Optogenetic inhibition of the limbic corticothalamic circuit does not alter spontaneous oscillatory activity, auditory-evoked oscillations, and deviant detection.

Aberrant neuronal circuit dynamics are at the core of complex neuropsychiatric disorders, such as schizophrenia (SZ). Clinical assessment of the integrity of neuronal circuits in SZ has consistently described aberrant resting-state gamma oscillatory activity, decreased auditory-evoked gamma responses, and abnormal mismatch responses. We hypothesized that corticothalamic circuit manipulation could recapitulate SZ circuit phenotypes in rodent models. In this study, we optogenetically inhibited the mediodorsal thalamus-to-prefrontal cortex (MDT-to-PFC) or the PFC-to-MDT projection in rats and assessed circuit function through electrophysiological readouts. We found that MDT-PFC perturbation could not recapitulate SZ-linked phenotypes such as broadband gamma disruption, altered evoked oscillatory activity, and diminished mismatch negativity responses. Therefore, the induced functional impairment of the MDT-PFC pathways cannot account for the oscillatory abnormalities described in SZ.

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells.

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.

Blue light-mediated gene expression as a promising strategy to reduce antibiotic resistance in Escherichia coli.

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of ß-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.

Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Optogenetic therapeutic strategies for diabetes mellitus.

Diabetes mellitus (DM) is a common chronic disease affecting humans globally. It is characterized by abnormally elevated blood glucose levels due to the failure of insulin production or reduction of insulin sensitivity and functionality. Insulin and glucagon-like peptide (GLP)-1 replenishment or improvement of insulin resistance are the two major strategies to treat diabetes. Recently, optogenetics that uses genetically encoded light-sensitive proteins to precisely control cell functions has been regarded as a novel therapeutic strategy for diabetes. Here, we summarize the latest development of optogenetics and its integration with synthetic biology approaches to produce light-responsive cells for insulin/GLP-1 production, amelioration of insulin resistance and neuromodulation of insulin secretion. In addition, we introduce the development of cell encapsulation and delivery methods and smart bioelectronic devices for the in vivo application of optogenetics-based cell therapy in diabetes. The remaining challenges for optogenetics-based cell therapy in the clinical translational study are also discussed.

Removing direct photocurrent artifacts in optogenetic connectivity mapping data via constrained matrix factorization.

Monosynaptic connectivity mapping is crucial for building circuit-level models of neural computation. Two-photon optogenetic stimulation, when combined with whole-cell recording, enables large-scale mapping of physiological circuit parameters. In this experimental setup, recorded postsynaptic currents are used to infer the presence and strength of connections. For many cell types, nearby connections are those we expect to be strongest. However, when the postsynaptic cell expresses opsin, optical excitation of nearby cells can induce direct photocurrents in the postsynaptic cell. These photocurrent artifacts contaminate synaptic currents, making it difficult or impossible to probe connectivity for nearby cells. To overcome this problem, we developed a computational tool, Photocurrent Removal with Constraints (PhoRC). Our method is based on a constrained matrix factorization model which leverages the fact that photocurrent kinetics are less variable than those of synaptic currents. We demonstrate on real and simulated data that PhoRC consistently removes photocurrents while preserving synaptic currents, despite variations in photocurrent kinetics across datasets. Our method allows the discovery of synaptic connections which would have been otherwise obscured by photocurrent artifacts, and may thus reveal a more complete picture of synaptic connectivity. PhoRC runs faster than real time and is available as open source software.

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer.

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Closed-loop optogenetic neuromodulation enables high-fidelity fatigue-resistant muscle control.

Closed-loop neuroprostheses show promise in restoring motion in individuals with neurological conditions. However, conventional activation strategies based on functional electrical stimulation (FES) fail to accurately modulate muscle force and exhibit rapid fatigue because of their unphysiological recruitment mechanism. Here, we present a closed-loop control framework that leverages physiological force modulation under functional optogenetic stimulation (FOS) to enable high-fidelity muscle control for extended periods of time (>60 minutes) in vivo. We first uncovered the force modulation characteristic of FOS, showing more physiological recruitment and significantly higher modulation ranges (>320%) compared with FES. Second, we developed a neuromuscular model that accurately describes the highly nonlinear dynamics of optogenetically stimulated muscle. Third, on the basis of the optogenetic model, we demonstrated real-time control of muscle force with improved performance and fatigue resistance compared with FES. This work lays the foundation for fatigue-resistant neuroprostheses and optogenetically controlled biohybrid robots with high-fidelity force modulation.

Systems for Targeted Silencing of Gene Expression and Their Application in Plants and Animals.

At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes-plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.

Heliorhodopsin-mediated light-modulation of ABC transporter.

