Optogenetic Technology Research Articles

Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media.

Blue wavelength light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue wavelength light in cultured primary rat cortical cells. Exposure to blue light (470 nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1, Cry1, Cry2, Clock, or Per2. IEG expression was increased following 4 h of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Induction of IEGs by blue light was maintained in cortical cultures treated with AraC to block glial proliferation, indicating that induction occurred selectively in postmitotic neurons. Importantly, changes in gene expression induced by blue wavelength light were prevented when cultures were maintained in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.

Molecular Dynamics Simulation of Transmembrane Transport of Chloride Ions in Mutants of Channelrhodopsin.

Channelrhodopsins (ChRs) are light-gated transmembrane cation channels which are widely used for optogenetic technology. Replacing glutamate located at the central gate of the ion channel with positively charged amino acid residues will reverse ion selectivity and allow anion conduction. The structures and properties of the ion channel, the transport of chloride, and potential of mean force (PMF) of the chimera protein (C1C2) and its mutants, EK-TC, ER-TC and iChloC, were investigated by molecular dynamics simulation. The results show that the five-fold mutation in E122Q-E129R-E140S-D195N-T198C (iChloC) increases the flexibility of the transmembrane channel protein better than the double mutations in EK-TC and ER-TC, and results in an expanded ion channel pore size and decreased steric resistance. The iChloC mutant was also found to have a higher affinity for chloride ions and, based on surface electrostatic potential analysis, provides a favorable electrostatic environment for anion conduction. The PMF free energy curves revealed that high affinity Cl− binding sites are generated near the central gate of the three mutant proteins. The energy barriers for the EK-TC and ER-TC were found to be much higher than that of iChloC. The results suggest that the transmembrane ion channel of iChloC protein is better at facilitating the capture and transport of chloride ions.

A Protocol for Transverse Cardiac Slicing and Optical Mapping in Murine Heart.

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or β-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.

Regulation of Feeding-Related Behaviors by Arcuate Neuropeptide Y Neurons.

Research over recent decades has established neuropeptide Y (NPY) neurons in the arcuate nucleus (Arc) of the hypothalamus as a group of powerful orexigenic acting neurons in the brain. However, genetic mouse models in combination with novel neuron-controlling chemogenetic and optogenetic technologies have also uncovered additional functions for this Arc NPY population that go beyond the simple food intake stimulatory action and link these NPY neurons to the control of energy expenditure, thermogenesis, physical activity, food-seeking behavior, and anxiety. This control is achieved by complex neuronal networks connecting these Arc NPY neurons with other vital neuronal centers in the brain, including the paraventricular nucleus, ventral tegmental area, amygdala, and brainstem. In addition, single-cell sequencing approaches have revealed that a greater heterogeneity of NPY neurons actually exists, giving rise to various subsets of NPY neuronal populations that are distinguished by the profile of other neurotransmitters that they coexpress. In this review we will focus on aspects of food intake-associated behaviors and shed more light on the integrative role of NPY neurons in potential interaction pathways of individual survival circuits.

Tailoring optogenetic tools for AF treatment.

A novel bioelectronic system that combines automated arrhythmia detection with optogenetic engineering for arrhythmia termination is able to stop atrial fibrillation without the need of electrical shock in a closed-chest experimental model.

Study on optogenetics technology and the design of its script for virtual simulation experiment

Optogenetics is one of the biggest breakthroughs in neurobiology in recent decades, which is a revolutionary approach to precise intervention of specific cell functions through the combination of optical and genetic engineering techniques. However, this technique involves the interdisciplinary theoretical knowledge and methods, and it requires expensive equipments and long experimental period. Thus, it is not convenient to carry out widely in experiment teaching for undergraduates except the virtual simulation teaching. In present virtual simulation experiment, we aim to learn the basic principle, operation and application of optogenetics and achieve the desired teaching effects by simulating the process of the virus package, simulating the process of the animal selection, surgery and virus stereotactic microinjection, simulating the expression process of photosensitive channel, the extracellular verification process of photosensitive channel function in vivo, and simulating the observation process that optogenetic inhibition of glutamate neurons in the amygdala affect animal fear behavior. In this paper, the content design of the script making of the virtual experiment has been discussed in the above ways. Key words: Virtual simulation experiment; Experimental teaching; Optogenetics; Script

New Pioneers of Optogenetics in Neuroscience.

