Molecular Diversity of Photosensitive Protein Opsins and Their High Potential for Optogenetic Applications.

Because G protein coupled receptors (GPCRs) represent the largest family of drug targets in clinical trials, GPCR signaling cascades are closely related to various physiological phenomena, attracting significant attention in pharmaceutical science. Opsins (also known as animal rhodopsins) are photoreceptive proteins containing retinal as a chromophore, which function as GPCRs and underlie the molecular basis of photoreception in animals. Recently, opsins have been progressively applied in an innovative technology called optogenetics to regulate biological activities using light. A wide variety of opsins have been identified in metazoans and characterized at the molecular and physiological levels, providing a foundation for their optogenetic applications. In this review, I briefly introduce the diversity of opsins in terms of their molecular functions, including G protein selectivity and photoreaction properties. This diversity provides a significant advantage for optically manipulating a wide variety of GPCR signaling cascades with high temporal resolution. Additionally, I discuss the rich array of opsin-based optogenetic tools used to control various physiological processes and their potential as therapeutic tools for vision restoration. Based on the introduction, I expect that the optogenetic approach will offer powerful tools to provide valuable insights into the molecular mechanisms of various physiological phenomena and next-generation treatment options for diseases beyond the capacity of traditional drugs.

Comparison of Nocifensive Behavior in NaV1.7-, NaV1.8-, and NaV1.9-Channelrhodopsin-2 Mice by Selective Optogenetic Activation of Targeted Sodium Channel Subtype-Expressing Afferents.

Voltage-gated sodium channels, including NaV1.7, NaV1.8, and NaV1.9, play important roles in pain transmission and chronic pain development. However, the specific mechanisms of their action remain unclear, highlighting the need for in vivo stimulation studies of these channels. Optogenetics, a novel technique for targeting the activation or inhibition of specific neural circuits using light, offers a promising solution. In our previous study, we used optogenetics to selectively excite NaV1.7-expressing neurons in the dorsal root ganglion of mice to induce nocifensive behavior. Here, we further characterize the impact of nocifensive behavior by activation of NaV1.7, NaV1.8, or NaV1.9-expressing neurons. Using CRISPR/Cas9-mediated homologous recombination, NaV1.7-iCre, NaV1.8-iCre, or NaV1.9-iCre mice expressing iCre recombinase under the control of the endogenous NaV1.7, NaV1.8, or NaV1.9 gene promoter were produced. These mice were then bred with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7-ChR2, NaV1.8-ChR2, or NaV1.9-ChR2 mice. Blue light exposure triggered paw withdrawal in all mice, with the strongest response in NaV1.8-ChR2 mice. These light sensitivity differences observed across NaV1.x-ChR2 mice may be dependent on ChR2 expression or reflect the inherent disparities in their pain transmission roles. In conclusion, we have generated noninvasive pain models, with optically activated peripheral nociceptors. We believe that studies using optogenetics will further elucidate the role of sodium channel subtypes in pain transmission.

Differential impact of optogenetic stimulation of direct and indirect pathways from dorsolateral and dorsomedial striatum on motor symptoms in Huntington's disease mice.

The alterations in the basal ganglia circuitry are core pathological hallmark in Huntington's Disease (HD) and traditionally linked to its sever motor symptoms. Recently it was shown that optogenetic stimulation of cortical afferences to the striatum is able to reverse motor symptoms in HD mice. However, the specific contribution of the direct and indirect striatal output pathways from the dorsolateral (DLS) and dorsomedial striatum (DMS) to the motor phenotype is still not clear. Here, we aim to uncover the contributions of these striatal subcircuits to motor control in wild type (WT) and HD mice by using the symptomatic R6/1 mice. We systematically evaluated locomotion, exploratory behavior, and motor learning effects of the selective optogenetic stimulation of D1 or A2A expressing neurons (direct and indirect pathway, respectively), in DLS or DMS. Bilateral optogenetic stimulation of the direct pathway from DLS and the indirect pathway from DMS resulted in subtle locomotor enhancements, while unaltering exploratory behavior. Additionally, bilateral stimulation of the indirect pathway from the DLS improved performance in the accelerated rotarod task, suggesting a role in motor learning. In contrast, in HD mice, stimulation of these pathways did not modulate any of these behaviors. Overall, this study highlights that selective stimulation of direct and indirect pathways from DLS and DMS have subtle impact in locomotion, exploratory activity nor motor learning. The lack of responses in HD mice also suggests that strategies involving cortico-striatal circuits rather than striatal output circuits might be a better strategy for managing motor symptoms in movement disorders.

