Optogenetic Applications Research Articles

The clinical potential of optogenetic interrogation of pathogenesis.

Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye. This work summarizes the progress of current clinical trials and provides a brief overview of basic structures and photophysics of commonly used photoactivable proteins. We highlight recent achievements such as optogenetic control of the chimeric antigen receptor, CRISPR-Cas system, gene expression, and organelle dynamics. We discuss conceptual innovation and technical challenges faced by current optogenetic research. In doing so, we provide a framework that showcases ever-growing applications of optogenetics in biomedical research and may inform novel precise medicine strategies based on this enabling technology.

Sub-1 Volt and high-bandwidth visible to near-infrared electro-optic modulators

Integrated electro-optic (EO) modulators are fundamental photonics components with utility in domains ranging from digital communications to quantum information processing. At telecommunication wavelengths, thin-film lithium niobate modulators exhibit state-of-the-art performance in voltage-length product (VπL), optical loss, and EO bandwidth. However, applications in optical imaging, optogenetics, and quantum science generally require devices operating in the visible-to-near-infrared (VNIR) wavelength range. Here, we realize VNIR amplitude and phase modulators featuring VπL’s of sub-1 V ⋅ cm, low optical loss, and high bandwidth EO response. Our Mach-Zehnder modulators exhibit a VπL as low as 0.55 V ⋅ cm at 738 nm, on-chip optical loss of ~0.7 dB/cm, and EO bandwidths in excess of 35 GHz. Furthermore, we highlight the opportunities these high-performance modulators offer by demonstrating integrated EO frequency combs operating at VNIR wavelengths, with over 50 lines and tunable spacing, and frequency shifting of pulsed light beyond its intrinsic bandwidth (up to 7x Fourier limit) by an EO shearing method.

Developments in application of optogenetics in pain and anxiety: a literature review

Optogenetic interventions have put forth unprecedented therapies that help us provide therapeutic treatment to various neurological conditions. This novel therapy allows us to target neuronal tissues in complex mammalian systems with a precise and specific impact using photoresponsive Microbial opsins ( modified to suit mammalian physiologies) that enable cell-type specific, precise biological processing on a millisecond scale. As a result, we have been able to explore numerous new directions as the field develops and becomes more diverse. The literature review that follows will try to examine pain and anxiety as neurological disorders and delve deeper into the advancements that allow optogenetic therapies to treat them, and assess them against the currently available therapies.

Wirelessly Powered 3-D Printed Headstage Based Neural Stimulation System for Optogenetic Neuromodulation Application

This work presents a miniaturized wireless power transfer (WPT) system integrated with a neuromodulation headstage for duty-cycled optical stimulation of freely moving rodents. The proposed WPT system is built using the commercially available off-the-shelf components (COTS) for the optogenetic neuromodulation system consisting of a bridge rectifier, a DC-DC converter, an oscillator circuit, an LED driver, and a μLED. The total power consumption of the stimulation system is 14 mW which is provided using the WPT method. The WPT system includes a novel transmitter (TX) coil implemented on a printed circuit board (PCB), and a solenoid receiver (RX) coil wrapped around a customized 3-D printed headstage. The proposed TX coil is designed in such a way that the magnetic field all across the TX coil is sufficient to provide the required power to the optical stimulation system that is worn as a headstage by the freely moving rat. The headstage device's dimension is 18.75 mm × 21.95 mm, weighing 4.75 g. The ratio of the weight of the headstage and rat is 4.75:300. The proposed system is able to achieve a maximum overall efficiency of ∼63% at 5 cm separation between the TX and RX coils, where the maximum power transfer efficiency (PTE) of the WPT system is ∼88% and the power conversion efficiency (PCE) of the rectifier is 71.6%. The proposed system with reconfigurable stimulation frequency is suitable for exciting different brain areas for long-term health monitoring.

Reaction Dynamics in the Chrimson Channelrhodopsin: Observation of Product-State Evolution and Slow Diffusive Protein Motions.