Optogenetics have recently increased in popularity as tools to study behavior in response to the brain and how these trends relate back to a neuronal circuit. Additionally, the high demand for human cerebral tissue in research has led to the generation of a new model to investigate human brain development and disease. Human Pluripotent Stem Cells (hPSCs) have been previously used to recapitulate the development of several tissues such as intestine, stomach and liver and to model disease in a human context, recently new improvements have been made in the field of hPSC-derived brain organoids to better understand overall brain development but more specifically, to mimic inter-neuronal communication. This review aims to highlight the recent advances in these two separate approaches of brain research and to emphasize the need for overlap. These two novel approaches would combine the study of behavior along with the specific circuits required to produce the signals causing such behavior. This review is focused on the current state of the field, as well as the development of novel optogenetic technologies and their potential for current scientific study and potential therapeutic use.

Проблемы и перспективы использования оптогенетических технологий в XXI веке

Проблемы и перспективы использования оптогенетических технологий в XXI веке

Brain-Machine Interface-Based Rat-Robot Behavior Control.

Brain-machine interface (BMI) provides a bidirectional pathway between the brain and external facilities. The machine-to-brain pathway makes it possible to send artificial information back into the biological brain, interfering neural activities and generating sensations. The idea of the BMI-assisted bio-robotic animal system is accomplished by stimulations on specific sites of the nervous system. With the technology of BMI, animals' locomotion behavior can be precisely controlled as robots, which made the animal turning into bio-robot. In this chapter, we reviewed our lab works focused on rat-robot navigation. The principles of rat-robot system have been briefly described first, including the target brain sites chosen for locomotion control and the design of remote control system. Some methodological advances made by optogenetic technologies for better modulation control have then been introduced. Besides, we also introduced our implementation of "mind-controlled" rat navigation system. Moreover, we have presented our efforts made on combining biological intelligence with artificial intelligence, with developments of automatic control and training system assisted with images or voices inputs. We concluded this chapter by discussing further developments to acquire environmental information as well as promising applications with write-in BMIs.

An Optogenetic Controllable T Cell System for Hepatocellular Carcinoma Immunotherapy.

Rationale: T-cell based immunotherapy increasingly shows broad application prospects in cancer treatment, but its performance in solid tumors is far from our expectation, partly due to the re-inhibition of infiltrated T cells by immunosuppressive tumor microenvironment. Here we presented an artificial synthetic optogenetic circuit to control the immune responses of engineered T cells on demand for promoting and enhancing the therapeutic efficiency of cancer immunotherapy.Methods: We designed and synthesized blue-light inducible artificial immune signaling circuit and transgene expression system. The blue light triggered transgene expression was investigated by luciferase activity assay, qPCR and ELISA. The in vitro cytotoxicity and proliferation assays were carried out on engineered T cells. The in vivo anti-tumor activity of engineered T cells was investigated on xenograft model of human hepatocellular carcinoma.Results: Blue light stimulation could spatiotemporally control gene expression of specific cytokines (IL2, IL15, and TNF-α) in both engineered 293T cells and human primary T cells. This optogenetic engineering strategy significantly enhanced the expansion ability and cytolytic activity of primary T cells upon light irradiation, and the light activated T cells showed high-efficiency of elimination against xenograft of hepatocellular carcinoma cells.Conclusions: The current study represented an engineered remotely control T cell system for solid tumor treatment, and provided a potential strategy to partially overcome the intrinsic shortages of current immune cell therapy.

Optogenetic Light Sensors in Human Retinal Organoids.

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.

Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome

Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome. VIDEO ABSTRACT.

Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)-derived and light-sensitive channelrhodopsin-2-induced MN spheroids from a patient with sporadic ALS. Each tissue was cultured in a different compartment of a microfluidic device. Axon outgrowth formed neuromuscular junctions on the muscle fiber bundles. Light was used to activate muscle contraction, which was measured on the basis of pillar deflections. Compared to a non-ALS motor unit, the ALS motor unit generated fewer muscle contractions, there was MN degradation, and apoptosis increased in the muscle. Furthermore, the muscle contractions were recovered by single treatments and cotreatment with rapamycin (a mechanistic target of rapamycin inhibitor) and bosutinib (an Src/c-Abl inhibitor). This recovery was associated with up-regulation of autophagy and degradation of TAR DNA binding protein-43 in the MNs. Moreover, administering the drugs via an endothelial cell barrier decreased the expression of P-glycoprotein (an efflux pump that transports bosutinib) in the endothelial cells, indicating that rapamycin and bosutinib cotreatment has considerable potential for ALS treatment. This ALS-on-a-chip and optogenetics technology could help to elucidate the pathogenesis of ALS and to screen for drug candidates.

