Blue Light Stimulation Research Articles

Luminescência opticamente estimulada em pastilhas de quartzo utilizando iluminação azul e verde

In this study, pellets fabricated from powdered rose quartz are investigated for their potential as ionizing radiation detectors, taking advantage of the abundant availability of quartz in the Earth's crust. For this purpose, optically stimulated luminescence (OSL) technique, using blue and green light stimulation, was used. The pellets were irradiated with a 90Sr/90Y beta source to doses from 80 to 550 mGy (dose rate = 0.08Gy/s). Analysis of the OSL decay curves showed a good fit with 3 exponential components (fast, medium, and slow). A linear dose-response relationship was observed within this interval of dose for both light stimulations. The repeatability of the OSL signal was found to be 3% for blue light and 2% for green light. Additionally, the fading experiment indicated that, after 1 hour, 41% and 35% of the OSL signal remains for blue and green stimulation, respectively. The results obtained highlight the promising usefulness of rose quartz-based pellets as effective detectors of ionizing radiation.

Light-responsive adipose-hypothalamus axis controls metabolic regulation

Light is fundamental for biological life, with most mammals possessing light-sensing photoreceptors in various organs. Opsin3 is highly expressed in adipose tissue which has extensive communication with other organs, particularly with the brain through the sympathetic nervous system (SNS). Our study reveals a new light-triggered crosstalk between adipose tissue and the hypothalamus. Direct blue-light exposure to subcutaneous white fat improves high-fat diet-induced metabolic abnormalities in an Opsin3-dependent manner. Metabolomic analysis shows that blue light increases circulating levels of histidine, which activates histaminergic neurons in the hypothalamus and stimulates brown adipose tissue (BAT) via SNS. Blocking central actions of histidine and denervating peripheral BAT blunts the effects of blue light. Human white adipocytes respond to direct blue light stimulation in a cell-autonomous manner, highlighting the translational relevance of this pathway. Together, these data demonstrate a light-responsive metabolic circuit involving adipose-hypothalamus communication, offering a potential strategy to alleviate obesity-induced metabolic abnormalities.

Chromatic Light Therapy for Inhibiting Myopia Progression: Human Studies.

Myopia, a common refractive error, has been associated with various risk factors, but time outdoors has emerged as a significant protective factor against its onset. This association is believed to be mediated by the influence of sunlight on dopamine release, a neurotransmitter crucial for regulating eye growth. Recent research has explored the specific properties of light in order to identify potential interventions for myopia control in children. Low-level red light therapy has gained attention, and has shown promise in inhibiting myopia progression, although there are concerns about safety and rebound effects. Similarly, blue light stimulation aims to upregulate retinal dopamine activity, yet conclusive evidence supporting its efficacy is lacking. Moreover, researchers explored the use of the entire visible light spectrum by digitally imposing longitudinal chromatic aberration to adjust proper eye growth. Preliminary findings suggest that digitally simulated chromatic aberration could potentially serve as a myopia control strategy and highlights the need for further investigation into long-term effects. As research progresses, understanding the efficacy and safety of light-based interventions for myopia control remains crucial for informing clinical practice and optimizing patient outcomes.

Exploration of the Graphene Quantum Dots-Blue Light Combination: A Promising Treatment against Bacterial Infection.

Graphene Quantum Dots (GQDs) have shown the potential for antimicrobial photodynamic treatment, due to their particular physicochemical properties. Here, we investigated the activity of three differently functionalized GQDs-Blue Luminescent GQDs (L-GQDs), Aminated GQDs (NH2-GQDs), and Carboxylated GQDs (COOH-GQDs)-against E. coli. GQDs were administrated to bacterial suspensions that were treated with blue light. Antibacterial activity was evaluated by measuring colony forming units (CFUs) and metabolic activities, as well as reactive oxygen species stimulation (ROS). GQD cytotoxicity was then assessed on human colorectal adenocarcinoma cells (Caco-2), before setting in an in vitro infection model. Each GQD exhibits antibacterial activity inducing ROS and impairing bacterial metabolism without significantly affecting cell morphology. GQD activity was dependent on time of exposure to blue light. Finally, GQDs were able to reduce E. coli burden in infected Caco-2 cells, acting not only in the extracellular milieu but perturbating the eukaryotic cell membrane, enhancing antibiotic internalization. Our findings demonstrate that GQDs combined with blue light stimulation, due to photodynamic properties, have a promising antibacterial activity against E. coli. Nevertheless, we explored their action mechanism and toxicity on epithelial cells, fixing and standardizing these infection models.

Hypersensitivity of Intrinsically Photosensitive Retinal Ganglion Cells in Migraine Induces Cortical Spreading Depression.

Migraine is a complex disorder characterized by episodes of moderate-to-severe, often unilateral headaches and generally accompanied by nausea, vomiting, and increased sensitivity to light (photophobia), sound (phonophobia), and smell (hyperosmia). Photophobia is considered the most bothersome symptom of migraine attacks. Although the underlying mechanism remains unclear, the intrinsically photosensitive retinal ganglion cells (ipRGCs) are considered to be involved in photophobia associated with migraine. In this study, we investigated the association between the sensitivity of ipRGCs and migraines and cortical spreading depression (CSD), which may trigger migraine attacks. The pupillary responses closely associated with the function of ipRGCs in patients with migraine who were irradiated with lights were evaluated. Blue (486 nm) light irradiation elicited a response from ipRGCs; however, red light (560 nm) had no such effect. Melanopsin, a photosensitive protein, phototransduces in ipRGCs following blue light stimulation. Hypersensitivity of ipRGCs was observed in patients with migraine. CSD was more easily induced with blue light than with incandescent light using a mouse CSD model. Moreover, CSD was suppressed, even in the presence of blue light, after injecting opsinamide, a melanopsin inhibitor. The hypersensitivity of ipRGCs in patients with migraine may induce CSD, resulting in migraine attacks.

Investigating luminescence signals of pharmaceuticals and dietary supplements for emergency dosimetry

This study investigates the potential use of dietary supplements and pharmaceuticals containing magnesium, calcium or potassium for emergency dosimetry applications using the phenomenon of optically stimulated luminescence (OSL). Signal measurements were carried out using different stimulation wavelengths, and blue light stimulation was found to be the most efficient. More than half of the samples exhibited a measurable OSL signal and relatively high radiation sensitivity compared to other previously measured emergency detectors. Moreover, samples generally demonstrated a linear dose response. Possible causes of their high zero-dose signal were investigated: mechanical processing and UV light excitation. As variability in sensitivity was observed, the test-dose protocol was used during measurements. Furthermore, the study showed a significant loss of OSL signal intensity within 24 h after irradiation, which suggests the necessity for a fading correction. Finally, a dose recovery test was performed to evaluate the materials and the test-dose protocol under realistic conditions. The findings indicate the potential for using pharmaceuticals and dietary supplements in the event of a radiation emergency due to their dosimetric properties and ease of obtaining.

Intracellular tau fragment droplets serve as seeds for tau fibrils

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils invitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Constitutive Androstane Receptor and Peroxisome Proliferator-Activated Receptor α Do Not Perform Liquid-Liquid Phase Separation in Cells.

Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are members of the nuclear receptor superfamily, which regulates various physiologic and pathologic processes. Phase separation is a dynamic biophysical process in which biomacromolecules form liquid-like condensates, which have been identified as contributors to many cellular functions, such as signal transduction and transcription regulation. However, the possibility of phase separation for CAR and PPARα remains unknown. This study explored the potential phase separation of CAR and PPARα The computational analysis utilizing algorithm tools examining the intrinsically disordered regions of CAR and PPARα suggested a limited likelihood of undergoing phase separation. Experimental assays under varying conditions of hyperosmotic stress and agonist treatments confirmed the absence of phase separation for these receptors. Additionally, the optoDroplets assay, which utilizes blue light stimulation to induce condensate formation, showed that there was no condensate formation of the fusion protein of Cry2 with CAR or PPARα Furthermore, phase separation of CAR or PPARα did not occur despite reduced target expression under hyperosmotic stress. In conclusion, these findings revealed that neither the activation of CAR and PPARα nor hyperosmotic stress induces phase separation of CAR and PPARα in cells. SIGNIFICANCE STATEMENT: Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are key regulators of various functions in the body. This study showed that CAR and PPARα do not exhibit phase separation under hyperosmotic stress or after agonist-induced activation. These findings provide new insights into the CAR and PPARα biology and physiology.

Optical control of cell-surface and endomembrane-exclusive β-adrenergic receptor signaling

Beta-adrenergic receptors (βARs) are G protein-coupled receptors (GPCRs) that mediate catecholamine hormone-induced stress responses, such as elevation of heart rate. Besides those that are plasma membrane-bound, endomembrane βARs are also signaling competent. Dysregulation of βAR pathways underlies severe pathological conditions. Emerging evidence indicates pathological molecular signatures in deeper endomembrane βARs signaling, likely contributing to conditions such as cardiomyocyte hypertrophy and apoptosis. However, the lack of approaches to control endomembrane β1ARs has impeded linking signaling with pathology. Informed by the β1AR-catecholamine interactions, we engineered an efficient photolabile proligand (OptoIso) to trigger βAR signaling exclusively in endomembrane regions using blue light stimulation. Not only does OptoIso undergo blue light deprotection in seconds, but also efficiently enters cells and allows examination of G protein heterotrimer activation exclusively at endomembranes. OptoIso also allows optical activation of plasma membrane βAR signaling in selected single cells with native fidelity, which can be reversed by terminating blue light. Thus, OptoIso will be a valuable experimental tool to elicit spatial and temporal control of βAR signaling in user-defined endomembrane or plasma membrane regions in unmodified cells with native fidelity.

Cell Ablation Techniques for the Larval Drosophila Neuromuscular System.

Tissue development requires local and long-distance communication between cells. Cell ablation experiments have provided critical insights into the functions of specific cell types and the tissue surrounding the dead cells. In the Drosophila neuromuscular system, ablation of motor neurons and muscles has revealed the roles of the ablated cells in axon pathfinding and circuit wiring. For example, when muscles are denervated due to laser ablation of their motor neuron inputs, they receive ectopic innervation from neighboring motor neurons. Here, we describe two methods of specific cell ablation. The first is a genetic ablation approach that uses GAL4 (ideally expressed in a small subset of cells) to drive expression of cell death genes reaper and head involution defective The second method relies on reactive oxygen species produced by light activation of the Arabidopsis-derived Singlet Oxygen Generator, miniSOG2, expressed in a subset of cells. For the latter, the precision stems from both the GAL4 and the restricting of the blue-light stimulation area.

Increase in choroidal thickness after blue light stimulation of the blind spot in young adults.

Blue light activates melanopsin, a photopigment that is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs). The axons of ipRGCs converge on the optic disc, which corresponds to the physiological blind spot in the visual field. Thus, a blue light stimulus aligned with the blind spot captures the ipRGCs axons at the optic disc. This study examined the potential changes in choroidal thickness and axial length associated with blue light stimulation of melanopsin-expressing ipRGCs at the blind spot. It was hypothesized that blue light stimulation at the blind spot in adults increases choroidal thickness. The blind spots of both eyes of 10 emmetropes and 10 myopes, with a mean age of 28 ± 6 years (SD), were stimulated locally for 1-minute with blue flickering light with a 460nm peak wavelength. Measurements of choroidal thickness and axial length were collected from the left eye before stimulation and over a 60-minute poststimulation period. At a similar time of day, choroidal thickness and axial length were measured under sham control condition in all participants, while a subset of 3 emmetropes and 3 myopes were measured after 1-minute of red flickering light stimulation of the blind spot with a peak wavelength of 620nm. Linear mixed model analyses were performed to examine the light-induced changes in choroidal thickness and axial length over time and between refractive groups. Compared with sham control (2 ± 1μm, n = 20) and red light (-1 ± 2μm, n = 6) stimulation, subfoveal choroidal thickness increased within 60min after blue light stimulation of the blind spot (7 ± 1μm, n = 20; main effect of light, p < 0.001). Significant choroidal thickening after blue light stimulation occurred in emmetropes (10 ± 2μm, p < 0.001) but not in myopes (4 ± 2μm, p > 0.05). Choroidal thickening after blue light stimulation was greater in the fovea, diminishing in the parafoveal and perifoveal regions. There was no significant main effect of light, or light by refractive error interaction on the axial length after blind spot stimulation. These findings demonstrate that stimulating melanopsin-expressing axons of ipRGCs at the blind spot with blue light increases choroidal thickness in young adults. This has potential implications for regulating eye growth.

