Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

A dark quencher genetically encodable voltage indicator (dqGEVI) exhibits high fidelity and speed.

Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

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.

Cortical Correlates of Psychedelic-Induced Shaking Behavior Revealed by Voltage Imaging.

(1) From mouse to man, shaking behavior (head twitches and/or wet dog shakes) is a reliable readout of psychedelic drug action. Shaking behavior like psychedelia is thought to be mediated by serotonin 2A receptors on cortical pyramidal cells. The involvement of pyramidal cells in psychedelic-induced shaking behavior remains hypothetical, though, as experimental in vivo evidence is limited. (2) Here, we use cell type-specific voltage imaging in awake mice to address this issue. We intersectionally express the genetically encoded voltage indicator VSFP Butterfly 1.2 in layer 2/3 pyramidal neurons. We simultaneously capture cortical hemodynamics and cell type-specific voltage activity while mice display psychedelic shaking behavior. (3) Shaking behavior is preceded by high-frequency oscillations and overlaps with low-frequency oscillations in the motor cortex. Oscillations spectrally mirror the rhythmics of shaking behavior and reflect layer 2/3 pyramidal cell activity complemented by hemodynamics. (4) Our results reveal a clear cortical fingerprint of serotonin-2A-receptor-mediated shaking behavior and open a promising methodological avenue relating a cross-mammalian psychedelic effect to cell-type specific brain dynamics.

Cortex-Wide Dynamics of Intrinsic Electrical Activities: Propagating Waves and Their Interactions.

Cortical circuits generate patterned activities that reflect intrinsic brain dynamics that lay the foundation for any, including stimuli-evoked, cognition and behavior. However, the spatiotemporal organization properties and principles of this intrinsic activity have only been partially elucidated because of previous poor resolution of experimental data and limited analysis methods. Here we investigated continuous wave patterns in the 0.5-4 Hz (delta band) frequency range on data from high-spatiotemporal resolution optical voltage imaging of the upper cortical layers in anesthetized mice. Waves of population activities propagate in heterogeneous directions to coordinate neuronal activities between different brain regions. The complex wave patterns show characteristics of both stereotypy and variety. The location and type of wave patterns determine the dynamical evolution when different waves interact with each other. Local wave patterns of source, sink, or saddle emerge at preferred spatial locations. Specifically, "source" patterns are predominantly found in cortical regions with low multimodal hierarchy such as the primary somatosensory cortex. Our findings reveal principles that govern the spatiotemporal dynamics of spontaneous cortical activities and associate them with the structural architecture across the cortex.SIGNIFICANCE STATEMENT Intrinsic brain activities, as opposed to external stimulus-evoked responses, have increasingly gained attention, but it remains unclear how these intrinsic activities are spatiotemporally organized at the cortex-wide scale. By taking advantage of the high spatiotemporal resolution of optical voltage imaging, we identified five wave pattern types, and revealed the organization properties of different wave patterns and the dynamical mechanisms when they interact with each other. Moreover, we found a relationship between the emergence probability of local wave patterns and the multimodal structure hierarchy across cortical areas. Our findings reveal the principles of spatiotemporal wave dynamics of spontaneous activities and associate them with the underlying hierarchical architecture across the cortex.

Combining Cortical Voltage Imaging and Hippocampal Electrophysiology for Investigating Global, Multi-Timescale Activity Interactions in the Brain.

A new generation of optogenetic tools for analyzing neural activity has been contributing to the elucidation of classical open questions in neuroscience. Specifically, voltage imaging technologies using enhanced genetically encoded voltage indicators have been increasingly used to observe the dynamics of large circuits at the mesoscale. Here, we describe how to combine cortical wide-field voltage imaging with hippocampal electrophysiology in awake, behaving mice. Furthermore, we highlight how this method can be useful for different possible investigations, using the characterization of hippocampal-neocortical interactions as a case study.

Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange.

