Name: Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish (Video) | JoVE Journal
Uploaded: 2021-02-10T15:00:00.000+00:00
Duration: 10 min 34 s
Description: Learn patch electrophysiology to record afferent neuron activity in zebrafish larvae during swimming. This method reveals hair cell circuit properties relevant to human hearing disorders.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.&#160;We would like to thank past and present members of the Liao Lab for stimulating discussions.

<ol><li>Trapani, J. G., Nicolson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+spontaneous+activity+in+afferent+neurons+of+the+zebrafish+lateral-line+organ%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Mechanism of spontaneous activity in afferent neurons of the zebrafish lateral-line organ.</a> The Journal of Neuroscience. 31 (5), 1614-1623 (2011).</li>
<li>Haehnel-Taguchi, M., Akanyeti, O., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Afferent+and+motorneuron+activity+in+response+to+single+neuromast+stimulation+in+the+posterior+lateral+line+of+larval+zebrafish%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Afferent and motorneuron activity in response to single neuromast stimulation in the posterior lateral line of larval zebrafish.</a> Journal of Neurophysiology. 112 (6), 1329-1339 (2014).</li>
<li>Levi, R., Akanyeti, O., Ballo, A., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency+response+properties+of+primary+afferent+neurons+in+the+posterior+lateral+line+system+of+larval+zebrafish%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Frequency response properties of primary afferent neurons in the posterior lateral line system of larval zebrafish.</a> Journal of Neurophysiology. 113 (2), 657-668 (2015).</li>
<li>Harris, G. G., Fishkopf, L. S., Flock, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Receptor+potentials+from+hair+cells+of+the+lateral+line%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Receptor potentials from hair cells of the lateral line.</a> Science. 167 (3914), 76-79 (1970).</li>
<li>Dow, E., Jacobo, A., Hossain, S., Siletti, K., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connectomics+of+the+zebrafish%27s+lateral+line+neuromast+reveals+wiring+and+miswiring+in+a+simple+microcircuit%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Connectomics of the zebrafish's lateral line neuromast reveals wiring and miswiring in a simple microcircuit.</a> eLife. 7, 33988(2018).</li>
<li>Obholzer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicular+glutamate+transporter+3+is+required+for+synaptic+transmission+in+zebrafish+hair+cells%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells.</a> The Journal of Neuroscience. 28 (9), 2110-2118 (2008).</li>
<li>Keen, E. C., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+characteristics+of+the+hair+cell%27s+afferent+synapse%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Transfer characteristics of the hair cell's afferent synapse.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (14), 5537-5542 (2006).</li>
<li>Li, G., Keen, E., Andor-Ardó, D., Hudspeth, A. J., von Gersdorff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+unitary+event+underlying+multiquantal+EPSCs+at+a+hair+cell%27s+ribbon+synapse%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">The unitary event underlying multiquantal EPSCs at a hair cell's ribbon synapse.</a> The Journal of Neuroscience. 29 (23), 7558-7568 (2009).</li>
<li>Manley, G. A., Robertson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+spontaneous+activity+of+auditory+neurons+in+the+spiral+ganglion+of+the+guinea-pig+cochlea%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Analysis of spontaneous activity of auditory neurons in the spiral ganglion of the guinea-pig cochlea.</a> The Journal of Physiology. 258 (2), 323-336 (1976).</li>
<li>Kiang, N. Y. S., Watanabe, T., Thomas, E., Clark, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKiang%2C+NYS+%22Discharge+patterns+of+single+fibers+in+the+cat%27s+auditory+nerve%22">Discharge patterns of single fibers in the cat's auditory nerve</a>. , MIT Press. Cambridge MA. (1965).</li>
<li>Corey, D. P., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ionic+basis+of+the+receptor+potential+in+a+vertebrate+hair+cell%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ionic basis of the receptor potential in a vertebrate hair cell.</a> Nature. 281 (5733), 675-677 (1979).</li>
<li>Trapani, J. G., Nicolson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+recordings+from+zebrafish+lateral-line+hair+cells+and+afferent+neurons%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Physiological recordings from zebrafish lateral-line hair cells and afferent neurons.</a> Methods in Cell Biology. 100, 219-231 (2010).</li>
<li>Reinig, S., Driever, W., Arrenberg, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+descending+diencephalic+dopamine+system+is+tuned+to+sensory+stimuli%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The descending diencephalic dopamine system is tuned to sensory stimuli.</a> Current Biology. 27 (3), 318-333 (2017).</li>
<li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptically+silent+sensory+hair+cells+in+zebrafish+are+recruited+after+damage%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Synaptically silent sensory hair cells in zebrafish are recruited after damage.</a> Nature Communications. 9 (1), 1388(2018).</li>
<li>Pichler, P., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+behavior+selectively+inhibits+hair+cells+activated+forward+motion+in+the+lateral+line+of+zebrafish%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Motor behavior selectively inhibits hair cells activated forward motion in the lateral line of zebrafish.</a> Current Biology. 30 (1), 150-157 (2020).</li>
<li>Olszewski, J., Haehnel, M., Taguchi, M., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+larvae+exhibit+rheotaxis+and+can+escape+a+continuous+suction+source+using+their+lateral+line%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Zebrafish larvae exhibit rheotaxis and can escape a continuous suction source using their lateral line.</a> PloS One. 7 (5), 36661(2012).</li>
<li>Suli, A., Watson, G. M., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheotaxis+in+larval+zebrafish+is+mediated+by+lateral+line+mechanosensory+hair+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells.</a> PLoS One. 7 (2), 29727(2012).</li>
<li>Oteiza, P., Odstcil, I., Lauder, G., Portugues, R., Engert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mechanism+for+mechanosensory-based+rheotaxis+in+larval+zebrafish%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A novel mechanism for mechanosensory-based rheotaxis in larval zebrafish.</a> Nature. 547 (7664), 445-448 (2017).</li>
<li>McHenry, M. J., Feitl, K. E., Strother, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Larval+zebrafish+rapidly+sense+the+water+flow+of+a+predator%27s+strike%5BTitle%5D)+AND+%22Biology+Letters%22%5BJournal%5D)">Larval zebrafish rapidly sense the water flow of a predator's strike.</a> Biology Letters. 5 (4), 477-479 (2009).</li>
<li>Stewart, W. J., Cardenas, G. S., McHenry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+larvae+evade+predators+by+sensing+water+flow%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Zebrafish larvae evade predators by sensing water flow.</a> The Journal of Experimental Biology. 216, Pt 3 388-398 (2013).</li>
<li>Mekdara, P. J., Schwalbe, M. A. B., Coughlin, L. L., Tytell, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+lateral+line+ablation+and+regeneration+in+schooling+giant+danios%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">The effects of lateral line ablation and regeneration in schooling giant danios.</a> The Journal of Experimental Biology. 221, Pt 8 175166(2018).</li>
<li>Palmer, L. M., Giuffrida, B. A., Mensinger, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+recordings+from+the+lateral+line+in+free-swimming+toadfish%2C+Opsanus+tau%5BTitle%5D)+AND+%22The+Biological+Bulletin%22%5BJournal%5D)">Neural recordings from the lateral line in free-swimming toadfish, Opsanus tau.</a> The Biological Bulletin. 205 (2), 216-218 (2003).</li>
<li>Ayali, A., Gelman, S., Tytell, E. D., Cohen, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lateral+line+activity+during+undulatory+body+motions+suggests+a+feedback+link+in+closed-loop+control+of+sea+lamprey+swimming%5BTitle%5D)+AND+%22Canadian+Journal+of+Zoology%22%5BJournal%5D)">Lateral line activity during undulatory body motions suggests a feedback link in closed-loop control of sea lamprey swimming.</a> Canadian Journal of Zoology. 87 (8), 671-683 (2009).</li>
<li>Mensinger, A. F., Van Wert, J. C., Rogers, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lateral+line+sensitivity+in+free-swimming+toad+fish+Opsanus+tau%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Lateral line sensitivity in free-swimming toad fish Opsanus tau.</a> The Journal of Experimental Biology. 222, Pt 2 190587(2019).</li>
<li>Montgomery, J., Bodznick, D., Halstead, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hindbrain+signal+processing+in+the+lateral+line+system+of+the+dwarf+scorpionfish+Scopeana+papillosus%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Hindbrain signal processing in the lateral line system of the dwarf scorpionfish Scopeana papillosus.</a> The Journal of Experimental Biology. 199, Pt 4 893-899 (1996).</li>
<li>Montgomery, J. C., Bodznick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+adaptive+filter+that+cancels+self-induced+noise+in+the+electrosensory+and+lateral+line+mechanosensory+systems+of+fish%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">An adaptive filter that cancels self-induced noise in the electrosensory and lateral line mechanosensory systems of fish.</a> Neuroscience Letters. 174 (2), 145-148 (1994).</li>
<li>Palmer, L. M., Deffenbaugh, M., Mensinger, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitivity+of+the+anterior+lateral+line+to+natural+stimuli+in+the+oyster+toadfish%2C+Opsanus+tau+%28Linnaeus%29%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Sensitivity of the anterior lateral line to natural stimuli in the oyster toadfish, Opsanus tau (Linnaeus).</a> The Journal of Experimental Biology. 208, Pt 18 3441-3450 (2005).</li>
<li>Russell, I. J., Roberts, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+spontaneous+lateral-line+activity+of+efferent+nerve+stimulation%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">Inhibition of spontaneous lateral-line activity of efferent nerve stimulation.</a> The Journal of Experimental Biology. 57, 77-82 (1972).</li>
<li>Lunsford, E. T., Skandalis, D. A., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efferent+modulation+of+spontaneous+lateral+line+activity+during+and+after+zebrafish+motor+commands%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Efferent modulation of spontaneous lateral line activity during and after zebrafish motor commands.</a> Journal of Neurophysiology. 122 (6), 2438-2448 (2019).</li>
<li>Russell, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pharmacology+of+efferent+synapses+in+the+lateral-line+system+of+Xenopus+laevis%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">The pharmacology of efferent synapses in the lateral-line system of Xenopus laevis.</a> The Journal of Experimental Biology. 54 (3), 643-659 (1971).</li>
<li>Roberts, B. L., Russell, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+activity+of+lateral-line+efferent+neurons+in+stationary+and+swimming+dogfish%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Biology%22%5BJournal%5D)">The activity of lateral-line efferent neurons in stationary and swimming dogfish.</a> The Journal of Experimental Biology. 57 (2), 435-448 (1972).</li>
<li>Flock, A., Russell, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+post-synaptic+action+of+efferent+fibres+in+the+lateral+line+organ+of+the+burbot+Lota+lota%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">The post-synaptic action of efferent fibres in the lateral line organ of the burbot Lota lota.</a> The Journal of Physiology. 235 (3), 591-605 (1973).</li>
<li>Montgomery, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noise+cancellation+in+the+electrosensory+system+of+the+thornback+ray%3B+common+mode+rejection+of+input+produced+by+the+animal%27s+own+ventilatory+movement%5BTitle%5D)+AND+%22Journal+of+Comparative+Physiology%22%5BJournal%5D)">Noise cancellation in the electrosensory system of the thornback ray; common mode rejection of input produced by the animal's own ventilatory movement.</a> Journal of Comparative Physiology. 155, 103-111 (1984).</li>
<li>Tricas, T. C., Highstein, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Action+of+the+octavolateralis+efferent+system+upon+the+lateral+line+of+free-swimming+toadfish%2C+Opsanus+tau%5BTitle%5D)+AND+%22Journal+of+Comparative+Physiology%22%5BJournal%5D)">Action of the octavolateralis efferent system upon the lateral line of free-swimming toadfish, Opsanus tau.</a> Journal of Comparative Physiology. 169 (1), 25-37 (1991).</li>
<li>Weeg, M. S., Land, B. R., Bass, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vocal+pathways+modulate+efferent+neurons+to+the+inner+ear+and+lateral+line%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Vocal pathways modulate efferent neurons to the inner ear and lateral line.</a> The Journal of Neuroscience. 25 (25), 5967-5974 (2005).</li>
<li>Elgoyhen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E., Heinemann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B19%3A+an+acetylcholine+receptor+with+novel+pharmacological+properties+expressed+in+rat+cochlear+hair+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">α9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.</a> Cell. 79 (4), 705-715 (1994).</li>
<li>Masino, M. A., Fetcho, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fictive+swimming+motor+patterns+in+wild+type+and+mutant+larval+zebrafish%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Fictive swimming motor patterns in wild type and mutant larval zebrafish.</a> Journal of Neurophysiology. 93 (6), 3177-3188 (2005).</li>
<li>Hentschke, H. abfload. 1.4.0.0. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHentschke%2C+H+%22MATLAB+Central+File+Exchange%22">MATLAB Central File Exchange</a>. , (2020).</li>
<li>Harris, G. G., Milne, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Input-output+characteristics+of+the+lateral-line+sense+organs+of+Xenopus+laevis%5BTitle%5D)+AND+%22The+Journal+of+the+Acoustical+Society+of+America%22%5BJournal%5D)">Input-output characteristics of the lateral-line sense organs of Xenopus laevis.</a> The Journal of the Acoustical Society of America. 40 (1), 32-42 (1966).</li>
<li>Liao, J. C., Haehnel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiology+of+afferent+neurons+in+larval+zebrafish+provides+a+functional+framework+for+lateral+line+somatotopy%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Physiology of afferent neurons in larval zebrafish provides a functional framework for lateral line somatotopy.</a> Journal of Neurophysiology. 107 (10), 2615-2623 (2012).</li>
<li>Song, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mathematical+modeling+and+analyses+of+interspike-intervals+of+spontaneous+activity+in+afferent+neurons+of+the+zebrafish+lateral+line%5BTitle%5D)+AND+%22Nature+Science+Reports%22%5BJournal%5D)">Mathematical modeling and analyses of interspike-intervals of spontaneous activity in afferent neurons of the zebrafish lateral line.</a> Nature Science Reports. 8, 14851(2018).</li>
<li>Liao, J. C., Fetcho, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shared+versus+specialized+glycinergic+spinal+interneurons+in+axial+motor+circuits+of+larval+zebrafish%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Shared versus specialized glycinergic spinal interneurons in axial motor circuits of larval zebrafish.</a> The Journal of Neuroscience. 28 (48), 12982-12992 (2008).</li>
<li>von Holst, E., Mittelstaedt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+principle+of+reafference%3A+interactions+between+the+central+nervous+system+and+the+peripheral+organs%5BTitle%5D)+AND+%22Die+Naturwissenschften%22%5BJournal%5D)">The principle of reafference: interactions between the central nervous system and the peripheral organs.</a> Die Naturwissenschften. 37, 463(1950).</li>
<li>Crapse, T. B., Sommer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corollary+discharge+across+the+animal+kingdom%5BTitle%5D)+AND+%22Nature+Reviews.+Neuroscience%22%5BJournal%5D)">Corollary discharge across the animal kingdom.</a> Nature Reviews. Neuroscience. 9 (8), 587-600 (2008).</li>
<li>Brichta, A. M., Goldberg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responses+to+efferent+activation+and+excitatory+response-intensity+relations+of+turtle+posterior-crista+afferents%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Responses to efferent activation and excitatory response-intensity relations of turtle posterior-crista afferents.</a> Journal of Neurophysiology. 83 (3), 1224-1242 (2000).</li>
</ol>

The authors declare no competing financial interests.

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,<sup class="x...

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 neurons4,5,6. In the absence of external stimuli, afferent neurons also spike spontaneously due to glutamate leak from the hair cells on to afferent post-synaptic terminals7,8, and have been shown to contribute toward maintaining sensitivity9,10. Patch clamp recording of afferent activity enables observation of hair cell sensitivity and signal dynamics that are not possible using techniques with lower temporal resolution, such as in microphonics11,12 or functional calcium imaging13,14,15. The following protocol will allow the recording of afferent activity concurrent with motor commands to reveal instantaneous changes in hair cell sensitivity.
Zebrafish (Danio rerio) use hair cells contained in neuromasts that compose the lateral line system to detect water movement relative to their body, which is translated into neural signals essential for navigation16,17,18, predator avoidance, prey capture19,20, and schooling21. Water flow can also be self-generated by the motions of swimming22,23,24, respiration22,25,26, and feeding27. These behaviors comprise repetitive movements that can fatigue hair cells and impair sensing. Therefore, it is critical that the lateral line system differentiates between external (exafferent) and self-generated (reafferent) flow stimuli. A corollary discharge attenuates self-generated flow signals during locomotion in zebrafish. This inhibitory predictive motor signal is relayed via descending neurons to the sensory receptors to modify the input or interrupt the processing of the reafferent feedback28,29. Seminal work contributing to our early understanding of this feedforward system relied on in vitro preparations where the connectivity and endogenous activity of the neural circuit were not maintained28,30,31,32,33,34,35. This protocol describes an approach to preserving an intact neural circuit where endogenous feedback dynamics are maintained thus enabling better understanding of the corollary discharge in vivo.
The protocol outlined here describes how to monitor posterior lateral line afferent neuron and motor neuron activity simultaneously in larval zebrafish. Characterizing afferent signal dynamics before, during, and after motor commands provides insights into real-time, endogenous feedback from the central nervous system that modulates hair cell sensitivity during locomotion. This protocol outlines what materials will need to be prepared prior to experiments and then describes how to paralyze and prepare zebrafish larvae. The protocol will describe how to establish a stable loose patch recording of afferent neurons and extracellular ventral root (VR) recordings of motor neurons. Representative data that can be obtained using this protocol are presented from an exemplar individual and analysis was performed on multiple replicates of the experimental protocol. Pre-processing of data is performed using custom written scripts in MATLAB. Overall, this in vivo experimental paradigm is poised to provide a better understanding of sensory feedback during locomotion in a model vertebrate hair cell system.

