Astrocyte-mediated regulation of BLAWFS1 neurons alleviates risk-assessment deficits in DISC1-N mice.

Assessing and responding to threats is vital in everyday life. Unfortunately, many mental illnesses involve impaired risk assessment, affecting patients, families, and society. The brain processes behind these behaviors are not well understood. We developed a transgenic mouse model (disrupted-in-schizophrenia 1 [DISC1]-N) with a disrupted avoidance response in risky settings. Our study utilized single-nucleus RNA sequencing and path-clamp coupling with real-time RT-PCR to uncover a previously undescribed group of glutamatergic neurons in the basolateral amygdala (BLA) marked by Wolfram syndrome 1 (WFS1) expression, whose activity is modulated by adjacent astrocytes. These neurons in DISC1-N mice exhibited diminished firing ability and impaired communication with the astrocytes. Remarkably, optogenetic activation of these astrocytes reinstated neuronal excitability via D-serine acting on BLAWFS1 neurons' NMDA receptors, leading to improved risk-assessment behavior in the DISC1-N mice. Our findings point to BLA astrocytes as a promising target for treating risk-assessment dysfunctions in mental disorders.

Optogenetic Reporters Delivered as mRNA Facilitate Repeatable Action Potential and Calcium Handling Assessment in Human iPSC-Derived Cardiomyocytes.

Electrical activity and intracellular Ca2+ transients are key features of cardiomyocytes. They can be measured using organic voltage- and Ca2+-sensitive dyes but their photostability and phototoxicity mean they are unsuitable for long-term measurements. Here, we investigated whether genetically encoded voltage and Ca2+ indicators (GEVIs and GECIs) delivered as modified mRNA (modRNA) into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be accurate alternatives allowing measurements over long periods. These indicators were detected in hiPSC-CMs for up to 7 days after transfection and did not affect responses to proarrhythmic compounds. Furthermore, using the GEVI ASAP2f we observed action potential prolongation in long QT syndrome models, while the GECI jRCaMP1b facilitated the repeated evaluation of Ca2+ handling responses for various tyrosine kinase inhibitors. This study demonstrated that modRNAs encoding optogenetic constructs report cardiac physiology in hiPSC-CMs without toxicity or the need for stable integration, illustrating their value as alternatives to organic dyes or other gene delivery methods for expressing transgenes.

Low-cost and easy-fabrication lightweight drivable electrode array for multiple-regions electrophysiological recording in free-moving mice.

Objective.Extracellular electrophysiology has been widely applied to neural circuit dissections. However, long-term multiregional recording in free-moving mice remains a challenge. Low-cost and easy-fabrication of elaborate drivable electrodes is required for their prevalence.Approach.A three-layer nested construct (outside diameter, OD ∼ 1.80 mm, length ∼10 mm, <0.1 g) was recruited as a drivable component, which consisted of an ethylene-vinyl acetate copolymer heat-shrinkable tube, non-closed loop ceramic bushing, and stainless ferrule with a bulge twining silver wire. The supporting and working components were equipped with drivable components to be assembled into a drivable microwire electrode array with a nested structure (drivable MEANS). Two drivable microwire electrode arrays were independently implanted for chronic recording in different brain areas at respective angles. An optic fiber was easily loaded into the drivable MEANS to achieve optogenetic modulation and electrophysiological recording simultaneously.Main results.The drivable MEANS had lightweight (∼0.37 g), small (∼15 mm × 15 mm × 4 mm), and low cost (⩽$64.62). Two drivable MEANS were simultaneously implanted in mice, and high-quality electrophysiological recordings could be applied ⩾5 months after implantation in freely behaving animals. Electrophysiological recordings and analysis of the lateral septum (LS) and lateral hypothalamus in food-seeking behavior demonstrated that our drivable MEANS can be used to dissect the function of neural circuits. An optical fiber-integrated drivable MEANS (∼0.47 g) was used to stimulate and record LS neurons, which suggested that changes in working components can achieve more functions than electrophysiological recordings, such as optical stimulation, drug release, and calcium imaging.Significance.Drivable MEANS is an easily fabricated, lightweight drivable microwire electrode array for multiple-region electrophysiological recording in free-moving mice. Our design is likely to be a valuable platform for both current and prospective users, as well as for developers of multifunctional electrodes for free-moving mice.

