Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by ...all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.
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
. 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.
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.
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•A PCR-based electroporation screen yielded an improved voltage indicator, ASAP3•ASAP3 shows larger voltage responses than other fluorescent protein-based sensors•Ultrafast local volume excitation (ULoVE) boosts random-access two-photon signals•ASAP3 and ULoVE report subthreshold and spiking potentials in deep brain regions
The genetically encoded voltage indicator, ASAP3, provides improved voltage responses and activation kinetics that enable single-trial tracking of action potentials and subthreshold events with subcellular resolution deep in the mouse brain during behavioral tasks when imaged with ULoVE, a kilohertz-rate two-photon sampling method with increased stability and sensitivity.
•Genetically encoded voltage indicators show great promise as tools for probing neuronal activity with millisecond-timescale resolution.•To develop voltage indicators, protein engineers have utilized ...proteins whose conformational or photophysical states are sensitive to voltage.•Further improvement in the kinetics, dynamic range and/or brightness are needed to facilitate broader deployment of voltage sensors in vivo.
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.
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.
Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track ...underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions, report an advancing gradient of dendritic theta phase along the basal-tuft axis, and describe a predominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find that spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.Neurons receive their input in three dimensions via their dendrites, but how electrical activity in dendrites is organized is unknown. Here, the authors work out the distinct rules that govern activity across this 3D structure in different brain states.
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium ...indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access ...multiphoton microscopy (RAMP) and the latest two-photon-compatible high-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.
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•Kilohertz-order two-photon voltage imaging with single-trial acquisition•Readout spontaneous activity from multi-cells or subcellular regions simultaneously•Long-duration voltage imaging insuring functional studies of neuron circuit•Functional connections analysis with greater precision than calcium imaging
Optical Imaging; Developmental Neuroscience; Cellular Neuroscience; Techniques in Neuroscience
Large-scale, high-throughput specificity assays to characterize binding properties within a competitive and complex environment of potential binder-target pairs remain challenging and cost ...prohibitive. Barcode cycle sequencing (BCS) is a molecular binding assay for proteins, peptides, and other small molecules that is built on a next-generation sequencing (NGS) chip. BCS uses a binder library and targets labeled with unique DNA barcodes. Upon binding, binder barcodes are ligated to target barcodes and sequenced to identify encoded binding events.
For complete details on the use and execution of this protocol, please refer to Hong et al. (2022).
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•Binding events are encoded in DNA barcodes that identify both binders and targets•Binding events occur directly on a DNA sequencing chip for direct sequencing•Binding events can be multiplexed and scaled to record billions of events•Colocalization of a target and foundation allows multiple rounds of BCS on a chip
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Large-scale, high-throughput specificity assays to characterize binding properties within a competitive and complex environment of potential binder-target pairs remain challenging and cost prohibitive. Barcode cycle sequencing (BCS) is a molecular binding assay for proteins, peptides, and other small molecules that is built on a next-generation sequencing (NGS) chip. BCS uses a binder library and targets labeled with unique DNA barcodes. Upon binding, binder barcodes are ligated to target barcodes and sequenced to identify encoded binding events.
We demonstrate early progress toward constructing a high-throughput, single-molecule protein sequencing technology utilizing barcoded DNA aptamers (binders) to recognize terminal amino acids of ...peptides (targets) tethered on a next-generation sequencing chip. DNA binders deposit unique, amino acid-identifying barcodes on the chip. The end goal is that, over multiple binding cycles, a sequential chain of DNA barcodes will identify the amino acid sequence of a peptide. Toward this, we demonstrate successful target identification with two sets of target-binder pairs: DNA-DNA and Peptide-Protein. For DNA-DNA binding, we show assembly and sequencing of DNA barcodes over six consecutive binding cycles. Intriguingly, our computational simulation predicts that a small set of semi-selective DNA binders offers significant coverage of the human proteome. Toward this end, we introduce a binder discovery pipeline that ultimately could merge with the chip assay into a technology called ProtSeq, for future high-throughput, single-molecule protein sequencing.
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•Designed ProtSeq protein sequencing method compatible with widely used NGS technology•Built Target-Switch SELEX to isolate aptamers specific to N-terminal amino acids (AAs)•Showed binding, ligation, cleavage, and NGS of six DNA binders in ordered barcode chain•Developed pipeline to deconvolve AAs from DNA barcodes to identify putative proteins
Biochemistry; Biochemistry applications; Sequence analysis; Proteomics; Transcriptomics
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