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Method Article

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

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July 8th, 2025

In This Article

Abstract

Source: Benedetti, L., et al. Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos. 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.

Protocol

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 th....

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Disclosures

No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Low Melting Point AgaroseSigma-AldrichA9414
DMSOSigma-AldrichW387520
RiluzoleSigma-AldrichR116
Pfu Ultra HQ DNA polymeraseAgilent Technologies - Stratagene Products Division600389
T3 Universal primerSigma-Aldrich
Wizard SV Gel and PCR Clean-Up systemPromegaA9280
Universal SmaI primerEurofins
StrataClone Mammalian Expression Vector System / pCMV-SC blunt vectorAgilent Technologies - Stratagene Products Division240228
SmaINew England BiolabsR0141S
T4 DNA ligasePromegaM1801
SalINew England BiolabsR0138S
EcoRVNew England BiolabsR0195S
35 mm, glass-bottomed imaging dishIbidi81151
forcepsSigma-AldrichF6521
StereomicroscopeLeica MicrosystemsM10 F
Digital cameraLeica MicrosystemsDFC 310 FX
Leica Application Suite 4.7.1 softwareLeica Microsystems
QuickTime Player, v10.4Apple
Confocal microscope (inverted)Leica MicrosystemsTCS SP5
MicroinjectorEppendorfFemtojet
ImageJ macro Biosensor_FRET
GraphPad Prism 6.0cGraphPad Software, Inc

Tags

Motor Neuron ElectrophysiologyTail Coiling AnalysisSodium Channel BlockerFluorescence MicroscopyConfocal MicroscopyFRET MeasurementVoltage-sensing BiosensorRiluzole TreatmentSpontaneous Depolarization