This work was supported by the World Class Institute (WCI) program of the National Research Foundation of Korea funded by Ministry of Education, Science, and Technology of Korea Grant WCI 2009-003 and Korea Institute of Science and Technology Institutional Program Project 2E24210. Sungmoo Lee was supported by Global Ph.D. Fellowship program (NRF-2013H1A2A1033344) of the National Research Foundation (NRF) under the Ministry of Education (MOE, Korea).

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Die Autoren haben nichts zu offenbaren.

Das Nervensystem verwendet Spannung auf mehrere verschiedene Arten, Hemmung verursacht eine leichte Hyperpolarisation, synaptischen Eingangs bewirkt eine leichte Depolarisation und ein Aktionspotential führt zu einer relativ großen Spannungsänderung. Die Fähigkeit von GeViS Änderungen des Membranpotentials zu messen bietet das vielversprechende Potential von gleichzeitig mehreren Komponenten neuronaler Schaltkreise analysieren. In diesem Bericht zeigen wir eine grundlegende Methode zur Bildgebung Änderungen des Me...

Der Schwerpunkt dieses Aufsatzes ist das optische Abbildungsveränderungsmembranpotentiale in vitro zu zeigen, unter Verwendung von genetisch kodierten fluoreszierenden Proteinen. Imaging Änderungen des Membranpotentials bietet die aufregende Möglichkeit, die Aktivität von neuronalen studieren. Wenn Änderungen des Membranpotentials führen zu einer Veränderung der Fluoreszenzintensität, wird jedes Pixel der Kamera ein Surrogat Elektrode eingriffsfreien Messung der neuronalen Aktivität ermöglicht. Seit über vierzig Jahren, organische spannungsabhängigen Farbstoffen wurden zur Beobachtung der Veränderungen des Membranpotentials 4.1 nützlich. Jedoch fehlt diesen Farbstoffen zelluläre Spezifität. Darüber hinaus sind einige Zelltypen schwierig zu färben. Genetisch kodierte Spannungsanzeigen (GeViS) überwinden diese Einschränkungen durch die Zellen spezifisch ausdrücken die fluoreszierende spannungsempfindliche Sonde untersucht werden müssen. Es gibt drei Klassen von GeViS. Die erste Klasse von GEVI verwendet die vorsorgungs--Sensing-Domain von der Spannungsfühl Phosphatase entweder mit einem einzelnen fluoreszierenden Protein (FP) 5-9 oder ein Förster-Resonanzenergietransfer (FRET) Paar 10-12. Die zweite Klasse von Sensoren verwendet mikrobiellen Rhodopsin als Fluoreszenzindikator direkt 13-15 oder über elektrochromen FRET 16,17. Die dritte Klasse verwendet zwei Komponenten, die genetische Komponente eine Membran verankert FP und eine zweite Komponente, die eine membrangebundene Abschrecken Farbstoff ist 18-20 ist. Während die zweiten und dritten Klassen, die für in vitro und Scheibe Experimente sind 19,20, nur die erste Klasse von Sensoren liegen noch nützlich für die in vivo 6 analysiert. In diesem Bericht werden wir die Darstellung von Membranpotential unter Verwendung der ersten Klasse von GeViS (Abbildung 1) in vitro zeigen. Diese erste Klasse von Spannungssensoren ist am einfachsten zu in-vivo-Bildgebung übergeht. Seit GeViS utilizing eine Spannung fühlenden Domain auf einen FP verschmolzen sind etwa 50-fach heller als das Rhodopsin Klasse von Sensoren, können sie eher als einen extrem leistungsfähigen Laser erfordern mit Bogenlampe Beleuchtung abgebildet werden. Eine weitere Folge der Unterschiede in der Helligkeit ist, dass die erste Klasse von GeViS leicht die Autofluoreszenz des Gehirns übersteigen kann. Die Rhodopsin-basierte Sonden kann es nicht. Die dritte Klasse von Sensor ist ebenso hell wie die erste Klasse erfordert aber die Zugabe eines chemischen Quencher, die schwierig in vivo zu verabreichen. Wir werden daher zeigen die Übernahme einer Sonde mit einem einzigen FP (Bongwoori) 8 und eine Sonde, bestehend aus einem FRET-Paar (Nabi 2) 12. Die FRET-Konstrukte in diesem Bericht sind Schmetterling Versionen VSFP-CR (spannungssensitiven Fluoreszenzproteine ​​- Klee-mRuby2) 11, bestehend aus einem grün fluoreszierenden Spender, Klee, und ein rot fluoreszierendes Akzeptor, mRuby2 namens Nabi 2.242 und 2.244 Nabi <sbis&gt; 12. Die Einführung auf diese Arten von Aufnahmen sollten Forscher geben ein besseres Verständnis der Art der Informationen GeViS bereitstellen kann. <img alt="Abbildung 1" src="/files/ftp_upload/53566/53566fig1.jpg" /> Abbildung 1. Zwei Arten von genetisch kodierte Spannungsanzeiger (GeViS) abgebildet in diesem Bericht (A) ein FP Mono basierend GEVI ein Transmembranspannung-Sensing-Domäne und ein fluoreszierendes Protein mit. (B) Ein FRET basierten GEVI bestehend aus einem Transmembranspannung-Sensing-Domäne, einer FRET-Donor und Akzeptor. <a href="https://www.jove.com/files/ftp_upload/53566/53566fig1large.jpg" target="_blank">Bitte hier klicken, um eine größere Version dieser Figur zu sehen.</a>

