All-optical physiology resolves a synaptic basis for behavioral timescale plasticity.

Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.

Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans.

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact C. elegans.

Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (Ik1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking Ik1-like currents with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1's role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology.

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium from all wells in parallel. The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 394-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated deployment of optogenetic cell spheroids for point pacing to study conduction in 96-well format, and the use of temporal multiplexing to register voltage and calcium simultaneously on a single camera in this stand-alone platform. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates, in the various configurations. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

CRISPRi Gene Modulation and All-Optical Electrophysiology in Post-Differentiated Human iPSC-Cardiomyocytes.

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all-optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all-optical tools. More potent CRISPRi effectors, e.g. Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology.

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium (parallel recordings from up to 100 wells in 384-well plates are demonstrated). The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 384-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated optogenetic point pacing via cell spheroids to study conduction in 96-well format, as well as temporal multiplexing to register voltage and calcium simultaneously on a single camera. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

Near-infrared and far-red genetically encoded indicators of neuronal activity.

Genetically encoded fluorescent indicators of neuronal activity are ultimately developed to dissect functions of neuronal ensembles during behavior in living animals. Recent development of near-infrared shifted calcium and voltage indicators moved us closer to this goal and enabled crosstalk-free combination with blue light-controlled optogenetic tools for all-optical control and readout. Here I discuss designs of recent near-infrared and far-red calcium and voltage indicators, compare their properties and performance, and overview their applications to spectral multiplexing and in vivo imaging. I also provide perspectives for further development.

Integration of Engineered "Spark-Cell" Spheroids for Optical Pacing of Cardiac Tissue.

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional "spark cells" spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These "spark-cell" spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm2) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.

Screening and Cellular Characterization of Genetically Encoded Voltage Indicators Based on Near-Infrared Fluorescent Proteins.

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS.

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, "Optopatch," pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales.

Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescent proteins (FPs) engineered from bacterial phytochromes have become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.