Optogenetic Peripheral Nerve Immunogenicity

Optogenetic technologies have been the subject of great excitement within the scientific community for their ability to demystify complex neurophysiological pathways in the central (CNS) and peripheral nervous systems (PNS). The excitement surrounding optogenetics has also extended to the clinic with a trial for ChR2 in the treatment of retinitis pigmentosa currently underway and additional trials anticipated for the near future. In this work, we identify the cause of loss-of-expression in response to transdermal illumination of an optogenetically active peroneal nerve following an anterior compartment (AC) injection of AAV6-hSyn-ChR2(H134R) with and without a fluorescent reporter. Using Sprague Dawley Rag2−/− rats and appropriate controls, we discover optogenetic loss-of-expression is chiefly elicited by ChR2-mediated immunogenicity in the spinal cord, resulting in both CNS motor neuron death and ipsilateral muscle atrophy in both low and high Adeno-Associated Virus (AAV) dosages. We further employ pharmacological immunosuppression using a slow-release tacrolimus pellet to demonstrate sustained transdermal optogenetic expression up to 12 weeks. These results suggest that all dosages of AAV-mediated optogenetic expression within the PNS may be unsafe. Clinical optogenetics for both PNS and CNS applications should take extreme caution when employing opsins to treat disease and may require concurrent immunosuppression. Future work in optogenetics should focus on designing opsins with lesser immunogenicity.

Recent advances on application of gene technology and optogenetics combined with calcium imaging in pain study

Background The mechanisms and circuits of pain are complex and involve multiple levels in the periphery, spinal cord, and supraspinal brain regions. To solve the pain problem, it is necessary to have a clear understanding of the pain circuits and mechanisms. Objective With the development of optogenetics technology, in vivo fiber photometry and miniaturized microscopic calcium imaging, the joint use of these methods is expecting to provide an opportunity for exploring the mechanisms and circuits of pain. Content This study reviews the research progress in calcium imaging technology, as well as gene technology, optogenetics technology, most recently-developed in vivo fiber photometry and miniaturized microscopic calcium imaging techniques in pain research. Trend This review summarizes new advances of pain circuitry and mechanisms using joint use of these new technologies. The combined use of these methods provides an unprecedented opportunity to explore complex spinal pain circuits and mechanisms. With the continuous advancement of technology, it is believed that people will eventually solve unknown problems related to pain. Key words: Gene recombination; Calcium ion imaging; Optogenetic; Spinal cord; In vivo fiber photometry; In vivo miniaturized microscopic calcium imaging

Optogenetic interrogation of neurovascular coupling in the cerebral cortex of transgenic mice

Objective. We introduce an engineering approach to study spatiotemporal correlations between vasodynamics and the nearby neural activity in open-loop and closed-loop paradigms. Approach. We integrated optogenetic technology with optical coherence tomography to apply spatiotemporal patterns of optical neurostimulation to the cortex of transgenic optogenetic mice and measure blood flow-rate, velocity, and diameter changes of selected middle cerebral artery branches. Main results. The spatiotemporal characteristics of blood flow-rate, velocity, and vessel diameter responses to localized neurostimulation light pulses were measured. It was observed that the location of stimulation relative to the surrounding vascular topology had notable effects on temporal patterns of vasodynamic responses. This effect was studied by creating velocity, flow-rate, and diameter sensitivity maps for selected arteries. Generally, neural stimulation in the vicinity of downstream capillaries of an artery evoked a fast transient increase in the blood flow-rate, velocity, and vessel diameter which was followed by a long-lasting secondary peak-response. The temporal span of the flow-rate response was quasi-linearly proportional to the length of stimulation. When neural stimulation was delivered to the area in the vicinity of one daughter branch of an artery, in other branches, we observed some drop in blood velocity and/or flow-rate and concurring increase of the vessel diameter. To examine the reliability of the coupling between neural activity and regional blood flow, a closed-loop feedback controller was implemented which is capable of maintaining blood flow-rate at any desired level for relatively longer periods by continuously adjusting the width of stimulation pulses. Significance. The proposed approach opens new lines of research with potential applications in understanding the role of different cell types in the cerebrovascular regulatory mechanisms and the study of the adaptive process of angiogenesis in the cerebral cortex. The observation of incoherent responses of vessel diameter, blood flow-rate, and velocity suggests that such detailed information is necessary to obtain an accurate interpretation of the data acquired via hemodynamic based functional imaging techniques.

