconverted are routinely used to label cell subpopulations but have only been used so far for anatomical tracing or cell migration studies. In a recent study published in JACS, Campbell and coworkers introduce a photoconvertible genetically-encoded calcium indicator, providing the possibility to easily monitor the activity of subsets of cells of a given cell type.
This dual-function Ca2+ indicator was made by combining two of the most powerful implementations of fluorescent protein (FP) technology: the “highlighters” that can be converted from non-fluorescent to fluorescent or from one … Continue reading
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
In a recent paper published in PLoS One, Ohkura et al. introduce R-CaMP1.07, an improved variant of a recently developed red fluorescent Ca2+ indicator protein called R-GECO1. While the sensitivity of R-CaMP1.07 is similar to that of R-GECO1 (Kd for Ca2+ is around 200 nM), R-CaMP1.07 shows 1.5–2-fold greater signals than R-GECO1 due to its enhanced dynamic range (Fmax/Fmin is near 30). The greatest advantage of R-CaMP1.07 is that its excitation wavelength ranges from 500 to 580 nm, which rarely overlaps with the photo-stimulation range of channelrhodopsin-2 (ChR2). Taking this merit of R-CaMP1.07, the authors demonstrate an application example of this indicator in hippocampal pyramidal neurons expressing ChR2: the successful detection of Ca2+ signals in response to action potentials evoked by the photo-stimulation of ChR2. Needless to say, this red fluorescent R-CaMP1.07 can be used for Ca2+ imaging of cells expressing blue, cyan or green fluorescent proteins.
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.
As already anticipated by many, the next big step of optogenetics will consist in multiplexing optogenetic tools in the same experiment (see the page Combining Optogenetic Tools). Tools used in parallel will need to have well-separated spectral characteristics, a requirement that FRET sensors will have a hard time meeting. The problems of FRET sensors might run deeper than their bandwidth of operation, with issues including differential donor/acceptor photobleaching, worse 2P performance & more scattering of the excitation wavelength (usually blue-shifted), the difficulty to fuse them with other proteins and their signal-to-noise ratio being lower than for 1-FP sensors. The race for diversifying 1-FP sensors has already begun, with new hue-variants of the GCaMP scaffold (the GECOs series and RCaMP), of kinase activity sensors (Cyan Sinphos) and of voltage-sensitive fluorescent proteins (VSFP3s).
The lab of Richard Tsien has just added a new member to this emerging family of hue-shifted single-FP sensors: a novel red pH-sensitive red fluorescent protein called pHTomato. In a nicely-done 2-author paper published in Nature Neuroscience, Li and Tsien demonstrate the usefulness and efficiency of pHTomato by fusing it to the vesicular membrane protein synaptophysin. The resulting protein, SypHTomato, can report vesicle fusion and recycling as well as its … Continue reading
A Nature Methods article from the cohen lab shows how a microbial light-driven proton pump, Archeorhodopsin-3 (Arch), usually used to silence neuronal activity, can be used to efficiently image the membrane potential of individual neurons. Like many other microbial rhodopsins, Arch is weakly fluorescent. When excited using green light (550 nm), Arch emits light in the far red (680 nm). This fluorescence process is poorly efficient (quantum yield < 0.001) but sensitive to the membrane potential. When expressed in mammalian neurons in vitro, the fluorescence change of Arch in response to steps of potential was extremely fast (< 0.5 ms) with a high signal-to-noise ratio, allowing the detection of single action potentials in single trials. However, the fact that Arch generates an outward proton current when illuminated seriously challenges the relevance of its use as a voltage sensor. A mutated form of Arch (D95N) which does not generate any current was also tested. Arch(D95N) retains the ability to report single action potentials but with a significantly slower response time (40 ms).