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Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions.

During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila. This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Working memory, the process through which information is transiently maintained and manipulated over a brief period, is essential for most cognitive functions1-4. However, the mechanisms underlying the generation and evolution of working-memory neuronal representations at the population level over long timescales remain unclear. Here, to identify these mechanisms, we trained head-fixed mice to perform an olfactory delayed-association task in which the mice made decisions depending on the sequential identity of two odours separated by a 5 s delay. Optogenetic inhibition of secondary motor neurons during the late-delay and choice epochs strongly impaired the task performance of the mice. Mesoscopic calcium imaging of large neuronal populations of the secondary motor cortex (M2), retrosplenial cortex (RSA) and primary motor cortex (M1) showed that many late-delay-epoch-selective neurons emerged in M2 as the mice learned the task. Working-memory late-delay decoding accuracy substantially improved in the M2, but not in the M1 or RSA, as the mice became experts. During the early expert phase, working-memory representations during the late-delay epoch drifted across days, while the stimulus and choice representations stabilized. In contrast to single-plane layer 2/3 (L2/3) imaging, simultaneous volumetric calcium imaging of up to 73,307 M2 neurons, which included superficial L5 neurons, also revealed stabilization of late-delay working-memory representations with continued practice. Thus, delay- and choice-related activities that are essential for working-memory performance drift during learning and stabilize only after several days of expert performance.

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.

SIGNIFICANCE: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AIM: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. APPROACH: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. RESULTS: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. CONCLUSIONS: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Establishing reliable intravenous catheterization in mice with optical implants allows the combination of neural manipulations and recordings with rapid, time-locked delivery of pharmacological agents. Here we present a procedure for handmade jugular vein catheters designed for head-mounted intravenous access and provide surgical and postoperative guidance for improved survival and patency. A head-mounted vascular access point eliminates the need for a back-mounted button in animals already receiving neural implants, thereby reducing sites of implantation. This protocol, which is readily adoptable by experimenters with previous training and experience in mouse surgery, enables repeated fiber photometry recordings or optogenetic manipulation during drug delivery in adult mice that are awake and behaving, whether head fixed or freely moving. With practice, an experienced surgeon requires ~30 min to perform catheterization on each mouse. Altogether, these techniques facilitate the reliable and repeated delivery of pharmacological agents in mouse models while simultaneously recording at high temporal resolution and/or manipulating neural populations.

Technical innovation in neuroscience introduced powerful tools for measuring and manipulating neuronal activity via optical, chemogenetic, and calcium-imaging tools. These tools were initially tested primarily in male animals but are now increasingly being used in females as well. In this review, we consider how these tools may work differently in males and females. For example, we review sex differences in the metabolism of chemogenetic ligands and their downstream signaling effects. Optical tools more directly alter depolarization or hyperpolarization of neurons, but biological sex and gonadal hormones modulate synaptic inputs and intrinsic excitability. We review studies demonstrating that optogenetic manipulations are sometimes consistent across the rodent estrous cycle but within certain circuits; manipulations can vary across the ovarian cycle. Finally, calcium-imaging methods utilize genetically encoded calcium indicators to measure neuronal activity. Testosterone and estradiol can directly modulate calcium influx, and we consider these implications for interpreting the results of calcium-imaging studies. Together, our findings suggest that these neuroscientific tools may sometimes work differently in males and females and that users should be aware of these differences when applying these methods.

BACKGROUND & AIMS: Intestinal 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. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation 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, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation 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 acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Identifying the properties of the rapid eye movement (REM) sleep circuitry and its relation to diseases has been challenging due to the neuronal heterogeneity of the brainstem. Here, we show in mice that neurons in the pontine sublaterodorsal tegmentum (SubLDT) that express corticotropin-releasing hormone-binding protein (Crhbp + neurons) and project to the medulla promote REM sleep. Within the medullary area receiving projections from Crhbp + neurons, neurons expressing nitric oxide synthase 1 (Nos1 + neurons) project to the SubLDT and promote REM sleep, suggesting a positively interacting loop between the pons and the medulla operating as a core REM sleep circuit. Nos1 + neurons also project to areas that control wide forebrain activity. Ablating Crhbp + neurons reduces sleep and impairs REM sleep atonia. In Parkinson's disease patients with REM sleep behavior disorders, CRHBP-immunoreactive neurons are largely reduced and contain pathologic α-synuclein, providing insight into the mechanisms underlying the sleep deficits characterizing this disease. [Display omitted] • Crhbp + SubLDT→Gi neurons promote REM sleep and muscle atonia • Nos1 + DPGi,Pr neurons promote REM sleep and project to areas regulating forebrain activity • The above 2 neuronal populations constitute a pontine-medullary loop circuit • CRHBP+ SubLDT neurons are lost in human patients with Parkinson's disease and RBD A neuronal circuit originating in the brainstem pons and medulla strongly induces REM sleep, even in awake mice. Moreover, a subpopulation of neurons within this circuit is lost in patients with Parkinson's disease, a neurodegenerative disease characterized by severe REM sleep abnormalities. [ABSTRACT FROM AUTHOR]