Luminescence properties of SrB6O10:Ce,Gd phosphor for applications in OSL dosimetry and lighting

Strontium hexaborate (SrB6O10) doped with different metals and/or lanthanides has been studied widely as the thermoluminescence (TL) dosimeter. However, there is almost no dosimetry study concerning the utility of Optically Stimulated Luminescence (OSL) on SrB6O10. This preliminary study aims to examine the luminescence properties of SrB6O10 phosphor for their application in the areas of dosimetry and lighting technologies. Undoped and doped material to get an OSL dosimeter were synthesized by incorporating the dopants Ce3+, in the range of 0.1–0.7 % wt, and Gd3+, in the range of 0.1–1 % wt into the SrB6O10 structure using the solution combustion (SCS) method. The structural and morphological characterizations were performed using X-ray diffraction (XRD) and Scanning Electron Microscope (SEM) methods, respectively. Two TL peaks of SrB6O10:Ce0.1%,Gd0.5% occurred at 70 °C and 182 °C after 1 Gy exposure to beta rays with a heating rate of 2 °C/s. The step annealing measurements were carried out to determine the traps responsible for the OSL signal. Various properties of the luminophore including dose-response and reusability were investigated using blue light stimulation. The phosphor showed a little variation of response up to 20 cycles within 2 % deviation (STD) from the first OSL readout. The OSL output within the range of 2–20 Gy was determined as linear. Radioluminescence (RL) measurement was investigated by emission in the range λem = 200–1000 nm. The parent compound displayed UV emission subdivided into UVA (315–400 nm), and UVB (280–315 nm) emissions at room temperature. The Chromaticity Coordinates (CIE) of SrB6O10:Ce0.1%,Gd0.5% were calculated as x = 0.2165, y = 0.1767. These remarkable characteristics show that, if developed, SrB6O10 could be used as an OSL dosimeter.

Wirkmechanismus der Blaulicht-Stimulation des blinden Flecks bei progredienter Myopie

Purpose: The beneficial effects of time spent outdoors and the seasonal increase of progression in winter emphasize the major role of daylight in myopia. Based upon the patho­physiology of myopia and the physiological effects of daylight, the mechanism of action of a targeted stimulation of melanopsin by blue light at the blind spot for the treatment of progressive myopia is investigated. Material and Methods: The manuscript is a subjective review of the mechanism of action of blue light stimulation at the blind spot for the treatment of progressive myopia. The literature search in PubMed covered a period from 1980 through 2023. Results: Blue light stimulation at the blind spot bases upon the clinical experience that daylight protects from the development of progressive myopia. Parameters and site of light stimulation are mainly defined by the photopigment melanopsin, that is sensitive to blue light and localized on intrinsically photosensitive, retinal ganglion cells (ipRGC) whose axons converge to the blind spot. The blind spot as stimulation site provides on the one hand the opportunity to activate the vast majority of ipRGC and hence dopaminergic amacrine cells, and on the other hand avoids additional involvement of rods and cones which may exert inhibitory effects on dopamine release. Furthermore, the proposed dopaminergic and potentially antimyopic effect can be applied invisibly to the patient. Conclusion: Preclinical and human experimental studies indicate that after a short-term blue light stimulation the choroidal thickness transiently increases and the axial length of the eye decreases. These temporal changes of clinically relevant biomarkers after short-term blue light stimulation suggest potential antimyopic effects which are investigated in current clinical trials in children.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

Exogenous Pregnane X Receptor Does Not Undergo Liquid-Liquid Phase Separation in Nucleus under Cell-Based In Vitro Conditions.

