Name: activity of posterior lateral line afferent neurons during swimming in zebrafish
Uploaded: 2021-02-10T15:00:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish at JoVE.com

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.

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 activity1,2,39,40,41. Without monitoring the presence of motor activity with ventral root recordings, deciphering afferent activity will likely be underestimated due to the influence of efferent inhibition during, and even after, spontaneous swimming.
In vivo electrophysiological recordings are inherently challenging. In our experience, maintaining a healthy preparation is the single greatest factor to achieving successful, long-lasting recordings for afferent neurons and ventral motor roots. To do this, it is important to not only identify and monitor fast blood flow, but also recognize the texture of the skin and underlying musculature. We recommend observing several paralyzed larvae under a microscope before further handling to become familiar with the intrinsic blood flow and skin state of healthy larvae. A successful ventral root recording through the skin requires a smooth, healthy skin surface in order for the recording electrode to generate a tight seal. This approach circumvents traditional protocols37,42 that are invasive and time-consuming, which call for dissecting away the epithelium to expose the underlying musculature. An inconvenience of recording through the skin is the potential variability in time before signals are realized. Optimizing the magnitude and duration of applied negative pressure will decrease the time required to establish a signal and potentially improve the signal-to-noise ratio. Recordings from a healthy, active preparation should yield spontaneous afferent spike rates between 5-10 Hz with fictive swim bouts occurring every few seconds.
In addition to revealing motor activity state, ventral root recordings can serve as a proxy for monitoring efferent activity that discharges parallel to motor commands to attenuate lateral line activity30,31,32 as well as activity in homologous hair cell systems (e.g., the auditory and vestibular system35,43,44,45). Efferent neurons reside deep in the hindbrain, making electrophysiological recordings of them exceedingly challenging. Zebrafish are a model genetic system, and our electrophysiology protocol can be complemented by transgenic lines to powerfully investigate aspects of corollary discharge, hair cell sensitivity, excitotoxicity, and beyond.