Degenerative eye diseases cause partial or complete blindness due to photoreceptor degeneration. Optogenetic gene therapy is a revolutionary technique combining genetics and optical methods to control the function of neurons. Due to the inherent risk of photochemical damage, the light intensity necessary to activate Opto-mGluR6 surpasses the safe threshold for retinal illumination. Conversely, red-shifted lights pose a significantly lower risk of inducing such damage compared to blue lights. We designed red-shifted Opto-mGluR6 photopigments with a wide, red-shifted working spectrum compared to Opto-mGluR6 and examined their excitation capability in vitro. ROM19, ROM18 and ROM17, red-shifted variants of Opto-mGluR6, were designed by careful bioinformatics/computational studies. The predicted molecules with the best scores were selected, synthesised and cloned into the pAAV-CMV-IRES-EGFP vector. Expression of constructs was confirmed by functional assessment in engineered HEK-GIRK cells. Spectrophotometry and patch clamp experiments demonstrated that the candidate molecules were sensitive to the desired wavelengths of the light and directly coupled light stimuli to G-protein signalling. Herein, we introduce ROM17, ROM18 and ROM19 as newly generated, red-shifted variants with maximum excitation red-shifted of ~ 40nm, 70 nm and 126 nm compared to Opto-mGluR6. [ABSTRACT FROM AUTHOR]

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. [Display omitted] • Vinculin is mechanosensitively recruited to tricellular junctions • Vinculin's actin-binding function is needed for tricellular actomyosin organization • Vinculin reinforces tricellular junction integrity in response to increased tension • Vinculin is required to maintain barrier function at tricellular junctions Epithelial tricellular junctions are vulnerable to disruption. Here, van den Goor et al. reveal that Vinculin is mechanosensitively recruited to vertebrate tricellular junctions, where it regulates actomyosin organization and barrier function. Vinculin reinforces tricellular junction integrity when epithelia experience increased mechanical force. [ABSTRACT FROM AUTHOR]

Inhibitory interneurons are pivotal components of cortical circuits. Beyond providing inhibition, they have been proposed to coordinate the firing of excitatory neurons within cell assemblies. While the roles of specific interneuron subtypes have been extensively studied, their influence on pyramidal cell synchrony in vivo remains elusive. Employing an all-optical approach in mice, we simultaneously recorded hippocampal interneurons and pyramidal cells and probed the network influence of individual interneurons using optogenetics. We demonstrate that CA1 interneurons form a functionally interconnected network that promotes synchrony through disinhibition during awake immobility, while preserving endogenous cell assemblies. Our network model underscores the importance of both cell assemblies and dense, unspecific interneuron connectivity in explaining our experimental findings, suggesting that interneurons may operate not only via division of labor but also through concerted activity. Interneurons control the timing of activation of excitatory neurons, but how single interneurons affect synchrony in vivo is unclear. This study shows that CA1 hippocampus interneurons form a functionally connected network that promotes synchrony between pyramidal cells at the single neuron level. [ABSTRACT FROM AUTHOR]

Focused ultrasound can non-invasively modulate neural activity, but whether effective stimulation parameters generalize across brain regions and cell types remains unknown. We used focused ultrasound coupled with fiber photometry to identify optimal neuromodulation parameters for four different arousal centers of the brain in an effort to yield overt changes in behavior. Applying coordinate descent, we found that optimal parameters for excitation or inhibition are highly distinct, the effects of which are generally conserved across brain regions and cell types. Optimized stimulations induced clear, target-specific behavioral effects, whereas non-optimized protocols of equivalent energy resulted in substantially less or no change in behavior. These outcomes were independent of auditory confounds and, contrary to expectation, accompanied by a cyclooxygenase-dependent and prolonged reduction in local blood flow and temperature with brain-region-specific scaling. These findings demonstrate that carefully tuned and targeted ultrasound can exhibit powerful effects on complex behavior and physiology. [Display omitted] • Optimal ultrasound neuromodulation parameters generalize across neuronal cell types • Distinct parameters for excitation and inhibition are identified • Targeted stimulation induces site-specific behavioral changes • COX2-dependent vasoconstriction and brain cooling occur with excitatory protocols The field of focused ultrasound (FUS) neuromodulation is undergoing rapid growth but is limited by our understanding of effective waveform parameters and cell-type effects. Murphy et al. combine deep-brain optical readouts with FUS technology to uncover waveforms for inducing powerful, bidirectional neuromodulation across a collection of therapeutically important brain cell types. [ABSTRACT FROM AUTHOR]

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion. [Display omitted] • Optogenetics-driven TANGO1S-Sec23A binding at ERES inhibits cargo export • Sustained TANGO1S-Sec23A-binding clusters ERES • 47% of ERES handle bulky cargo export, while small cargoes use ERES indiscriminately • ERES specialization balances the simultaneous export of both small and bulky cargoes Using optogenetics, Saxena et al. demonstrated that prolonged binding of TANGO1S-Sec23A stalls cargoes at ERES. Under these conditions, bulky cargoes like collagen concentrate at about 47% of these stalled ERES, whereas small cargoes accumulate uniformly, supporting segregation of export sites based on cargo size. [ABSTRACT FROM AUTHOR]

Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors. [Display omitted] • Late endosomes (LEs) surf over the ER network in continuous contact with Protrudin • LEs hop and glide along microtubules to pass mitochondria • Bidirectionally moving LEs do not exchange among Rab7 effectors or motor types • MAPs control the directionality of LE transport through dynein-dynactin assembly Microtubule-linked endosomes move bidirectionally through the crowded cytoplasm, maintaining contacts with the ER. Using endogenously tagged proteins, Jongsma et al. show that bidirectional transport control via the Rab7-effector-motor axis is provided by microtubule-associated proteins (MAPs). [ABSTRACT FROM AUTHOR]