Optogenetic estimation of synaptic connections in brain slices.

BACKGROUND: Detection of synaptic connections is essential for understanding neural circuits. By using optogenetics, current injection, and glutamate uncaging to activate presynaptic cells and simultaneously recording the subsequent response of postsynaptic cells, the presence of synaptic connections can be confirmed. However, these methods present throughput challenges, such as the need for simultaneous multicellular patch-clamp recording and two-photon microscopy. These challenges lead to a trade-off between sacrificing resolution and experimental throughput. NEW METHOD: We adopted the laser, typically used for local field ablation, and combined this with post hoc analysis. We successfully approximated the synaptic connection probabilities using only an epi-fluorescence microscope and single-cell recordings. RESULTS: We sequentially stimulated the channelrhodopsin 2-expressing cells surrounding the recorded cell and approximated the synaptic connection probabilities. This probability value was comparable to that obtained from simultaneous multi-cell patch-clamp recordings, which included more than 600 pairs. COMPARISON WITH EXISTING METHODS: Our setup allows us to estimate connection probabilities within 100 s, outperforming existing methods. We successfully estimated synaptic connection probabilities using only the optical path typically used by an epi-fluorescence microscope and single-cell recordings. It may also be suitable for dendritic ablation experiments. CONCLUSIONS: The proposed method simplifies the estimation of connection probabilities, which is expected to advance the study of neural circuits in conditions such as autism and schizophrenia where connection probabilities vary. Furthermore, this approach is applicable not only to local circuits but also to long-range connections, thus increasing experimental throughput.

Novel aspect of oxytocin neurons mediating parental behavior and aversive burying behavior under the control of melanin-concentrating hormone neurons.

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.

Strength of activation and temporal dynamics of bioluminescent-optogenetics in response to systemic injections of the luciferin.

BioLuminescent OptoGenetics ("BL-OG") is a chemogenetic method that can evoke optogenetic reactions in the brain non-invasively. In BL-OG, an enzyme that catalyzes a light producing reaction (i.e., a luciferase) is tethered to an optogenetic element that is activated in response to bioluminescent light. Bioluminescence is generated by injecting a chemical substrate (luciferin, e.g., h-Coelenterazine; h-CTZ) that is catalyzed by the luciferase. By directly injecting the luciferin into the brain, we show that bioluminescent light is proportional to spiking activity, and this relationship scales as a function of luciferin dosage. Here, we build on these previous observations by characterizing the temporal dynamics and dose response curves of bioluminescence generated by luminopsins (LMOs), a proxy of BL-OG effects, to intravenous (IV) injections of the luciferin. We imaged bioluminescence through a thinned skull of mice running on a wheel, while delivering h-CTZ via the tail vein with different dosage concentrations and injection rates. The data reveal a systematic relationship between strength of bioluminescence and h-CTZ dosage, with higher concentration generating stronger bioluminescence. We also found that bioluminescent activity occurs rapidly (< 60 s after IV injection) regardless of concentration dosage. However, as expected, the onset time of bioluminescence is delayed as the injection rate decreases. Notably, the strength and time decay of bioluminescence is invariant to the injection rate of h-CTZ. Taken together, these data show that BL-OG effects are highly consistent across injection parameters of h-CTZ, highlighting the reliability of BL-OG as a minimally invasive neuromodulation method.

