In silico voltage-sensitive dye imaging reveals the emergent dynamics of cortical populations.

Voltage-sensitive dye imaging (VSDI) is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but with effective resolution limits that confound interpretation. To address this limitation, we developed an in silico model of VSDI in a biologically faithful digital reconstruction of rodent neocortical microcircuitry. Using this model, we extend previous experimental observations regarding the cellular origins of VSDI, finding that the signal is driven primarily by neurons in layers 2/3 and 5, and that VSDI measurements do not capture individual spikes. Furthermore, we test the capacity of VSD image sequences to discriminate between afferent thalamic inputs at various spatial locations to estimate a lower bound on the functional resolution of VSDI. Our approach underscores the power of a bottom-up computational approach for relating scales of cortical processing.

Cardiac optogenetics: a decade of enlightenment.

The electromechanical function of the heart involves complex, coordinated activity over time and space. Life-threatening cardiac arrhythmias arise from asynchrony in these space-time events; therefore, therapies for prevention and treatment require fundamental understanding and the ability to visualize, perturb and control cardiac activity. Optogenetics combines optical and molecular biology (genetic) approaches for light-enabled sensing and actuation of electrical activity with unprecedented spatiotemporal resolution and parallelism. The year 2020 marks a decade of developments in cardiac optogenetics since this technology was adopted from neuroscience and applied to the heart. In this Review, we appraise a decade of advances that define near-term (immediate) translation based on all-optical electrophysiology, including high-throughput screening, cardiotoxicity testing and personalized medicine assays, and long-term (aspirational) prospects for clinical translation of cardiac optogenetics, including new optical therapies for rhythm control. The main translational opportunities and challenges for optogenetics to be fully embraced in cardiology are also discussed.

Effects of high-frequency biphasic shocks on ventricular vulnerability and defibrillation outcomes through synchronized virtual electrode responses.

Electrical defibrillation is a well-established treatment for cardiac dysrhythmias. Studies have suggested that shock-induced spatial sawtooth patterns and virtual electrodes are responsible for defibrillation efficacy. We hypothesize that high-frequency shocks enhance defibrillation efficacy by generating temporal sawtooth patterns and using rapid virtual electrodes synchronized with shock frequency. High-speed optical mapping was performed on isolated rat hearts at 2000 frames/s. Two defibrillation electrodes were placed on opposite sides of the ventricles. An S1-S2 pacing protocol was used to induce ventricular tachyarrhythmia (VTA). High-frequency shocks of equal energy but varying frequencies of 125-1000 Hz were used to evaluate VTA vulnerability and defibrillation success rate. The 1000-Hz shock had the highest VTA induction rate in the shorter S1-S2 intervals (50 and 100 ms) and the highest VTA defibrillation rate (70%) among all frequencies. Temporal sawtooth patterns and synchronous shock-induced virtual electrode responses could be observed with frequencies of up to 1000 Hz. The improved defibrillation outcome with high-frequency shocks suggests a lower energy requirement than that of low-frequency shocks for successful ventricular defibrillation.

Open-Source Multiparametric Optocardiography.

Since the 1970s fluorescence imaging has become a leading tool in the discovery of mechanisms of cardiac function and arrhythmias. Gradual improvements in fluorescent probes and multi-camera technology have increased the power of optical mapping and made a major impact on the field of cardiac electrophysiology. Tandem-lens optical mapping systems facilitated simultaneous recording of multiple parameters characterizing cardiac function. However, high cost and technological complexity restricted its proliferation to the wider biological community. We present here, an open-source solution for multiple-camera tandem-lens optical systems for multiparametric mapping of transmembrane potential, intracellular calcium dynamics and other parameters in intact mouse hearts and in rat heart slices. This 3D-printable hardware and Matlab-based RHYTHM 1.2 analysis software are distributed under an MIT open-source license. Rapid prototyping permits the development of inexpensive, customized systems with broad functionality, allowing wider application of this technology outside biomedical engineering laboratories.

In vivo one-photon confocal calcium imaging of neuronal activity from the mouse neocortex.

In vivo calcium imaging is a powerful tool used to record neuronal activity from living animals. For this purpose, two-photon excitation laser-scanning microscopy is commonly used because of the optical accessibility of deep tissues. In this study, we report that one-photon confocal scanning laser microscopy, when optimally tuned, is also applicable for in vivo calcium imaging from the superficial layer of the neocortex. By combining a Nipkow-disk confocal unit with a fluorescence stereo zoom microscope and a high numerical aperture objective, we succeeded in recording the fluorescence signal of individual cells at a depth of up to 160 µm in brain tissues, which corresponds to layer II of the mouse neocortex. In fact, we conducted in vivo functional multineuron calcium imaging and simultaneously recorded spontaneous activity from more than 100 neocortical layer II neurons. This one-photon confocal system provides a simple, low-cost experimental platform for time-lapse imaging from living animals.