Chrimson is a red-light absorbing channelrhodopsin useful for deep-tissue optogenetics applications. Here, we present the Chrimson reaction dynamics from femtoseconds to seconds, analyzed with target analysis methods to disentangle spectrally and temporally overlapping excited- and product-state dynamics. We found multiple phases ranging from ≈100 fs to ≈20 ps in the excited-state decay, where spectral features overlapping with stimulated emission components were assigned to early dynamics of K-like species on a 10 ps time scale. Selective excitation at the maximum or the blue edge of the absorption spectrum resulted in spectrally distinct but kinetically similar excited-state and product-state species, which gradually became indistinguishable on the μs to 100 μs time scales. Hence, by removing specific protein conformations within an inhomogeneously broadened ensemble, we resolved slow protein backbone and amino acid side-chain motions in the dark that underlie inhomogeneous broadening, demonstrating that the latter represents a dynamic interconversion between protein substates.

Diminishing neuronal acidification by channelrhodopsins with low proton conduction

Many channelrhodopsins are permeable to protons. We show that in electrically polarized cells, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification. From an initial intracellular pH of 7.3, cells acidified to pH 6.9 ± 0.24 (mean ± s.d., n = 34 cells) after 60 seconds of illumination. To determine whether this acidification arose via proton transport through the opsin vs through other voltage-dependent proton channels, we performed experiments with patterned optogenetic stimulation in monolayers of HEK293 cells expressing CheRiff, an inward-rectifier potassium channel, Kir2.1, and a red-shifted pH indicator, pHoran4. Illumination with stripes of blue light led to electrical depolarization that extended beyond the illuminated spots, due to gap junctional conduction; but acidification was localized to the illuminated spots. These observations established that the acidification required proton transport through the opsin itself. We then identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2 3M. These opsins were effective at evoking neural activity, but neither induced detectable acidification. PsCatCh2.0 was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of artifacts and disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.

Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis.

There is extensive research which explores the photochemistry of Blue Light Using FAD photoreceptors, however there is limited literature available on how the signal is transduced from the BLUF domain to the functional/output domain. A detailed understanding of their operation is needed to direct applications such as the development of optogenetic devices. Here, we elucidate the signal transduction pathway of the photoactivated adenylate cyclase OaPAC, in which a BLUF domain is fused to an adenylate cyclase domain that catalyzes the light-dependent formation of cAMP from ATP. OaPAC has very low dark state activity and is therefore a promising starting point for optogenetic applications. We used unnatural amino acid mutagenesis to explore how light absorption by the N-terminal BLUF domain on the fs timescale leads to activation of the C-terminal adenylate cyclase domain. We incorporated the IR probe azidophenylalanine at F103, W90 and F131 using orthogonal aminoacyl-tRNA-synthetases. The FTIR light minus dark difference spectrum shows that the environment around F103 and W90 residues changes upon light activation, however, there is no change in the environment around F131 upon photoactaction. The enzymatic activity of these mutants was not affected due to the IR probe incorporation. We also replaced Y125 with fluorotyrosine analogues to increase the acidity of the phenolic hydroxyl group and probe the role of Y125 in signal transduction which forms an intersubunit hydrogen bond with N256 from the other monomer in the OaPAC dimer. OaPAC with 3-fluorotyrosine, 3,5-difluorotyrosine or 2,3,5-trifluorotyrosine incorporated at Y125 showed 10 fold higher activity in the dark compared to wild-type OaPAC, whereas Y125F and Y125S showed no dark state activity, implying that the intersubunit hydrogen bond between Y125 and N256 is critical for signal transduction.

Ion-pumping microbial rhodopsin protein classification by machine learning approach

BackgroundRhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype.ResultsThis study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server (https://bioinfo.imtech.res.in/servers/rhodopred) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers.ConclusionThe overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers.

Distribution and inflammatory cell response to intracranial delivery of radioluminescent Y2(SiO4)O:Ce particles.