Frequency modulation of entorhinal cortex neuronal activity drives distinct frequency-dependent states of brain-wide dynamics.

Human neuroimaging studies have shown that, during cognitive processing, the brain undergoes dynamic transitions between multiple, frequency-tuned states of activity. Although different states may emerge from distinct sources of neural activity, it remains unclear whether single-area neuronal spiking can also drive multiple dynamic states. In mice, we ask whether frequency modulation of the entorhinal cortex activity causes dynamic states to emerge and whether these states respond to distinct stimulation frequencies. Using hidden Markov modeling, we perform unsupervised detection of transient states in mouse brain-wide fMRI fluctuations induced via optogenetic frequency modulation of excitatory neurons. We unveil the existence of multiple, frequency-dependent dynamic states, invisible through standard static fMRI analyses. These states are linked to different anatomical circuits and disrupted in a frequency-dependent fashion in a transgenic model of cognitive disease directly related to entorhinal cortex dysfunction. These findings provide cross-scale insight into basic neuronal mechanisms that may underpin flexibility in brain-wide dynamics.

Modeling optical design parameters for fine stimulation in sciatic nerve of optogenetic mice.

Optogenetics presents an alternative method for interfacing with the nervous system over the gold-standard of electrical stimulation. While electrical stimulation requires electrodes to be surgically embedded in tissue for in vivo studies, optical stimulation offers a less-invasive approach that may yield more specific, localized stimulation. The advent of optogenetic laboratory animals-whose motor neurons can be activated when illuminated with blue light-enables research into refining optical stimulation of the mammalian nervous system where subsets of nerve fibers within a nerve may be stimulated without embedding any device directly into the nerve itself. However, optical stimulation has a major drawback in that light is readily scattered and absorbed in tissue thereby limiting the depth with which a single emission source can penetrate. We hypothesize that the use of multiple, focused light emissions deployed around the circumference of a nerve can overcome these light-scattering limitations. To understand the physical parameters necessary to produce pinpointed light stimulation within a single nerve, we employed a simplified Monte Carlo simulation to estimate the size of nerves where this technique may be successful, as well as the necessary optical lens design for emitters to be used during future in vivo studies. By modeling multiple focused beams, we find that only fascicles within a nerve diameter less than 1 mm are fully accessible to focused optical stimulation; a minimum of 4 light sources is required to generate a photon intensity at a point in a nerve over the initial contact along its surface. To elicit the same effect in larger nerves, focusing lenses would require a numerical aperture [Formula: see text]. These simulations inform on the design of instrumentation capable of stimulating disparate motor neurons in mouse sciatic nerve to control hindlimb movement.

An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans.

BACKGROUND: Optogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm. RESULTS: The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm's power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing. CONCLUSION: We have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

mem-iLID, a fast and economic protein purification method.

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Perturbation of amygdala-cortical projections reduces ensemble coherence of palatability coding in gustatory cortex.

Taste palatability is centrally involved in consumption decisions-we ingest foods that taste good and reject those that don't. Gustatory cortex (GC) and basolateral amygdala (BLA) almost certainly work together to mediate palatability-driven behavior, but the precise nature of their interplay during taste decision-making is still unknown. To probe this issue, we discretely perturbed (with optogenetics) activity in rats' BLA→GC axons during taste deliveries. This perturbation strongly altered GC taste responses, but while the perturbation itself was tonic (2.5 s), the alterations were not-changes preferentially aligned with the onset times of previously-described taste response epochs, and reduced evidence of palatability-related activity in the 'late-epoch' of the responses without reducing the amount of taste identity information available in the 'middle epoch.' Finally, BLA→GC perturbations changed behavior-linked taste response dynamics themselves, distinctively diminishing the abruptness of ensemble transitions into the late epoch. These results suggest that BLA 'organizes' behavior-related GC taste dynamics.