BACKGROUND: Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (Vmem) and intracellular pH (pHi). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation. RESULTS: We expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K+channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium. CONCLUSIONS: The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow.

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca2+ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening.

Genetically Encoded Fluorescent Calcium and Voltage Indicators.

Fluorescent probes that indicate biologically important quantities are widely used for many different types of biological experiments across life sciences. During recent years, limitations of small molecule-based indicators have been overcome by the development of genetically encoded indicators. Here we focus on fluorescent calcium and voltage indicators and point to their applications mainly in neurosciences.

Genetically Encoded Voltage Indicators: Opportunities and Challenges.

UNLABELLED: A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. SIGNIFICANCE STATEMENT: Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions.

Voltage imaging to understand connections and functions of neuronal circuits.

Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems.

Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators.

Neural circuits are non-linear dynamical systems that transform information based on the pattern of input, current state and functional connectivity. To understand how a given stimulus is processed, one would ideally record neural activity across the entire brain of a behaving animal, at cellular or even subcellular resolution, in addition to characterizing anatomical connectivity. Given their transparency and relatively small size, larval zebrafish provide a powerful system for brain-wide monitoring of neural activity. Genetically encoded calcium indicators have been used for this purpose, but cannot directly report hyperpolarization or sub-threshold activity. Voltage indicators, in contrast, have this capability. Here, we test whether two different genetically encoded voltage reporters, ASAP1 and Bongwoori, can be expressed and report activity in the zebrafish brain, using widefield, two-photon and light sheet microscopy. We were unable to express ASAP1 in neurons. Bongwoori, in contrast expressed well, and because of its membrane localization, allowed visualization of axon trajectories in 3D. Bongwoori displayed stimulus-evoked changes in fluorescence, which could be detected in single trials. However, under high laser illumination, puncta on neural membranes underwent spontaneous fluctuations in intensity, suggesting that the probe is susceptible to blinking artefacts. These data indicate that larval zebrafish can be used to image electrical activity in the brain of an intact vertebrate at high resolution, although care is needed in imaging and analysis. Recording activity across the whole brain will benefit from further developments in imaging hardware and indicators.

Genetically Encoded Voltage Indicators in Circulation Research.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

Physiological features of parvalbumin-expressing GABAergic interneurons contributing to high-frequency oscillations in the cerebral cortex.

Parvalbumin-expressing (PV+) inhibitory interneurons drive gamma oscillations (30-80 Hz), which underlie higher cognitive functions. In this review, we discuss two groups/aspects of fundamental properties of PV+ interneurons. In the first group (dubbed Before Axon), we list properties representing optimal synaptic integration in PV+ interneurons designed to support fast oscillations. For example: [i] Information can neither enter nor leave the neocortex without the engagement of fast PV+ -mediated inhibition; [ii] Voltage responses in PV+ interneuron dendrites integrate linearly to reduce impact of the fluctuations in the afferent drive; and [iii] Reversed somatodendritic Rm gradient accelerates the time courses of synaptic potentials arriving at the soma. In the second group (dubbed After Axon), we list morphological and biophysical properties responsible for (a) short synaptic delays, and (b) efficient postsynaptic outcomes. For example: [i] Fast-spiking ability that allows PV+ interneurons to outpace other cortical neurons (pyramidal neurons). [ii] Myelinated axon (which is only found in the PV+ subclass of interneurons) to secure fast-spiking at the initial axon segment; and [iii] Inhibitory autapses - autoinhibition, which assures brief biphasic voltage transients and supports postinhibitory rebounds. Recent advent of scientific tools, such as viral strategies to target PV cells and the ability to monitor PV cells via in vivo imaging during behavior, will aid in defining the role of PV cells in the CNS. Given the link between PV+ interneurons and cognition, in the future, it would be useful to carry out physiological recordings in the PV+ cell type selectively and characterize if and how psychiatric and neurological diseases affect initiation and propagation of electrical signals in this cortical sub-circuit. Voltage imaging may allow fast recordings of electrical signals from many PV+ interneurons simultaneously.

Current Practice in Using Voltage Imaging to Record Fast Neuronal Activity: Successful Examples from Invertebrate to Mammalian Studies.

The optical imaging of neuronal activity with potentiometric probes has been credited with being able to address key questions in neuroscience via the simultaneous recording of many neurons. This technique, which was pioneered 50 years ago, has allowed researchers to study the dynamics of neural activity, from tiny subthreshold synaptic events in the axon and dendrites at the subcellular level to the fluctuation of field potentials and how they spread across large areas of the brain. Initially, synthetic voltage-sensitive dyes (VSDs) were applied directly to brain tissue via staining, but recent advances in transgenic methods now allow the expression of genetically encoded voltage indicators (GEVIs), specifically in selected neuron types. However, voltage imaging is technically difficult and limited by several methodological constraints that determine its applicability in a given type of experiment. The prevalence of this method is far from being comparable to patch clamp voltage recording or similar routine methods in neuroscience research. There are more than twice as many studies on VSDs as there are on GEVIs. As can be seen from the majority of the papers, most of them are either methodological ones or reviews. However, potentiometric imaging is able to address key questions in neuroscience by recording most or many neurons simultaneously, thus providing unique information that cannot be obtained via other methods. Different types of optical voltage indicators have their advantages and limitations, which we focus on in detail. Here, we summarize the experience of the scientific community in the application of voltage imaging and try to evaluate the contribution of this method to neuroscience research.

Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model.

Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Conserved Amino Acids Residing Outside the Voltage Field Can Shift the Voltage Sensitivity and Increase the Signal Speed and Size of Ciona Based GEVIs.

To identify potential regions of the voltage-sensing domain that could shift the voltage sensitivity of Ciona intestinalis based Genetically Encoded Voltage Indicators (GEVIs), we aligned the amino acid sequences of voltage-gated sodium channels from different organisms. Conserved polar residues were identified at multiple transmembrane/loop junctions in the voltage sensing domain. Similar conservation of polar amino acids was found in the voltage-sensing domain of the voltage-sensing phosphatase gene family. These conserved residues were mutated to nonpolar or oppositely charged amino acids in a GEVI that utilizes the voltage sensing domain of the voltage sensing phosphatase from Ciona fused to the fluorescent protein, super ecliptic pHluorin (A227D). Different mutations shifted the voltage sensitivity to more positive or more negative membrane potentials. Double mutants were then created by selecting constructs that shifted the optical signal to a more physiologically relevant voltage range. Introduction of these mutations into previously developed GEVIs resulted in Plos6-v2 which improved the dynamic range to 40% ΔF/F/100 mV, a 25% increase over the parent, ArcLight. The onset time constant of Plos6-v2 is also 50% faster than ArcLight. Thus, Plos6-v2 appears to be the GEVI of choice.

Thalamic bursting and the role of timing and synchrony in thalamocortical signaling in the awake mouse.

The thalamus controls transmission of sensory signals from periphery to cortex, ultimately shaping perception. Despite this significant role, dynamic thalamic gating and the consequences for downstream cortical sensory representations have not been well studied in the awake brain. We optogenetically modulated the ventro-posterior-medial thalamus in the vibrissa pathway of the awake mouse and measured spiking activity in the thalamus and activity in primary somatosensory cortex (S1) using extracellular electrophysiology and genetically encoded voltage imaging. Thalamic hyperpolarization significantly enhanced thalamic sensory-evoked bursting; however, surprisingly, the S1 cortical response was not amplified, but instead, timing precision was significantly increased, spatial activation more focused, and there was an increased synchronization of cortical inhibitory neurons. A thalamocortical network model implicates the modulation of precise timing of feedforward thalamic population spiking, presenting a highly sensitive, timing-based gating of sensory signaling to the cortex.