Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function

Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators

Machine Learning Establishes Single-Cell Calcium Dynamics as an Early Indicator of Antibiotic Response

Changes in bacterial physiology necessarily precede cell death in response to antibiotics. Herein we investigate the early disruption of Ca2+ homeostasis as a marker for antibiotic response. Using a machine learning framework, we quantify the temporal information encoded in single-cell Ca2+ dynamics. We find Ca2+ dynamics distinguish kanamycin sensitive and resistant cells before changes in gross cell phenotypes such as cell growth or protein stability. The onset time (pharmacokinetics) and probability (pharmacodynamics) of these aberrant Ca2+ dynamics are dose and time-dependent, even at the resolution of single-cells. Of the compounds profiled, we find Ca2+ dynamics are also an indicator of Polymyxin B activity. In Polymyxin B treated cells, we find aberrant Ca2+ dynamics precedes the entry of propidium iodide marking membrane permeabilization. Additionally, we find modifying membrane voltage and external Ca2+ concentration alters the time between these aberrant dynamics and membrane breakdown suggesting a previously unappreciated role of Ca2+ in the membrane destabilization during Polymyxin B treatment. In conclusion, leveraging live, single-cell, Ca2+ imaging coupled with machine learning, we have demonstrated the discriminative capacity of Ca2+ dynamics in identifying antibiotic-resistant bacteria. &nbsp;</p

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Machine Learning Establishes Single-Cell Calcium Dynamics as an Early Indicator of Antibiotic Response

Changes in bacterial physiology necessarily precede cell death in response to antibiotics. Herein we investigate the early disruption of Ca2+ homeostasis as a marker for antibiotic response. Using a machine learning framework, we quantify the temporal information encoded in single-cell Ca2+ dynamics. We find Ca2+ dynamics distinguish kanamycin sensitive and resistant cells before changes in gross cell phenotypes such as cell growth or protein stability. The onset time (pharmacokinetics) and probability (pharmacodynamics) of these aberrant Ca2+ dynamics are dose and time-dependent, even at the resolution of single-cells. Of the compounds profiled, we find Ca2+ dynamics are also an indicator of Polymyxin B activity. In Polymyxin B treated cells, we find aberrant Ca2+ dynamics precedes the entry of propidium iodide marking membrane permeabilization. Additionally, we find modifying membrane voltage and external Ca2+ concentration alters the time between these aberrant dynamics and membrane breakdown suggesting a previously unappreciated role of Ca2+ in the membrane destabilization during Polymyxin B treatment. In conclusion, leveraging live, single-cell, Ca2+ imaging coupled with machine learning, we have demonstrated the discriminative capacity of Ca2+ dynamics in identifying antibiotic-resistant bacteria