Person: Cohen, Adam
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Cohen, Adam
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Publication Bioelectrical Domain Walls in Homogeneous Tissues(Springer Science and Business Media LLC, 2020-01-20) McNamara, Harold M.; Salegame, Rajath; Tanoury, Ziad Al; Xu, Haitan; Begum, Shahinoor; Ortiz, Gloria; Pourquie, Olivier; Cohen, AdamElectrical signaling in biology is typically associated with action potentials, transient spikes in membrane voltage that return to baseline. Hodgkin-Huxley and related conductance-based models of electrophysiology belong to a more general class of reaction-diffusion equations which could, in principle, support spontaneous emergence of patterns of membrane voltage which are stable in time but structured in space. Here we show theoretically and experimentally that homogeneous or nearly homogeneous tissues can undergo spontaneous spatial symmetry breaking through a purely electrophysiological mechanism, leading to formation of domains with different resting potentials separated by stable bioelectrical domain walls. Transitions from one resting potential to another can occur through long-range migration of these domain walls. We map bioelectrical domain wall motion using all-optical electrophysiology in an engineered cell line and in human induced pluripotent stem cell (iPSC)-derived myoblasts. Bioelectrical domain wall migration may occur during embryonic development and during physiological signaling processes in polarized tissues. These results demonstrate that nominally homogeneous tissues can undergo spontaneous bioelectrical symmetry breaking.Publication Ultrawidefield Microscope for High-Speed Fluorescence Imaging and Targeted Optogenetic Stimulation(The Optical Society, 2017-11-29) Werley, Christopher; Chien, Miao-ping; Cohen, AdamThe rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.Publication A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store(Public Library of Science, 2015) Henderson, Mark J.; Baldwin, Heather A.; Werley, Christopher A.; Boccardo, Stefano; Whitaker, Leslie R.; Yan, Xiaokang; Holt, Graham T.; Schreiter, Eric R.; Looger, Loren L.; Cohen, Adam; Kim, Douglas S.; Harvey, Brandon K.Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.Publication Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology(Public Library of Science, 2017) Ogden, Kevin K.; Chen, Wenjuan; Swanger, Sharon A.; McDaniel, Miranda J.; Fan, Linlin; Hu, Chun; Tankovic, Anel; Kusumoto, Hirofumi; Kosobucki, Gabrielle J.; Schulien, Anthony J.; Su, Zhuocheng; Pecha, Joseph; Bhattacharya, Subhrajit; Petrovski, Slavé; Cohen, Adam; Aizenman, Elias; Traynelis, Stephen F.; Yuan, HongjieN-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs.Publication Bringing Bioelectricity to Light(Annual Reviews, 2014) Cohen, Adam; Venkatachalam, VeenaAny bilayer lipid membrane can support a membrane voltage. The combination of optical perturbation and optical readout of membrane voltage opens the door to studies of electrophysiology in a huge variety of systems previously inaccessible to electrode-based measurements. Yet, the application of optogenetic electrophysiology requires careful reconsideration of the fundamentals of bioelectricity. Rules of thumb appropriate for neuroscience and cardiology may not apply in systems with dramatically different sizes, lipid compositions, charge carriers, or protein machinery. Optogenetic tools are not electrodes; thus, optical and electrode-based measurements have different quirks. Here we review the fundamental aspects of bioelectricity with the aim of laying a conceptual framework for all-optical electrophysiology.Publication All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS(Elsevier, 2018) Kiskinis, Evangelos; Kralj, Joel M.; Zou, Peng; Weinstein, Eli; Zhang, Hongkang; Tsioras, Konstantinos; Wiskow, Ole; Ortega, J. Alberto; Eggan, Kevin; Cohen, AdamSummary Human 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.Publication Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells(Public Library of Science, 2013) Park, Jeehae; Werley, Christopher A.; Venkatachalam, Veena; Kralj, Joel; Dib-Hajj, Sulayman D.; Waxman, Stephen G.; Cohen, AdamDevelopment 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.Publication Euler buckling and nonlinear kinking of double-stranded DNA(Oxford University Press, 2013) Fields, Alexander P.; Meyer, Elisabeth; Cohen, AdamThe bending stiffness of double-stranded DNA (dsDNA) at high curvatures is fundamental to its biological activity, yet this regime has been difficult to probe experimentally, and literature results have not been consistent. We created a ‘molecular vise’ in which base-pairing interactions generated a compressive force on sub-persistence length segments of dsDNA. Short dsDNA strands (<41 base pairs) resisted this force and remained straight; longer strands became bent, a phenomenon called ‘Euler buckling’. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any ‘kinked’ states. Greater concentrations of monovalent salts or 1 mM Mg2+ induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.Publication Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents(Frontiers Media S.A., 2014) Hou, Jennifer H.; Kralj, Joel M.; Douglass, Adam D.; Engert, Florian; Cohen, AdamThe 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.Publication In Honor of W.E. Moerner: Confining Molecules for Single-Molecule Spectroscopy(Wiley-Blackwell, 2009) Cohen, Adam; Fields, Alexander Preston; Hou, Jennifer; Leslie, Sabrina R.; Shon, Min JuSingle-molecule spectroscopy provides a wealth of information on the dynamics and interactions of complex biological molecules. Yet these measurements are extremely challenging, partly because Brownian motion prevents molecules in free solution from remaining stationary. Here we describe several techniques that our lab has developed for confining single molecules for optical spectroscopy. These alternatives to surface immobilization provide confinement that is gentle enough to minimize perturbations to the molecule, but strong enough to allow long-time imaging of single fluorescent molecules, often in the presence of a high fluorescent background.