Due to increasing advances in their manufacture and functionalization, nanoparticle-based systems have become a popular tool for in vivo drug delivery and biodetection. Recently, scintillating nanoparticles such as yttrium orthosilicate doped with cerium (Y2(SiO4)O:Ce) have come under study for their potential utility in optogenetic applications, as they emit photons upon low levels of stimulation from remote x-ray sources. The utility of such nanoparticles in vivo is hampered by rapid clearance from circulation by the mononuclear phagocytic system, which heavily restricts nanoparticle accumulation at target tissues. Local transcranial injection of nanoparticles may deliver scintillating nanoparticles to highly specific brain regions by circumventing the blood-brain barrier and avoiding phagocytic clearance. Few studies to date have examined the distribution and response to nanoparticles following localized delivery to cerebral cortex, a crucial step in understanding the therapeutic potential of nanoparticle-based biodetection in the brain. Following the synthesis and surface modification of these nanoparticles, two doses (1 and 3 mg/ml) were introduced into mouse secondary motor cortex (M2). This region was chosen as the site for RLP delivery, as it represents a common target for optogenetic manipulations of mouse behavior, and RLPs could eventually serve as an injectable x-ray inducible light delivery system. The spread of particles through the target tissue was assessed 24 hours, 72 hours, and 9 days post-injection. Y2(SiO4)O:Ce nanoparticles were found to be detectable in the brain for up to 9 days, initially diffusing through the tissue until 72 hours before achieving partial clearance by the final endpoint. Small transient increases in the presence of IBA-1+ microglia and GFAP+ astrocytic cell populations were detected near nanoparticle injection sites of both doses tested 24 hours after surgery. Taken together, these data provide evidence that Y2(SiO4)O:Ce nanoparticles coated with BSA can be injected directly into mouse cortex in vivo, where they persist for days and are broadly tolerated, such that they may be potentially utilized for remote x-ray activated stimulation and photon emission for optogenetic experiments in the near future.

Advances in Optogenetics Applications for Central Nervous System Injuries.

Injuries to the central nervous system (CNS) often lead to severe neurological dysfunction and even death. However, there are still no effective measures to improve functional recovery following CNS injuries. Optogenetics, an ideal method to modulate neural activity, has shown various advantages in controlling neural circuits, promoting neural remapping, and improving cell survival. In particular, the emerging technique of optogenetics has exhibited promising therapeutic methods for CNS injuries. In this review, we introduce the light-sensitive proteins and light stimulation system that are important components of optogenetic technology in detail and summarize the development trends. In addition, we construct a comprehensive picture of the current application of optogenetics in CNS injuries and highlight recent advances for the treatment and functional recovery of neurological deficits. Finally, we discuss the therapeutic challenges and prospective uses of optogenetics therapy by photostimulation/photoinhibition modalities that would be suitable for clinical applications.

High-Performance Zwitterionic Organohydrogel Fiber in Bioelectronics for Monitoring Bioinformation

Bioinformation plays an imperative role in day-to-day life. Wearable bioelectronics are important for sensing bioinformation in real-time and conductive hydrogel fibers are a key component in next generation wearable bioelectronics. However, current conductive hydrogel fibers have remarkable disadvantages such as insufficient conductivity, stability, and bioinformation sensing ability. Here, we report the synthesis of a zwitterionic organohydrogel (ZOH) fiber by the combination of the mold method and solvent replacement strategy. The ZOH fiber shows transparency (92.1%), stretchability (905.8%), long-term stability, anti-freezing ability (-35-60 °C), and low light transmission loss (0.17 dB/cm). Then, we integrate the ZOH fiber into fabric for use as a bioinformation sensor, the results prove its capability as a bioinformation monitor, monitoring information such as motion and bioelectric signals. In addition, the potential of the ZOH fiber in optogenetic applications is also confirmed.

Effect of tryptophan mutation on the structure of LOV1 domain of phototropin1 protein of Ostreococcus tauri: A combined molecular dynamics simulation and biophysical approach