A neural circuit for lavender-essential-oil-induced antinociception.

Lavender essential oil (LEO) has been shown to relieve pain in humans, but the underlying neural mechanisms remain unknown. Here, we found that inhalation exposure to 0.1% LEO confers antinociceptive effects in mice with complete Freund adjuvant (CFA)-induced inflammatory pain through activation of projections from the anterior piriform cortex (aPir) to the insular cortex (IC). Specifically, in vivo fiber photometry recordings and viral tracing data show that glutamatergic projections from the aPir (aPirGlu) innervate GABAergic neurons in the IC (ICGABA) to inhibit local glutamatergic neurons (ICGlu) that are hyperactivated in inflammatory pain. Optogenetic or chemogenetic activation of this aPirGlu→ICGABA→Glu pathway can recapitulate the antinociceptive effects of LEO inhalation in CFA mice. Conversely, artificial inhibition of IC-projecting aPirGlu neurons abolishes LEO-induced antinociception. Our study thus depicts an LEO-responsive olfactory system circuit mechanism for alleviating inflammatory pain via aPir→IC neural connections, providing evidence to support development of aroma-based treatments for alleviating pain.

Light-driven synchronization of optogenetic clocks.

Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here, we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli, with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system's response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level.

α-Synuclein aggregation decreases cortico-amygdala connectivity and impairs social behavior in mice.

Abnormal accumulation of insoluble α-synuclein (α-Syn) inclusions in neurons, neurites, and glial cells is the defining neuropathology of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. Accumulation of α-Syn inclusions in the amygdala has been well-documented in post-mortem studies of PD and DLB brains, as well as preclinical animal models of these conditions. Though α-Syn pathology is closely associated with neurodegeneration, there is a poor correlation between neuronal loss in the amygdala and the clinical features of PD and DLB. Moreover, functional interaction between the cerebral cortex and the amygdala is critical to regulating emotion, motivation, and social behaviors. The cortico-amygdala functional interaction is likely to be disrupted by the development of α-Syn pathology in the brain. Thus, we hypothesize that neuronal α-Syn inclusions disrupt cortical modulation of the amygdala circuits and are sufficient to drive social behavioral deficits. In the present work, we designed a series of longitudinal studies to rigorously measure the time courses of neurodegeneration, functional impairment of cortico-amygdala connectivity, and development of amygdala-dependent social behavioral deficits to test this hypothesis. We injected α-Syn preformed fibrils (PFFs) into the dorsal striatum to induce α-Syn aggregation in the amygdala and the medial prefrontal cortex (mPFC) of C57BL6 mice of both sexes, followed by a detailed analysis of temporal development of α-Syn pathology, synaptic deficits, and neuronal loss in the amygdala, as well as behavioral deficits at 3-12 months post injections. Development of α-Syn inclusions caused losses of cortical axon terminals and cell death in the basolateral amygdala (BLA) at 6- and 12-months post injections, respectively. At a relatively early stage of 3 months post injections, the connection strength of the mPFC-BLA synapse was decreased in PFFs-injection mice compared to controls. Meanwhile, the PFFs-injected mice showed impaired social interaction behavior, which was rescued by chemogenetic stimulation of mPFC-BLA connections. Altogether, we presented a series of evidence to delineate circuit events in the amygdala associated with the accumulation of α-Syn inclusions in the mouse brain, highlighting that functional impairment of the amygdala is sufficient to cause social behavior deficits. The present work further suggests that early circuit modulation could be an effective approach to alleviate symptoms associated with α-Syn pathology, necessitating studies of functional consequences of α-Syn aggregation.

Precise Control of Intracellular Trafficking and Receptor-Mediated Endocytosis in Living Cells and Behaving Animals.

Intracellular trafficking, an extremely complex network, dynamically orchestrates nearly all cellular activities. A versatile method that enables the manipulation of target transport pathways with high spatiotemporal accuracy in vitro and in vivo is required to study how this network coordinates its functions. Here, a new method called RIVET (Rapid Immobilization of target Vesicles on Engaged Tracks) is presented. Utilizing inducible dimerization between target vesicles and selective cytoskeletons, RIVET can spatiotemporally halt numerous intracellular trafficking pathways within seconds in a reversible manner. Its highly specific perturbations allow for the real-time dissection of the dynamic relationships among different trafficking pathways. Moreover, RIVET is capable of inhibiting receptor-mediated endocytosis. This versatile system can be applied from the cellular level to whole organisms. RIVET opens up new avenues for studying intracellular trafficking under various physiological and pathological conditions and offers potential strategies for treating trafficking-related disorders.

Homeostatic regulation of brain activity: from endogenous mechanisms to homeostatic nanomachines.

After the initial concepts of the constancy of the internal milieu or homeostasis, put forward by Claude Bernard and Walter Cannon, homeostasis emerged as a mechanism to control oscillations of biologically meaningful variables within narrow physiological ranges. This is a primary need in the central nervous system that is continuously subjected to a multitude of stimuli from the internal and external environments that affect its function and structure, allowing to adapt the individual to the ever-changing daily conditions. Preserving physiological levels of activity despite disturbances that could either depress neural computation or excessively stimulate neural activity is fundamental, and failure of these homeostatic mechanisms can lead to brain diseases. In this review, we cover the role and main mechanisms of homeostatic plasticity involving the regulation of excitability and synaptic strength from the single neuron to the network level. We analyze the relationships between homeostatic and Hebbian plasticity and the conditions under which the preservation of the excitatory/inhibitory balance fails, triggering epileptogenesis and eventually epilepsy. Several therapeutic strategies to cure epilepsy have been designed to strengthen homeostasis when endogenous homeostatic plasticity mechanisms have become insufficient or ineffective to contrast hyperactivity. We describe "on demand" gene therapy strategies including optogenetics, chemogenetics, and chemo-optogenetics, and particularly focus on new closed loop sensor-actuator strategies mimicking homeostatic plasticity that can be endogenously expressed to strengthen the homeostatic defenses against brain diseases.

Robust optogenetic inhibition with red-light-sensitive anion-conducting channelrhodopsins.

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. The Mantoniella squamata ACR (MsACR1) showed high sensitivity to yellow-green light (λmax at 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light-sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm toward red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neurons in vitro and in vivo, while raACR offers a higher temporal resolution.

Basolateral amygdala inputs to the nucleus accumbens shell modulate the consolidation of cued-response and inhibitory avoidance learning.

The basolateral amygdala (BLA) modulates different types of memory consolidation via distinct projections to downstream brain regions in multiple memory systems. Prior studies indicate that the BLA projects to the nucleus accumbens shell (NAshell) and that these regions interact to influence some types of behavior. Moreover, previous pharmacological work suggests the BLA and NAshell interact to influence memory. However, the precise role of the BLA-NAshell pathway has never been directly investigated in the consolidation of different types of memory including cued-response, spatial, or inhibitory avoidance (IA) learning. To address this, male and female Sprague-Dawley rats received optogenetic manipulations of the BLA or BLA-NAshell pathway immediately following training in different learning tasks. An initial experiment found that optogenetically inhibiting the BLA itself immediately after training impaired cued-response retention in a Barnes maze task in males and females, confirming earlier pharmacological work in males alone. Subsequent experiments found that BLA-NAshell pathway inhibition impaired retention of cued-response and IA learning but had no effect on retention of spatial learning. However, the present work did not observe any effects of pathway stimulation immediately after cued-response or IA learning. Together, the present findings suggest the BLA modulates the consolidation of cued-response and IA, but not spatial, memory consolidation via NAshell projections.

Optogenetic Control of the Mitochondrial Protein Import in Mammalian Cells.

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.

Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation.

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that trigger either nuclear export or degradation of Dorsal under blue light. Nuclear export of Dorsal leads to loss of the high-threshold, ventrally expressed target gene snail (sna), while the low-threshold, laterally expressed target gene short-gastrulation (sog) is retained. In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels compared to when Dorsal is exported from the nucleus. To understand why nuclear export causes loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found that it rapidly reenters the nucleus even under blue-light conditions favoring export. The associated kinetics of Dorsal being rapidly imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results indicate that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention.

Optogenetic targeting of cortical astrocytes selectively improves NREM sleep in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative condition marked by memory impairments and distinct histopathological features such as amyloid-beta (Aß) 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.

The elevated open platform stress suppresses excitatory synaptic transmission in the layer V anterior cingulate cortex.

There are various forms of stress including; physical, psychological and social stress. Exposure to physical stress can lead to physical sensations (e.g. hyperalgesia) and negative emotions including anxiety and depression in animals and humans. Recently, our studies in mice have shown that acute physical stress induced by the elevated open platform (EOP) can provoke long-lasting mechanical hypersensitivity. This effect appears to be related to activity in the anterior cingulate cortex (ACC) at the synapse level. Indeed, the EOP exposure induces synaptic plasticity in layer II/III pyramidal neurons from the ACC. However, it is still unclear whether or not the EOP alters synaptic transmission in layer V pyramidal neurons. This is essential because these neurons are known to be a primary output to subcortical structures which may ultimately impact the behavioral stress response. Here, we studied both intrinsic properties and excitatory/inhibitory synaptic transmission by using whole-cell patch-clamp method in brain slice preparations. The EOP exposure did not change intrinsic properties including resting membrane potentials and action potentials. In contrast, the EOP suppressed the frequency of miniature and spontaneous excitatory synaptic transmission with an alteration of the kinetics of AMPA/GluK receptors. The EOP also reduced evoked synaptic transmission induced by electrical stimulation. Furthermore, we investigated projection-selective responses of the mediodorsal thalamus to the layer V ACC neurons. The EOP produced short-term depression in excitatory synaptic transmission on thalamo-ACC projections. These results suggest that the acute stress, induced by the EOP, provokes abnormal excitatory synaptic transmission in layer V pyramidal neurons of the ACC.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions.

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 electro-pathophysiology. 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 non-invasive 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.

Two-photon optogenetics-based assessment of neuronal connectivity in healthy and chronic hypoperfusion mice.

Significance: Two-photon optogenetics and simultaneous calcium imaging can be used to visualize the response of surrounding neurons with respect to the activity of an optically stimulated target neuron, providing a direct method to assess neuronal connectivity. Aim: We aim to develop a two-photon optogenetics-based method for evaluating neuronal connectivity, compare it to the existing indirect resting-state synchrony method, and investigate the application of the method to brain pathophysiology. Approach: C1V1-mScarlet was introduced into GCaMP6s-expressing transgenic mice with an adeno-associated virus. Optical stimulation of a single target neuron and simultaneous calcium imaging of the target and surrounding cells were performed. Neuronal connectivity was evaluated from the correlation between the fluorescence intensity of the target and surrounding cells. Results: The neuronal connectivity in the living brain was evaluated using two-photon optogenetics. However, resting-state synchrony was not always consistent with two-photon optogenetics-based connectivity. Comparison with neuronal synchrony measured during sensory stimulation suggested that the disagreement was due to external sensory input. Two-photon optogenetics-based connectivity significantly decreased in the common carotid artery occlusion model, whereas there was no significant change in the control group. Conclusions: We successfully developed a direct method to evaluate neuronal connectivity in the living brain using two-photon optogenetics. The technique was successful in detecting connectivity impairment in hypoperfusion model mice.

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions.

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.