For organisms tracking a chemical cue to its source, the motion of their surrounding fluid provides crucial information for success. Swimming and flying animals engaged in olfaction-driven search often start by turning into the direction of an oncoming wind or water current. However, it is unclear how organisms adjust their strategies when directional cues are absent or unreliable, as is often the case in nature. Here, we use the genetic toolkit of Drosophila melanogaster to develop an optogenetic paradigm to deliver temporally precise "virtual" olfactory experiences for free-flying animals in either laminar wind or still air. We first confirm that in laminar wind flies turn upwind. Furthermore, we show that they achieve this using a rapid (∼ 100 ms) turn, implying that flies estimate the ambient wind direction prior to "surging" upwind. In still air, flies adopt a remarkably stereotyped "sink and circle" search state characterized by ∼ 60° turns at 3–4 Hz, biased in a consistent direction. Together, our results show that Drosophila melanogaster assesses the presence and direction of ambient wind prior to deploying a distinct search strategy. In both laminar wind and still air, immediately after odor onset, flies decelerate and often perform a rapid turn. Both maneuvers are consistent with predictions from recent control theoretic analyses for how insects may estimate properties of wind while in flight. We suggest that flies may use their deceleration and "anemometric" turn as active sensing maneuvers to rapidly gauge properties of their wind environment before initiating a proximal or upwind search routine. [Display omitted] • Activating Orco+ neurons in flying Drosophila elicits stereotyped plume tracking • In still air, flies use a novel search strategy: sink and circle • In wind, flies orient upwind with rapid ∼100-ms turns • We propose that flies use active sensing maneuvers to gauge wind properties Here, Stupski and van Breugel develop an optogenetic paradigm to precisely control the olfactory experiences of freely flying Drosophila melanogaster. They then investigate olfactory search in the absence of wind and describe "sink and circle." Finally, they describe an active sensing maneuver for how flies may measure the wind while flying. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative condition marked by memory impairments and distinct histopathological features such as amyloid-beta (Aβ) accumulations. Alzheimer's patients experience sleep disturbances at early stages of the disease. APPswe/PS1dE9 (APP) mice exhibit sleep disruptions, including reductions in non-rapid eye movement (NREM) sleep, that contribute to their disease progression. In addition, astrocytic calcium transients associated with a sleep-dependent brain rhythm, slow oscillations prevalent during NREM sleep, are disrupted in APP mice. However, at present it is unclear whether restoration of circuit function by targeting astrocytic activity could improve sleep in APP mice. To that end, APP mice expressing channelrhodopsin-2 (ChR2) targeted to astrocytes underwent optogenetic stimulation at the slow oscillation frequency. Optogenetic stimulation of astrocytes significantly increased NREM sleep duration but not duration of rapid eye movement (REM) sleep. Optogenetic treatment increased delta power and reduced sleep fragmentation in APP mice. Thus, optogenetic activation of astrocytes increased sleep quantity and improved sleep quality in an AD mouse model. Astrocytic activity provides a novel therapeutic avenue to pursue for enhancing sleep and slowing AD progression. [ABSTRACT FROM AUTHOR]

Parental behavior comprises a set of crucial actions essential for offspring survival. In this study, a double transgenic mouse model engineered to specifically express channelrhodopsin-2 (ChR2) in paraventricular hypothalamic nucleus (PVN)-oxytocin neurons and ablate lateral hypothalamic area (LHA)-melanin-concentrating hormone (MCH) neurons was used to determine the relationship between PVN-oxytocin neurons and LHA-MCH neurons associated with parental behavior. Optogenetic stimulation of ChR2-expressing PVN-oxytocin neurons induces typical parental behavior with intact LHA-MCH neurons. However, after the partial ablation of LHA-MCH neurons, even optogenetic stimulation of PVN-oxytocin neurons failed to induce parental behavior in virgin male mice, resulting in neglect rather than parental behavior. Furthermore, approximately half of the subjects exhibited burying behavior toward pups, suggesting that pups became aversive stimuli, and male mice actively performed burying behavior to avoid these aversive stimuli. This study emphasizes the novel aspect of oxytocin neurons that could result in neglect in the absence of LHA-MCH neurons regulation. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane‐less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane‐less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo‐mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in‐depth insights into phase separation‐associated biochemical processes, bio‐metabolism, and diseases. [ABSTRACT FROM AUTHOR]

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway. [ABSTRACT FROM AUTHOR]