BackgroundLight, oxygen and voltage (LOV) proteins detect blue light by formation of a covalent ‘photoadduct’ between the flavin chromophore and the neighboring conserved cysteine residue. LOV proteins devoid of this conserved photoactive cysteine are unable to form this ‘photoadduct’ upon light illumination, but they can still elicit functional response via the formation of neutral flavin radical. Recently, tryptophan residue has been shown to be the primary electron donors to the flavin excited state. MethodsPhotoactive cysteine (Cys42) and tryptophan (Trp68) residues in the LOV1 domain of phototropin1 of Ostreococcus tauri (OtLOV1) was mutated to alanine and threonine respectively. Effect of these mutations have been studied using molecular dynamics simulation and spectroscopic techniques. ResultsMolecular dynamics simulation indicated that W68T did not affect the structure of OtLOV1 protein, but C42A leads to some structural changes. An increase in the fluorescence lifetime and quantum yield values was observed for the Trp68 mutant. ConclusionsAn increase in the fluorescence lifetime and quantum yield of Trp68 mutant compared to the wild type protein suggests that Trp68 residue participates in quenching of the flavin excited state followed by photoexcitation. General significanceEnhanced photo-physical properties of Trp68 OtLOV1 mutant might enable its use for the optogenetic and microscopic applications.

Ultrasound-Triggered In Situ Photon Emission for Noninvasive Optogenetics.

Optogenetics has revolutionized neuroscience understanding by allowing spatiotemporal control over cell-type specific neurons in neural circuits. However, the sluggish development of noninvasive photon delivery in the brain has limited the clinical application of optogenetics. Focused ultrasound (FUS)-derived mechanoluminescence has emerged as a promising tool for in situ photon emission, but there is not yet a biocompatible liquid-phase mechanoluminescence system for spatiotemporal optogenetics. To achieve noninvasive optogenetics with a high temporal resolution and desirable biocompatibility, we have developed liposome (Lipo@IR780/L012) nanoparticles for FUS-triggered mechanoluminescence in brain photon delivery. Synchronized and stable blue light emission was generated in solution under FUS irradiation due to the cascade reactions in liposomes. In vitro tests revealed that Lipo@IR780/L012 could be triggered by FUS for light emission at different stimulation frequencies, resulting in activation of opsin-expressing spiking HEK cells under the FUS irradiation. In vivo optogenetic stimulation further demonstrated that motor cortex neurons could be noninvasively and reversibly activated under the repetitive FUS irradiation after intravenous injection of lipid nanoparticles to achieve limb movements.

Pushing the Frontiers: Optogenetics for Illuminating the Neural Pathophysiology of Bipolar Disorder.

Bipolar disorder (BD), a disabling mental disorder, is featured by the oscillation between episodes of depression and mania, along with disturbance in the biological rhythms. It is on an urgent demand to identify the intricate mechanisms of BD pathophysiology. Based on the continuous progression of neural science techniques, the dysfunction of circuits in the central nervous system was currently thought to be tightly associated with BD development. Yet, challenge exists since it depends on techniques that can manipulate spatiotemporal dynamics of neuron activity. Notably, the emergence of optogenetics has empowered researchers with precise timing and local manipulation, providing a possible approach for deciphering the pathological underpinnings of mental disorders. Although the application of optogenetics in BD research remains preliminary due to the scarcity of valid animal models, this technique will advance the psychiatric research at neural circuit level. In this review, we summarized the crucial aberrant brain activity and function pertaining to emotion and rhythm abnormities, thereby elucidating the underlying neural substrates of BD, and highlighted the importance of optogenetics in the pursuit of BD research.

Structural mechanism of signal transduction in a phytochrome histidine kinase

Phytochrome proteins detect red/far-red light to guide the growth, motion, development and reproduction in plants, fungi, and bacteria. Bacterial phytochromes commonly function as an entrance signal in two-component sensory systems. Despite the availability of three-dimensional structures of phytochromes and other two-component proteins, the conformational changes, which lead to activation of the protein, are not understood. We reveal cryo electron microscopy structures of the complete phytochrome from Deinoccocus radiodurans in its resting and photoactivated states at 3.6 Å and 3.5 Å resolution, respectively. Upon photoactivation, the photosensory core module hardly changes its tertiary domain arrangement, but the connector helices between the photosensory and the histidine kinase modules open up like a zipper, causing asymmetry and disorder in the effector domains. The structures provide a framework for atom-scale understanding of signaling in phytochromes, visualize allosteric communication over several nanometers, and suggest that disorder in the dimeric arrangement of the effector domains is important for phosphatase activity in a two-component system. The results have implications for the development of optogenetic applications.

