Widefield imaging of rapid pan-cortical voltage dynamics with an indicator evolved for one-photon microscopy.

Widefield imaging with genetically encoded voltage indicators (GEVIs) is a promising approach for understanding the role of large cortical networks in the neural coding of behavior. However, the limited performance of current GEVIs restricts their deployment for single-trial imaging of rapid neuronal voltage dynamics. Here, we developed a high-throughput platform to screen for GEVIs that combine fast kinetics with high brightness, sensitivity, and photostability under widefield one-photon illumination. Rounds of directed evolution produced JEDI-1P, a green-emitting fluorescent indicator with enhanced performance across all metrics. Next, we optimized a neonatal intracerebroventricular delivery method to achieve cost-effective and wide-spread JEDI-1P expression in mice. We also developed an approach to correct optical measurements from hemodynamic and motion artifacts effectively. Finally, we achieved stable brain-wide voltage imaging and successfully tracked gamma-frequency whisker and visual stimulations in awake mice in single trials, opening the door to investigating the role of high-frequency signals in brain computations.

Scanless two-photon voltage imaging.

Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.

Automating the High-Throughput Screening of Protein-Based Optical Indicators and Actuators.

Over the last 25 years, protein engineers have developed an impressive collection of optical tools to interface with biological systems: indicators to eavesdrop on cellular activity and actuators to poke and prod native processes. To reach the performance level required for their downstream applications, protein-based tools are usually sculpted by iterative rounds of mutagenesis. In each round, libraries of variants are made and evaluated, and the most promising hits are then retrieved, sequenced, and further characterized. Early efforts to engineer protein-based optical tools were largely manual, suffering from low throughput, human error, and tedium. Here, we describe approaches to automating the screening of libraries generated as colonies on agar, multiwell plates, and pooled populations of single-cell variants. We also briefly discuss emerging approaches for screening, including cell-free systems and machine learning.

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.

Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition.

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of 23 makes H. catenoides KCR1 (HcKCR1) a powerful hyperpolarizing tool to suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. HcKCR1 enables optogenetic control of K+ gradients, which is promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinson's disease and long-QT syndrome and other cardiac arrhythmias.

A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3.

A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.

Retinal horizontal cells use different synaptic sites for global feedforward and local feedback signaling.

In the outer plexiform layer (OPL) of the mammalian retina, cone photoreceptors (cones) provide input to more than a dozen types of cone bipolar cells (CBCs). In the mouse, this transmission is modulated by a single horizontal cell (HC) type. HCs perform global signaling within their laterally coupled network but also provide local, cone-specific feedback. However, it is unknown how HCs provide local feedback to cones at the same time as global forward signaling to CBCs and where the underlying synapses are located. To assess how HCs simultaneously perform different modes of signaling, we reconstructed the dendritic trees of five HCs as well as cone axon terminals and CBC dendrites in a serial block-face electron microscopy volume and analyzed their connectivity. In addition to the fine HC dendritic tips invaginating cone axon terminals, we also identified "bulbs," short segments of increased dendritic diameter on the primary dendrites of HCs. These bulbs are in an OPL stratum well below the cone axon terminal base and make contacts with other HCs and CBCs. Our results from immunolabeling, electron microscopy, and glutamate imaging suggest that HC bulbs represent GABAergic synapses that do not receive any direct photoreceptor input. Together, our data suggest the existence of two synaptic strata in the mouse OPL, spatially separating cone-specific feedback and feedforward signaling to CBCs. A biophysical model of a HC dendritic branch and voltage imaging support the hypothesis that this spatial arrangement of synaptic contacts allows for simultaneous local feedback and global feedforward signaling by HCs.

Ancestral circuits for vertebrate color vision emerge at the first retinal synapse.

For color vision, retinal circuits separate information about intensity and wavelength. In vertebrates that use the full complement of four "ancestral" cone types, the nature and implementation of this computation remain poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying color processing in larval zebrafish. We find that the synaptic outputs of red and green cones efficiently rotate the encoding of natural daylight in a principal components analysis­like manner to yield primary achromatic and spectrally opponent axes, respectively. Blue cones are tuned to capture most remaining variance when opposed to green cones, while UV cone present a UV achromatic axis for prey capture. We note that fruitflies use essentially the same strategy. Therefore, rotating color space into primary achromatic and chromatic axes at the eye's first synapse may thus be a fundamental principle of color vision when using more than two spectrally well-separated photoreceptor types.

A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

Versatile phenotype-activated cell sorting.

Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight's ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

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.

Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice.

Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.

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.

Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.

A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

Designs and sensing mechanisms of genetically encoded fluorescent voltage indicators.

Neurons tightly regulate the electrical potential difference across the plasma membrane with millivolt accuracy and millisecond resolution. Membrane voltage dynamics underlie the generation of an impulse, the transduction of impulses from one end of the neuron to the other, and the release of neurotransmitters. Imaging these voltage dynamics in multiple neurons simultaneously is therefore crucial for understanding how neurons function together within circuits in intact brains. Genetically encoded fluorescent voltage sensors have long been desired to report voltage in defined subsets of neurons with optical readout. In this review, we discuss the diverse strategies used to design and optimize protein-based voltage sensors, and highlight the chemical mechanisms by which different classes of reporters sense voltage. To guide neuroscientists in choosing an appropriate sensor for their applications, we also describe operating trade-offs of each class of voltage indicators.

High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor.

Accurate optical reporting of electrical activity in genetically defined neuronal populations is a long-standing goal in neuroscience. We developed Accelerated Sensor of Action Potentials 1 (ASAP1), a voltage sensor design in which a circularly permuted green fluorescent protein is inserted in an extracellular loop of a voltage-sensing domain, rendering fluorescence responsive to membrane potential. ASAP1 demonstrated on and off kinetics of ∼ 2 ms, reliably detected single action potentials and subthreshold potential changes, and tracked trains of action potential waveforms up to 200 Hz in single trials. With a favorable combination of brightness, dynamic range and speed, ASAP1 enables continuous monitoring of membrane potential in neurons at kilohertz frame rates using standard epifluorescence microscopy.

One-step cloning and chromosomal integration of DNA.

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.