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Theoretical prediction of broadband ambient light optogenetic vision restoration with ChRmine and its mutants.

Vision restoration is one of the most promising applications of optogenetics. However, it is limited due to the poor-sensitivity, slow-kinetics and narrow band absorption spectra of opsins. Here, a detailed theoretical study of retinal ganglion neurons (RGNs) expressed with ChRmine, ReaChR, CoChR, CatCh and their mutants, with near monochromatic LEDs, and broadband sunlight, halogen lamp, RGB LED light, and pure white light sources has been presented. All the opsins exhibit improved light sensitivity and larger photocurrent on illuminating with broadband light sources compared to narrow band LEDs. ChRmine allows firing at ambient sunlight (1.5 nW/mm2) and pure white light (1.2 nW/mm2), which is lowest among the opsins considered. The broadband activation spectrum of ChRmine and its mutants is also useful to restore color sensitivity. Although ChRmine exhibits slower turn-off kinetics with broadband light, high-fidelity spikes can be evoked upto 50 Hz. This limit extends upto 80 Hz with the improved hsChRmine mutant although it requires double the irradiance compared to ChRmine. The present study shows that ChRmine and its mutants allow activation of RGNs with ambient light which is useful for goggle-free white light optogenetic retinal prostheses with improved quality of restored vision.

Nano-optogenetics for Disease Therapies.

Optogenetic, known as the method of 21 centuries, combines optic and genetic engineering to precisely control photosensitive proteins for manipulation of a broad range of cellular functions, such as flux of ions, protein oligomerization and dissociation, cellular intercommunication, and so on. In this technique, light is conventionally delivered to targeted cells through optical fibers or micro light-emitting diodes, always suffering from high invasiveness, wide-field illumination facula, strong absorption, and scattering by nontargeted endogenous substance. Light-transducing nanomaterials with advantages of high spatiotemporal resolution, abundant wireless-excitation manners, and easy functionalization for recognition of specific cells, recently have been widely explored in the field of optogenetics; however, there remain a few challenges to restrain its clinical applications. This review summarized recent progress on light-responsive genetically encoded proteins and the myriad of activation strategies by use of light-transducing nanomaterials and their disease-treatment applications, which is expected for sparking helpful thought to push forward its preclinical and translational uses.

Photoacoustic: A Versatile Nongenetic Method for High-Precision Neuromodulation.

High-precision neuromodulation plays a pivotal role in elucidating fundamental principles of neuroscience and treating specific neurological disorders. Optical neuromodulation, enabled by spatial resolution defined by the diffraction limit at the submicrometer scale, is a general strategy to achieve such precision. Optogenetics offers single-neuron spatial resolution with cellular specificity, whereas the requirement of genetic transfection hinders its clinical application. Direct photothermal modulation, an alternative nongenetic optical approach, often associates a large temperature increase with the risk of thermal damage to surrounding tissues.Photoacoustic (also called optoacoustic) neural stimulation is an emerging technology for neural stimulation with the following key features demonstrated. First, the photoacoustic approach demonstrated high efficacy without the need for genetic modification. The generated pulsed ultrasound upon ns laser pulses with energy ranging from a few µJ to tens of µJ is sufficient to activate wild-type neurons. Second, the photoacoustic approach provides sub-100-µm spatial precision. It overcomes the fundamental wave diffraction limit of ultrasound by harnessing the localized ultrasound field generated through light absorption. A spatial precision of 400 µm has been achieved in rodent brains using a fiber-based photoacoustic emitter. Single-cell stimulation in neuronal cultures in vitro and in brain slices ex vivo is achieved using tapered fiber-based photoacoustic emitters. This precision is 10 to 100 times better than that for piezo-based low-frequency ultrasound and is essential to pinpoint a specific region or cell population in a living brain. Third, compared to direct photothermal stimulation via temperature increase, photoacoustic stimulation requires 40 times less laser energy dose to evoke neuron activities and is associated with a minimal temperature increase of less than 1 °C, preventing potential thermal damage to neurons. Fourth, photoacoustics is a versatile approach and can be designed in various platforms aiming at specific applications. Our team has shown the design of fiber-based photoacoustic emitters, photoacoustic nanotransducers, soft biocompatible photoacoustic films, and soft photoacoustic lenses. Since they interact with neurons through ultrasound without the need for direct contact, photoacoustic enables noninvasive transcranial and dura-penetrating brain stimulation without compromising high precision.In this Account, we will first review the basic principles of photoacoustic and discuss the key design elements of PA transducers for neural modulation guided by the principle. We will also highlight how these design goals were achieved from a materials chemistry perspective. The design of different PA interfaces, their unique capability, and their applications in neural systems will be reviewed. In the end, we will discuss the remaining challenges and future perspectives for this technology.

