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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A modular single-construct tool for optical control of cell signaling

The study of cell signaling has revealed that cells can be particularly sensitive to spatiotemporal properties of the signaling dynamics. For instance, a cell can choose divergent fates (proliferation vs. differentiation, senescence vs. apoptosis, etc.) depending on the duration and strength of a protein signal. The development of fluorescent reporters has enabled us to observe these dynamics, but researchers still lack appropriate, general tools to perturb cells on the second- and minute-timescales on which signaling dynamics can occur.

Coupling light to protein activation may offer a powerful solution due to its fast, reversible, and highly tunable nature. In the March 2013 issue of Nature Methods, Bugaj, Kane, Schaffer and colleagues accomplish this by developing a method for light inducible homo-oligomerization of proteins. In this study, the authors report a genetically encodable, generalizable system that can activate multiple signaling proteins and networks in mammalian cells.

To do this, the authors leveraged the interesting ability of the Arabidopsis protein Cryptochrome 2 (Cry2) to form oligomers in response to blue light. To date, this clustering ability had only been shown in plant cells, but upon transfection into HEK 293Ts, the authors demonstrated that Cry2 can be clustered in mammalian cells as well (fig. … 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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The optopatcher: an electrode holder allowing the insertion of an optical fiber into a patch pipette

In order to perform simultaneous intracellular recording and light stimulation of a single neuron, two separate positioning systems are often needed (one to position the recording electrode, one to position a waveguide near the recorded neuron). More sophisticated solutions for single neuron photostimulation involve light patterning techniques which are not suited for deep in vivo recordings. Katz et al. came up with a simple and affordable solution for this problem, by designing a patch pipette holder containing an additional port for the insertion of an optical fiber into the pipette.

This device, which they called “OptoPatcher” allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. Moreover, the presence of the bare fiber within an aqueous solution instead of the brain can prevent tissue damage due to heating of the brain. In addition, replacement of standard pipettes is done as easily as with the available commercial holders.

The OptoPatcher was used successfully in vivo for intracellular recordings from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter and it was also … Continue reading

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Micro-electrocorticogram electrodes for simultaneous recording and optical stimulation

Wen Li’s group at Michigan State University recently presented preliminary results on the development of an epidural micro-electrocorticogram (μECoG) array combining microelectrodes and light emitting diodes (LEDs) for optical neural stimulation. This work follows-up on a new line of research aiming at providing μECoG arrays with versatile optical stimulation capabilities (see for example the work of Ledochowitsch et al). The “Opto-μECoG” arrays developped in Wen Li’s group were especially designed to address three major limitations of current designs, in particular the limited cortical area and spatial resolution available for optical stimulation. The key features of these Opto-μECoG arrays are the following:

Untethered system: integration of surface mounted μ-LED light sources (220 × 270 ×50 μm3, wavelength peak at 460nm, Cree® TR2227TM) on the Opto-μECoG array allows the possibility to achieve a truly untethered system. Maximized target cortical area available for optical stimulation: optically transparent indium tin oxide (ITO) [2] epidural electrodes of the Opto-μECoG array allow maximum exposure of the target cortical area for optical stimulation. Maximized spatial resolution of optical stimulation: the embedded light sources, placed on top of the ITO electrodes, were preciously arranged based on a recent study of the optimal spacing of subdural, epidural, and scalp … 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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A multimodal microimplant for combined light/liquid delivery and multi-site electrical recording

As optogenetic neuronal control strategies develop and get widely adopted in neurobiology labs, the demand for devices allowing combined light delivery and electrophysiological recording is growing. These devices, commonly called opto-electrodes or optrodes, already exist under a wide variety of forms, from the simple home made single optical fiber + single electrode to more complex microfabricated multi-fiber/multi-electrode systems. In the past years, Zhang et al. integrated an optical fibre in a Utah Array, and NeuroNexus assembled an optical fiber on their standard silicon shaft. Other groups implemented waveguides directly into the optrode fabrication process: Cho et al. integrated a microstructured SU-8 waveguide on the shaft of a Michigan Probe and the group of Ed Boyden integrated 12 silicon oxynitride waveguides on silicon shafts (paper1, paper2). Royer et al. in the Buszaki lab managed to establish silicon based shaft arrays with integrated optical fibers.

In a recent paper published in “Lab on a Chip”, the Stieglitz and Lüthi labs introduced a novel optrode which also comprises a microfluidic channel for liquid delivery at the tip of the probe. This channel can be used for example to inject a solution containing a virus right under the waveguide tip and around the electrical … Continue reading

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A photoconvertible genetically-encoded calcium indicator

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

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A 3D multiwaveguide array for delivering light to multiple brain circuits

The ability to deliver light into the brain for the purpose of controlling neural activity or other biological processes, optogenetics, has opened up new frontiers in both basic neuroscience and neuroengineering. One arena of great activity is in the use of microbial opsins such as channelrhodopsins, halorhodopsins, and archaerhodopsins to make neurons activatable or silenceable by light, thus enabling assessment of the causal contribution of specific neurons, brain regions, and neural pathways to normal and abnormal behaviors and neural computations. To date, numerous in vivo studies have used optical fibers, and fiber-electrode hybrids, to deliver visible light into brain targets in which neurons express opsins, but an optical fiber can target just a single region. An implantable probe capable of delivering light to arbitrary points in a 3-dimensional volume would enable more versatile optical control, opening up the ability to deliver patterned light to manipulate neural activity in distributed brain circuits.

The Boyden lab recently developed a linear probe comprising a set of integrated microwaveguides running in parallel to each other, microfabricated on a single substrate and capable of delivering light independently to multiple brain targets along the probe axis. In a paper published in Optics Letters, they now extend … Continue reading

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A generalizable method for controlling protein activities with light

If we could control the activity of any intracellular protein of interest with light, it would revolutionize how we study biology and engineer gene- or cell-based therapies. For instance, optical protein control would allow testing of protein functions in cells with high spatiotemporal precision, so we can observe how protein activation at specific times and places affects downstream biochemical reactions and cellular behavior, without secondary effects from chronic or ubiquitous activation. Optical protein control would also allow four-dimensional regulation of cellular proliferation, survival, or differentiation in tissues or animals for basic research or therapy. Thus controlling a wide variety of proteins with light is one of the major long-term goals of optogenetics.

Meeting this challenge will require the development of easily generalizable methods for optical control of protein activity. Considerable efforts have been made to adapt natural light-responsive signaling proteins to regulate specific proteins of interest. In recent years, phytochrome-PIF, cryptochrome-CIB, and FKF-gigantea light-dependent interactions from plants have been adapted to control heterodimerization of proteins in mammalian cells. The LOV domain from phototropin has also been used to create single-chain photoactivatable proteins via light-modulated allosteric mechanisms. However, these methods have certain disadvantages. Light-controlled heterodimerization can not effectively control activities of … Continue reading

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