Tag Archives: Opsins

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

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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

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Stop lights for temporal lobe seizures

Unlike electrical stimulation, optogenetics allows neuronal manipulation with great cell-type specificity, with light directly affecting only those cells expressing opsins. In a recent report in Nature Communications, Krook-Magnuson et al harnessed this specificity to stop seizures in vivo in a mouse model of temporal lobe epilepsy. Mice were implanted with electrodes to record brain activity and 200µm thick optical fibers to deliver light to the brain. A closed-loop, on-demand responsive system detected seizures in real time, allowing temporal specificity, in addition to the cell-type specificity achieved through selective opsin expression. Specifically, the authors either selectively inhibited excitatory principal cells or, alternatively, excited a subpopulation of GABAergic inhibitory neurons in the hippocampus by delivering light at the time of a seizure. Both approaches proved successful, despite the less than 5% of illuminated neurons expressing opsins in the latter approach. Light arrested ongoing electrical seizure activity and reduced the incidence of events progressing to overt behavioral seizures.

Epilepsy, a condition of recurrent, spontaneous seizures, is a prevalent disorder, with 1 out of 26 people developing epilepsy during their lifetime. Unfortunately, for over 40% of patients, seizures cannot be controlled with current treatment options. Temporal lobe epilepsy, the most common form of epilepsy … Continue reading

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KENGE-tet System for Expanding the Repertoire of Optogenetically Targeted Cells

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

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Expanding the color palette of channelrhodopsins

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.

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A reason to prefer Arch over Halo?

In the last few years, several strategies for silencing neurons optogenetically have emerged. The most popular approaches make use of two types of microbial light-driven ion pumps. One type is an optimized version of halorhodopsin, an inward chloride pump isolated from the halophilic bacterium Natronomonas pharaonis. Over the past couple years, halorhodopsin was tweaked by adding a series of trafficking signals to improve its addressing to the cytoplasmic membrane (Gradinaru et al, 2010). The latest version is eNpHR3.0. The other type corresponds to archeorhodopsins, which are outward proton pumps isolated from halophilic archaebacteria of the genus Halorubrum. The two popular archeorhodopsins are Arch from Halorubrum sodomense and ArchT from Halorubrum genus (Chow et al., 2010, Han et al., 2011). The same optimization tricks used for halorhodopsin also worked for Arch and ArchT. Both types of pumps generate a hyperpolarizing current in mammalian neurons in response to 500-600 nm light and are effective optogenetic silencers of neuronal activity.

A recent head-to-head comparison performed in the lab of K. Deisseroth has shown that, under matched experiments conditions, archeorhodopsins seem to perform slightly better than the best available halorhodopsin mutant (Mattis et al., 2012). In particular eArchT3.0 (an enhanced version of ArchT) generates about … Continue reading

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A systematic comparison of microbial opsins

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.

Tools compared:

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.

Experimental conditions:

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).

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Using Arch for voltage sensing

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).

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The first opsin gene fusions

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).

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