Optogenetically controlled inflammasome activation demonstrates two phases of cell swelling during pyroptosis.

Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

Postoperatively Noninvasive Optogenetic Stimulation via Upconversion Nanoparticles Enhancing Sciatic Nerve Repair.

The efficacy of electrical stimulation facilitating peripheral nerve regeneration is evidenced extensively, while the associated secondary damage resulting from repeated electrode invasion and indiscriminate stimulation is inevitable. Here, we present an optogenetics strategy that utilizes upconversion nanoparticles (UCNPs) to convert deeply penetrating near-infrared excitation into blue emission, which activates an adeno-associated virus-encoding ChR2 photoresponsive ion channel on cell membranes. The induced Ca2+ flux, similar to the ion flux in the electrical stimulation approach, efficiently regulates viability and proliferation, secretion of nerve growth factor, and neural function of RSC96 cells. Furthermore, deep near-infrared excitation is harnessed to stimulate autologous Schwann cells in situ via a UCNP-composited scaffold, which enhances nerve sprouting and myelination, consequently promoting functional recovery, electrophysiological restoration, and reinnervation of damaged nerves. This developed postoperatively noninvasive optogenetics strategy presents a novel, minimally traumatic, and enduring therapeutic stimulus to effectively promote peripheral nerve repair.

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro.

The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive split transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.

Controlling neural activity: LPV modelling of optogenetically actuated Wilson-Cowan model.

Objective.This paper aims to bridge the gap between neurophysiology and automatic control methodologies by redefining the Wilson-Cowan (WC) model as a control-oriented linear parameter-varying (LPV) system. A novel approach is presented that allows for the application of a control strategy to modulate and track neural activity.Approach.The WC model is redefined as a control-oriented LPV system in this study. The LPV modelling framework is leveraged to design an LPV controller, which is used to regulate and manipulate neural dynamics.Main results.Promising outcomes, in understanding and controlling neural processes through the synergistic combination of control-oriented modelling and estimation, are obtained in this study. An LPV controller demonstrates to be effective in regulating neural activity.Significance.The presented methodology effectively induces neural patterns, taking into account optogenetic actuation. The combination of control strategies with neurophysiology provides valuable insights into neural dynamics. The proposed approach opens up new possibilities for using control techniques to study and influence brain functions, which can have key implications in neuroscience and medicine. By means of a model-based controller which accounts for non-linearities, noise and uncertainty, neural signals can be induced on brain structures.

Control of polymers' amorphous-crystalline transition enables miniaturization and multifunctional integration for hydrogel bioelectronics.

Soft bioelectronic devices exhibit motion-adaptive properties for neural interfaces to investigate complex neural circuits. Here, we develop a fabrication approach through the control of metamorphic polymers' amorphous-crystalline transition to miniaturize and integrate multiple components into hydrogel bioelectronics. We attain an about 80% diameter reduction in chemically cross-linked polyvinyl alcohol hydrogel fibers in a fully hydrated state. This strategy allows regulation of hydrogel properties, including refractive index (1.37-1.40 at 480 nm), light transmission (>96%), stretchability (139-169%), bending stiffness (4.6 ± 1.4 N/m), and elastic modulus (2.8-9.3 MPa). To exploit the applications, we apply step-index hydrogel optical probes in the mouse ventral tegmental area, coupled with fiber photometry recordings and social behavioral assays. Additionally, we fabricate carbon nanotubes-PVA hydrogel microelectrodes by incorporating conductive nanomaterials in hydrogel for spontaneous neural activities recording. We enable simultaneous optogenetic stimulation and electrophysiological recordings of light-triggered neural activities in Channelrhodopsin-2 transgenic mice.

Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.

Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aß clearance in an autophagy-dependent manner in cellular models, and alleviates Aß-induced paralysis in the Caenorhabditis elegans model of Alzheimer's disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.

Recent clinical trials and optical control as a potential strategy to develop microtubule-targeting drugs in colorectal cancer management.

Colorectal cancer (CRC) has remained the second and the third leading cause of cancer-related death worldwide and in the United States, respectively. Although significant improvement in overall survival has been achieved, death in adult populations under the age of 55 appears to have increased in the past decades. Although new classes of therapeutic strategies such as immunotherapy have emerged, their application is very limited in CRC so far. Microtubule (MT) inhibitors such as taxanes, are not generally successful in CRC. There may be some way to make MT inhibitors work effectively in CRC. One potential advantage that we can take to treat CRC may be the combination of optical techniques coupled to an endoscope or other fiber optics-based devices. A combination of optical devices and photo-activatable drugs may allow us to locally target advanced CRC cells with highly potent MT-targeting drugs. In this Editorial review, we would like to discuss the potential of optogenetic approaches in CRC management.