Synergistic Ensemble of Optogenetic Actuators and Dynamic Indicators in Cell Biology.

Discovery of the naturally evolved fluorescent proteins and their genetically engineered biosensors have enormously contributed to current bioimaging techniques. These reporters to trace dynamic changes of intracellular protein activities have continuously transformed according to the various demands in biological studies. Along with that, light-inducible optogenetic technologies have offered scientists to perturb, control and analyze the function of intracellular machineries in spatiotemporal manner. In this review, we present an overview of the molecular strategies that have been exploited for producing genetically encoded protein reporters and various optogenetic modules. Finally, in particular, we discuss the current efforts for combined use of these reporters and optogenetic modules as a powerful tactic for the control and imaging of signaling events in cells and tissues.

Feasibility of Joint Application of Techniques of Optogenetics and Neuroelectrophysiology to Research of Acupuncture Analgesia

Since the invention of optogenetic technology, it has greatly promoted the development of neuroscience. Currently, optogenetic approaches have been mostly used to map neural circuits and new neuropharmacology but are rarely seen in the research field of acupuncture analgesia. The mechanism of neural circuits contributing to acupuncture analgesia, an important research hotspot in recent years, has not been fully determined. The optogenetic techniques can be used to modulate and control specific cells, provides highly precise spatial and temporal resolution, is repeatable, and may functionally dissect neuronal networks in vivo. The neuronal activities and their information transmission, processing and storage in intercluster neural networks in different brain regions, and the correlation between behavioral changes and electrical activities of neurons in vivo studies are mainly captured by the implanted microelectropode array, etc. If these two (or more) approaches are combined together, it is definitely and highly helpful to reveal the driving dynamics of neural circuits, plasticity and temporal-spatial activity mode of neurons, as well as behavioral reactions of animals with chronic pain during acupuncture analgesia and may open a new prospect for the application of acupuncture analgesia study.

Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base

Optogenetic technologies have often been used as tools for neuronal activation or silencing by light. Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven chloride ion pump. Upon light absorption, a chloride ion passes through the cell membrane, which is accompanied by the temporary binding of a chloride ion with Thr126 at binding site-1 (BS1) near the protonated Schiff base in NpHR. However, the mechanism of stabilization of the binding state between a chloride ion and BS1 has not been investigated. Therefore, to identify a key component of the chloride ion transport pathway as well as to acquire dynamic information about the chloride ion-BS1 binding state, we performed a rough analysis of the chloride ion pathway shape followed by molecular dynamics (MD) simulations for both wild-type and mutant NpHR structures. The MD simulations showed that the hydrogen bond between Thr126 and the chloride ion was retained in the wild-type protein, while the chloride ion could not be retained at and tended to leave BS1 in the S81A mutant. We found that the direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81 through a hydrogen bond and that Thr126 bound to a chloride ion in the wild-type protein, while this interaction was lost in the S81A mutant, resulting in rotation of the Thr126 side chain and reduction in the interaction between Thr126 and a chloride ion. To confirm the role of S81, patch clamp recordings were performed using cells expressing NpHR S81A mutant protein. Considered together with the results that the NpHR S81A-expressing cells did not undergo hyperpolarization under light stimulation, our results indicate that Ser81 plays a key role in chloride migration. Our findings might be relevant to ongoing clinical trials using optogenetic gene therapy in blind patients.

Photoactivation of Extracellular‐Signal‐Regulated Kinase Signaling in Target Cells by Femtosecond Laser

AbstractOptogenetics employs light to manipulate cellular events such as activation of molecular channels, signaling molecules, and gene transcription, by transfection with engineered light‐sensitive proteins. In the last decade, great progresses in life science have been made by optogenetics, but the gene transfection limits its further medical applications. In this study, it is reported that a short flash of femtosecond‐laser stimulation can directly activate extracellular signal‐regulated kinase (ERK), an essential signaling pathway, in target cells, without any optogenetic technology. This all‐optical approach can be easily accomplished by a two‐photon or confocal microscope coupled with a femtosecond laser. The photostimulation can initiate immediate ERK translocation into nucleus and exit back to cytosol. The nuclear ERK level can be regulated by combination of multiple photostimulations. Subsequent up‐regulation of phosphorylated endogenous eIF4E indicates successful activation of the ERK signaling pathway. Occasionally, ERK activation in the surrounding cells of the stimulation is observed. Some key signaling cascades in the ERK2 pathway activated by photostimulation is further clarified. Altogether, this all‐optical method can activate ERK without any exogenous chemicals or proteins and thus be with promissing potential for related research.