Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds.

Converting a Natural-Light-Driven Outward Proton Pump Rhodopsin into an Artificial Inward Proton Pump.

Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H) pumps, new subfamilies of microbial rhodopsins transporting H inwardly, i.e.

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design.

Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins.

Multimodal Functional Analysis Platform: 4. Optogenetics-Induced Oscillatory Activation to Explore Neural Circuits.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent.

Application of Optogenetics for Muscle Cells and Stem Cells.

This chapter describes the current progress of basic research, and potential therapeutic applications primarily focused on the optical manipulation of muscle cells and neural stem cells using microbial rhodopsin as a light-sensitive molecule. Since the contractions of skeletal, cardiac, and smooth muscle cells are mainly regulated through their membrane potential, several studies have been demonstrated to up- or downregulate the muscle contraction directly or indirectly using optogenetic actuators or silencers with defined stimulation patterns and intensities. Light-dependent oscillation of membrane potential also facilitates the maturation of myocytes with the development of T tubules and sarcomere structures, tandem arrays of minimum contractile units consists of contractile proteins and cytoskeletal proteins.

Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR.

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all- retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown.

Ultraflexible organic light-emitting diodes for optogenetic nerve stimulation.

Organic electronic devices implemented on flexible thin films are attracting increased attention for biomedical applications because they possess extraordinary conformity to curved surfaces. A neuronal device equipped with an organic light-emitting diode (OLED), used in combination with animals that are genetically engineered to include a light-gated ion channel, would enable cell type-specific stimulation to neurons as well as conformal contact to brain tissue and peripheral soft tissue. This potential application of the OLEDs requires strong luminescence, well over the neuronal excitation threshold in addition to flexibility.

Physical disuse contributes to widespread chronic mechanical hyperalgesia, tactile allodynia, and cold allodynia through neurogenic inflammation and spino-parabrachio-amygdaloid pathway activation.

Physical disuse could lead to a state of chronic pain typified by complex regional pain syndrome type I due to fear of pain through movement (kinesiophobia) or inappropriate resting procedures. However, the mechanisms by which physical disuse is associated with acute/chronic pain and other pathological signs remain unresolved. We have previously reported that inflammatory signs, contractures, disuse muscle atrophy, spontaneous pain-like behaviors, and chronic widespread mechanical hyperalgesia based on central plasticity occurred after 2 weeks of cast immobilization in chronic post-cast pain (CPCP) rat model.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.

Driving Neurogenesis in Neural Stem Cells with High Sensitivity Optogenetics.

Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems.

Expanding the Toolbox of Upconversion Nanoparticles for In Vivo Optogenetics and Neuromodulation.

Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio-temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near-infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation.

Optogenetic study of the response interaction among multi-afferent inputs in the barrel cortex of rats.

We investigated the relationship between whisker mechanoreceptive inputs and the neural responses to optical stimulation in layer 2/upper 3 (L2/U3) of the barrel cortex using optogenetics since, ideally, we should investigate interactions among inputs with spatiotemporal acuity. Sixteen whisker points of a transgenic rat (W-TChR2V4), that expresses channelrhodopsin 2 (ChR2)-Venus conjugate (ChR2V) in the peripheral nerve endings surrounding the whisker follicles, were respectively connected one-by-one with 16 LED-coupled optical fibres, which illuminated the targets according to a certain pattern in order to evaluate interactions among the inputs in L2/U3. We found that the individual L2/U3 neurons frequently received excitatory inputs from multiple whiskers that were arrayed in a row.

Analysis of Neuro-Neuronal Synapses Using Embryonic Chick Ciliary Ganglion via Single-Axon Tracing, Electrophysiology, and Optogenetic Techniques.

The calyx-type synapse is a giant synaptic structure in which a presynaptic terminal wraps around a postsynaptic neuron in a one-to-one manner. It has been used for decades as an experimental model system of the synapse due to its simplicity and high accessibility in physiological recording methods. In particular, the calyx of the embryonic chick ciliary ganglion (CG) has enormous potential for synapse science because more flexible genetic manipulations are available compared with other synapses.

Large Timescale Interrogation of Neuronal Function by Fiberless Optogenetics Using Lanthanide Micro-particles.

Optogenetics requires implantation of light-delivering optical fibers, as current light-sensitive opsins are activated by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photostimulation inherently damages brain tissue, and fiber tethering can restrict animal behavior. To overcome these technical limitations, we developed minimally invasive "fiberless" optogenetics using lanthanide micro-particles (LMPs), which emit up-conversion luminescence in the visible spectrum in response to irradiation with tissue-penetrating near-infrared light.

Quantitative study of the somatosensory sensitization underlying cross-modal plasticity.

Loss of one sensory modality can cause other types to become more perceptive (cross-modal plasticity). To test the hypothesis that the loss of vision changes the perceptual threshold in the somatosensory system, we applied optogenetics to directly manipulate the afferent inputs involved in the whisker-barrel system using a transgenic rat (W-TChR2V4) that expresses channelrhodopsin-2 (ChR2) selectively in the large mechanoreceptive neurons in the trigeminal ganglion (TG) and their peripheral nerve terminals. The licking behavior of W-TChR2V4 rat was conditioned to a blue LED light cue on the whisker area while the magnitude and duration of light pulses were varied.

Organelle Optogenetics: Direct Manipulation of Intracellular Ca Dynamics by Light.

As one of the ubiquitous second messengers, the intracellular Ca, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca-dependent signals: influx from the extracellular space and release from the intracellular Ca stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii).

Functional emergence of a column-like architecture in layer 5 of mouse somatosensory cortex in vivo.

To investigate how the functional architecture is organized in layer 5 (L5) of the somatosensory cortex of a mouse in vivo, the input-output relationship was investigated using an all-optical approach. The neural activity in L5 was optically recorded using a Ca sensor, R-CaMP2, through a microprism inserted in the cortex under two-photon microscopy, while the L5 was regionally excited using optogenetics. The excitability was spread around the blue-light irradiated region, but the horizontal propagation was limited to within a certain distance (λ < 130 μm from the center of the illumination spot).

Targeted expression of step-function opsins in transgenic rats for optogenetic studies.

Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase.

Optogenetic Activation of Non-Nociceptive Aβ Fibers Induces Neuropathic Pain-Like Sensory and Emotional Behaviors after Nerve Injury in Rats.

Neuropathic pain is caused by peripheral nerve injury (PNI). One hallmark symptom is allodynia (pain caused by normally innocuous stimuli), but its mechanistic underpinning remains elusive. Notably, whether selective stimulation of non-nociceptive primary afferent Aβ fibers indeed evokes neuropathic pain-like sensory and emotional behaviors after PNI is unknown, because of the lack of tools to manipulate Aβ fiber function in awake, freely moving animals.

Optogenetic conditioning of paradigm and pattern discrimination in the rat somatosensory system.

The rodent whisker-barrel cortical system is a model for studying somatosensory discrimination at high spatiotemporal precision. Here, we applied optogenetics to produce somatosensory inputs in the whisker area using one of transgenic rat lines, W-TChR2V4, which expresses channelrhodopsin-2 (ChR2) in the mechanoreceptive nerve endings around whisker follicles. An awake W-TChR2V4 rat was head-fixed and irradiated by blue LED light on the whisker area with a paradigm conditioned with a reward.

Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins.

Functional characterization of sodium-pumping rhodopsins with different pumping properties.

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization.