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Tag Archives: ChRs
The electrical cochlear implant (CI) is considered the most successful neuroprosthesis. Implanted in more than 200,000 hearing-impaired subjects worldwide, CIs enable open speech comprehension in the majority of users. A major drawback of current cochlear prostheses is their low frequency resolution due to current spread of electrical stimulation, limiting their use in music enjoyment and prosody. Thus, there is need for spatially confined stimulation to improve frequency resolution. In a recent Journal of Clinical Investigation article, Tobias Moser and his colleagues demonstrate the feasibility of optogenetic activation of the auditory pathway by stimulating the auditory nerve inside the cochlea (Hernandez et al., 2014).
Two animal models were employed: a transgenic model that expresses ChR2 under control of the Thy1.2 promoter in auditory neurons and an adeno-associated virus-mediated model that expressed the ChR2 variant CatCh (AAV2/6-hSyn-CatCh-YFP). Optogenetic stimulation of spiral ganglion neurons (SGN) evoked auditory brainstem responses both in normal hearing mice and in mouse models of deafness, suggesting the general feasability of cochlear optogenetics for restoring auditory activity.
Approximation of the spatial spread of cochlear excitation by several means, including local field potential recordings in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that even … Continue reading
A recently developed smartphone application allows estimating the required optical power for a given in vivo optogenetic stimulation experiment or any other experimental approach that includes light delivery to deep brain areas via optical fibers. Different brain areas have different optical properties, which determine how light scatters and distributes (and how deeply it penetrates the tissue), once it exits the fiber. The application has a complete mouse brain atlas included that can be used to determine the optical properties of any brain area in the mouse brain (the data on which the calculations are based on was recently published in PlosONE: Aj-Juboori et al, 2013). The user can find the brain areas of his choice, mark it on the atlas, then tell the application what type of optogenetic protein he/she wants to use, as well as the type of optical fiber, desired optical power, and desired protein activation ratio. The application then estimates how far the light will spread in this particular experimental situation (and thus, up to which distance from the fiber tip optogenetic protein activation can be expected). The APP comes in two versions, a free version and a Pro Version that costs $1.99. The two versions are … Continue reading
Sensory information is typically represented in distributed patterns of activity across large populations of neurons. Therefore, many emerging neuro-photonic applications, such as optogenetic retinal prostheses require systems capable of delivering intense, parallel and dynamic light patterns. Such a system will ideally allow photo-control with single-cell selectivity of large neural populations expressing optogenetic probes, rather than nonspecific flashed illumination of the whole population (as provided by many current optogenetic light delivery systems). In a recent Nature Communications report, Reutsky-Gefen, Shoham and colleagues demonstrate holographic optogenetic control of retinal neural activity which is shown to provide rapid cellular-resolution, massively parallel excitation across macroscopic (millimeter-scale) coverage areas. The study illustrates that diffractive wavefront shaping (holographic) tools offer a powerful modality for dynamic patterned photo-stimulation as they naturally combine the high intensity, efficiency and resolution that are characteristic of sequential laser deflectors (like acousto-optical deflectors) with the simultaneous scan-less parallel illumination of multiple locations of microdisplay array projectors, but without their respective limitations. Holographic tools were previously shown to allow structured excitation of dendritic arbors and neurons using neurotransmitter photolysis, as well as two-photon optogenetic stimulation of proximal neurons in brain slices.
The study’s main goal was to develop a prosthetic system that would … Continue reading
Luminopsins: bioluminescent opsins allowing combined opto- and chemogenetic control of neuronal activity
In optogenetic experiments aiming at controlling neuronal activity light-sensitive molecules such as channelrhodopsins or proton pumps are activated through application of light from a physical light source, such as arc lamps, lasers or LEDs. An interesting extension of such experiments could be achieved by controlling light-sensing molecules with a biological light source. Luciferases are such light producing proteins; they are enzymes, used by a variety of bioluminescent organisms, which produce light by oxidizing a substrate molecule. Replacing the physical light source with a biological one, i.e. a light-emitting protein, should allow non-invasive activation of light-sensitive molecules. In addition, the advantage of genetically encoding both light production and light sensing introduces unique combinatorial possibilities.
Using bioluminescence in combination with optogenetic actuators hinges on the ability of luciferase to activate these actuators. In a recent paper published in PLoS ONE, Berglund and colleagues demonstrate that this is indeed possible. In this study the authors engineered fusion proteins of a luciferase from the marine copepod Gaussia princeps (GLuc) and channelrhodopsins, creating luminescent opsins, or “luminopsins”. Their study brings the proof-of-concept that light produced upon supplying the luciferase with its substrate, coelenterazine (CTZ), can activate the fused channelrhodopsin, thereby modulating neuronal activity.
Because luminopsins … Continue reading
Long-term channelrhodopsin-2 expression can induce abnormal axonal morphology and targeting in cerebral cortex
When using optogenetics to study circuit function or animal behavior, a critical prerequisite is that optogenetic protein expression does not, in itself, perturb the circuit being studied. While short-term expression is very commonly used without observable circuit disruption, whether this is also true for long-term expression is less clear. A recent paper by Miyashita et al. in Frontiers in Neural Circuits shows that long-term, high-level expression of ChR2 can induce abnormal axonal morphology and targeting in cerebral cortex. This underscores the importance of using the lowest expression possible, particularly for long-term studies.
Miyashita et al. expressed a common construct, CAG::hChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal neurons in rat somatosensory cortex via in utero electroporation (IUE). This same strategy was used in several prior studies of S1 circuit function, with one important difference: Miyashita et al. expressed hChR2 that was codon-optimized for mammalian expression, while prior studies expressed native ChR2 (discussed below). This strategy successfully conferred light-evoked spiking in vivo and in in vitro brain slices. However, long-term expression (> 40 d) also caused major abnormalities in axonal morphology, which included cylinders of axonal membrane that enveloped pyramidal cell proximal dendrites, and spherical, calyx-like axonal swellings that surrounded neuron cell bodies. These … Continue reading
Optogenetics has proven to be a powerful tool capable of manipulating the activity of a specific population of cells in a complex multicellular organism. This approach is enthusiastically pursued in recent neuroscience field and the causal relationship between neural activity and behavior is finally starting to become unveiled. However, most studies utilize virus mediated gene transfer for the induction of light-sensitive proteins, such as channelrhodopsin-2 (ChR2), and such method inevitably introduces surgical injuries and variability of expression between trials. Therefore, transgenic approach has long been sought, however, satisfying the demands of the specificity as well as the abundance of expression were difficult.
In a recent paper published in the Cell Reports, Tanaka and Matsui and their colleagues at the National Institute for Physiological Sciences (Okazaki, Japan) established Knockin-mediated ENhanced Gene Expression by improved tetracycline-controlled gene induction system (KENGE-tet). The authors found that high levels of tTA-mediated transcription can be achieved by knocking in tetO-ChR2 cassette into a locus at a housekeeping gene, beta-actin. The authors crossed this tetO-ChR2 knockin mouse with 7 different tTA lines and achieved ChR2 expression in specific cell-types including sub-populations of neurons, astrocytes, oligodendrocytes, and microglial cells. In all cases, the level of ChR2 expression was … Continue reading
In a recent paper published in the Journal of Biological Chemistry, the Hegemann and Deisseroth labs introduced several color-shifted Channelrhodopsins (ChRs) with different absorption and kinetic properties. Prigge et al. mutated several key amino acids in ChR2 and C1V1 to further separate action spectra of those two existing ChRs. The resulting colour-variants are separated by 30 nm from each other and show peak absorption at 460 ,490, 520 and 550 nm respectively (Fig. 1). All color-variants exhibit two times larger photocurrents in HEK-cells then the wild type ChR2. Further engineering yielded off-kinetics spanning the range from ms to s for each colour variant. The two most spectrally separated variants (ChR2 T159C and C1V1-triple) were used to show the feasibility of a separate, wavelength-dependent activation of a HEK cell population expressing on those variants (Fig. 2). In addition the blue absorbing mutant ChR2 T159C-L132C exhibits 3 times larger photocurrents then ChR2 H134R, has a small inactivation and a reduced proton permeation making this variant the most efficient ChR for blue activation so far.
The team of Karl Deisseroth conducted a series of experiments under matched conditions in order to draw a systematic comparison of several microbial opsins. Experiments aimed at comparing action spectra, peak photocurrents, steady-state/peak ratios, time-to-peak, off kinetics, desensitization kinetics, kinetics of recovery from desensitization in darkness and 50% effective light power density. The report is published in an upcoming issue of Nature Methods.
11 ChR variants were tested: ChR2, ChR2(H134R), ChR2(E123A), ChR2(T159C), ChR2(E123T/T159C), ChR2(L132C), ChIEF, channelrhodopsin-fast receiver, channelrhodopsin-green receiver, C1V1(E162T) and C1V1(E122T/E162T). 4 ultrafast control tools were compared: ChR2(E123A), ChR2(E123A/H134R), ChR2(E123T) and ChR2(E123T/H134R). 7 light-driven pumps were compared: eNpHR3.0, Arch1.0, eArch3.0, ArchT1.0, eArchT3.0, Mac1.0 and eMac3.0.
all opsin genes were packaged identically in a lentiviral backbone under the control of the mouse excitatory neuron–specific CaMKIIα promoter. all opsin coding sequences were fused in frame with the gene encoding enhanced YFP (eYFP). electrophysiological measurements were performed on transfected cultured hippocampal pyramidal neurons with matched light power densities across experiments (5 mW/mm2).
The labs of Ernst Bamberg and Ed Boyden introduced a gene-fusion approach to achieve co-localized and stoichiometric expression of synergistic or antagonistic opsin pairs. The method relies on a genetic tandem cassette which intercalates the coding sequence of a transmembrane helix (from the β subunit of the rat gastric H+,K+-ATPase) between two opsin genes. The resulting fusion proteins allow strict ratiometric activation of rhodopsin pairs. Fusing ChR2 and NphR or Arch can be used for example to activate or silence all neurons in a field of view to a similar extent or to precisely simulate subthreshold events across the somatodendritic tree of a neuron (as each patch of membrane will preserve the stoichiometry of expression).