<table><tbody><tr><td>Inverted Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>60X objective lens (numerical aperture = 1.35)</td><td>Olympus</td><td>UPLSAPO 60XO</td><td></td></tr><tr><td>Excitation filter</td><td>Semrock</td><td>&amp;nbsp;FF02-472/30</td><td>For voltage imaging of super ecliptic pHluorin in Bongwoori</td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>FF495-Di03-25x36</td><td></td></tr><tr><td>Emission filter</td><td>Semrock</td><td>FF01-497/LP</td><td></td></tr><tr><td>75W Xenon arc lamp</td><td>CAIRN</td><td>OptoSource Illuminator</td><td>LEDs and lasers are also effective light sources</td></tr><tr><td>Slow speed CCD camera</td><td>Hitachi</td><td>KP-D20BU</td><td></td></tr><tr><td>Dual port camera adaptor</td><td>Olympus</td><td>U-DPCAD</td><td></td></tr><tr><td>High speed CCD camera</td><td>RedShirtImaging, LLC</td><td>NeuroCCD-SM</td><td></td></tr><tr><td>Image splitter</td><td>CAIRN</td><td>Optosplit 2</td><td></td></tr><tr><td>Excitation filter</td><td>Semrock</td><td>FF01-475/23-25</td><td>For voltage imaging of FRET pair based GEVI consisting of Clover and mRuby2)</td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>FF495-Di03-25x36</td><td></td></tr><tr><td>Emission filter</td><td>Chroma</td><td>ET520/40</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>FF560-FDi01-25X36</td><td></td></tr><tr><td>Emission filter</td><td>Chroma</td><td>ET645/75</td><td></td></tr><tr><td>Vibration isolation system</td><td>Kinetic systems</td><td>250BM-IC, 5702E-3036-31</td><td></td></tr><tr><td>Patching chamber</td><td>Warner instruments</td><td>RC-26G, 64-0235</td><td></td></tr><tr><td>#0 Micro Coverglass (22 x 40 mm)</td><td>Electron Microscopy Sciences</td><td>72198-20</td><td></td></tr><tr><td>Temperature controller</td><td>Warner instruments</td><td>TC-344B</td><td></td></tr><tr><td>#0 (0.08 ~ 0.13 mm) - 10 mm diameter glass coverslip</td><td>Ted Pella</td><td>260366</td><td></td></tr><tr><td>Lipofection agent</td><td>Life Technologies</td><td>11668-027</td><td></td></tr><tr><td>Calcium phosphate reagent</td><td>Clontech - Takara</td><td>631312</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>HEKA</td><td>EPC 10 USB amplifier</td><td></td></tr><tr><td>Multi-channel data acquisition software</td><td>HEKA</td><td>Patchmaster</td><td></td></tr><tr><td>Image acquisition and analysis software</td><td>RedShirtImaging</td><td>Neuroplex</td><td></td></tr><tr><td>Spreadsheet application software</td><td>Microsoft</td><td>Microsoft Excel 2010</td><td></td></tr><tr><td>Data analysis software</td><td>OriginLab</td><td>OriginPro 8.6.0</td><td></td></tr><tr><td>Demagnifier</td><td>Qioptiq LINOS</td><td>Optem standard camera coupler 0.38x SC38 J clamp</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Nikon A1R confocal microscope</td><td></td></tr><tr><td>Anti-fade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr></tbody></table>

imaging membrane potential with two types of genetically encoded fluorescent voltage sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