Optical Action Potential Mapping in Acute Models of Ischemia-Reperfusion Injury: Probing the Arrhythmogenic Role of the Mitochondrial Translocator Protein.

Ischemia-reperfusion (I/R) injury causes dynamic changes in electrophysiological properties that promote the incidence of post-ischemic arrhythmias. High-resolution optical action potential mapping allows for a quantitative assessment of the electrophysiological substrate at a cellular resolution within the intact heart, which is critical for elucidation of arrhythmia mechanisms. We and others have found that pharmacological inhibition of the translocator protein (TSPO) is highly effective against postischemic arrhythmias. A major hurdle that has limited the translation of this approach to patients is the fact that available TSPO ligands have several confounding effects, including a potent negative ionotropic property. To circumvent such limitations we developed an in vivo cardiac specific TSPO gene silencing approach as an alternative. Here, we provide the methodological details of our optical action potential mapping studies that were designed to probe the effects of TSPO silencing in hearts from spontaneously hypertensive rats (SHR) that are prone to I/R injury.

A high-throughput model for investigating neuronal function and synaptic transmission in cultured neuronal networks.

Loss of synapses or alteration of synaptic activity is associated with cognitive impairment observed in a number of psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. Therefore successful development of in vitro methods that can investigate synaptic function in a high-throughput format could be highly impactful for neuroscience drug discovery. We present here the development, characterisation and validation of a novel high-throughput in vitro model for assessing neuronal function and synaptic transmission in primary rodent neurons. The novelty of our approach resides in the combination of the electrical field stimulation (EFS) with data acquisition in spatially separated areas of an interconnected neuronal network. We integrated our methodology with state of the art drug discovery instrumentation (FLIPR Tetra) and used selective tool compounds to perform a systematic pharmacological validation of the model. We investigated pharmacological modulators targeting pre- and post-synaptic receptors (AMPA, NMDA, GABA-A, mGluR2/3 receptors and Nav, Cav voltage-gated ion channels) and demonstrated the ability of our model to discriminate and measure synaptic transmission in cultured neuronal networks. Application of the model described here as an unbiased phenotypic screening approach will help with our long term goals of discovering novel therapeutic strategies for treating neurological disorders.

Design and validation of a tissue bath 3-D printed with PLA for optically mapping suspended whole heart preparations.

With the sudden increase in affordable manufacturing technologies, the relationship between experimentalists and the designing process for laboratory equipment is rapidly changing. While experimentalists are still dependent on engineers and manufacturers for precision electrical, mechanical, and optical equipment, it has become a realistic option for in house manufacturing of other laboratory equipment with less precise design requirements. This is possible due to decreasing costs and increasing functionality of desktop three-dimensional (3-D) printers and 3-D design software. With traditional manufacturing methods, iterative design processes are expensive and time consuming, and making more than one copy of a custom piece of equipment is prohibitive. Here, we provide an overview to design a tissue bath and stabilizer for a customizable, suspended, whole heart optical mapping apparatus that can be produced significantly faster and less expensive than conventional manufacturing techniques. This was accomplished through a series of design steps to prevent fluid leakage in the areas where the optical imaging glass was attached to the 3-D printed bath. A combination of an acetone dip along with adhesive was found to create a water tight bath. Optical mapping was used to quantify cardiac conduction velocity and action potential duration to compare 3-D printed baths to a bath that was designed and manufactured in a machine shop. Importantly, the manufacturing method did not significantly affect conduction, action potential duration, or contraction, suggesting that 3-D printed baths are equally effective for optical mapping experiments.NEW & NOTEWORTHY This article details three-dimensional printable equipment for use in suspended whole heart optical mapping experiments. This equipment is less expensive than conventional manufactured equipment as well as easily customizable to the experimentalist. The baths can be waterproofed using only a three-dimensional printer, acetone, a glass microscope slide, c-clamps, and adhesive.

Skin suturing and cortical surface viral infusion improves imaging of neuronal ensemble activity with head-mounted miniature microscopes.

BACKGROUND: In vivo optical imaging of neural activity provides important insights into brain functions at the single-cell level. Cranial windows and virally delivered calcium indicators are commonly used for imaging cortical activity through two-photon microscopes in head-fixed animals. Recently, head-mounted one-photon microscopes have been developed for freely behaving animals. However, minimizing tissue damage from the virus injection procedure and maintaining window clarity for imaging can be technically challenging. NEW METHOD: We used a wide-diameter glass pipette at the cortical surface for infusing the viral calcium reporter AAV-GCaMP6 into the cortex. After infusion, the scalp skin over the implanted optical window was sutured to facilitate postoperative recovery. The sutured scalp was removed approximately two weeks later and a miniature microscope was attached above the window to image neuronal activity in freely moving mice. RESULTS: We found that cortical surface virus infusion efficiently labeled neurons in superficial layers, and scalp skin suturing helped to maintain the long-term clarity of optical windows. As a result, several hundred neurons could be recorded in freely moving animals. COMPARISON WITH EXISTING METHODS: Compared to intracortical virus injection and open-scalp postoperative recovery, our methods minimized tissue damage and dura overgrowth underneath the optical window, and significantly increased the experimental success rate and the yield of identified neurons. CONCLUSION: Our improved cranial surgery technique allows for high-yield calcium imaging of cortical neurons with head-mounted microscopes in freely behaving animals. This technique may be beneficial for other optical applications such as two-photon microscopy, multi-site imaging, and optogenetic modulation.

An Integrated Circuit for Simultaneous Extracellular Electrophysiology Recording and Optogenetic Neural Manipulation.

OBJECTIVE: The ability to record and to control action potential firing in neuronal circuits is critical to understand how the brain functions. The objective of this study is to develop a monolithic integrated circuit (IC) to record action potentials and simultaneously control action potential firing using optogenetics. METHODS: A low-noise and high input impedance (or low input capacitance) neural recording amplifier is combined with a high current laser/light-emitting diode (LED) driver in a single IC. RESULTS: The low input capacitance of the amplifier (9.7 pF) was achieved by adding a dedicated unity gain stage optimized for high impedance metal electrodes. The input referred noise of the amplifier is [Formula: see text], which is lower than the estimated thermal noise of the metal electrode. Thus, the action potentials originating from a single neuron can be recorded with a signal-to-noise ratio of at least 6.6. The LED/laser current driver delivers a maximum current of 330 mA, which is adequate for optogenetic control. The functionality of the IC was tested with an anesthetized Mongolian gerbil and auditory stimulated action potentials were recorded from the inferior colliculus. Spontaneous firings of fifth (trigeminal) nerve fibers were also inhibited using the optogenetic protein Halorhodopsin. Moreover, a noise model of the system was derived to guide the design. SIGNIFICANCE: A single IC to measure and control action potentials using optogenetic proteins is realized so that more complicated behavioral neuroscience research and the translational neural disorder treatments become possible in the future.

FIM$

In vivo whole-body imaging of small animals plays an important role for biomedical studies. In particular, animals like the fruit fly Drosophila melanogaster or the nematode Caenorhabditis elegans are popular model organisms for preclinical research since they offer sophisticated genetic tool-kits. Recording these translucent animals with high contrast in a large arena is however not trivial. Furthermore, fluorescent proteins are widely used to mark cells in vivo and report their functions. This paper introduces a novel optical imaging technique called FIM2c enabling simultaneous detection of the animals posture and movement as well as fluorescent markers like green fluorescent protein (GFP). FIM2c utilizes frustrated total internal reflection of two distinct wavelengths and captures both, reflected and emitted light. The resultant two-color high-contrast images are superb compared to other imaging systems for larvae or worms. This multipurpose method enables a large variety of different experimental approaches. For example, FIM2c can be used to image GFP positive cells/tissues/animals and supports the integration of fluorescent tracers into multitarget tracking paradigms. Moreover, optogenetic tools can be applied in large-scale behavioral analysis to manipulate and study neuronal functions. To demonstrate the benefit of our system, we use FIM2c to resolve colliding larvae in a high-throughput approach, which was impossible given the existing tools. Finally, we present a comprehensive database including images and locomotion features of more than 1300 resolved collisions available for the community. In conclusion, FIM2c is a versatile tool for advanced imaging and locomotion analysis for a variety of different model organisms.

Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain.

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ∼1 mm(2), precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm(2)) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

High-resolution non-contact measurement of the electrical activity of plants in situ using optical recording.

The limitations of conventional extracellular recording and intracellular recording make high-resolution multisite recording of plant bioelectrical activity in situ challenging. By combining a cooled charge-coupled device camera with a voltage-sensitive dye, we recorded the action potentials in the stem of Helianthus annuus and variation potentials at multiple sites simultaneously with high spatial resolution. The method of signal processing using coherence analysis was used to determine the synchronization of the selected signals. Our results provide direct visualization of the phloem, which is the distribution region of the electrical activities in the stem and leaf of H. annuus, and verify that the phloem is the main action potential transmission route in the stems of higher plants. Finally, the method of optical recording offers a unique opportunity to map the dynamic bioelectrical activity and provides an insight into the mechanisms of long-distance electrical signal transmission in higher plants.

Historical Overview and General Methods of Membrane Potential Imaging.

Voltage imaging was first conceived in the late 1960s and efforts to find better organic voltage sensitive dyes began in the 1970s and continue until today. At the beginning it was difficult to measure an action potential signal from a squid giant axon in a single trial. Now it is possible to measure the action potential in an individual spine. Other chapters will discuss advances in voltage imaging technology and applications in a variety of biological preparations. The development of genetically encoded voltage sensors has started. A genetically encoded sensor could provide cell type specific expression and voltage recording (see Chap. 20). Optimizing the signal-to-noise ratio of an optical recording requires attention to several aspects of the recording apparatus. These include the light source, the optics and the recording device. All three have improved substantially in recent years. Arc lamp, LED, and laser sources are now stable, more powerful, and less expensive. Cameras for recording activity have frames rates above 1 kHz and quantum efficiencies near 1.0 although they remain expensive. The sources of noise in optical recordings are well understood. Both the apparatus and the noise sources are discussed in this chapter.

Design and Use of Organic Voltage Sensitive Dyes.

The chemistry and the physics of voltage sensitive dyes (VSDs) should be understood and appreciated as a prerequisite for their optimal application to problems in neuroscience cardiology. This chapter provides a basic understanding of the properties of the large variety of available organic VSDs. The mechanisms by which the dyes respond to voltage guides the best set up of the optics for recording or imaging electrophysiological activity. The physical and chemical properties of the dyes can be tuned to optimize delivery to and staining of the cells in different experimental preparations. The aim of this chapter is to arm the experimentalists who use the dyes with enough information and data to be able to intelligently choose the best dye for their specific requirements.

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines.

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly signals are integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts on distal dendrites) and summate at particular locations to influence action potential initiation. It became possible recently to carry out this type of measurement using high-resolution multisite recording of membrane potential changes with intracellular voltage-sensitive dyes. This chapter reviews the development and foundation of the method of voltage-sensitive dye recording from individual neurons. Presently, this approach allows monitoring membrane potential transients from all parts of the dendritic tree as well as from axon collaterals and individual dendritic spines.

Combining Membrane Potential Imaging with Other Optical Techniques.

Membrane potential imaging using voltage-sensitive dyes can be combined with other optical techniques for a variety of applications. Combining voltage imaging with Ca2+ imaging allows correlating membrane potential changes with intracellular Ca2+ signals or with Ca2+ currents. Combining voltage imaging with uncaging techniques allows analyzing electrical signals elicited by photorelease of a particular molecule. This approach is also a useful tool to calibrate the change in fluorescence intensity in terms of membrane potential changes from different sites permitting spatial mapping of electrical activity. Finally, combining voltage imaging with optogenetics, in particular with channelrhodopsin stimulation, opens the gate to novel investigations of brain circuitries by allowing measurements of synaptic signals mediated by specific sets of neurons. Here we describe in detail the methods of membrane potential imaging in combination with other optical techniques and discus some important applications.

Monitoring Spiking Activity of Many Individual Neurons in Invertebrate Ganglia.

Optical recording with fast voltage sensitive dyes makes it possible, in suitable preparations, to simultaneously monitor the action potentials of large numbers of individual neurons. Here we describe methods for doing this, including considerations of different dyes and imaging systems, methods for correlating the optical signals with their source neurons, procedures for getting good signals, and the use of Independent Component Analysis for spike-sorting raw optical data into single neuron traces. These combined tools represent a powerful approach for large-scale recording of neural networks with high temporal and spatial resolution.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

Monitoring Population Membrane Potential Signals from Neocortex.

Voltage-sensitive dyes (VSDs) and optical imaging are useful tools for studying spatiotemporal patterns of population neuronal activity in cortex. Because fast VSDs respond to membrane potential changes with microsecond temporal resolution, these are better suited than calcium indicators for recording rapid neural signals. Here we describe methods for using a 464 element photodiode array and fast VSDs to record signals ranging from large scale network activity in brain slices and in vivo mammalian preparations with sensitivity comparable to local field potential (LFP) recordings. With careful control of dye bleaching and phototoxicity, long recording times can be achieved. Absorption dyes have less photo-toxicity than fluorescent dyes. In brain slices, the total recording time in each slice can be 1,000-2,000 s, which can be divided into hundreds of short recording trials over several hours. In intact brains when fluorescent dyes are used, reduced light intensity can also increase recording time. In this chapter, we will discuss technical details for the methods to achieve reliable VSD imaging with high sensitivity and long recording time.