Category Archives: Journal Club

Hearing the light

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

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Injectable optoelectronics with wireless control

The ability to wirelessly control neural circuitry has been a long-standing goal in neuroscience. Recent advances have put this goal in site using optogenetic approaches. In 2011, multiple groups presented the first attempts at making wireless light delivery application for optogenetics (Iwai et al., 2011; Wentz et al., 2011). These advances, however, were constrained to particular environments or apparatuses to power the devices. The radiofrequency power scavenging approach presented in Kim et al., 2013 frees the experimenter from these constraints. In the recent report, the Bruchas (Washington University at St. Louis) and Rogers (University of Illinois at Urbana-Champaign) labs present ultrathin, microscale optoelectronics and sensors that can be used for the optogenetic manipulations. Unlike other wireless approaches this system can be used with any behavioral apparatus or paradigm, which should allow researchers to explore more complex behaviors while perturbing neural circuitry.

The wireless µILED devices can incorporate wired cellular-scale components that can all be inserted into the brain using a combination of a silk-based biodissolvable adhesive (Kim et al., 2010) and an injection needle similar in concept to electrode delivery presented previously (Kozai and Kipke, 2009). These components include, but are presumably not limited to, temperature sensors, electrodes for … Continue reading

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Combining up to 16 individually adjustable electrodes with optical stimulation in mice

Simultaneously recording and perturbing neural circuits with millisecond-scale temporal precision is a cornerstone of optogenetics research, but the methods for doing so are not always easily accessible. A variety of labs have come up with ad hoc ways to incorpoate fiber optic cables into existing multielectrode implant designs. Very few of these solutions have been documented or published, even though this is becoming an increasingly popular technique. Fortunately, the Moore Lab at Brown University recently published a manuscript on their “flexDrive,” a lightweight implant that can hold multiple fiber optic cables and 16 electrodes (Voigts et al., 2013).

The basic concept is similar to the designs from Matt Wilson’s lab involving a ring of electrodes, each driven by its own screw (Kloosterman et al., 2009). But it introduces a novel spring-based drive mechanism that significantly reduces both the weight of the implant and the time it takes to build. In contrast to previously published designs (Anikeeva et al., 2011), each of the electrodes on the flexDrive can be moved independently—a feature that is essential for maximizing the number of well-isolated single units that can be recorded.

The authors have put a lot of effort into making their designs as accessible … Continue reading

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Genetically encoded sensors for simultaneous monitoring of Cl− and H+

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

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Thermal and optical characterization of a micro-LED probe

Over the last few years, there has been a significant drive to improve light sources for in vivo optogenetic control of neuronal activity. In particular, recent work has focused on the design and microfabrication of compact devices that exhibit multiple optical stimulation sites in order to gain control over closely spaced regions within the brain (see here a list of posts related to this). In a recent Optics Letters paper, McAlinden, Massoubre and colleagues presented a novel microprobe device with integrated light sources. The probe produces sufficient light for optogenetic stimulation without causing significant heating in local brain tissue. The device (see figure below) consists of a 100 µm wide, 50-100 µm thick probe with 5 individually addressable microLEDs. Each LED has a diameter of 40 µm, but can be reduced to 10 µm if higher density probes are required. The LED probes are p-n diodes made from quantum well structures using GaN on Sapphire material. The spacing between LEDs is fixed to allow 1mm of neural tissue to be addressed.

The light output from the LEDs was measured, with scattering and absorption accounted for by experimentally measuring the light transmitted through varying thicknesses of brain tissue. The recorded light … Continue reading

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Photoswitchable protein degradation using a LOV2 domain-based single construct

Plants have developed elaborate light-sensing pathways to be able to adequately react on the quality and quantity of light. These signal transduction pathways regulate among others growth directionality (phototropism), flowering, stomatal opening, and chloroplast movement. Light-signalling pathways in plants converge on the ubiquitin-protein ligase COP1 that regulates stability of many transcription factors involved in light-dependent responses. This puts regulated proteolysis by the ubiquitin proteasome system at the heart of light-depending signaling. Recently, Renicke et al., reported in Chemistry & Biology on the engineering of a synthetic tool that concentrates light-regulated proteolysis into a single construct. The module developed by the authors comprises the light-sensing LOV2 domain of Arabidopsis thaliana phot1 and the ornithine decarboxylase (ODC) like degradation sequence (degron) cODC1. Activity of this so-called photo-sensitive degron (psd) module depends on the presence of blue light: the conformational changes within the LOV2 domain expose the degron, which induces proteasomal degradation. In contrast to the majority of proteasomal substrates, the cODC1 degron is directly recognized by the proteasome, without polyubiquitylation by an ubiquitin-protein ligase. Thus, the psd module is a minimized plant light-signalling pathway that comprises a light-responsive domain fused with a degradation-inducing sequence. The module can be fused to target proteins … Continue reading

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Holographic optogenetic control of retinal neural activity

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

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A multimodal micro-optrode

Microelectrodes are powerful tools for in vivo functional studies. However they are limited in the number of information they provide. In January 2011, LeChasseur and colleagues (Nature Methods 8(4), 319-325, 2011) developed a glass microelectrode which was integrating an optical micro-channel for light delivery and fluorescence collection. The probe serves for specific cellular fluorescence optical detection and activation/inhibition. In a recent issue of PLOS ONE, Dufour et al. extended the multimodal aspect of this micro-optrode. They introduce a, aluminum-coated, fibre optic-based glass microprobe (diameter ≤ 10 μm) with multiple electrical and optical detection capabilities. The probe enables optical separation from individual cells in transgenic mice expressing multiple fluorescent proteins in distinct populations of neurons within the same deep brain nucleus. It also enables color conversion of photoswitchable fluorescent proteins, which can be used for post-hoc identification of the recorded cells and finally it enables dual electrical recordings. Figure 1 shows a representation of the microp-optrode and the modalities described in this paper.

These modalities are in addition to the calcium monitoring and optogenetic cellular activation previously reported (Nature Methods 8(4), 319-325, 2011). In this study, two different excitation sources and detection pathways were used simultaneously to differentiate two different populations … Continue reading

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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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