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

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 that best results and accurate spike detection were products of recordings that achieved a signal-to-noise ratio of at least 0.5. Custom written pre-processing scripts generate plots to assist in visualization of afferent and VR spike detection. Spontaneous afferent spikes are identified using a combination of spike parameters such as threshold, minimum duration (0.01 ms), and minimum inter-spike interval (ISI; 1 ms). Increasing negative pressure while establishing the recording often yields signal detection from multiple afferent units at once. Filtering by amplitude allows for distinguishing between signal dynamics of independent afferents. Isolating signals can be achieved by adjusting the lower-bound and upper-bound detection variables in the pre-processing script (Figure 2A). Aggressive suction to achieve multi-unit recordings can lead to unstable recordings, mechanical noise, degradation of afferent health, and ultimately a loss of signal. Therefore, it is important to slowly dial back suction to atmospheric pressure once the desired signal is achieved. Ventral root spike detection follows identical parameters to afferent spike detection but requires additional inputs to define distinct fictive swim bouts. Bursts within a motor command are defined by VR activity with a minimum of two spikes within 0.1 ms of each other and lasted a minimum of 5 ms. All swim bouts are then delineated by a minimum of three bursts with inter-burst intervals of &#60;200 ms (Figure 2B).
Afferent activity is difficult to interpret when looking at a recording in its entirety. Pre-processing scripts will overlay sections of afferent activity centered on a well-defined period of interest, in this case, the onset of a swim bout (n = 33, Figure 2C) to assist in visualizing trends in signal dynamics. Instantaneous afferent activity is calculated using a moving average filter and a 100 ms sampling window. Mean spontaneous activity shows dramatic changes in response to the onset of motor activity (Figure 2C). To better dissect and analyze afferent activity, periods before and after the swim are set to match the time interval of the corresponding swim bout. In the pre-processing script and representative analyzed results these periods are termed &#34;pre-swim&#34; and &#34;post-swim&#34;. Pre-swim, swim, and post-swim spike rates were calculated by taking the number of spikes within the respective period over its duration. The precision of estimates for each individual is partly a function of the number of swims, so we analyzed variable relationships using weighted regressions, with individual weights equal to the square root of the number of swims.
Differences in afferent spike rates across the various periods of interest (pre-swim, swim, and post-swim) were tested by a two-way analysis of variance (ANOVA). Tukey&#39;s post-hoc test detected significant differences in spike rates between swimming spike rates and spike rates of both pre-swim (8.94 &#177; 0.2 Hz, relative decrease 57%) and post-swim (5.34 &#177; 0.2 Hz, relative decrease 40%) periods. The spike rate did not immediately return to the baseline given&#160;we also found that post-swim spike rate was lower than the pre-swim spike rate (Tukey post-hoc tests across groups, p &#60; 0.001; Figure 3A). Linear models were used to detect relationships between relative spike rate and fictive swim parameters. Relative spike rate was calculated by taking the swim spike rate over the pre-swim spike rate. Fictive swim parameters included swim duration, swim frequency (i.e., number of bursts within a swim bout over the duration of the swim bout), and duty cycle (i.e., sum of the swim burst durations over the swim bout total duration). In our hands, the mean and variance of relative spike rate was correlated, so it was necessary for the data to be log transformed for analysis. Afferent spike rate was negatively correlated with swim duration meaning that the lateral line experiences&#160;greater inhibition during swims of longer duration (r2 = 0.186, F2,26 = 2.971, p = 0.045; Figure 3B). There was no correlation detected between relative spike rate and neither swim frequency nor duty cycle ((r2 = 0.099, F2,26 = 1.431, p = 0.231, and r2 = 0.047, F2,26 = 0.645, p = 0.932, respectively; Figure 3C,D). All analyses of variable relationships were weighted by the number of swims per individual and all the variables were then averaged by each individual (n = 29).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62233/62233fig01.jpg" /> 
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) recording electrodes. Scale bars represent 50 &#181;m. (B) Larval zebrafish are paralyzed and pinned in four locations (cross symbols) to a Sylgard dish for recording stability. Bold crosses represent insertion points for pins. (C) The electrophysiology rig is mounted on a vibration-isolation table and consists of an upright fixed stage microscope on a motorized controller capable of 40x magnification. Dual current clamp and voltage clamp head stages are mounted on micromanipulators. (D) The myomeres of the body musculature are separated by myosepta that serve as recording landmarks for motor neuron arborizations. The ventral motor root electrode approaches the ventral body (left) and is centered and lowered on top of a myoseptum (arrowhead). Scale bar represents 50 &#181;m. (E) Screen capture of electrophysiological recording in real-time allowing visualization of the spontaneous afferent activity (channel 1) and bursting ventral root activity indicative of fictive swim bout (channel 2). The Record and Play buttons are denoted with red arrows. (F) The posterior lateral line afferent ganglion (dashed line) lies just under the skin and can be identified by a tight cluster of afferent soma. The ganglion can be located by following the lateral line nerve past the cleithrum bone (arrowhead) to where it connects to the ganglion. Scale bar represents 30 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62233/62233fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62233/62233fig02.jpg" /> 
Figure 2: Pre-processing figure outputs visualize accurate spike detection. (A) Extracellular, loose-patch recording of posterior lateral line afferent neurons.&#160;Discrete spikes (labeled with red dots) are detected with a minimum inter-spike interval of 1 ms. Baseline noise and activity from other units are filtered out by thresholding to only include spike amplitudes within 50% of the maximum. (B) Ventral motor root recording (VR) of fictive swim bouts reveal voluntary motor commands throughout the duration of the recording. VR spikes (red dots) are detected using a similar threshold filter and then binned into a single swim bout (green) by detecting a burst of activity within 200 ms of one another (see insert; scale bar represents 200 ms). Spikes detected outside the defined swim bout do not occur within the inter-spike interval of stereotyped burst activity and are therefore excluded. (C) Mean spontaneous afferent spike rate centered on the onset of each swim bout (time = 0 s) illustrating spike rate before, during, and after swimming. Error bars represent &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/62233/62233fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62233/62233fig03.jpg" /> 
Figure 3: Quantification of afferent activity before, during, and after fictive swimming. (A) Afferent spike rate is significantly reduced during swimming and this effect persists even afterwards. Statistically similar groupings are denoted by a and b. (B) Longer swim duration is correlated to decreased afferent spike rate. (C-D) Swim frequency and swim duty cycle show no correlation to afferent spike rate. All values represent mean &#177; SEM. Outlying individuals with low statistical weight were omitted. <a href="https://www.jove.com/files/ftp_upload/62233/62233fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish at JoVE.com

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.7K Views.  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.

Video: Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

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

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

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.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Developmental Biology

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

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

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.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

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

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

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

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, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

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.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Biology

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

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.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

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.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

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

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

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

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...

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

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

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.

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) ...

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

Source: Cresci, A., et al. <a href="https://www.jove.com/v/59229/" target="_blank">Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish</a>.&#160;J. Vis. Exp.&#160;(2019)
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.

Source: Cresci, A., et al. Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish.&#160;J. Vis. Exp.&#160;(2019)
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Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Summary

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Chapters in this video

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

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

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

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

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

An Assay for Lateral Line Regeneration in Adult Zebrafish

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

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

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

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