Name: biosensing motor neuron membrane potential in live zebrafish embryos
Uploaded: 2017-06-26T13:00:00
Description: Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

The authors would like to thank Simona Rodighiero for her priceless support with the FRET imaging analysis.

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)5 voltage sensor containing phosphatase (Ci-VSP) with the 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 ...

<ol>
	<li>Kn&#246;pfel, T., Gallero-Salas, Y., Song, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+voltage+indicators+for+large+scale+cortical+imaging+come+of+age.">Genetically encoded voltage indicators for large scale cortical imaging come of age.</a> Curr Opin Chem Biol. 27, 75-83 (2015).</li><li>Chemla, S., Chavane, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+imaging:+Technique+review+and+models.">Voltage-sensitive dye imaging: Technique review and models.</a> J Physiol Paris. 104 (1-2), 40-50 (2010).</li><li>Tsutsui, H., Karasawa, S., Okamura, Y., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+membrane+voltage+measurements+using+FRET+with+new+fluorescent+proteins.">Improving membrane voltage measurements using FRET with new fluorescent proteins.</a> Nat Methods. 5 (8), 683-685 (2008).</li><li>Benedetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INaP+selective+inhibition+reverts+precocious+inter-+and+motorneurons+hyperexcitability+in+the+Sod1-G93R+zebrafish+ALS+model.">INaP selective inhibition reverts precocious inter- and motorneurons hyperexcitability in the Sod1-G93R zebrafish ALS model.</a> Sci Rep. 6, 24515 (2016).</li><li>Pennati, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differences+between+larvae+of+the+Ciona+intestinalis+species+complex:+hints+for+a+valid+taxonomic+definition+of+distinct+species.">Morphological differences between larvae of the Ciona intestinalis species complex: hints for a valid taxonomic definition of distinct species.</a> PLoS ONE. 10 (5), 0122879 (2015).</li><li>Park, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+upstream+elements+in+the+HuC+promoter+leads+to+the+establishment+of+transgenic+zebrafish+with+fluorescent+neurons.">Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons.</a> Dev Biol. 227 (2), 279-293 (2000).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Drapeau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+locomotor+network+in+zebrafish.">Development of the locomotor network in zebrafish.</a> Prog Neurobiol. 68 (2), 85-111 (2002).</li><li>Brustein, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steps+during+the+development+of+the+zebrafish+locomotor+network.">Steps during the development of the zebrafish locomotor network.</a> J Physiol Paris. 97 (1), 77-86 (2003).</li><li>Drapeau, P., Ali, D. W., Buss, R. R., Saint-Amant, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+recording+from+identifiable+neurons+of+the+locomotor+network+in+the+developing+zebrafish.">In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish.</a> J Neurosci Methods. 88, 1-13 (1999).</li><li>Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2:+a+versatile+gene+transfer+vector+in+vertebrates.">Tol2: a versatile gene transfer vector in vertebrates.</a> Genome Biol. 8, (2007).</li><li>Sungmoo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Membrane+Potential+with+Two+Types+of+Genetically+Encoded+Fluorescent+Voltage+Sensors.">Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.</a> J Vis Exp. (108), e53566 (2016).</li></ol>

The protocol presented here allowed us to explore the association between the electrical properties of zebrafish embryo spinal motor neurons and the spontaneous coiling behavior, the earliest stereotypic motor activity, which appears around 17 hpf of embryonic development and lasts until 24 hpf10.
Our approach provides researchers with a tool to study the neural system of intact embryos, fully preserving the complexity of the interactions between cells in a developing f...

In vivo systemic component analysis allows scientists to investigate cellular behavior in the most reliable way. This is particularly true when the activity under scrutiny is heavily influenced by cell-cell interactions (both contact- and non-contact-dependent), as in the nervous system, where membrane voltage changes drive the communication among excitable cells. The comprehension of the information encoded by these electrical signals is the key to understanding the way the nervous system works in both physiological and disease states.
In order to study cell electrical properties in the most non-invasive physiological conditions, several genetically encoded voltage indicators have been recently developed1. As opposed to the previous generations of optical voltage sensors (mainly voltage-sensitive dyes)2, GEVIs allow for in vivo analyses of the intact neural system, and their expression can be limited to specific cell types or populations.
The zebrafish embryo is the in vivo &#34;substrate&#34; of choice to take advantage of the great potential attributed to GEVIs. In fact, thanks to its optical clarity and its simplified yet evolutionarily conserved nervous system, the zebrafish model allows for the straightforward identification and manipulation of every cellular component in a network. Indeed, the employment of the FRET-based GEVI Mermaid3 led to the identification of pre-symptomatic alterations in spinal motor neuron behavior in a zebrafish model of amyotrophic lateral sclerosis (ALS)4.
The following in vivo protocol describes how to monitor the electrical properties of spinal motor neurons in intact zebrafish embryos expressing Mermaid in a neuronal-specific manner. Moreover, it demonstrates how pharmacologically induced changes in such electrical properties can be associated with alterations in the frequency of embryonic spontaneous coilings, the stereotypic motor activity that characterizes the movement behavior of the zebrafish at very early stages of development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Low Melting Point Agarose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
		</tr>
		<tr>
			<td>Riluzole</td>
			<td>Sigma-Aldrich</td>
			<td>R116</td>
		</tr>
		<tr>
			<td>Pfu Ultra HQ DNA polymerase&#160;</td>
			<td>Agilent Technologies - Stratagene Products Division</td>
			<td>600389</td>
		</tr>
		<tr>
			<td>T3 Universal primer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up system</td>
			<td>Promega</td>
			<td>A9280</td>
		</tr>
		<tr>
			<td>Universal SmaI primer&#160;</td>
			<td>Eurofins</td>
			<td></td>
		</tr>
		<tr>
			<td>StrataClone Mammalian Expression Vector System / pCMV-SC blunt vector&#160;</td>
			<td>Agilent Technologies - Stratagene Products Division&#160;</td>
			<td>240228</td>
		</tr>
		<tr>
			<td>SmaI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0141S</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Promega</td>
			<td>M1801</td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>New England Biolabs</td>
			<td>R0138S</td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England Biolabs</td>
			<td>R0195S</td>
		</tr>
		<tr>
			<td>35 mm, glass-bottomed imaging dish&#160;</td>
			<td>Ibidi</td>
			<td>81151</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>M10 F</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Leica Microsystems</td>
			<td>DFC 310 FX</td>
		</tr>
		<tr>
			<td>Leica Application Suite 4.7.1 software&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickTime Player, v10.4</td>
			<td>Apple</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope (inverted)</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
		</tr>
		<tr>
			<td>Microinjector&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>Femtojet</td>
		</tr>
		<tr>
			<td>ImageJ macro Biosensor_FRET&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 6.0c&#160;</td>
			<td>GraphPad Software, Inc</td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

An expression vector carrying the FRET-based Mermaid biosensor coding sequence under the control of the pHuC pan-neuronal promoter, which drives the synthesis of the protein exclusively in the nervous system, was delivered into single-cell fertilized eggs by means of a microinjection in order to obtain transient transgenic embryos (Figure 1, left panel). After mastering the microinjection technique, the percentage of Mermaid-positive embryos was close to ...

Watch this Scientific Journal Video about Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos at JoVE.com

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

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.

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

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Developmental Biology

Unit 1

The Nervous System and Nervous Tissue

SCIENTISTS IN ACTION

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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 ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...