Precise modulation of embryonic development through optogenetics.

The past decade has witnessed enormous progress in optogenetics, which uses photo-sensitive proteins to control signal transduction in live cells and animals. The ever-increasing amount of optogenetic tools, however, could overwhelm the selection of appropriate optogenetic strategies. In this work, we summarize recent progress in this emerging field and highlight the application of opsin-free optogenetics in studying embryonic development, focusing on new insights gained into optical induction of morphogenesis, cell polarity, cell fate determination, tissue differentiation, neuronal regeneration, synaptic plasticity, and removal of cells during development.

Structural insights into light-driven anion pumping in cyanobacteria

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

Dynamical regulation of epileptiform discharges caused by abnormal astrocyte function with optogenetic stimulation

A simple network model consisting of a pyramidal neuron, an interneuron, and an astrocyte is constructed to simulate epileptiform discharges, focusing on the role of the interneuron in the pathological state. Simulation results show that with the change of the parameters related to abnormal glutamate degradation, the system can be transformed from bursting discharges or subthreshold oscillations to seizure-like discharges containing depolarization block. Meanwhile, the proposal of optogenetics has made it possible to target specific cells to modulate seizures, however, discoveries remain to be made regarding the specific effects limited by light mechanisms, stimulation patterns, and other factors. Hence, based on the constructed model, firstly, the experimental phenomenon that different types of light mechanisms are required to target the interneuron to control seizures under different situations is verified, and further, the effect of blue light targeting the astrocyte on seizure thresholds is revealed. The results demonstrate that the choice of stimulation frequency for seizure control varies in different situations, but the pulse width must be larger to be more conducive to control. In particular, the inhibitory photostimulation may change bursting discharges into spike discharges or subthreshold oscillations, in addition to eliminating the depolarization block part of the bursting discharges. Due to the slow-scale variation of calcium dynamics, stimulation with the same duty cycle does not have a consistent effect on thresholds for the appearance of epileptiform discharges. More importantly, by means of dynamical changes in the calcium signal near the bifurcation point from oscillation to resting, the effect of different stimulation patterns on the onset threshold can be explained. Our results may provide theoretical insight for the application of optogenetics in epileptic disorders caused by abnormal astrocyte function.

P‐8.6: Elimination of Nanorods by Tetramethylammonium Hydroxide for the Fabrication of AlGaN‐based UV‐C Micro‐LED Array

Short‐wavelength ultraviolet (UV‐C) micro light‐emitting diode (Micro‐LED) array greatly benefits the optogenetic applications in neuroscience, optical communications, and maskless lithography. In this work, UV‐C Micro‐LED arrays with each pixel size of 10×10 μm2 were fabricated, and the nanorods introduced by inductively coupled plasma (ICP) etching were eliminated by post‐ICP tetramethylammonium hydroxide (TMAH) treatment. The pixel‐level electrical characteristics were analyzed, and the peak wavelength stability under variable injected current densities was demonstrated. The results indicated the potential of applying the TMAH treatment in the fabrication of the UV‐C Micro‐LED array.

Applications and challenges of rhodopsin-based optogenetics in biomedicine.

Optogenetics is an emerging bioengineering technology that has been rapidly developed in recent years by cross-integrating optics, genetic engineering, electrophysiology, software control, and other disciplines. Since the first demonstration of the millisecond neuromodulation ability of the channelrhodopsin-2 (ChR2), the application of optogenetic technology in basic life science research has been rapidly progressed, especially in neurobiology, which has driven the development of the discipline. As the optogenetic tool protein, microbial rhodopsins have been continuously explored, modified, and optimized, with many variants becoming available, with structural characteristics and functions that are highly diversified. Their applicability has been broadened, encouraging more researchers and clinicians to utilize optogenetics technology in research. In this review, we summarize the species and variant types of the most important class of tool proteins in optogenetic techniques, the microbial rhodopsins, and review the current applications of optogenetics based on rhodopsin qualitative light in biology and other fields. We also review the challenges facing this technology, to ultimately provide an in-depth technical reference to support the application of optogenetics in translational and clinical research.