Assessment of optogenetically-driven strategies for prosthetic restoration of cortical vision in large-scale neural simulation of V1.

The neural encoding of visual features in primary visual cortex (V1) is well understood, with strong correlates to low-level perception, making V1 a strong candidate for vision restoration through neuroprosthetics. However, the functional relevance of neural dynamics evoked through external stimulation directly imposed at the cortical level is poorly understood. Furthermore, protocols for designing cortical stimulation patterns that would induce a naturalistic perception of the encoded stimuli have not yet been established. Here, we demonstrate a proof of concept by solving these issues through a computational model, combining (1) a large-scale spiking neural network model of cat V1 and (2) a virtual prosthetic system transcoding the visual input into tailored light-stimulation patterns which drive in situ the optogenetically modified cortical tissue. Using such virtual experiments, we design a protocol for translating simple Fourier contrasted stimuli (gratings) into activation patterns of the optogenetic matrix stimulator. We then quantify the relationship between spatial configuration of the imposed light pattern and the induced cortical activity. Our simulations in the absence of visual drive (simulated blindness) show that optogenetic stimulation with a spatial resolution as low as 100 [Formula: see text]m, and light intensity as weak as [Formula: see text] photons/s/cm[Formula: see text] is sufficient to evoke activity patterns in V1 close to those evoked by normal vision.

Breathing-driven prefrontal oscillations regulate maintenance of conditioned-fear evoked freezing independently of initiation.

Brain-body interactions are thought to be essential in emotions but their physiological basis remains poorly understood. In mice, regular 4 Hz breathing appears during freezing after cue-fear conditioning. Here we show that the olfactory bulb (OB) transmits this rhythm to the dorsomedial prefrontal cortex (dmPFC) where it organizes neural activity. Reduction of the respiratory-related 4 Hz oscillation, via bulbectomy or optogenetic perturbation of the OB, reduces freezing. Behavioural modelling shows that this is due to a specific reduction in freezing maintenance without impacting its initiation, thus dissociating these two phenomena. dmPFC LFP and firing patterns support the region's specific function in freezing maintenance. In particular, population analysis reveals that network activity tracks 4 Hz power dynamics during freezing and reaches a stable state at 4 Hz peak that lasts until freezing termination. These results provide a potential mechanism and a functional role for bodily feedback in emotions and therefore shed light on the historical James-Cannon debate.

A low-cost microfluidic platform coupled with light emitting diode for optogenetic analysis of neuronal response in C. elegans.

Optogenetic method is widely used for dissecting the neuronal function and connectivity in a specific neural circuit, which can help understanding how the animal process information and generate behavior. The nematode C. elegans has a simple but complete nervous system, making it an attractive model to study the dynamics signals of neural circuits. However, in vivo analysis on neural circuits usually rely on the complex and expensive optical equipment to allow optogenetic stimulating the neuron while recording its activities in such a freely moving animal. Hence, in this paper we reported a portable optofluidic platform that works based on optical fiber illumination and functional imaging for worm optogenetic manipulation. A light beam from LED laser pen crossing the 3D-printed optical fiber channel is used to activate the neurons specific-expressed with light sensitive proteins ChR-2. The imaging light path is perpendicular to the stimulation light, which allows activating neuron precisely and measuring cellular signals simultaneously. By using such an easy-to-assemble device, optical stimulation of the specific neurons and detection of dynamic calcium responses of other neurons could be proceeded simultaneously. Thus, the developed microfluidic platform puts forward a simple, rapid and low-cost strategy for further neural circuits studies.

Histological assessment of optogenetic tools to study fronto-visual and fronto-parietal cortical networks in the rhesus macaque.

Optogenetics offers unprecedented possibilities to investigate cortical networks. Yet, the number of successful optogenetic applications in non-human primates is still low, and the consequences of opsin expression in the primate brain are not well documented. We assessed histologically if we can target cerebrocortical networks with three common optogenetic constructs (AAV2/5-CaMKIIα-eNpHR3.0-mCherry, -ChR2-eYFP, -C1V1-mCherry). The frontal eye field or the dorsal premotor area of rhesus macaques were virally injected, and the resulting transduction spread, expression specificity, and opsin trafficking into axons projecting to parietal and visual areas were examined. After variable periods (2-24 months), expression was robust for all constructs at the injection sites. The CaMKIIα promoter driven-expression was predominant, but not exclusive, in excitatory neurons. In the case of eNpHR3.0-mCherry and ChR2-eYFP, opsins were present in axonal projections to target areas, in which sparse, retrogradely transduced neurons could also be found. Finally, the intracellular distribution of opsins differed: ChR2-eYFP had almost exclusive membrane localization, while eNpHR3.0-mCherry and C1V1-mCherry showed additional intracellular accumulations, which might affect neuronal survival in the long-term. Results indicate that all three constructs can be used for local neuronal modulation, but axonal stimulation and long-term use require additional considerations of construct selection and verification.

Innovations in the Neurosurgical Management of Epilepsy.

Technical limitations and clinical challenges have historically limited the diagnostic tools and treatment methods available for surgical approaches to the management of epilepsy. By contrast, recent technological innovations in several areas hold significant promise in improving outcomes and decreasing morbidity. We review innovations in the neurosurgical management of epilepsy in several areas, including wireless recording and stimulation systems (particularly responsive neurostimulation [NeuroPace]), conformal electrodes for high-resolution electrocorticography, robot-assisted stereotactic surgery, optogenetics and optical imaging methods, novel positron emission tomography ligands, and new applications of focused ultrasonography. Investigation into genetic causes of and susceptibilities to epilepsy has introduced a new era of precision medicine, enabling the understanding of cell signaling mechanisms underlying epileptic activity as well as patient-specific molecularly targeted treatment options. We discuss the emerging path to individualized treatment plans, predicted outcomes, and improved selection of effective interventions, on the basis of these developments.

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

Neural mechanisms of economic choices in mice.

Economic choices entail computing and comparing subjective values. Evidence from primates indicates that this behavior relies on the orbitofrontal cortex. Conversely, previous work in rodents provided conflicting results. Here we present a mouse model of economic choice behavior, and we show that the lateral orbital (LO) area is intimately related to the decision process. In the experiments, mice chose between different juices offered in variable amounts. Choice patterns closely resembled those measured in primates. Optogenetic inactivation of LO dramatically disrupted choices by inducing erratic changes of relative value and by increasing choice variability. Neuronal recordings revealed that different groups of cells encoded the values of individual options, the binary choice outcome and the chosen value. These groups match those previously identified in primates, except that the neuronal representation in mice is spatial (in monkeys it is good-based). Our results lay the foundations for a circuit-level analysis of economic decisions.

How technical progress reshaped behavioral neuroendocrinology during the last 50 years and some methodological remarks.

The first issue of Hormones and Behavior was published 50 years ago in 1969, a time when most of the techniques we currently use in Behavioral Endocrinology were not available. Researchers have during the last 5 decades developed techniques that allow measuring hormones in small volumes of biological samples, identify the sites where steroids act in the brain to activate sexual behavior, characterize and quantify gene expression correlated with behavior expression, modify this expression in a specific manner, and manipulate the activity of selected neuronal populations by chemogenetic and optogenetic techniques. This technical progress has considerably transformed the field and has been very beneficial for our understanding of the endocrine controls of behavior in general, but it did also come with some caveats. The facilitation of scientific investigations came with some relaxation of methodological exigency. Some critical controls are no longer performed on a regular basis and complex techniques supplied as ready to use kits are implemented without precise knowledge of their limitations. We present here a selective review of the most important of these new techniques, their potential problems and how they changed our view of the hormonal control of behavior. Fortunately, the scientific endeavor is a self-correcting process. The problems have been identified and corrections have been proposed. The next decades will obviously be filled with exciting discoveries in behavioral neuroendocrinology.

Optogenetic assessment of VIP, PV, SOM and NOS inhibitory neuron activity and cerebral blood flow regulation in mouse somato-sensory cortex.

The impact of different neuronal populations on local cerebral blood flow (CBF) regulation is not well known and insight into these relationships could enhance the interpretation of brain function and dysfunction from brain imaging data. We investigated the role of sub-types of inhibitory neuron activity on the regulation of CBF using optogenetics, laser Doppler flowmetry and different transgenic mouse models (parvalbumin (PV), vasoactive intestinal peptide (VIP), somatostatin (SOM) and nitric oxide synthase (NOS)). Whisker stimulation was used to verify that typical CBF responses were obtained in all mice. Photo-stimulation of SOM-cre and NOS-cre mice produced significant increases in CBF that were similar to whisker responses. In NOS-cre mice, CBF responses scaled with the photo-stimulus pulse duration and frequency. In SOM-cre mice, CBF increases were followed by decreases. In VIP-cre mice, photo-stimulation did not consistently produce significant changes in CBF, while slower increases in CBF that peaked 14-18 s after stimulation onset were observed in PV-cre mice. Control experiments performed in non-expressing regions showed no changes in CBF. These findings suggest that dysfunction in NOS or SOM neurons can have a significant impact on vascular responses that are detected by brain imaging methods like functional magnetic resonance imaging (fMRI).

Real-Time Value Integration during Economic Choice Is Regulated by Orbitofrontal Cortex.

Neural correlates implicate the orbitofrontal cortex (OFC) in value-based or economic decision making [1-3]. Yet inactivation of OFC in rats performing a rodent version of the standard economic choice task is without effect [4, 5], a finding more in accord with ideas that the OFC is primarily necessary for behavior when new information must be taken into account [6-9]. Neural activity in the OFC spontaneously updates to reflect new information, particularly about outcomes [10-16], and the OFC is necessary for adjustments to learned behavior only under these conditions [4, 16-26]. Here, we merge these two independent lines of research by inactivating lateral OFC during an economic choice that requires new information about the value of the predicted outcomes to be incorporated into an already established choice. Outcome value was changed by pre-feeding the rats one of two food options before testing. In control rats, this pre-feeding resulted in divergent changes in choice behavior that depended on the rats' prior preference for the pre-fed food. Optogenetic inactivation of the OFC disrupted this bi-directional effect of pre-feeding without affecting other measures that describe the underlying choice behavior. This finding unifies the role of the OFC in economic choice with its role in a host of other behaviors, causally demonstrating that the OFC is not necessary for economic choice per se-unless that choice incorporates new information about the outcomes.

Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Assessment of Proarrhythmic Potential of Drugs in Optogenetically Paced Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiac side-effects are one of the major reasons for failure of drugs during preclinical development. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been proposed as a model for predicting drug-induced arrhythmias under the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Field potential duration (FPD) in spontaneously beating iPSC-CMs is commonly corrected for beating rate using formulas originally derived from the clinical QT-RR relationship that have not been thoroughly validated for use with iPSC-CMs. In this study, channelrhodopsin-2 was expressed in iPSC-CMs allowing for recordings in both spontaneously beating and optically paced (0.8, 1, and 1.5 Hz pacing rate) iPSC-CMs using a microelectrode array system (Maestro, Axion Biosystems). After optimizing the intensity (>1 mW/mm2), duration (15 ms) and frequency of the stimulating light pulses, we recorded iPSC-CMs' responses to 28 blinded CiPA compounds with clinically characterized risk of causing ventricular arrhythmia (Torsade de Pointes or TdP). Drug-induced FPD prolongation data along with drug-induced arrhythmia-like events were used to build a logistic regression model, separating high or intermediate TdP risk drugs from low-or-no TdP risk drugs. The area under the receiver operator characteristic curve for drug TdP risk prediction was identical for spontaneously beating and 0.8 Hz-paced iPSC-CMs (AUC = 0.96; 95% CI [0.9, 1]), while it was slightly lower for 1 and 1.5 Hz pacing (AUC = 0.88; 95% CI [0.76, 1] and 0.93; 95% CI [0.84, 1], respectively). In this study, optical pacing did not offer substantial improvement in proarrhythmic risk prediction when compared with nonpaced iPSC-CMs in the sample of 28 drugs.