a non-invasive approach to study the electrophysiology of motor neurons in zebrafish embryos

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

For spontaneous tail-coiling analysis, transfer the embryos to a 90-millimeter round Petri dish filled with fish water containing 0.2% DMSO, and manually dechorionate them using two jeweler&#39;s forceps with sharp tips or needles. Afterward, incubate the embryos for 5 minutes at 28 degrees Celsius. Using a digital camera mounted on a stereomicroscope, detect the tail coiling at room temperature during a one-minute video recording.
Next, acquire the time series at a time resolution of 30 frames per second. Then, calculate the frequency of spontaneous tail coiling by counting the number of bends per time unit. To evaluate the effect of the drug riluzole, transfer the embryos to a new 90-millimeter Petri dish filled with fish water containing 5 micromolar riluzole. Then, incubate the embryos for 5 minutes at 28 degrees Celsius before recording a one-minute video and performing the behavioral analysis.
Now, mount the 20 to 24 hpf embryos in 1% low melting point agarose in fish water inside a 35-millimeter glass-bottomed imaging dish at 37 degrees Celsius. Orient the embryos on their sides and wait until the agarose solidifies at room temperature.
Next, transfer the imaging dish to the stage of a confocal microscope. Identify the motor neurons expressing the biosensor by exciting monomeric Umikinoko-Green with 488-nanometer argon laser. Then, set its emission between 495 and 525 nanometers and press Record.
For a FRET measurement, excite mUKG, the donor of the FRET pair with a 488-nanometer laser. Simultaneously, using two photomultipliers, detect the fluorescence emitted by the donor and the fluorescence emitted by the monomeric Kusabira-Orange acceptor. At the beginning of the experiment, optimize the gain and offset of photomultiplier 1, where the mUKG fluorescence is recorded, and keep them constant throughout the session.
To set the offset, change the color of the image to the intensity values by using the Q-lookup table. While scanning with a laser off, use the offset slider so that the background pixels have an intensity slightly higher than zero. Using the same lookup table, switch the laser on. While scanning, use the gain slider to maximize the signal-to-noise ratio, being careful to avoid saturated pixels.
In the software acquisition window, select the xyt acquisition mode and an image field size of 512-by-64 pixels from the dropdown menu. Then, set the recording of the changes in embryo spinal neuron voltage to acquire a single xy plane every 30 milliseconds for one minute. Evaluate the effect of riluzole administration on membrane depolarization in the same neuron, acquire a new data set five minutes after the addition of fish water containing 5 micromolar riluzole.