Pregnane X receptor (PXR) belongs to the nuclear receptor superfamily that plays a crucial role in hepatic physiologic and pathologic conditions. Phase separation is a process in which biomacromolecules aggregate and condense into a dense phase as liquid condensates and coexist with a dilute phase, contributing to various cellular and biologic functions. Until now, whether PXR could undergo phase separation remains unclear. This study aimed to investigate whether PXR undergoes phase separation. Analysis of the intrinsically disordered regions (IDRs) using algorithm tools indicated a low propensity of PXR to undergo phase separation. Experimental assays such as hyperosmotic stress, agonist treatment, and optoDroplets assay demonstrated the absence of phase separation for PXR. OptoDroplets assay revealed the inability of the fusion protein of Cry2 with PXR to form condensates upon blue light stimulation. Moreover, phase separation of PXR did not occur even though the mRNA and protein expression levels of PXR target, cytochrome P450 3A4, changed after sorbitol treatment. In conclusion, for the first time, these findings suggested that exogenous PXR does not undergo phase separation following activation or under hyperosmotic stress in nucleus of cells. SIGNIFICANCE STATEMENT: PXR plays a critical role in hepatic physiological and pathological processes. The present study clearly demonstrated that exogenous PXR does not undergo phase separation after activation by agonist or under hyperosmotic stress in nucleus. These findings may help understand PXR biology.

Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation.

Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis

Background & AimsIntestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease (IBD). MethodsMice were fed 2.5% dextran sodium sulfate (DSS) for seven days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the mid-colon of ChAT::Cre;R26-iDTR (ChAT-iDTR) mice by local injection of diphtheria toxin prior to colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 (ChAT-ChR2) mice, in which ChAT+ neurons express channelrhodopsin-2 (ChR2), with daily blue light stimulation (BLS) delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, qPCR, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. ResultsAblation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both ACh and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in LPS-stimulated muscularis macrophages. ConclusionsThese findings demonstrate that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential IBD treatment.

Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.

Optogenetic Activation of Peripheral Somatosensory Neurons in Transgenic Mice as a Neuropathic Pain Model for Assessing the Therapeutic Efficacy of Analgesics.

Optogenetics is a novel biotechnology widely used to precisely manipulate a specific peripheral sensory neuron or neural circuit. However, the use of optogenetics to assess the therapeutic efficacy of analgesics is elusive. In this study, we generated a transgenic mouse stain in which all primary somatosensory neurons can be optogenetically activated to mimic neuronal hyperactivation in the neuropathic pain state for the assessment of analgesic effects of drugs. A transgenic mouse was generated using the advillin-Cre line mated with the Ai32 strain, in which channelrhodopsin-2 fused to enhanced yellow fluorescence protein (ChR2-EYFP) was conditionally expressed in all types of primary somatosensory neurons (advillincre/ChR2+/+). Immunofluorescence and transdermal photostimulation on the hindpaws were used to verify the transgenic mice. Optical stimulation to evoke pain-like paw withdrawal latency was used to assess the analgesic effects of a series of drugs. Injury- and pain-related molecular biomarkers were investigated with immunohistofluorescence. We found that the expression of ChR2-EYFP was observed in many primary afferents of paw skin and sciatic nerves and in primary sensory neurons and laminae I and II of the spinal dorsal horns in advillincre/ChR2+/+ mice. Transdermal blue light stimulation of the transgenic mouse hindpaw evoked nocifensive paw withdrawal behavior. Treatment with gabapentin, some channel blockers, and local anesthetics, but not opioids or COX-1/2 inhibitors, prolonged the paw withdrawal latency in the transgenic mice. The analgesic effect of gabapentin was also verified by the decreased expression of injury- and pain-related molecular biomarkers. These optogenetic mice provide a promising model for assessing the therapeutic efficacy of analgesics in neuropathic pain.

Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics

Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.