Optogenetic Research Articles

Bioluminescence-Optogenetics: A Practical Guide.

Manipulation of neural activity in genetically predefined populations of neurons through genetic techniques is an essential tool in the field of neuroscience as well as a potential avenue in treating a vast assortment of neurological and psychiatric diseases. Here, we describe an emerging methodology of molecular neuromodulation termed bioluminescence-optogenetics (BL-OG) where BL is harnessed to activate bacterial light-driven channels and pumps expressed in neurons to control their activity. BL-OG is realized through opsin-luciferase fusion proteins called luminopsins (LMOs). In this chapter, we will provide a practical guide for applying BL-OG and LMOs in vitro using a cell line and primary cells in culture. In the following chapter, we will turn our focus towards BL-OG applications in ex vivo and in vivo rodent models of the nervous system.

Forum: Optogenetics roundtable with Deisseroth and Gradinaru

Forum: Optogenetics roundtable with Deisseroth and Gradinaru

Optogenetics in pigeons

Optogenetics in pigeons

Color Sensing and Signal Transmission Diversity of Cyanobacterial Phytochromes and Cyanobacteriochromes.

To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.

An Introduction to Optogenetics: Novel Tools for Physiological Psychology Research

光遺伝学（オプトジェネティクス）は近年，神経科学分野において非常に注目を集めている実験技術であり，自由行動中の動物の脳内に光を照射することで目的の神経細胞集団および神経投射経路に限局した人工的な神経活動操作をミリ秒単位の時間分解能で行うことができる。本稿では，(1)光遺伝学において重要な役割を果たす光感受性チャネル・ポンプの仕組み，(2)およびこれらを標的脳領域に発現させ，特定の神経細胞集団や神経回路を活性化/不活性化する方法について紹介する。また，光遺伝学を用いて具体的に明らかになった最新の知見について，特に計算論的神経科学の研究に焦点を当てながら紹介する。

Application and research progress of optogenetic technology in retinal diseases

Optogenetics is a technique combining optics and the power of light with genetics; it uses light-mediated protein-protein interactions to control the open/closed state of channels or the activation/inactivation states of signaling components within live cells.Recently developed optogenetic tools offer exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution.The eye is a light-bioelectric conversion system.Compared with diseases of other organs, there are great advantages to apply optogenetics to the treatment of ophthalmic conditions, as new optical genetic tools are rapidly emerging.This article will focus on the main methods of optogenetic control of cells and the current status and outlook of its application in retinal diseases. Key words: Optogenetics; Opsins; Retinal diseases

Activation of parvalbumin-expressing neurons reconfigures neuronal ensembles in murine striatal microcircuits.

The striatum is the largest entrance to the basal ganglia. Diverse neuron classes make up striatal microcircuit activity, consisting in the sequential activation of neuronal ensembles. How different neuron classes participate in generating ensemble sequences is unknown. In control mus musculus brain slices in vitro, providing excitatory drive generates ensemble sequences. In Parkinsonian microcircuits captured by a highly recurrent ensemble, a cortical stimulus causes a transitory reconfiguration of neuronal groups alleviating Parkinsonism. Alternation between neuronal ensembles needs interconnectivity, in part due to interneurons, preferentially innervated by incoming afferents. One main class of interneuron expresses parvalbumin (PV+ neurons) and mediates feed-forward inhibition. However, its more global actions within the microcircuit are unknown. Using calcium imaging in ex vivo brain slices simultaneously recording dozens of neurons, we aimed to observe the actions of PV+ neurons within the striatal microcircuit. PV+ neurons in active microcircuits are 5%-11% of the active neurons even if, anatomically, they are <1% of the total neuronal population. In resting microcircuits, optogenetic activation of PV+ neurons turns on circuit activity by activating or disinhibiting, more neurons than those actually inhibited, showing that feed-forward inhibition is not their only function. Optostimulation of PV+ neurons in active microcircuits inhibits and activates different neuron sets, resulting in the reconfiguration of neuronal ensembles by changing their functional connections and ensemble membership, showing that neurons may belong to different ensembles at different situations. Our results show that PV+ neurons participate in the mechanisms that generate alternation of neuronal ensembles, therefore provoking ensemble sequences.

Research progress on ophthalmologic optogenetics

Optogenetics is a genetic technique that applies illumination with certain wavelength to modulate the biological activity of cells or subcellular components accurately.This technique is friendly to researchers of ophthalmology due to characteristics of the eyes including transparency, accessibility and comparative independence.Optogenetics can be used in retinal neurons, such as retinal ganglion cells (RGCs), even extended to vascular endothelial cells, immune cells and other ocular cells or cell substructures, which can further our understanding of ocular physiology and provide potential, therapeutic approaches for neurodegenerative diseases, vascular diseases, inflammation and other eye-related diseases.Improvement in effectiveness, safety and comfort is pivotal for this technique to expand application in ophthalmology and for its function to reach the physiology state of nomal eyes.In this review, a comprehensive analysis of optogenetics progress in ophthalmology was performed.Challenges in imaging including light sensitivity, spatial resolution and temporal resolution, and problems in expression involving local and systemic safety, specificity and persistence were reviewed. Key words: Ophthalmology; Optogenetics; Neurodegenerative disease

The BioLuminescent-OptoGenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex.

BioLuminescent (BL) light production can modulate neural activity and behavior through co-expressed OptoGenetic (OG) elements, an approach termed "BL-OG." Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and nontoxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically nonsensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

Focus on the treatment of inherited retinal diseases

Inherited retinal diseases (IRDs) are rare and incurable eye diseases.Gene therapy has become a new method for the treatment of IRDs.At present, at least 26 clinical trials of gene therapy involving at least 16 different IRDs genes are in progress or imminent.Such as autosomal recessive retinitis pigmentosa (RP, MERTK mutation), choroideremia (CHM mutation), Stargardt’s disease (ABCA4 mutation), Usher syndrome 1B subtype (Myo7a mutation), X-linked retinoschisis (RS1 mutation), mitochondrial-related Leber hereditary optic neuropathy (ND4 mutation), panchromatic blindness (CNGA3 mutation and CNNGB3 mutation), sex-linked RP (RPGR mutation). Adeno-associated viral (AAV) vector-mediated gene expression is a conventional method for gene transduction, which has showed efficacies in autosomal recessive IRDs caused by small gene mutations, especially for those IRDs, which have the original lesions in retinal pigment epithelial (RPE) cells and photoreceptor cells.Novel progress has been made in animal experiments by gene editing technologies for autosomal dominant IRDs or autosomal recessive IRDs with large gene mutations, which is going to clinical trial.Oligonucleotide therapy based on RNA has also shown good efficacy for the IRDs caused by large genes, which beyond the AAV capacity.These research achievements are attacking more and more attentions in these fields.Although there are still some efficacy and safety issues on the way to treat IRDs, it is reasonable to expect that more and more patients with IRDs will be treated in the near future. Key words: Inherited retinal diseases; Gene therapy; Clinical trials; Optogenetic gene therapy; Antisense Oliogonucleotide therapy

Response-contingent optogenetics to discover the mechanisms of nicotine-cue associations.

Response-contingent optogenetics to discover the mechanisms of nicotine-cue associations.

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

Optogenetics and Vision

Optogenetics and Vision

Optogenetics Therapy of Human Cancers

Optogenetics Therapy of Human Cancers

Advances of optogenetics in the treatment of retinitis pigmentosa

Retinitis pigmentosa (RP) is a disease that seriously affects vision. It mainly affects rod cells and causes night blindness. At the end of the disease, due to the simultaneous involvement of cone cells, the patient’s central vision and peripheral vision loss are not effective. There is no effective treatment method. However, some studies have found that although the function of photoreceptors is lost in the pathological process of RP, the function of bipolar cells and ganglion cells and the neural connection with the visual center are preserved, which provides a condition of therapeutic application in optogenetics for optogenetics. Optogenetics controls the excitability of neurons by expressing the light-sensitive protein represented by rhodopsin ion channel protein-2 on neurons, and has shown great application prospects in reshaping the photoreceptor function of the retina. The treatment of a type of retinal degenerative disease provides an effective treatment option. Key words: Retinitis pigmentosa/therapy; Optogenetics; Review

Modeling temperature during optogenetic illumination.

Modeling temperature during optogenetic illumination.

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

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.

Application of new techniques in mechanistic study of general anesthesia

Background The mechanisms underlying the actions of general anesthetics are poorly understood. The application of breakthrough techniques in neuroscience research could advance our understanding of general anesthesia. Objective To review the application of research techniques to study the mechanisms underlying the actions of general anesthetics. Content Studies using conventional neuroscience research techniques revealed that general anesthetics may cause deep anesthesia by targeting at least three parts, thalamocortical pathway, classical sleep-wake circuitry, and cortical fragmentation circuits. The combination of viral vector-assisted retrograde and anterograde neuronal tracing and transgenic Cre-mouse lines allows tracing neural pathways involving particular neuron types. Application of optogenetics, chemogenetics, in vivo calcium imaging, and microdialysis and neuroimaging techniques would overcome the obstacles unconquerable to conventional techniques and fulfill in vivo real-time neuroimaging, neurochemical and behavioral monitoring, and neuromodulations as well. Trend More investigations incorporate the multidisciplinary techniques to systemically study the potential targets of general anesthetics, and evaluate the involvement of the targets in various aspects of anesthesia. These studies could pave the avenue to eventually solve the mystery of general anesthesia. Key words: Anesthetics, general; Mechanism; New techniques

Research advances in the role of dopaminergic neurons involved in emergence from general anesthesia

Background Dopamine plays an important role in learning and memory, arousal, emotion and motor control. Recent studies found that dopaminergic (DA) neurons are also involved in both induction and emergence of general anesthesia. But the underlying mechanism is unclear. Objective To review the potential mechanisms underlying DA regulation of the emergence from general anesthesia. Content Midbrain DA neurons are densely distributed in the ventral tegmental area(VTA) and the substantia nigra pars compacta(SNc), where mesolimbic/mesocortical and mesostriatal DA pathways respectively originate. Genetic and pharmacological enhancement of the DA pathways promotes arousal. In contrast, knock-out of dopamine receptors or ablation of VTA abolishes the arousal effect of dopamine. Recordings of the activity of VTA DA neurons show an increase or a decrease in neuronal activity when subjects are in awake or sleeping state. It is putatively accepted that general anesthesia shares similar electroencephalographic signatures with sleep, so does emergence of general anesthesia with arousal. Several optogenetic studies reveal that activating VTA DA neurons facilitates the emergence of general anesthesia. Similarly, electrical stimuli in the VTA, instead of in the SNc, shortens the delay of the emergence from general anesthesia. Therefore, VTA DA neurons may have greater impact on the recovery from general anesthesia than SNc DA neurons. Trend DA are involved in the emergence from general anesthesia, and stimulating VTA DA neurons may provide a means to accelerate the recovery of general anesthesia. Key words: Dopaminergic neuron; Anesthesia, general; Consciousness recovery; Arousal