<table><tbody><tr><td>100 mL beaker</td><td>PYREX</td><td>1000</td><td>resceptacle for etchant</td></tr><tr><td>10x water immersion objective</td><td>Olympus</td><td>UMPLFLN10xW</td><td>low magnification for positioning larvae and recording electrode</td></tr><tr><td>40x water immersion objective</td><td>Olympus</td><td>LUMPLFLN40XW</td><td>higher magnification for position electrode tip and establishing patch-clamp</td></tr><tr><td>abfload.m</td><td></td><td>supplemental coding file</td><td>custom written MATLAB script for converting raw electrophysiology recordings to .mat files</td></tr><tr><td>AffVR_preprocess.m</td><td></td><td>supplemental coding file</td><td>custom written MATLAB script for preprocessing recording data</td></tr><tr><td>BNC coaxial cables</td><td>ThorLabs</td><td>2249-C-12</td><td>connecting amplifier and digitizer channels; require 4</td></tr><tr><td>borosilicate glass capillaries w/ filament</td><td>Warner Instruments</td><td>G150F-3</td><td>inner diameter: 0.86, outer diameter: 1.50; capillary glass used to form recording electrodes</td></tr><tr><td>burst_detect</td><td></td><td>supplemental coding file</td><td>custom written MATLAB function necessary to run AffVR_preprocess.m</td></tr><tr><td>computer</td><td>N/A</td><td>N/A</td><td>any computer should work</td></tr><tr><td>DC Power Supply</td><td>Tenma</td><td>72-420</td><td>used for electrically etching dissection pins</td></tr><tr><td>electrophysiology digitizer</td><td>Axon Instruments, Molecular Devices</td><td>Axon DigiData 1440A</td><td>enables acquisition of patch-clamp data</td></tr><tr><td>filament</td><td>Sutter Instrument Company</td><td>FB255B</td><td>2.5 mm box filament used in micropipette puller</td></tr><tr><td>fine forceps</td><td>Fine Science Tools</td><td>Dumont #5 (0.05 x 0.02 mm) Item No. 11295-10</td><td>used to manipulate larvae and insert pins</td></tr><tr><td>fixed stage DIC microscope</td><td>Olympus</td><td>BX51WI</td><td>microscope used to visualize and establish patch-clamp recordings</td></tr><tr><td>flexible, tapered pipette tip</td><td>Fisher Scientific</td><td>02-707-169</td><td>flexible tips enable insertion into recording electrode to dispense extracellular solution at the tip</td></tr><tr><td>FluoroDish</td><td>World Precision Instruments Inc.</td><td>FD3510-100</td><td>cover glass bottomed dish recording dish</td></tr><tr><td>KimWipe</td><td>KimTech</td><td>34155</td><td>task wipe used for wicking away excess fluid from larvae</td></tr><tr><td>Kwik-Gard</td><td>World Precision Instruments Inc.</td><td>710172</td><td>self-mixing sylgard elastomer</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>R2020b</td><td>command line software for preprocessing data</td></tr><tr><td>microelectrode amplifier</td><td>Axon Instruments, Molecular Devices</td><td>MultiClamp 700B</td><td>patch clamp amplifier for dual channel recordings</td></tr><tr><td>microforge</td><td>Narishige</td><td>MF-830 microforge</td><td>to polish recording electrode</td></tr><tr><td>micromanipulator control unit</td><td>Siskiyou</td><td>MC1000-eR/T</td><td>4-axis dial coordinator for controlling micromanipulator</td></tr><tr><td>micropipette puller</td><td>Sutter Instrument Company</td><td>Flaming/Brown P-97</td><td>for pulling capillary glass into recording electrodes</td></tr><tr><td>microscope control unit</td><td>Siskiyou</td><td>MC1000e</td><td>positions the microscope around the fixed stage and preparation</td></tr><tr><td>motorized micromanipulator</td><td>Siskiyou</td><td>MX7600</td><td>positions the headstage and attached recording electrode for patch-clamp recording</td></tr><tr><td>MultiClamp Commander</td><td>Molecular Devices</td><td>2.2.2</td><td>downloadable from Axon MultiClamp 700B Commander download page</td></tr><tr><td>optical air table</td><td>Newport Corporation</td><td>VH3036W-OPT</td><td>breadboard isolation table to float microscope and minimize vibrations during recordings</td></tr><tr><td>pCLAMP</td><td>Molecular Devices</td><td>10.7.0</td><td>downloadable from Axon pCLAMP 10 Electrophysiology Data Acquisition &amp;amp; Analysis Software Download page</td></tr><tr><td>permanent ink marker</td><td>Sharpie</td><td>order from amazon.com</td><td>for marking the leading edge side of the VR electrode to ensure proper orientation when inserting into pipette holder</td></tr><tr><td>petri-dish</td><td>Falcon</td><td>35-3001</td><td>used to immerse larvae in paralytic</td></tr><tr><td>pipette holder</td><td>Molecular Devices</td><td>1-HL-U</td><td>hold recording electrode and connect to the headstage</td></tr><tr><td>pneumatic transducer</td><td>Fluke Biomedical Instruments</td><td>DPM1B</td><td>for controlling recording electrode internal pressure</td></tr><tr><td>potassium hydroxide</td><td>Sigma-Aldrich</td><td>221473-25G</td><td>etchant for etching dissection pins</td></tr><tr><td>silicone tubing</td><td>Tygon</td><td>14-169-1A</td><td>tubing to connect pneumatic transducer to pipette holder</td></tr><tr><td>spike_detect</td><td></td><td>supplemental coding file</td><td>custom written MATLAB function necessary to run AffVR_preprocess.m</td></tr><tr><td>stereomicroscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td>used to visualize pin tips and during preparation of larvae</td></tr><tr><td>straight edge razor blade</td><td>Canopus</td><td>order from amazon.com</td><td>cuts the tungsten wire while making dissection pins</td></tr><tr><td>swimbout_detect</td><td></td><td>supplemental coding file</td><td>custom written MATLAB function necessary to run AffVR_preprocess.m</td></tr><tr><td>syringe</td><td>Becton Dickinson Compoany</td><td>309602</td><td>filled with extracellular solution to inject into recording electrodes</td></tr><tr><td>transfer pipette</td><td>Sigma-Aldrich</td><td>Z135003-500EA</td><td>single use, non-sterile pipette for transfering larvae</td></tr><tr><td>tricaine methanesulfonate</td><td>Syndel</td><td>12854</td><td>pharmaceutical aneasthetic used to euthanize larvae with high dosage.</td></tr><tr><td>tungsten wire</td><td>World Precision Instruments Inc.</td><td>715500</td><td>0.002 inch, 50.8 &amp;mu;m diameter; used to make dissection pins</td></tr><tr><td>vacuum filtration unit</td><td>Sigma-Aldrich</td><td>SCGVU11RE</td><td>single use, sterile, vacuum filtration units used to sterilize extracellular solution used for electrophysiology electrode ringer</td></tr><tr><td>voltage-clamp current-clamp headstage</td><td>Molecular Devices</td><td>CV-7B</td><td>supplied with MultiClamp 700B amplifier used as left and right headstages</td></tr><tr><td>&amp;alpha;-bungarotoxin</td><td>ThermoFisher</td><td>B1601</td><td>for immobilizing the larvae prior to recording</td></tr></tbody></table>

activity of posterior lateral line afferent neurons during swimming in zebrafish

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

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.

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...

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 ...

activity-posterior-lateral-line-afferent-neurons-during-swimming

Research

JoVE Journal

Neuroscience

This video describes the rheotaxis assay in zebrafish. The method involves the measurement of orientation behavior of zebrafish in response to different water flow rates under the influence of different magnetic fields.

Zebrafish Rheotaxis Assay: A Method for Measuring Orientation Behavior in Response to Different Water Flow Rates

This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

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.

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

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

An Assay for Lateral Line Regeneration in Adult Zebrafish

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Learn patch electrophysiology to record afferent neuron activity in zebrafish larvae during swimming. This method reveals hair cell circuit properties relevant to human hearing disorders.

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Explore More Videos