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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Published: February 10th, 2021

DOI:

10.3791/62233

1Department of Biology, University of Florida, The Whitney Laboratory for Marine Bioscience

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

Afferent neurons of mechanosensory systems transmit information from hair cells to the brain during hearing and balance. Electrophysiology can reveal the sensitivity of afferent neurons through direct recordings. While whole cell patching from hair cells can be challenging, recording from downstream afferent neurons is easier and allows assessment of action potentials in response to controlled stimulations1,2,3. Stimulating hair cells lead to their deflection, which modifies mechanosensory structures, thus triggering an increase in action potentials (spikes) in afferent neuro....

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All animal care and experiments were performed in accordance with protocols approved by the University of Florida's Institutional Animal Care and Use Committee.

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 shallow well. Approximately 0.5.......

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After zebrafish larvae are properly immobilized and the posterior lateral line afferent ganglion and VR recording is achieved, activity in both afferent and motor neurons can be measured simultaneously. Recording channels are displayed using gap-free recording protocols (step 1.4) for continuous monitoring of afferent and VR activity. In real-time, decreases in spontaneous afferent spike rate can be observed concurrent with VR activity indicative of fictive swim bouts (Figure 1E). We found t.......

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The experimental protocol described provides the potential to monitor endogenous changes in sensory input across motor behaviors in an intact, behaving vertebrate. Specifically, it details an in vivo approach to performing simultaneous extracellular recordings of lateral line afferent neurons and ventral motor roots in larval zebrafish. Spontaneous afferent activity has been previously characterized in zebrafish without consideration of potential concurrent motor activity1,

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We gratefully acknowledge support from the National Institute of Health (DC010809), National Science Foundation (IOS1257150, 1856237), and the Whitney Laboratory for Marine Biosciences to J.C.L. We would like to thank past and present members of the Liao Lab for stimulating discussions.

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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
abfload.m supplemental coding file custom written MATLAB script for converting raw electrophysiology recordings to .mat files
AffVR_preprocess.m supplemental coding file custom written MATLAB script for preprocessing recording data
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
burst_detect supplemental coding file custom written MATLAB function necessary to run AffVR_preprocess.m
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
MATLAB MathWorks R2020b command line software for preprocessing data
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
permanent ink marker Sharpie order from amazon.com for marking the leading edge side of the VR electrode to ensure proper orientation when inserting into pipette holder
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
spike_detect supplemental coding file custom written MATLAB function necessary to run AffVR_preprocess.m
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
swimbout_detect supplemental coding file custom written MATLAB function necessary to run AffVR_preprocess.m
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

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