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JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. pHuC_Mermaid Plasmid Generation
NOTE: Mermaid is a biosensor developed by pairing the voltage-sensing domain (VSD) of the Ciona intestinalis (now Ciona robusta) voltage sensor containing phosphatase (Ci-VSP) with the fluorescence resonance energy transfer (FRET) partner fluorophores Umi-Kinoko Green (mUKG: donor) and a monomeric version of the orange-emitting fluorescent protein Kusabira Orange (mKOk: acceptor). For this biosensor, conformational changes of the VSD domain, induced by membrane depolarization, increase the proximity of the donor and acceptor fluorescent proteins, thus increasing the energy transfer between them (increasing the FRET Ratio). The VSD assures the efficient localization of the biosensor at the plasma membrane. The neuronal expression of the biosensor (in the spinal cord, the signal is detectable in both interneurons and motor neurons) is achieved by cloning the Mermaid open reading frame (ORF) under the zebrafish pan-neural promoter HuC.
<ol>
	<li>Amplify by polymerase chain reaction (PCR) the Mermaid open reading frame (ORF) from the pCS4+ Mermaid plasmid with Pfu (proofreading) DNA polymerase using the T3 Universal primer and the Mermaid SmaI primer (5&#8242;TATCCCGGGATTCGACGGTTCAGATTTTA) in order to insert a SmaI restriction site upstream of the Mermaid ORF.&#8203;
	<ol>
		<li>Use the following PCR mixture: 0.5 &#181;L of Pfu DNA polymerase, 7.5 &#181;L of the specific 10x buffer, 1 &#181;L of 10 mM deoxynucleotide triphosphates (dNTPs), 2.5 &#181;L of dimethyl sulfoxide (DMSO), 0.5 &#181;L (50 ng) of the plasmid preparation, and 1 &#181;L of 20-&#181;M stock solutions of each primer in a total volume of 50 &#181;L.</li>
		<li>Heat the PCR mixture for 15 s at 95 &#176;C, prime it for 15 s at 50 &#176;C, and elongate it for 4 min at 72 &#176;C for a total of 35 cycles.</li>
	</ol>
	</li>
	<li>Gel-purify the specific blunt PCR product using a commercial gel purification kit, following the manufacturer&#39;s protocol.</li>
	<li>Clone the DNA fragment into the pCMV-SC blunt vector following the manufacturer&#39;s protocol.</li>
	<li>Linearize the Mermaid-positive plasmid (named pCMV-SC_Mermaid) with the SmaI restriction enzyme for the following insertion of the HuC promoter.
	<ol>
		<li>Set up the restriction reaction as follow: 0.5 &#181;L (10 U) of SmaI restriction enzyme, 2 &#181;L of the kit 10x buffer, and 5 &#181;g of plasmid DNA in a total volume of 20 &#181;L. Incubate the reaction at 25 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Gel-purify the digested plasmid using a commercial gel purification kit following the manufacturer&#39;s protocol.</li>
	<li>PCR-amplify the HuC promoter (pHuC), with Pfu DNA polymerase and zebrafish genomic DNA as template, using a pair of HuC-specific primers (HuCprom-forw1_SalI: 5&#8242;-GTAGTCGACCAGACTTGTCAAAAGGGTCCA and HuCprom-rev1: 5&#8242;-TCCATTCTTGACGTACAAAGATG) and spanning a 3,150-bp region upstream of the ATG.
	<ol>
		<li>Set up the PCR mixture using the following scheme: 0.5 &#181;L of Pfu DNA polymerase, 7.5 &#181;L of the specific 10x buffer, 1 &#181;L of 10 mM dNTPs, 2.5 &#181;L of DMSO, 200 ng of genomic DNA, and 1 &#181;L of 20-&#181;M stock solutions of each primer in a total volume of 50 &#181;L.</li>
		<li>After an initial step of 2 min at 95 &#176;C, heat the PCR mixture for 15 s at 95 &#176;C, prime it for 15 s at 50&#176;C, and elongate it for 4 min at 72 &#176;C for a total of 35 cycles.</li>
	</ol>
	</li>
	<li>Gel-purify the specific blunt PCR product using a commercial gel purification kit following the manufacturer&#39;s protocol.</li>
	<li>Ligate an equimolar amount of the purified pCMV-SC_Mermaid (step 1.5) and the pHuC DNA (step 1.7) using 1 &#181;L of T4 DNA ligase and 1 &#181;L of the specific 10X buffer in a total volume of 10 &#181;L. Incubate the reaction for 16 h at 4 &#176;C.</li>
	<li>Transform an aliquot of competent cells with 5 &#181;L of the ligation reaction (step 1.8) following the manufacturer&#39;s instructions.</li>
	<li>Select the pHuC-positive clones (pHuC_Mermaid) with the promoter inserted in the proper orientation by a SalI-EcoRV double digestion (the SalI restriction site has been inserted upstream of the promoter fragment in step 1.6, while the EcoRV restriction site is positioned downstream of the polyadenylation region, PolyA, of the pCMV-SC plasmid).
	<ol>
		<li>Set up the restriction reaction as follow: 0.5 &#181;L (10 U) of both SalI and EcoRV restriction enzymes, 2 &#181;L of the kit10X buffer, and 1 &#181;g of pHuC_Mermaid DNA in a total volume of 20 &#181;L. Incubate the reaction at 37 &#176;C for 1 h. Run the reactions onto an agarose gel.</li>
	</ol>
	</li>
</ol>
2. Embryo Microinjection
<ol>
	<li>Transfer the fertilized eggs obtained from wild-type (AB strain) or Sod1-G93R adult zebrafish to a 10 mm Petri dish using a plastic pipette.</li>