The islets of Langerhans reside within the endocrine pancreas as highly vascularized microorgans that are responsible for the secretion of key hormones, such as insulin and glucagon. Islet function relies on a range of dynamic molecular processes that include Ca2+ waves, hormone pulses, and complex interactions between islet cell types. Dysfunction of these processes results in poor maintenance of blood glucose homeostasis and is a hallmark of diabetes. Recently, the development of optogenetic methods that rely on light-sensitive molecular actuators has allowed perturbation of islet function with near physiological spatiotemporal acuity. These actuators harness natural photoreceptor proteins and their engineered variants to manipulate mouse and human cells that are not normally light-responsive. Until recently, optogenetics in islet biology has primarily focused on controlling hormone production and secretion; however, studies on further aspects of islet function, including paracrine regulation between islet cell types and dynamics within intracellular signaling pathways, are emerging. Here, we discuss the applicability of optogenetics to islets cells and comprehensively review seminal as well as recent work on optogenetic actuators and their effects in islet function and diabetes mellitus. Article Highlights: Optogenetics harnesses light-sensitive actuators to perturb biological systems with unparalleled spatiotemporal precision. Here, we comprehensively review recent and seminal applications of optogenetics to islets and designer islet-like cells. We cover hormone production and secretion, cell-cell communication, and intracellular signaling dynamics, as well as discuss the merits and limitations of the technique. Future opportunities focus on a broad applicability of the method and potential clinical perspectives. [ABSTRACT FROM AUTHOR]

Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electropathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide noninvasive and high-throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D-culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process. [ABSTRACT FROM AUTHOR]

Small-conductance Ca2+-activated K+ channels (SK, KCa²) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias. [ABSTRACT FROM AUTHOR]

Bioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life‐altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here we report an integrated optogenetic and bioelectronic platform for stable and long‐term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose‐dependent and time‐limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi‐electrode array that measures real‐time electrophysiological responses at 32 spatially‐distinct locations. Irradiation at 27 µW mm−2 results in a 14% elevation of the beating rate within 20–25 min, which remains stable for at least 2 h. The beating rate can be cycled through "on" and "off" light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed‐loop systems for cardiac regulation and intervention, for example, in the context of arrythmias. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cytoplasmic fragile X messenger ribonucleoprotein 1 (FMR1)‐interacting protein 2 (CYFIP2), as a component of the Wiskott–Aldrich syndrome protein family verprolin‐homologous protein (WAVE) regulatory complex, is involved in actin polymerization, contributing to neuronal development and structural plasticity. Mutating serine‐968 to phenylalanine (S968F) in CYFIP2 causes an altered cocaine response in mice. The neuronal mechanisms underlying this response remain unknown. Experimental Approach: We performed cocaine reward‐related behavioural tests and examined changes in synaptic protein phenotypes and neuronal morphology in the nucleus accumbens (NAc), using CYFIP2 S968F knock‐in mice to investigate the role of CYFIP2 in regulating cocaine reward. Key Results: CYFIP2 S968F mutation attenuated cocaine‐induced behavioural sensitization and conditioned place preference. Cocaine‐induced c‐Fos was not observed in the NAc of CYFIP2 S968F knock‐in mice. However, c‐Fos induction was still evident in the medial prefrontal cortex (mPFC). CYFIP2 S968F mutation altered cocaine‐associated CYFIP2 signalling, glutamatergic protein expression and synaptic density in the NAc following cocaine exposure. To further determine the role of CYFIP2 in NAc neuronal activity and the mPFC projecting to the NAc activity‐mediating reward response, we used optogenetic tools to stimulate the NAc or mPFC‐NAc pathway and observed that optogenetic activation of the NAc or mPFC‐NAc pathway induced reward‐related behaviours. This effect was not observed in the S968F mutation in CYFIP2. Conclusion and Implications: These results suggest that CYFIP2 plays a role in controlling cocaine‐mediated neuronal function and structural plasticity in the NAc, and that CYFIP2 could serve as a target for regulating cocaine reward. [ABSTRACT FROM AUTHOR]

Although the locus ceruleus (LC) is recognized as a crucial modulator for attention and perception by releasing norepinephrine into various cortical regions, the impact of LC-noradrenergic (LC-NE) modulation on auditory discrimination behavior remains elusive. In this study, we firstly recorded local field potential and single-unit activity in multiple cortical regions associated with auditory- motor processing, including the auditory cortex, posterior parietal cortex, secondary motor cortex, anterior cingulate cortex, prefrontal cortex, and orbitofrontal cortex (OFC), in response to optogenetic activation (40 Hz and 0.5 s) of the LC-NE neurons in awake mice (male). We found that phasic LC stimulation induced a persistent high gamma oscillation (50-80 Hz) in the OFC. Phasic activation of LC-NE neurons also resulted in a corresponding increase in norepinephrine levels in the OFC, accompanied by a pupillary dilation response. Furthermore, when mice were performing a go/no-go auditory discrimination task, we optogeneticaly activated the neural projections from LC to OFC and revealed a shortened latency in behavioral responses to sound stimuli and an increased false alarm rate. These impulsive behavioral responses may be associated with the gamma neural activity in the OFC. These findings have broadened our understanding of the neural mechanisms involved in the role of LC in auditory-motor processing. [ABSTRACT FROM AUTHOR]

Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools. [ABSTRACT FROM AUTHOR]

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator--Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins. [ABSTRACT FROM AUTHOR]

The continuous exchange between the neuroscience and neuroengineering communities that took place over the past decades has uncovered a multitude of technological solutions to interface with the brain. In this framework, a fascinating approach relies on the integration of multiple activation and monitoring capabilities in the same implantable neural probe to better study the multifaceted nature of neural signaling and related functions in the deep brain regions. We highlight current challenges and perspectives on technological developments that could potentially enable the integration of multiple functionalities on optical fiber-based non-planar implantable neurophotonics probes. [ABSTRACT FROM AUTHOR]

Optogenetics opened the door to a new era of neuroscience. New optical developments are under way to enable high-resolution neuronal activity imaging and selective photostimulation of neuronal ensembles in freely moving animals. These advancements could allow researchers to interrogate, with cellular precision, functionally relevant neuronal circuits in the framework of naturalistic brain activity. We provide an overview of the current state-of-the-art of imaging and photostimulation in freely moving rodents and present a road map for future optical and engineering developments toward miniaturized microscopes that could reach beyond the currently existing systems. [ABSTRACT FROM AUTHOR]

Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca2+ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex. [ABSTRACT FROM AUTHOR]

Holding still and aiming reaches to spatial targets may depend on distinct neural circuits. Using automated homecage training and a sensitive joystick, we trained freely moving mice to contact a joystick, hold their forelimb still, and then reach to rewarded target locations. Mice learned the task by initiating forelimb sequences with clearly resolved submillimeter-scale micromovements followed by millimeter-scale reaches to learned spatial targets. Hundreds of thousands of trajectories were decomposed into millions of kinematic submovements, while photoinhibition was used to test roles of motor cortical areas. Inactivation of both caudal and rostral forelimb areas preserved the ability to produce aimed reaches, but reduced reach speed. Inactivation specifically of contralateral caudal forelimb area (CFA) additionally impaired the ability to aim corrective submovements to remembered locations following target undershoots. Our findings show that motor cortical inactivations reduce the gain of forelimb movements but that inactivation specifically of contralateral CFA impairs corrective movements important for reaching a target location. NEW & NOTEWORTHY: To test the role of different cortical areas in holding still and reaching to targets, this study combined home-cage training with optogenetic silencing as mice engaged in a learned center-out-reach task. Inactivation specifically of contralateral caudal forelimb area (CFA) impaired corrective movements necessary to reach spatial targets to earn reward. [ABSTRACT FROM AUTHOR]

We demonstrated a wavelength discretely tunable laser with outputs at 572.6 nm, 589.1 nm, 603.8 nm, and 611.8 nm using external cavity Ng-cut KGW Raman conversion and intracavity frequency mixing. The multiple visible wavelengths were generated by utilizing this specially cut KGW crystal and the g[mm]g Raman configuration, as well as the temperature-dependent phase-matching characteristic of the LBO crystal. The maximum output powers at 572.6 nm, 589.1 nm, 603.8 nm, and 611.8 nm were measured to be 227 mW, 175 mW, 258 mW, and 103 mW respectively, with nearly fundamental Gaussian beam profiles. This work provides a monolithic setup capable of producing tunable yellow-red laser outputs through Raman and χ 2 frequency conversions for various applications such as air detection, optogenetics, biomedicine, and chemical analysis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phytochrome B variant phyB[G515E] with a slow dark reversion was generated. • phyB[G515E] forms warm temperature-insensitive nuclear photobodies. • PHYB[G515E] expression inhibited the phytochrome interacting factor4-auxin pathway. • PHYB[G515E] transgenic Arabidopsis and rice showed warming-insensitive growth. • phyB[G515E] is a powerful tool for engineering warm temperature-insensitive crops. Ambient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. This study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. The thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. At an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. Transient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants. [ABSTRACT FROM AUTHOR]

Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function. Recent advances in optogenetics, neuronal typing and labeling, and circuit tracing have facilitated the dissection of the neural circuitry involved in various important behaviors. The identification of monosynaptic connections, both upstream and downstream of specific neurons, serves as the foundation for understanding complex neural circuits and studying behavioral mechanisms. However, the practical implementation and mechanistic understanding of monosynaptic connection tracing techniques and functional identification remain challenging, particularly for inexperienced researchers. Improper application of these methods and misinterpretation of results can impede experimental progress and lead to erroneous conclusions. In this paper, we present a comprehensive description of the principles, specific operational details, and key steps involved in tracing anterograde and retrograde monosynaptic connections. We outline the process of functionally identifying monosynaptic connections through the integration of optogenetics and electrophysiological techniques, providing practical guidance for researchers. [ABSTRACT FROM AUTHOR]

Psychological stress is linked to infertility by suppressing the hypothalamic GnRH pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centers; however, their functional significance has not been determined. We hypothesize that MePD GABAergic signalling mediates psychological stress–induced suppression of pulsatile LH secretion. We selectively inhibited MePD GABA neurons during psychological stress in ovariectomized (OVX) Vgat-cre-tdTomato mice to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurons were virally infected with inhibitory hM4DGi-designer receptor exclusively activated by designer drugs (DREADDs) to selectively inhibit MePD GABA neurons. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurons in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fiber implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurons blocked predator odor and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20 Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress–induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator. [ABSTRACT FROM AUTHOR]

In retinitis pigmentosa (RP), rod and cone photoreceptors degenerate, depriving downstream neurons of light-sensitive input, leading to vision impairment or blindness. Although downstream neurons survive, some undergo morphological and physiological remodeling. Bipolar cells (BCs) link photoreceptors, which sense light, to retinal ganglion cells (RGCs), which send information to the brain. While photoreceptor loss disrupts input synapses to BCs, whether BC output synapses remodel has remained unknown. Here we report that synaptic output from BCs plummets in RP mouse models of both sexes owing to loss of voltage-gated Ca2+ channels. Remodeling reduces the reliability of synaptic output to repeated optogenetic stimuli, causing RGC firing to fail at high-stimulus frequencies. Fortunately, functional remodeling of BCs can be reversed by inhibiting the retinoic acid receptor (RAR). RAR inhibitors targeted to BCs present a new therapeutic opportunity for mitigating detrimental effects of remodeling on signals initiated either by surviving photoreceptors or by vision-restoring tools. [ABSTRACT FROM AUTHOR]

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control. It is known that channelrhodopsins use all-trans retinal as chromophore for light activation. Here, the authors find that different channelrhodopsins utilize various forms of retinal in mammalian cells, which could inspire next-generation optogenetic tools. [ABSTRACT FROM AUTHOR]

Visual perception of X-radiation is a well-documented, but poorly understood phenomenon. Scotopic rod cells and rhodopsin have been implicated in visual responses to X-rays, however, some evidence suggests that X-rays excite the retina via a different mechanism than visible light. While rhodopsin's role in X-ray perception is unclear, the possibility that it could function as an X-ray receptor has led to speculation that it could act as a transgenically expressed X-ray receptor. If so, it could be used to transduce transcranial X-ray signals and control the activity of genetically targeted populations of neurons in a less invasive version of optogenetics, X-genetics. Here we investigate whether human rhodopsin (hRho) is capable of transducing X-ray signals when expressed outside of the retinal environment. We use a live-cell cAMP GloSensor luminescence assay to measure cAMP decreases in hRho-expressing HEK293 cells in response to visible light and X-ray stimulation. We show that cAMP GloSensor luminescence decreases are not observed in hRho-expressing HEK293 cells in response to X-ray stimulation, despite the presence of robust responses to visible light. Additionally, irradiation had no significant effect on cAMP GloSensor responses to subsequent visible light stimulation. These results suggest that ectopically expressed rhodopsin does not function as an X-ray receptor and is not capable of transducing transcranial X-ray signals into neural activity for X-ray mediated, genetically targeted neuromodulation. [ABSTRACT FROM AUTHOR]

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5′-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics. [ABSTRACT FROM AUTHOR]

Recently developed optogenetic technology, which allows high‐fidelity control of neuronal activity, has been applied to investigate the neural circuits underlying sensory processing and behavior. The nasal cavity is innervated by the olfactory nerve and trigeminal nerve, which are closely related to common symptoms of rhinitis, such as impairment of smell, itching, and sneezing. The olfactory system has an amazing ability to distinguish thousands of odorant molecules at trace levels. However, there are many issues in olfactory sensing mechanisms that need to be addressed. Optogenetics offers a novel technical approach to solve this dilemma. Therefore, we review the recent advances in olfactory optogenetics to clarify the mechanisms of chemical sensing, which may help identify the mechanism of dysfunction and suggest possible treatments for impaired smell. Additionally, in rhinitis patients, alterations in the other nerve (trigeminal nerve) that innervates the nasal cavity can lead to hyperresponsiveness to various nociceptive stimuli and central sensitization, causing frequent and persistent itching and sneezing. In the last several years, the application of optogenetics in regulating nociceptive receptors, which are distributed in sensory nerve endings, and amino acid receptors, which are distributed in vital brain regions, to alleviate overreaction to nociceptive stimuli, has gained significant attention. Therefore, we focus on the progress in optogenetics and its application in neuromodulation of nociceptive stimuli and discuss the potential clinical translation for treating rhinitis in the future. [ABSTRACT FROM AUTHOR]

Melanopsin is a photopigment belonging to the G Protein-Coupled Receptor (GPCR) family expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) and responsible for a variety of processes. The bistability and, thus, the possibility to function under low retinal availability would make melanopsin a powerful optogenetic tool. Here, we aim to utilize mouse melanopsin to trigger macrophage migration by its subcellular optical activation with localized blue light, while simultaneously imaging the migration with red light. To reduce melanopsin's red light sensitivity, we employ a combination of in silico structure prediction and automated quantum mechanics/molecular mechanics modeling to predict minimally invasive mutations to shift its absorption spectrum towards the shorter wavelength region of the visible spectrum without compromising the signaling efficiency. The results demonstrate that it is possible to achieve melanopsin mutants that resist red light-induced activation but are activated by blue light and display properties indicating preserved bistability. Using the A333T mutant, we show that the blue light-induced subcellular melanopsin activation triggers localized PIP3 generation and macrophage migration, which we imaged using red light, demonstrating the optogenetic utility of minimally engineered melanopsins. [ABSTRACT FROM AUTHOR]

Cochlear implants (CI) represent incredible devices that restore hearing perception for those with moderate to profound sensorineural hearing loss. However, the ability of a CI to restore complex auditory function is limited by the number of perceptually independent spectral channels provided. A major contributor to this limitation is the physical gap between the CI electrodes and the target spiral ganglion neurons (SGNs). In order for CI electrodes to stimulate SGNs more precisely, and thus better approximate natural hearing, new methodologies need to be developed to decrease this gap, (i.e., transitioning CIs from a far-field to near-field device). In this review, strategies aimed at improving the neural-electrode interface are discussed in terms of the magnitude of impact they could have and the work needed to implement them. Ongoing research suggests current clinical efforts to limit the CI-related immune response holds great potential for improving device performance. This could eradicate the dense, fibrous capsule surrounding the electrode and enhance preservation of natural cochlear architecture, including SGNs. In the long term, however, optimized future devices will likely need to induce and guide the outgrowth of the peripheral process of SGNs to be in closer proximity to the CI electrode in order to better approximate natural hearing. This research is in its infancy; it remains to be seen which strategies (surface patterning, small molecule release, hydrogel coating, etc.) will be enable this approach. Additionally, these efforts aimed at optimizing CI function will likely translate to other neural prostheses, which face similar issues. [ABSTRACT FROM AUTHOR]

Gut peristaltic movements transport ingested materials along the gut axis, which is critical for food digestion and nutrient absorption. While a large amount of studies have been devoted to analyzing the physiological functions of peristalsis in adults, little is known about how the peristaltic system is established during embryogenesis. In recent years, the chicken developing gut has emerged as an excellent model, in which specific sites along the gut axis can be genetically labeled enabling live imaging and optogenetic analyses. This review provides an overview of recent progress in optogenetic studies of gut peristalsis. Analyses with an improved channelrhodopsin-2 variant demonstrated that the peristalsis can artificially be generated in the developing gut. These studies unveiled novel functional coordination between different regions along the gut axis. In addition, imaging with GCaMP6s, a genetically encoded calcium indicator, enabled a fine mapping of developmental changes in the peristaltic patterns as Ca2+ signals. These advanced techniques will broaden our knowledge of how embryonic peristalsis is established at the cellular and molecular level, leading to the understanding of physiological and pathological processes in adult peristalsis. [ABSTRACT FROM AUTHOR]

The potential role of astrocytes in lateral habenula (LHb) in modulating anxiety was explored in this study. The habenula are a pair of small nuclei located above the thalamus, known for their involvement in punishment avoidance and anxiety. Herein, we observed an increase in theta-band oscillations of local field potentials (LFPs) in the LHb when mice were exposed to anxiety-inducing environments. Electrical stimulation of LHb at theta-band frequency promoted anxiety-like behavior. Calcium (Ca2+) levels and pH in the cytosol of astrocytes and local brain blood volume changes were studied in mice expressing either a Ca2+ or a pH sensor protein specifically in astrocytes and mScarlet fluorescent protein in the blood plasma using fiber photometry. An acidification response to anxiety was observed. Photoactivation of archaerhopsin-T (ArchT), an optogenetic tool that acts as an outward proton pump, results in intracellular alkalinization. Photostimulation of LHb in astrocyte-specific ArchT-expressing mice resulted in dissipation of theta-band LFP oscillation in an anxiogenic environment and suppression of anxiety-like behavior. These findings provide evidence that LHb astrocytes modulate anxiety and may offer a new target for treatment of anxiety disorders. • Habenular astrocytes control the brain environment leading to anxiety modulation. • Theta band LFP activity increased and astrocytes became acidified with anxiety. • Optogenetic alkalinization of astrocytes resulted in the alleviation of anxiety. • Neuron-astrocyte reciprocal interaction amplifies theta band oscillation and anxiety. • Control of astrocyte activity could become a new therapeutic strategy for disorders. [ABSTRACT FROM AUTHOR]

The role of nociceptive nerves in modulating immune responses to harmful stimuli via pain or itch induction remains controversial. Compared to conventional surgery, various implant surgeries are more prone to infections even with low bacterial loads. In this study, an optogenetic technique is introduced for selectively activating peripheral nociceptive nerves using a fully implantable, wirelessly rechargeable optogenetic device. By targeting nociceptors in the limbs of awake, freely moving mice, it is found that activation induces anticipatory immunity in the innervated territory and enhances the adhesion of various host cells to the implant surface. This effect mediates acute immune cell‐mediated killing of Staphylococcus aureus on implants and enables the host to win “implant surface competition” against Staphylococcus aureus. This finding provides new strategies for preventing and treating implant‐associated infections. [ABSTRACT FROM AUTHOR]

During mild or moderate exercise, alveolar ventilation increases in direct proportion to metabolic rate, regulating arterial CO2 pressure near resting levels. Mechanisms giving rise to the hyperpnoea of exercise are unsettled despite over a century of investigation. In the past three decades, neuroscience has advanced tremendously, raising optimism that the 'exercise hyperpnoea dilemma' can finally be solved. In this review, new perspectives are offered in the hope of stimulating original ideas based on modern neuroscience methods and current understanding. We first describe the ventilatory control system and the challenge exercise places upon blood‐gas regulation. We highlight relevant system properties, including feedforward, feedback and adaptive (i.e., plasticity) control of breathing. We then elaborate a seldom explored hypothesis that the exercise ventilatory response continuously adapts (learns and relearns) throughout life and ponder if the memory 'engram' encoding the feedforward exercise ventilatory stimulus could reside within the cerebellum. Our hypotheses are based on accumulating evidence supporting the cerebellum's role in motor learning and the numerous direct and indirect projections from deep cerebellar nuclei to brainstem respiratory neurons. We propose that cerebellar learning may be obligatory for the accurate and adjustable exercise hyperpnoea capable of tracking changes in life conditions/experiences, and that learning arises from specific cerebellar microcircuits that can be interrogated using powerful techniques such as optogenetics and chemogenetics. Although this review is speculative, we consider it essential to reframe our perspective if we are to solve the till‐now intractable exercise hyperpnoea dilemma. What is the topic of this review?This review reframes our perspective on the control of breathing during mild or moderate exercise, emphasising concepts of learning, memory and plasticity.What advances does it highlight?We propose that exercise hyperpnoea continuously adapts to ever‐changing life conditions/experiences. We question if the memory 'engram' encoding exercise hyperpnoea resides within the cerebellum and outline powerful new methods to test this hypothesis. It is our hope that this review generates new ideas and renewed study of exercise hyperpnoea in the 21st century. [ABSTRACT FROM AUTHOR]

Optogenetic modulation of adenosine triphosphatase (ATPase) expression represents a novel approach to maximize bioprocess efficiency by leveraging enforced adenosine triphosphate (ATP) turnover. In this study, we experimentally implement a model-based open-loop optimization scheme for optogenetic modulation of the expression of ATPase. Increasing the intracellular concentration of ATPase, and thus the level of ATP turnover, in bioprocesses with product synthesis coupled with ATP generation, can lead to increased substrate uptrake and product formation. Previous simulation studies formulated optimal control problems using dynamic constraint-based models to find optimal light inputs in fermentations with optogenetically mediated ATPase expression. However, using these models poses challenges due to resulting bilevel optimizations and complex parameterization. Here, we outline a simplified unsegregated and quasi-unstructured kinetic modeling approach that reduces the number of dynamic states and leads to single-level optimizations. The models can be augmented with Gaussian processes to compensate for model uncertainties. We implement optimal control constrained by knowledge-based and hybrid models for optogenetic ATPase expression in Escherichia coli with lactate as the main product. To do so, we genetically engineer E. coli to obtain optogenetic expression of ATPase using the CcaS/CcaR system. This represents the first experimental implementation of model-based optimization of ATPase expression in bioprocesses. [Display omitted] • Experimental validation of model-based open-loop optimization of optogenetic ATPase expression in E. coli. • Simple macro-kinetic modeling can be augmented with Gaussian processes to address model uncertainties. • Adjustable batch-to-batch fermentation performance via optogenetic modulation of ATPase. • Validated optimization framework paves the way for advanced metabolic engineering applications. • Hybrid model-based optimization strategy with the potential for future metabolic cybergenetic implementations. [ABSTRACT FROM AUTHOR]

Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP mouse, in which humanized renilla green fluorescent protein (hrGFP) expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on several factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY on the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC. NEW & NOTEWORTHY: Across brain regions, neuropeptide Y (NPY) expression is dynamic and influenced by extrinsic and intrinsic factors. We previously showed that NPY is expressed by a class of inhibitory neurons in the auditory midbrain. Here, we find that this neuron class also includes neurons that previously expressed NPY, suggesting that NPY expression is dynamically regulated in the auditory midbrain. We also provide functional evidence that NPY neurons contribute to local inhibitory circuits in the auditory midbrain. [ABSTRACT FROM AUTHOR]

Aims: The parabrachial nucleus (PBN) promotes wakefulness states under general anesthesia. Recent studies have shown that glutamatergic neurons within the PBN play a crucial role in facilitating emergence from anesthesia. Our previous study indicates that vesicular glutamate transporter 2 (vglut2) expression neurons of the PBN extend into the extended amygdala (EA). However, the modulation of PBNvglut2‐EA in general anesthesia remains poorly understood. This study aims to investigate the role of PBNvglut2‐EA in alterations of consciousness during sevoflurane anesthesia. Methods: We first validated vglut2‐expressing neuron projections from the PBN to the EA using anterograde tracing. Then, we conducted immunofluorescence staining of c‐Fos to investigate the role of the EA involved in the regulation of consciousness during sevoflurane anesthesia. After, we performed calcium fiber photometry recordings to determine the changes in PBNvglut2‐EA activity. Lastly, we modulated PBNvglut2‐EA activity under sevoflurane anesthesia using optogenetics, and electroencephalogram (EEG) was recorded during specific optogenetic modulation. Results: The expression of vglut2 in PBN neurons projected to the EA, and c‐Fos expression in the EA was significantly reduced during sevoflurane anesthesia. Fiber photometry revealed that activity in the PBNvglut2‐EA pathway was suppressed during anesthesia induction but restored upon awakening. Optogenetic activation of the PBNvglut2‐EA delayed the induction of anesthesia. Meanwhile, EEG recordings showed significantly decreased δ oscillations and increased β and γ oscillations compared to the EYFP group. Furthermore, optogenetic activation of the PBNvglut2‐EA resulted in an acceleration of awakening from anesthesia, accompanied by decreased δ oscillations on EEG recordings. Optogenetic inhibition of PBNvglut2‐EA accelerated anesthesia induction. Surprisingly, we found a sex‐specific regulation of PBNvglut2‐EA in this study. The activity of PBNvglut2‐EA was lower in males during the induction of anesthesia and decreased more rapidly during sevoflurane anesthesia compared to females. Photoactivation of the PBNvglut2‐EA reduced the sensitivity of males to sevoflurane, showing more pronounced wakefulness behavior and EEG changes than females. Conclusions: PBNvglut2‐EA is involved in the promotion of wakefulness under sevoflurane anesthesia. Furthermore, PBNvglut2‐EA shows sex differences in the changes of consciousness induced by sevoflurane anesthesia. [ABSTRACT FROM AUTHOR]