Transient transfizierten Zellen können signifikante Veränderung in der Fluoreszenzintensität zeigen und den Grad der Plasmamembranexpression. Auch auf dem gleichen Deckglas haben einige Zellen Ebenen der inneren Fluoreszenz variiert. Dies ist höchstwahrscheinlich auf die Menge an Transfektionsmittel von der Zelle absorbiert. Gelegentlich zuviel Ausdruck der Zelle bewirkt, dass die entfaltete Protein-Reaktion, was zu Apoptose 27 zu erfahren (helle, runde Zellen mit hohen in...

Watch this Scientific Journal Video about Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors at JoVE.com

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Imaging Membrane Potential mit zwei Arten von genetisch kodierten Fluorescent Spannungssensoren

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

imaging-membrane-potential-with-two-types-genetically-encoded

Research

JoVE Journal

Neuroscience

10.6K Aufrufe.  Seoul National University. A method for imaging changes in membrane potential using genetically encoded voltage indicators is described. 

Serie: Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Biosensing Motor Neuron Membran Potenzial in Live Zebrafisch Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Forschung

Entwicklungsbiologie

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes 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 on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Voltage-sensitive Dye Aufnahme von Axonen, Dendriten und dendritischen Spines einzelner Neuronen in Hirnschnitten

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Neurowissenschaften

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualisierung von zellulären elektrische Aktivität im frühen Zebrafish Embryos und Tumoren

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Messung der induzierten Spannung Membran mit Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Biologie

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Optische Bildgebung von isolierten ventrikulären Myozyten

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Biotechnik

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Einzelzellige optische Aktionspotentialmessung in human induzierten pluripotenten Stammzell-abgeleiteten Kardiomyozyten

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

<meta charset="utf8"></head><body>Bewertung der Elektroporationseffekte auf gentechnisch veränderte Spiking-HEK-Zellen

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

<meta charset="utf8"></head><body>Überwachung von Elektroporations-induzierten Veränderungen der Aktionspotentialbildung in gentechnisch veränderten Tet-On-Spiking-HEK-Zellen

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Überwachung von Elektroporations-induzierten Veränderungen der Aktionspotentialbildung in gentechnisch veränderten Tet-On-Spiking-HEK-Zellen

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Echtzeit-Fluoreszenzmessung synaptischer Funktionen in Modellen der amyotrophen Lateralsklerose

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Imaging Membrane Potential mit zwei Arten von genetisch kodierten Fluorescent Spannungssensoren

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Biosensing Motor Neuron Membran Potenzial in Live Zebrafisch Embryos

Voltage-sensitive Dye Aufnahme von Axonen, Dendriten und dendritischen Spines einzelner Neuronen in Hirnschnitten

Visualisierung von zellulären elektrische Aktivität im frühen Zebrafish Embryos und Tumoren

Messung der induzierten Spannung Membran mit Di-8-ANEPPS

Optische Bildgebung von isolierten ventrikulären Myozyten

Einzelzellige optische Aktionspotentialmessung in human induzierten pluripotenten Stammzell-abgeleiteten Kardiomyozyten

Überwachung von Elektroporations-induzierten Veränderungen der Aktionspotentialbildung in gentechnisch veränderten Tet-On-Spiking-HEK-Zellen

Überwachung von Elektroporations-induzierten Veränderungen der Aktionspotentialbildung in gentechnisch veränderten Tet-On-Spiking-HEK-Zellen

Überwachung von Elektroporations-induzierten Veränderungen der Aktionspotentialbildung in gentechnisch veränderten Tet-On-Spiking-HEK-Zellen

Echtzeit-Fluoreszenzmessung synaptischer Funktionen in Modellen der amyotrophen Lateralsklerose