	<li>Rinse the embryos in cold (4 &#176;C) fish water and immediately microinject them into the yolk with 200 pg of pHuC_Mermaid plasmid using a microinjector (for an overall and detailed description of the microinjection procedure).</li>
	<li>Using a plastic pipette, transfer the embryos to a Petri dish and incubate them in fish water at 28 &#176;C until they reach the desired developmental stage (20-24 h post-fertilization, hpf) for the following analyses.</li>
</ol>
3. Spontaneous Tail Coiling Analysis
NOTE: Evaluate the spontaneous tail coiling behavior in 20-24 hpf embryos with or without the drug riluzole.
<ol>
	<li>Transfer an embryo to a 90-mm round Petri dish filled with fish water containing 0.2% DMSO (riluzole vehicle) and manually dechorionate it using two jeweler&#39;s forceps with sharp tips. Incubate the embryo for 5 min.</li>
	<li>Detect the tail coiling at room temperature (RT) during a 1 min video recording using a digital camera mounted on a stereomicroscope. Acquire time series at a time resolution of 30 frames/s.</li>
	<li>Calculate the frequency of spontaneous tail coilings by counting the number of bends (both contralateral and ipsilateral) per time unit.</li>
	<li>To evaluate the effect of the drug riluzole, gently use a plastic Pasteur pipette to transfer the embryo to a new 90 mm Petri dish filled with fish water containing 5 &#181;M riluzole.&#8203;
	<ol>
		<li>Incubate the embryo for 5 min before recording a 1-min video and performing the behavioral analysis as above.</li>
	</ol>
	</li>
</ol>
4. Imaging Setup for Mermaid Biosensor Visualization in Living Embryos: Simultaneous Detection of Donor and Acceptor Signals
<ol>
	<li>Mount the 20 - 24 hpf embryos in 1% low-melting-point agarose in fish water at 37 &#176;C inside a 35 mm glass-bottomed imaging dish. Orient the embryos on their sides. Wait until the agarose solidifies at room temperature.</li>
	<li>Transfer the imaging dish to the stage of an inverted confocal mounted on an inverted microscope. Identify motor neurons expressing the biosensor with a 20X objective (0.7 numerical aperture, NA) by exciting the mUKG with the 488 nm argon laser line and recording its emission between 495 and 525 nm.</li>
	<li>For a FRET measurement, excite mUKG, the donor of the FRET pair, with the 488 nm laser line. Simultaneously detect, with a resonant scanner operating at 8,000 Hz in the bidirectional mode, the fluorescence emitted by the donor (between 495 and 525 nm) and the fluorescence emitted by the mKOk acceptor (between 550 and 650 nm, FRET channel). If available, use the 473 nm laser. 
	NOTE: The excitation efficiency of the donor will be slightly reduced (85% instead of 93% with 488 nm), but the cross-excitation of the acceptor will be reduced as well (from 17% with the 488 nm to 9% with 473 nm laser line).</li>
	<li>To reduce phototoxicity and fluorophore bleaching, minimize the illumination of the sample by reducing the power of the laser line (on the beam path window of the acquisition software).</li>
	<li>Optimize the excitation to match gain and offset parameters that are set at the beginning of the experiment and kept constant throughout the session. To set the offset, change the color of the image to intensity values (by using the Q look-up table) and, while scanning with the laser off, turn the offset knob (smart offset) so that the background pixels have an intensity slightly higher than zero. With the same look-up table, by switching the laser on while scanning, turn the gain knob (smart gain) to maximize the signal-to-noise ratio, being careful to avoid saturated pixels.</li>
	<li>Using an opened pinhole (2 airy units), acquire 16-bit images to provide a sufficient dynamic range for quantitative analyses. Avoid averaging to increase the acquisition speed and to minimize photobleaching.</li>
	<li>In the software acquisition window, select an image field size of 512 x 64 pixels (pixel size: 605 nm) from the drop-down menu.</li>
	<li>From the acquisition mode window, select xyt (time lapse on a single xy plane) from the drop-down menu and record the changes in embryo spinal neuron voltage by acquiring a single xy plane, setting the acquisition parameters to record one image every 30 ms for 1 min.</li>
	<li>To evaluate the effect of riluzole administration on membrane depolarization in the same neuron, acquire a new dataset 5 min after the addition of fish water containing 5 &#181;M riluzole.</li>
	<li>For FRET analysis, use the ImageJ macro Biosensor_FRET (expressing single-chain FRET biosensors.) </li>
	<li>Evaluate the basal membrane FRET ratio of each neuron at t1 as ((FRET mean - FRET background)/(Donor mean - Donor background)), where FRET and Donor mean intensity is the mean fluorescence intensity calculated in the same region of interest (ROI) drawn around the cell for each channel acquired and FRET and Donor background is the mean fluorescence intensity calculated in an ROI of the field of view without the fluorescent sample. 
	NOTE: A detailed step-by-step description of the use of the plugin can be found at the www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/biosensor-fret website.</li>
	<li>Use a graphing software to compare the frequency, amplitude, and duration of depolarization between different experimental paradigms. Compare two groups using an unpaired Student&#39;s t-test and consider mean values as statistically different when P &#60;0.05.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Low Melting Point Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr>
			<td>Riluzole</td>
			<td>Sigma-Aldrich</td>
			<td>R116</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu Ultra HQ DNA polymerase</td>
			<td>Agilent Technologies - Stratagene Products Division</td>
			<td>600389</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 Universal primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up system</td>
			<td>Promega</td>
			<td>A9280</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal SmaI primer</td>
			<td>Eurofins</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>StrataClone Mammalian Expression Vector System / pCMV-SC blunt vector</td>
			<td>Agilent Technologies - Stratagene Products Division</td>
			<td>240228</td>
			<td></td>
		</tr>
		<tr>
			<td>SmaI</td>
			<td>New England Biolabs</td>
			<td>R0141S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Promega</td>
			<td>M1801</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>New England Biolabs</td>
			<td>R0138S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England Biolabs</td>
			<td>R0195S</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm, glass-bottomed imaging dish</td>
			<td>Ibidi</td>
			<td>81151</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>M10 F</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Leica Microsystems</td>
			<td>DFC 310 FX</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite 4.7.1 software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuickTime Player, v10.4</td>
			<td>Apple</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope (inverted)</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Eppendorf</td>
			<td>Femtojet</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ macro Biosensor_FRET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 6.0c</td>
			<td>GraphPad Software, Inc</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Benedetti, L., et al. <a href="https://app.jove.com/v/55297">Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates a non-invasive assay to study electrophysiology of motor neurons in zebrafish embryos. Upon measuring tail coiling in the embryos as a result of spontaneous depolarization of the spinal cord motor neurons, a sodium channel blocker is applied to reduce the coiling frequency. Next, the embryos are immobilized in agarose and placed under a fluorescence microscope. The genetically engineered motor neurons express a voltage-sensing biosensor with a donor and an acceptor fluorophore. A decrease in acceptor fluorophore emission indicates a reduction in the spontaneous depolarization of motor neurons due to the sodium channel blocker.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Place genetically engineered zebrafish embryos under a stereomicroscope and observe the spontaneous tail coiling triggered by spinal cord motor neurons.
The intrinsic properties of these neurons cause ion influx through sodium channels, leading to spontaneous membrane depolarization and the generation of action potentials that propagate to muscles, triggering tail coiling. Measure the coiling frequency.
Transfer some embryos to a sodium channel blocker to inhibit sodium influx and assess the reduced coiling frequency.
Mount untreated embryos in molten agarose; let agarose solidify to restrict movement, and observe under a fluorescence microscope.
The motor neurons express a biosensor with a membrane-bound voltage-sensing domain, or VSD, fused to donor and acceptor fluorophores.
Depolarization changes the VSD conformation, bringing the fluorophores closer.
Upon excitation, the donor transfers energy to the acceptor, causing acceptor fluorescence emission.
Add the blocker to inhibit depolarization, which reduces acceptor emission, confirming blocker-mediated inhibition of spontaneous depolarization in the neurons.

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Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

使用基于 FRET 的传感器 ATeam1.03YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

科研

JoVE Journal

神经科学

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

人诱导的多潜能干细胞源性心室心肌细胞亚型特异光学动作电位记录

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

生物学

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

成績單

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos