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Summary
Abstract
Introduction
Protocol
Ergebnisse
Discussion
Offenlegungen
Acknowledgements
Materials
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Aktivitet af Posterior Lateral Line Afferent Neuroner under svømning i zebrafisk

Published: February 10, 2021
doi:
10.3791/62233
,
1Department of Biology,University of Florida, The Whitney Laboratory for Marine Bioscience

Summary

Vi beskriver en protokol til at overvåge ændringer i den afferente neuron aktivitet under motoriske kommandoer i en model hvirveldyr hår celle system.

Abstract

Sensoriske systemer samler signaler, der er afgørende for at lede adfærd, men dyr skal dechifrere, hvilke oplysninger der er biologisk relevante. Bevægelse genererer reafferent stikord, at dyr skal adskille fra relevante sensoriske signaler i det omgivende miljø. For eksempel, når en fisk svømmer, opdages flow genereret fra kroppens bølger af de mekanoreceptive neuromasts, bestående af hårceller, der komponerer lateral linjesystemet. Hårcellerne overfører derefter flydende bevægelsesoplysninger fra sensoren til hjernen via de sensoriske afferente neuroner. Samtidig videresendes den naturlige udladning af motorkommandoen til hårceller for at forhindre sensorisk overbelastning. Det er derfor afgørende at tage højde for den hæmmende virkning af prædiktive motorsignaler under bevægelse, når følsomheden af sidelinjesystemet evalueres. Vi har udviklet en in vivo elektrofysiologisk tilgang til samtidig overvågning af posterior lateral linje afferent neuron og ventral motorrod aktivitet i zebrafisk larver (4-7 dage efter befrugtning), der kan vare i flere timer. Ekstracellulære optagelser af afferente neuroner opnås ved hjælp af den løse patch klemme teknik, som kan detektere aktivitet fra enkelt eller flere neuroner. Ventral rodoptagelser udføres gennem huden med glaselektroder for at detektere motorisk neuronaktivitet. Vores eksperimentelle protokol giver mulighed for at overvåge endogene eller fremkaldte ændringer i sensorisk input på tværs af motorisk adfærd i en intakt, opfører hvirveldyr.

Introduction

Afferente neuroner af mekanosensoriske systemer overfører information fra hårceller til hjernen under hørelse og balance. Elektrofysiologi kan afsløre følsomheden af afferente neuroner gennem direkte optagelser. Mens hele celle patching fra hårceller kan være udfordrende, registrering fra downstream afferent neuroner er lettere og giver mulighed for vurdering af handling potentialer som reaktion på kontrollerede stimulationer1,2,3. Stimulerende hårceller fører til deres afbøjning, som ændrer mekanosensoriske strukturer, hvilket udløser en stigning i handlingspotentialer (pigge) i afferente neuroner4,5,6. I mangel af eksterne stimuli spidser afferente neuroner også spontant på grund af glutamatlækage fra hårcellerne til afferente postsynaptiske terminaler7,8, og har vist sig at bidrage til at opretholde følsomhed9,10. Patch klemme registrering af afferent aktivitet gør det muligt observation af hår celle følsomhed og signal dynamik, der ikke er muligt ved hjælp af teknikker med lavere tidsmæssige opløsning, såsom i mikrofoni11,12 eller funktionelle calcium imaging13,14,15. Følgende protokol vil gøre det muligt at registrere afferent aktivitet samtidig med motoriske kommandoer til at afsløre øjeblikkelige ændringer i hår celle følsomhed.

Zebrafisk (Danio rerio) bruge hårceller indeholdt i neuromasts at komponere lateral linje system til at opdage vand bevægelse i forhold til deres krop, som er oversat til neurale signaler afgørende for navigation16,17,18,rovdyr undgåelse, bytte fange19,20, og skolegang21. Vandgennemstrømningen kan også være selvgenereret af bevægelserne ved svømning22,23,24, respiration22,25,26og fodring27. Disse adfærd omfatter gentagne bevægelser, der kan træthed hårceller og forringe sensing. Derfor er det afgørende, at det laterale linjesystem skelner mellem eksterne (exafferent) og selvgenererede (reafferent) flow stimuli. En naturlig følgeudladning dæmper selvgenererede strømningssignaler under bevægelse hos zebrafisk. Dette hæmmende prædiktivt motorsignal videresendes via faldende neuroner til de sensoriske receptorer for at ændre inputtet eller afbryde behandlingen af den reafferent feedback28,29. Skelsættende arbejde, der bidrager til vores tidlige forståelse af dette feedforward-system, var afhængig af in vitro-præparater, hvor forbindelsen og den endogene aktivitet i det neurale kredsløb ikke blev opretholdt28,30,31,32,33,34,35. Denne protokol beskriver en tilgang til at bevare et intakt neuralt kredsløb, hvor endogen feedbackdynamik opretholdes, hvilket muliggør bedre forståelse af den naturlige udledning in vivo.

Protokollen er skitseret her beskriver, hvordan man overvåger posterior lateral linje afferent neuron og motor neuron aktivitet samtidig i larve zebrafisk. Karakteriserer afferent signaldynamik før, under og efter motoriske kommandoer giver indsigt i realtid, endogen feedback fra centralnervesystemet, der modulerer hårcellefølsomhed under bevægelse. Denne protokol skitserer, hvilke materialer der skal udarbejdes forud for eksperimenter, og beskriver derefter, hvordan man lammer og forbereder zebrafisklarver. Protokollen vil beskrive, hvordan man etablerer en stabil løs patch optagelse af afferente neuroner og ekstracellulære ventral rod (VR) optagelser af motoriske neuroner. Repræsentative data, der kan fås ved hjælp af denne protokol, præsenteres fra en eksemplarisk person, og der blev udført analyse på flere replikater af forsøgsprotokollen. Forbehandling af data udføres ved hjælp af brugerdefinerede skrevne scripts i MATLAB. Samlet set er dette in vivo eksperimentelle paradigme klar til at give en bedre forståelse af sensorisk feedback under bevægelse i en model hvirveldyr hår celle system.

Protocol

Al dyrepleje og forsøg blev udført i overensstemmelse med protokoller godkendt af University of Florida’s Institutional Animal Care and Use Committee. 1. Fremstilling af materialer til elektrofysiologiske optagelser Lav en silikone elastomerbundet optageskål. Dispenser et tyndt lag af selvblandende silikone elastomerkomponenter (f.eks. Sylgard) i en dækglasbundet vævskulturskål, indtil den niveauer med kanten af lavvandet godt. Ca. 0,5 mL er tilstrækkeligt. …

Representative Results

Efter zebrafisk larver er korrekt immobiliseret og den bageste laterale linje afferent ganglion og VR optagelse er opnået, aktivitet i både afferent og motor neuroner kan måles samtidigt. Optagelseskanaler vises ved hjælp af gap-free optagelsesprotokoller (trin 1.4) til kontinuerlig overvågning af afferent- og VR-aktivitet. I realtid kan der observeres fald i spontan afferent spike rate samtidig med VR-aktivitet, der indikerer fiktive svømmekampe (figur 1E). Vi fandt ud af, at de bedst…

Discussion

Den beskrevne eksperimentelle protokol giver mulighed for at overvåge endogene ændringer i sensorisk input på tværs af motorisk adfærd i en intakt, barbering hvirveldyr. Specifikt beskriver den en in vivo-tilgang til udførelse af samtidige ekstracellulære optagelser af laterale linjeafferente neuroner og ventralmotoriske rødder i larve zebrafisk. Spontan afferent aktivitet har tidligere været karakteriseret ved zebrafisk uden hensyntagen til potentiel samtidig motoraktivitet1,<…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

Vi anerkender taknemmeligt støtte fra National Institute of Health (DC010809), National Science Foundation (IOS1257150, 1856237) og Whitney Laboratory for Marine Biosciences til J.C.L. Vi vil gerne takke tidligere og nuværende medlemmer af Liao Lab for at stimulere drøftelserne.

Materials

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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Referenzen

  1. Trapani, J. G., Nicolson, T. Mechanism of spontaneous activity in afferent neurons of the zebrafish lateral-line organ. The Journal of Neuroscience. 31 (5), 1614-1623 (2011).
  2. Haehnel-Taguchi, M., Akanyeti, O., Liao, J. C. Afferent and motorneuron activity in response to single neuromast stimulation in the posterior lateral line of larval zebrafish. Journal of Neurophysiology. 112 (6), 1329-1339 (2014).
  3. Levi, R., Akanyeti, O., Ballo, A., Liao, J. C. Frequency response properties of primary afferent neurons in the posterior lateral line system of larval zebrafish. Journal of Neurophysiology. 113 (2), 657-668 (2015).
  4. Harris, G. G., Fishkopf, L. S., Flock, A. Receptor potentials from hair cells of the lateral line. Science. 167 (3914), 76-79 (1970).
  5. Dow, E., Jacobo, A., Hossain, S., Siletti, K., Hudspeth, A. J. Connectomics of the zebrafish’s lateral line neuromast reveals wiring and miswiring in a simple microcircuit. eLife. 7, 33988 (2018).
  6. Obholzer, N., et al. Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells. The Journal of Neuroscience. 28 (9), 2110-2118 (2008).
  7. Keen, E. C., Hudspeth, A. J. Transfer characteristics of the hair cell’s afferent synapse. Proceedings of the National Academy of Sciences of the United States of America. 103 (14), 5537-5542 (2006).
  8. Li, G., Keen, E., Andor-Ardó, D., Hudspeth, A. J., von Gersdorff, H. The unitary event underlying multiquantal EPSCs at a hair cell’s ribbon synapse. The Journal of Neuroscience. 29 (23), 7558-7568 (2009).
  9. Manley, G. A., Robertson, D. Analysis of spontaneous activity of auditory neurons in the spiral ganglion of the guinea-pig cochlea. The Journal of Physiology. 258 (2), 323-336 (1976).
  10. Kiang, N. Y. S., Watanabe, T., Thomas, E., Clark, L. . Discharge patterns of single fibers in the cat’s auditory nerve. , (1965).
  11. Corey, D. P., Hudspeth, A. J. Ionic basis of the receptor potential in a vertebrate hair cell. Nature. 281 (5733), 675-677 (1979).
  12. Trapani, J. G., Nicolson, T. Physiological recordings from zebrafish lateral-line hair cells and afferent neurons. Methods in Cell Biology. 100, 219-231 (2010).
  13. Reinig, S., Driever, W., Arrenberg, A. B. The descending diencephalic dopamine system is tuned to sensory stimuli. Current Biology. 27 (3), 318-333 (2017).
  14. Zhang, Q., et al. Synaptically silent sensory hair cells in zebrafish are recruited after damage. Nature Communications. 9 (1), 1388 (2018).
  15. Pichler, P., Lagnado, L. Motor behavior selectively inhibits hair cells activated forward motion in the lateral line of zebrafish. Current Biology. 30 (1), 150-157 (2020).
  16. Olszewski, J., Haehnel, M., Taguchi, M., Liao, J. C. Zebrafish larvae exhibit rheotaxis and can escape a continuous suction source using their lateral line. PloS One. 7 (5), 36661 (2012).
  17. Suli, A., Watson, G. M., Rubel, E. W., Raible, D. W. Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells. PLoS One. 7 (2), 29727 (2012).
  18. Oteiza, P., Odstcil, I., Lauder, G., Portugues, R., Engert, F. A novel mechanism for mechanosensory-based rheotaxis in larval zebrafish. Nature. 547 (7664), 445-448 (2017).
  19. McHenry, M. J., Feitl, K. E., Strother, J. A. Larval zebrafish rapidly sense the water flow of a predator’s strike. Biology Letters. 5 (4), 477-479 (2009).
  20. Stewart, W. J., Cardenas, G. S., McHenry, M. J. Zebrafish larvae evade predators by sensing water flow. The Journal of Experimental Biology. 216, 388-398 (2013).
  21. Mekdara, P. J., Schwalbe, M. A. B., Coughlin, L. L., Tytell, E. D. The effects of lateral line ablation and regeneration in schooling giant danios. The Journal of Experimental Biology. 221, 175166 (2018).
  22. Palmer, L. M., Giuffrida, B. A., Mensinger, A. F. Neural recordings from the lateral line in free-swimming toadfish, Opsanus tau. The Biological Bulletin. 205 (2), 216-218 (2003).
  23. Ayali, A., Gelman, S., Tytell, E. D., Cohen, A. H. Lateral line activity during undulatory body motions suggests a feedback link in closed-loop control of sea lamprey swimming. Canadian Journal of Zoology. 87 (8), 671-683 (2009).
  24. Mensinger, A. F., Van Wert, J. C., Rogers, L. S. Lateral line sensitivity in free-swimming toad fish Opsanus tau. The Journal of Experimental Biology. 222, 190587 (2019).
  25. Montgomery, J., Bodznick, D., Halstead, M. Hindbrain signal processing in the lateral line system of the dwarf scorpionfish Scopeana papillosus. The Journal of Experimental Biology. 199, 893-899 (1996).
  26. Montgomery, J. C., Bodznick, D. An adaptive filter that cancels self-induced noise in the electrosensory and lateral line mechanosensory systems of fish. Neuroscience Letters. 174 (2), 145-148 (1994).
  27. Palmer, L. M., Deffenbaugh, M., Mensinger, A. F. Sensitivity of the anterior lateral line to natural stimuli in the oyster toadfish, Opsanus tau (Linnaeus). The Journal of Experimental Biology. 208, 3441-3450 (2005).
  28. Russell, I. J., Roberts, B. L. Inhibition of spontaneous lateral-line activity of efferent nerve stimulation. The Journal of Experimental Biology. 57, 77-82 (1972).
  29. Lunsford, E. T., Skandalis, D. A., Liao, J. C. Efferent modulation of spontaneous lateral line activity during and after zebrafish motor commands. Journal of Neurophysiology. 122 (6), 2438-2448 (2019).
  30. Russell, I. J. The pharmacology of efferent synapses in the lateral-line system of Xenopus laevis. The Journal of Experimental Biology. 54 (3), 643-659 (1971).
  31. Roberts, B. L., Russell, I. J. The activity of lateral-line efferent neurons in stationary and swimming dogfish. The Journal of Experimental Biology. 57 (2), 435-448 (1972).
  32. Flock, A., Russell, I. J. The post-synaptic action of efferent fibres in the lateral line organ of the burbot Lota lota. The Journal of Physiology. 235 (3), 591-605 (1973).
  33. Montgomery, J. C. Noise cancellation in the electrosensory system of the thornback ray; common mode rejection of input produced by the animal’s own ventilatory movement. Journal of Comparative Physiology. 155, 103-111 (1984).
  34. Tricas, T. C., Highstein, S. M. Action of the octavolateralis efferent system upon the lateral line of free-swimming toadfish, Opsanus tau. Journal of Comparative Physiology. 169 (1), 25-37 (1991).
  35. Weeg, M. S., Land, B. R., Bass, A. H. Vocal pathways modulate efferent neurons to the inner ear and lateral line. The Journal of Neuroscience. 25 (25), 5967-5974 (2005).
  36. Elgoyhen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E., Heinemann, S. α9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell. 79 (4), 705-715 (1994).
  37. Masino, M. A., Fetcho, J. R. Fictive swimming motor patterns in wild type and mutant larval zebrafish. Journal of Neurophysiology. 93 (6), 3177-3188 (2005).
  38. Hentschke, H. abfload. 1.4.0.0. MATLAB Central File Exchange. , (2020).
  39. Harris, G. G., Milne, D. C. Input-output characteristics of the lateral-line sense organs of Xenopus laevis. The Journal of the Acoustical Society of America. 40 (1), 32-42 (1966).
  40. Liao, J. C., Haehnel, M. Physiology of afferent neurons in larval zebrafish provides a functional framework for lateral line somatotopy. Journal of Neurophysiology. 107 (10), 2615-2623 (2012).
  41. Song, S., et al. Mathematical modeling and analyses of interspike-intervals of spontaneous activity in afferent neurons of the zebrafish lateral line. Nature Science Reports. 8, 14851 (2018).
  42. Liao, J. C., Fetcho, J. R. Shared versus specialized glycinergic spinal interneurons in axial motor circuits of larval zebrafish. The Journal of Neuroscience. 28 (48), 12982-12992 (2008).
  43. von Holst, E., Mittelstaedt, H. The principle of reafference: interactions between the central nervous system and the peripheral organs. Die Naturwissenschften. 37, 463 (1950).
  44. Crapse, T. B., Sommer, M. A. Corollary discharge across the animal kingdom. Nature Reviews. Neuroscience. 9 (8), 587-600 (2008).
  45. Brichta, A. M., Goldberg, J. M. Responses to efferent activation and excitatory response-intensity relations of turtle posterior-crista afferents. Journal of Neurophysiology. 83 (3), 1224-1242 (2000).

Tags

Keywords: Posterior Lateral LineAfferent NeuronsZebrafishPatch ElectrophysiologyHair CellsDissectionMicroelectrodeZebrafish LarvaeParalysisExtracellular Solution
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Lunsford, E. T., Liao, J. C. Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish. J. Vis. Exp. (168), e62233, doi:10.3791/62233 (2021).
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