Monitoring Electrical Signals from the Afferent Neurons in an Immobilized Zebrafish Larva

Abstract

Source: Lunsford, E. T., et al. Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish. J. Vis. Exp. (2021).

This video demonstrates the recording of electrical signals from the afferent neurons in a paralyzed zebrafish larva by positioning a micropipette near the lateral line ganglion and forming a loose seal for signal capture. The extracellular solution maintains neuronal viability during this process.

Protocol

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

1. Preparation of materials for electrophysiological recordings

1. Make a silicone elastomer-bottomed recording dish.
   1. Dispense a thin layer of self-mixing silicone elastomer components (e.g., Sylgard) into a cover glass-bottomed tissue culture dish until it levels with the rim of a shallow well. Approximately 0.5 mL is sufficient.
   2. Cover and cure the dish for a minimum of 48 h at room temperature.
2. Make dissection pins.
   ....

Results

Figure 1: Simultaneous electrophysiological recording of posterior lateral line afferent neuron and ventral motor root activity. (A). Example of a loose-patch afferent (i) and ventral motor root (ii) r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mL beaker	PYREX	1000	Resceptacle for etchant
10x water immersion objective	Olympus	UMPLFLN10xW	Low magnification for positioning larvae and recording electrode
40x water immersion objective	Olympus	LUMPLFLN40XW	Higher magnification for position electrode tip and establishing patch-clamp
BNC coaxial cables	ThorLabs	2249-C-12	Connecting amplifier and digitizer channels; require 4
Borosilicate glass capillaries w/ filament	Warner Instruments	G150F-3	Inner diameter: 0.86, outer diameter: 1.50; capillary glass used to form recording electrodes
Computer	N/A	N/A	Any computer should work
DC Power Supply	Tenma	72-420	Used for electrically etching dissection pins
Electrophysiology digitizer	Axon Instruments, Molecular Devices	Axon DigiData 1440A	Enables acquisition of patch-clamp data
Filament	Sutter Instrument Company	FB255B	2.5 mm box filament used in micropipette puller
Fine forceps	Fine Science Tools	Dumont #5 (0.05 x 0.02 mm) Item No. 11295-10	Used to manipulate larvae and insert pins
Fixed stage DIC microscope	Olympus	BX51WI	Microscope used to visualize and establish patch-clamp recordings
Flexible, tapered pipette tip	Fisher Scientific	02-707-169	Flexible tips enable insertion into recording electrode to dispense extracellular solution at the tip
FluoroDish	World Precision Instruments Inc.	FD3510-100	Cover glass bottomed dish recording dish
KimWipe	KimTech	34155	Task wipe used for wicking away excess fluid from larvae
Kwik-Gard	World Precision Instruments Inc.	710172	Self-mixing sylgard elastomer
Microelectrode amplifier	Axon Instruments, Molecular Devices	MultiClamp 700B	Patch clamp amplifier for dual channel recordings
Microforge	Narishige	MF-830 microforge	To polish recording electrode
Micromanipulator control unit	Siskiyou	MC1000-eR/T	4-axis dial coordinator for controlling micromanipulator
Micropipette puller	Sutter Instrument Company	Flaming/Brown P-97	For pulling capillary glass into recording electrodes
Microscope control unit	Siskiyou	MC1000e	Positions the microscope around the fixed stage and preparation
Motorized micromanipulator	Siskiyou	MX7600	Positions the headstage and attached recording electrode for patch-clamp recording
MultiClamp Commander	Molecular Devices	2.2.2	Downloadable from Axon MultiClamp 700B Commander download page
Optical air table	Newport Corporation	VH3036W-OPT	Breadboard isolation table to float microscope and minimize vibrations during recordings
pCLAMP	Molecular Devices	10.7.0	Downloadable from Axon pCLAMP 10 Electrophysiology Data Acquisition & Analysis Software Download page
Petri-dish	Falcon	35-3001	Used to immerse larvae in paralytic
Pipette holder	Molecular Devices	1-HL-U	Hold recording electrode and connect to the headstage
Pneumatic transducer	Fluke Biomedical Instruments	DPM1B	For controlling recording electrode internal pressure
Potassium hydroxide	Sigma-Aldrich	221473-25G	Etchant for etching dissection pins
Silicone tubing	Tygon	14-169-1A	Tubing to connect pneumatic transducer to pipette holder
Stereomicroscope	Carl Zeiss	Stemi 2000-C	Used to visualize pin tips and during preparation of larvae
Straight edge razor blade	Canopus	order from amazon.com	Cuts the tungsten wire while making dissection pins
syringe	Becton Dickinson Compoany	309602	Filled with extracellular solution to inject into recording electrodes
Transfer pipette	Sigma-Aldrich	Z135003-500EA	Single use, non-sterile pipette for transfering larvae
Tricaine methanesulfonate	Syndel	12854	Pharmaceutical aneasthetic used to euthanize larvae with high dosage.
Tungsten wire	World Precision Instruments Inc.	715500	0.002 inch, 50.8 μm diameter; used to make dissection pins
Vacuum filtration unit	Sigma-Aldrich	SCGVU11RE	Single use, sterile, vacuum filtration units used to sterilize extracellular solution used for electrophysiology electrode ringer
Voltage-clamp current-clamp headstage	Molecular Devices	CV-7B	Supplied with MultiClamp 700B amplifier used as left and right headstages
α-bungarotoxin	ThermoFisher	B1601	For immobilizing the larvae prior to recording