Optical Recording Of Action Potentials Research Articles

A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients.

Induced pluripotent stem cells (iPSCs) from type 1 long QT (LQT1) patients can differentiate into cardiomyocytes (CMs) including ventricular cells to recapitulate the disease phenotype. Although optical recordings using membrane potential dyes to monitor action potentials (APs) were reported, no study has investigated the disease phenotypes of cardiac channelopathy in association with the cardiac subtype at the single-cell level. We induced iPSC-CMs from three control and three LQT1 patients. Single-cell analysis using a fast-responding dye confirmed that ventricular cells were the dominant subtype (control-iPSC-CMs: 98%, 88%, 91%; LQT1-iPSC-CMs: 95%, 79%, 92%). In addition, LQT1-iPSC-ventricular cells displayed an increased frequency of early afterdepolarizations (pvalue = 0.031). Cardiomyocyte monolayers constituted mostly of ventricular cells derived from LQT1-iPSCs showed prolonged AP duration (APD) (pvalue = 0.000096). High-throughput assays using cardiomyocyte monolayers in 96-well plates demonstrated that IKr inhibitors prolonged APDs in both control- and LQT1-iPSC-CM monolayers. We confirmed that the optical recordings of APs in single cells and monolayers derived from control- and LQT1-iPSC-CMs can be used to assess arrhythmogenicity, supporting the feasibility of membrane potential dye-based high-throughput screening to study ventricular arrhythmias caused by genetic channelopathy or cardiotoxic drugs.

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

SummaryHuman 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.

In Vivo Optical Recording of Action Potentials In C. elegans Muscles using Arclight, a Genetically Expressed Voltage Sensitive Fluorescent Protein

The locomotory system of the roundworm C. elegans is an attractive model for elucidating the genetic, cellular, and neural circuit bases of behavior. How the motor neurons and body wall muscles (BWMs) coordinate the worm's locomotion remain poorly understood. Electrophysiological studies have found that BWMs fire action potentials, both spontaneously and in response to nerve stimulation (Liu et al., 2011, Gao and Zhen, 2011). However, these recordings require dissection of the animal and can only be obtained from one or at most two cells simultaneously. To measure electrical activity throughout the intact animal, we have introduced the voltage sensitive fluorescent protein, Arclight-A242 (Jin et al., 2012) into body wall muscle cells under the myo-3 promoter. Arclight is trafficked efficiently to the muscle cell membrane and does not cause any obvious behavioral defect. We record activity of muscles in immobilized worms at 100 frames per second throughout the worm by using a 5.5 megapixel sCMOS camera and LED illumination (475 nm), and find oscillations in fluorescence intensity similar to bursts of action potentials reported previously (Liu et al., 2010) with average frequency of approximately 3 Hz. These oscillations are particularly prominent in the head (anterior BWM). We observe that activity in dorsal and ventral muscle cells are anticorrelated, consistent with the muscles functioning in opposition to each other. These methods will allow us to investigate the mechanisms of nervous system coordination of BWM activity. Supported by U24 NS057631 (JRAW and BMS), R01 NS040966 (BMS), the University of Pennsylvania Comprehensive Neuroscience Center (LH), and the Alfred P. Sloan Research Foundation (CFY). We are grateful to Stanford Photonics, Inc. for the loan of the camera and Piper software. JRAW and LH contributed equally.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieve this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear two-fold increase in brightness between −150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio > 10. The mutant Arch(D95N) lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type, but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits, and electrophysiology in systems for which electrode-based techniques are challenging.

Genetic Targeting of a Voltage-Sensitive Dye by Enzymatic Activation of Phosphonooxymethyl-ammonium Derivative

Optical recording of action potentials in individual neurons requires cell-selective targeting with a fluorescent, voltage-sensitive probe. We report on a new labeling system that takes advantage of recent developments in prodrug-based chemistry and allows for the targeting of a lipophilic dye into the plasma membrane of genetically specified cells. With the introduction of two phosphonooxymethylammonium zwitterions into the hydrocarbon chains of an amphiphilic, voltage-sensitive hemicyanine dye, a precursor dye was formed that is water-soluble to an extent that it can no longer bind into cell membranes and hence prevents unspecific staining. Glycosylphosphatidylinositol anchored placental alkaline phosphatase expressed in HEK293 cells converted the precursor dye to a homologue of the widely used dye di-4-ANEPPS and gave rise to excellent levels of plasma membrane localized staining. The voltage sensitivity of the enzymatically activated dye was tested and shown to be similar to sensitivity reported for di-4-ANEPPS.

Optical Recording of Action Potentials and Other Discrete Physiological Events: A Perspective from Signal Detection Theory

Optical imaging of physiological events in real time can yield insights into biological function that would be difficult to obtain by other experimental means. However, the detection of all-or-none events, such as action potentials or vesicle fusion events, in noisy single-trial data often requires a careful balance of tradeoffs. The analysis of such experiments, as well as the design of optical reporters and instrumentation for them, is aided by an understanding of the principles of signal detection. This review illustrates these principles, using as an example action potential recording with optical voltage reporters.

Two-Photon Excitation of di-4-ANEPPS for Optical Recording of Action Potentials in Rabbit Heart

Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.

Two-Photon Excitation of di-4-ANEPPS for Optical Recording of Action Potentials in Rabbit Heart

Cardiac action potentials have been measured with single-photon excitation (SPE) of transmembrane voltage-sensitive fluorescent dye. Two-photon excitation (TPE) may have advantages for localization and depth of the tissue region from which the action potential is measured. However measurements of action potentials with SPE have not been demonstrated. We sought to develop a method for TPE of di-4-ANEPPS and test whether the method yields voltage-dependent fluorescence in cardiac tissue. We modified our SPE and ratio-metric fluorescence recording system to use a femtosecond pulsed near-infrared laser. Modifications were made to enhance fluorescence collection efficiency and to block infrared laser light from entering the fluorescence collection system. Fluorescence was collected simultaneously in green (510-570 nm) and red (590-700 nm) wavelength bands. Action potentials were observed in the ratio of the green signal to the red signal, but were not observed above the noise level in either of the individual signals. Incorporation of a common-mode noise subtraction method revealed action potentials in green and red signals. We also found that the di-4-ANEPPS fluorescence emission spectrum for TPE at 930 nm was similar to the emission spectrum for SPE at 488 nm. The multiphoton method may be beneficial for highly localized cardiac optical measurements.

Optical recording of action potentials with second-harmonic generation microscopy.

Nonlinear microscopy has proven to be essential for neuroscience investigations of thick tissue preparations. However, the optical recording of fast (approximately 1 msec) cellular electrical activity has never until now been successfully combined with this imaging modality. Through the use of second-harmonic generation microscopy of primary Aplysia neurons in culture labeled with 4-[4-(dihexylamino)phenyl][ethynyl]-1-(4-sulfobutyl)pyridinium (inner salt), we optically recorded action potentials with 0.833 msec temporal and 0.6 microm spatial resolution on soma and neurite membranes. Second-harmonic generation response as a function of change in membrane potential was found to be linear with a signal change of approximately 6%/100 mV. The signal-to-noise ratio was approximately 1 for single-trace action potential recordings but was readily increased to approximately 6-7 with temporal averaging of approximately 50 scans. Photodamage was determined to be negligible by observing action potential characteristics, cellular resting potential, and gross cellular morphology during and after laser illumination. High-resolution (micrometer scale) optical recording of membrane potential activity by previous techniques has been limited to imaging depths an order of magnitude less than nonlinear methods. Because second-harmonic generation is capable of imaging up to approximately 400 microm deep into intact tissue with submicron resolution and little out-of-focus photodamage or bleaching, its ability to record fast electrical activity should prove valuable to future electrophysiology studies.

Multi-Site Optical Recording of Action Potentials from <I>Aplysia</I> Neuronal Network and Analysis of Learning Mechanims

Multi-Site Optical Recording of Action Potentials from <I>Aplysia</I> Neuronal Network and Analysis of Learning Mechanims

Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes

To record post synaptic potentials or electrical activity from processes of single cells in a central nervous system (CNS) preparation in situ, voltage sensitive dyes can be injected intracellularly, thereby staining only the cell under investigation. We report the structure, evaluation, and synthesis of 11 fluorescent styryl dyes developed for iontophoretic injection. The optical signals that represent small synaptic potentials from single processes of iontophoretically injected cells are expected to be very small and, therefore, such measurements are not easy. We report the methodology that permitted the optical recording of action potentials from a 3-micron axon and the recording of small synaptic potentials from the processes of single cells in the segmental ganglia of the leech. The same dyes also proved useful for optical recording of action potentials of anterogradely labeled axons, following local extracellular injection at a remote site in a mammalian CNS preparation.

Conduction pattern of excitation in the amphibian atrium assessed by multiple-site optical recording of action potentials.

Spontaneous action potentials were monitored from multiple sites in the bullfrog atrium using a voltage-sensitive merocyanine-rhodanine dye together with a 100-element photodiode matrix array, and we have assessed the spread of the excitation from the pacemaker. Isochrone curves of conduction were obtained by timing the initiation of the action potential-related optical signals: we constructed maps of the spread. Excitatory waves appeared to conduct radially from the pacemaking area over the atrium, and the conduction velocity in the left atrium exceeded that in the right atrium.

Effects of calcium on electrical propagation in early embryonic precontractile heart as revealed by multiple-site optical recording of action potentials.

The effects of Ca2+ on electrical propagation in early embryonic precontractile chick hearts were studied optically using a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 25 separate regions of the eight-to-nine-somite embryonic primitive heart, using a square photodiode array. Electrical propagation was assessed by analyzing the timing of the signals obtained from different regions. Electrical propagation in the heart was suppressed by either lowering or raising extracellular Ca2+. Similar effects were produced by a Ca2+ ionophore (A23187). We have also found that electrical propagation across the primordial fusion line at the midline of the heart was enhanced by increasing, and depressed by lowering, external Ca2+. One possible interpretation is that intercellular communication in the embryonic precontractile heart is regulated by the level of the intracellular Ca2+ concentration, and it is suggested that intercellular communication across the primordial fusion line strongly depends on external Ca2+.

Multiple-site optical recording of action potentials

Multiple-site optical recording of action potentials

Optical recording of action potentials from vertebrate nerve terminals using potentiometric probes provides evidence for sodium and calcium components.

Optical methods are shown to monitor action potentials from a population of nerve terminals in the neurohypophysis of Xenopus. Calcium antagonists such as cadmium and nickel ions block a component of the action potential that probably reflects a calcium-mediated potassium conductance, and tetrodotoxin blocks an inward sodium current, revealing a calcium component to the action potential upstroke.

Flexibility of regional pacemaking priority in early embryonic heart monitored by simultaneous optical recording of action potentials from multiple sites.

The propagation of spontaneous action potentials in 7-9 somite embryonic pre-contractile chick hearts was measured optically using a potential-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 8-16 different sites of the primitive embryonic heart. Short delays were observed in the time of occurrence of optical signals obtained from the different regions. We have found (i) switching phenomena: the site exhibiting pacemaking priority was first situated in the right pre-atrium, double pacemakers: two different pacemaking areas were situated independently in the right and left pre-atrial portions of the heart. On the basis of analysis of such behavior, it was concluded that the regional priority of the pacemaking activity is not rigid but is flexible, that and the direction of the spread of excitation is adaptable to the circumstances in the early embryonic heart.

Improved fluorescent probes for the measurement of rapid changes in membrane potential

To improve the quality of fluorescent voltage-sensitive probes twenty new styryl dyes were synthesized. Some of the new probes are significantly better than any used in the past. A signal-to-noise ratio of 90 root mean square (rms) noise was obtained for an optical recording of action potentials from neuroblastoma cells maintained in monolayer culture. The fluorescence fractional change of the optical signal is as large as 14%/100 mV. Photodynamic damage and bleaching are much less significant with the new probes. These fluorescent probes can be used to measure small and rapid changes in membrane potential from single cells maintained in monolayer cultures, from single cells in invertebrate ganglia, from their arborization, and from other preparations. The optical measurement can be made with a standard fluorescent microscope equipped with DC mercury illumination. Guidelines for the design of even better fluorescent probes and more efficient instruments are suggested.