This work was supported by NIH/NIDCD intramural research funds 1ZIADC000085-01 (K.S.K.). We would like to acknowledge Candy Wong for her assistance in writing the Fiji macro. We would also like to thank Doris Wu and Candy Wong for their helpful suggestions with the protocol.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Cell+Patch-Clamp+Recording+of+Mouse+and+Rat+Inner+Hair+Cells+in+the+Intact+Organ+of+Corti.">Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.</a> Methods in Molecular Biology. 1427, 471-485 (2016).</li><li>Einarsson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recordings+in+Inner+Ear+Hair+Cells+Isolated+from+Zebrafish.">Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish.</a> Journal of Visualized Experiments. (68), (2012).</li><li>Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+and+intensity+coding+at+the+hair+cell's+ribbon+synapse.">Time and intensity coding at the hair cell's ribbon synapse.</a> The Journal of Physiology. 566, 7-12 (2005).</li><li>Moser, T., Brandt, A., Lysakowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+ribbon+synapses.">Hair cell ribbon synapses.</a> Cell and Tissue Research. 326 (2), 347-359 (2006).</li><li>Trapani, J. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+lateral+line+to+uncover+novel+mechanisms+of+action+and+prevention+in+drug-induced+hair+cell+death.">Using the zebrafish lateral line to uncover novel mechanisms of action and prevention in drug-induced hair cell death.</a> Frontiers in Cellular Neuroscience. 9, (2015).</li><li>Spinelli, K. J., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Intracellular+Calcium+Ion+Dynamics+in+Hair+Cell+Populations+with+Fluo-4+AM.">Monitoring Intracellular Calcium Ion Dynamics in Hair Cell Populations with Fluo-4 AM.</a> PLOS ONE. 7 (12), 51874 (2012).</li><li>Kwan, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+intracellular+calcium+regulation+is+integral+to+aminoglycoside-induced+hair+cell+death.">Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (17), 7513-7525 (2013).</li><li>Stengel, D., Zindler, F., Braunbeck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+to+assess+ototoxic+effects+in+the+lateral+line+of+zebrafish+(Danio+rerio)+embryos.">An optimized method to assess ototoxic effects in the lateral line of zebrafish (Danio rerio) embryos.</a> Comparative Biochemistry and Physiology Part C: Toxicology &amp; Pharmacology. 193, 18-29 (2017).</li><li>Fadero, T. 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The authors have nothing to disclose.

In vivo imaging in intact animals is inherently challenging. Several steps in this method are critical to obtaining reliable in vivo calcium measurements from lateral-line hair cells. For example, it is very important that the larva is pinned and paralyzed properly before imaging to minimize movement during imaging. Excess movement during imaging can lead to changes in GCaMP6s fluorescence that are not true signals and do not correspond to changes in calcium levels (e.g., Figures 4B1&#39;-B1&#39;&#39; and 4C1&#39;-C1&#39;&#39;). Tail pins can be placed more anteriorly to help minimize movement, though this may render more posterior neuromasts inaccessible. Furthermore, after heart injection, head pins can be rotated so that the horizontal portion of the pin lies across the yolk. In addition to altering the position of pins, it is also possible to use a brain-slice harp instead of pins to immobilize larvae34. When placed over the larvae properly, a harp is an additional, potentially less invasive method of immobilizing larvae. While significant movement can result from inadequate pinning, failure to properly perform the heart injection to deliver &#945;-bungarotoxin and paralyze larvae can result in incomplete paralysis, movement, and ultimately motion artifacts. Although commonly used anesthetics have been shown to affect the excitability of zebrafish hair cells, recent work has shown that the anesthetic benzocaine does not interfere with many aspects of hair-cell activity. Similarly, the more commonly used anesthetic MS-222 only interferes with certain aspects of hair-cell activity15. Therefore, due to the challenging nature of &#945;-bungarotoxin injection, benzocaine or MS-222 application may prove to be a useful alternative method of paralysis to prevent movement in the larva during functional calcium imaging.
In addition to the technical challenges involved in this protocol, even a perfectly mounted sample is useless if the larva and hair cells are not healthy prior to and during each imaging experiment. To ensure that larvae and hair cells are healthy, it is important that larvae are maintained in E3 buffer that is free of debris such as chorions (egg shells), waste, and microorganisms. Although the superficial location of lateral-line hair cells is advantageous for imaging, this location makes them more vulnerable to cellular damage when the E3 buffer is fouled. A clean, aqueous environment is particularly important for young larvae (2-4 dpf) or mutants that cannot maintain an upright swimming position and primarily lie on the bottom of the Petri dish. In these situations, lateral-line hair cells and the protective cupula surrounding the hair bundles can easily become compromised. Even when starting with healthy larvae and hair cells, throughout the course of each experiment, it is critical to ensure that the larva has a heartbeat and rapid blood flow. If blood flow slows or stops, the health of the hair cells can become compromised. In compromised preparations involving unhealthy larvae or loss of blood flow, dying hair cells can be identified several ways: first, by the appearance of karyopyknosis or nuclear condensation, which manifests as a bubble within the cell under DIC optics; second, by cell shrinkage and the presence of rapidly moving particles within the cytoplasm; and third, when kinocilia tips splay out in different directions35. When hair bundles are disrupted, the splayed kinocilia do not move cohesively together during stimulation.
This preparation has several minor limitations, one being that the preparation only remains robust for 1-3 hours after it is established. Modifications such as using smaller pins or a brain-slice harp to immobilize larvae and adding a perfusion system may extend the lifetime of this in vivo preparation. Another limitation is that photobleaching and phototoxicity can occur after repeated imaging trials. One exciting way to overcome this challenge is to adapt this protocol for light-sheet microscopy. Light-sheet microscopy is a powerful way to reduce out of focus light, leading to less photobleaching and phototoxicity36. Together, gentler immobilization and less photo-exposure may help prolong each imaging session. Longer imaging sessions can be used to examine the full duration of functional changes accompanying development and the processes underlying hair-cell clearance and regeneration after injury. It is important to point out that, in addition to light-sheet microscopy, this protocol can be adapted to other types confocal systems (point-scanning, 2-photon, and spinning disk) as well as relatively simple widefield systems28,34. Overall, its versatility makes this protocol a valuable tool that can be adapted and used with multiple imaging systems.
While this protocol can be adapted and used with many imaging systems, parts of this protocol can also be adapted and used (1) with other indicators besides GCaMP6s and (2) to image activity in other sensory cells and neurons within larval zebrafish. For example, in a previous study, we used this protocol to image activity using multiple genetically encoded indicators within lateral-line hair cells to detect cytosolic calcium (RGECO1), vesicle fusion (SypHy), membrane voltage (Bongwoori), and membrane calcium (jRCaMP1a-caax and GCaMP6s-caax), and within lateral-line afferent processes to detect membrane calcium (GCaMP6s-caax)5. Based on our experience using these indicators, the transgenic line Tg(myo6b:GCaMP6s-caax) described in this protocol offers an excellent start for imaging activity in lateral-line neuromasts. Of all the indicators listed above, we have found that GCaMP6s is the most sensitive and photostable. In addition to these features, we highlight the Tg(myo6b:GCaMP6s-caax) transgenic line because it can be used to make two distinct measurements: hair-cell mechanosensation and presynaptic calcium within a single transgenic line.
The technique outlined in this article demonstrates how calcium imaging in the zebrafish lateral line can be a powerful method to study how hair cells function in their native environment. This approach is complementary to studies of mammals in which hair-cell function is currently being studied in ex vivo explants. In addition, the zebrafish model can continue to be used as a platform to test the efficacy of genetically encoded indicators that can then be applied to examine activity in mammalian hair cells.

Functional calcium imaging is a powerful tool that can be used to monitor the activity of many cells simultaneously1. In particular, calcium imaging using genetically encoded calcium indicators (GECIs) has been shown to be advantageous because GECIs can be expressed in specific cell types and localized subcellularly2. In neuroscience research, these features have made calcium imaging using GECIs a powerful method to both define activity patterns within neuronal networks and measure calcium influx at individual synapses3,4. Taking advantage of these features, a recent study used confocal microscopy and GECIs to monitor subcellular activity within collections of sensory hair cells5.
Hair cells are the mechanoreceptors that detect sound and vestibular stimuli in the inner ear and local water movement in the lateral-line system in aquatic vetebrates6,7. Hair cells are often the target of damage or genetic mutations that result in the most common form of hearing loss in humans known as sensorineural hearing loss8,9. Therefore, it is critical to understand how these cells function in order to understand how to treat and prevent hearing loss. To properly function, hair cells utilize two specialized structures called mechanosensory-hair bundles and synaptic ribbons to detect and transmit stimuli, respectively. Hair bundles are located at the apex of hair cells and are made up primarily of fine, hair-like protrusions known as stereocilia (Figure 1A). In vestibular and lateral-line hair cells, each hair bundle also has a single long kinocilium (the cell&#39;s only true cilium), which can extend far above the stereocilia (Figure 1A). Mechanosensory stimuli deflect hair bundles, and deflection puts tension on linkages called &#34;tip-links&#34; that interconnect stereocilia10. This tension opens mechanotransduction (MET) channels located in the stereocilia, resulting in an apical influx of cations, including calcium11,12. This apical activity ultimately depolarizes the hair cell and opens voltage-gated calcium channels (Cav1.3) at the base of the cell. Cav1.3 channels are found adjacent to synaptic ribbons, a presynaptic structure that tethers vesicles at active zones. Basal calcium influx through Cav1.3 channels is required for vesicle fusion, neurotransmission, and activation of afferent neurons13,14.
For many years, electrophysiological techniques such as whole-cell patch clamping have been used to probe the functional properties of hair cells in many species, including zebrafish15,16,17,18,19,20. These electrophysiological recordings have been particularly valuable in the hearing and balance fields because they can be used to obtain extremely sensitive measurements from individual sensory cells, whose purpose is to encode extremely fast stimuli over a wide range of frequencies and intensities21,22. Unfortunately, whole-cell recordings cannot measure the activity of populations of hair cells. To study the activity of populations of cells in the zebrafish lateral-line, microphonic potentials and afferent action potentials have been used to measure the summed mechanosensitive and postsynaptic response properties of individual neuromasts23,24. Unfortunately, neither whole-cell recordings nor local field potential measurements have the spatial resolution to pinpoint where activity is occurring within individual cells or measure the activity of each cell within a population. More recently, calcium dyes and GECIs have been employed to bypass these challenges25,26.
In zebrafish, GECIs have proven to be a powerful approach to examining hair-cell function due to the relative ease of creating transgenic zebrafish and the optical clarity of larvae27. In zebrafish larvae, hair cells are present in the inner ear as well as the lateral-line system. The lateral line is made up of rosette-like clusters of hair cells called neuromasts that are used to detect local changes in water movement (Figure 1). The lateral line is particularly useful because it is located externally along the surface of the fish. This access has made it possible to stimulate hair cells and measure calcium signals optically in intact larvae. Overall, the ease of transgenesis, transparency of the larvae, and the unparalleled access of lateral-line hair cells have made zebrafish an invaluable model to study the activity of hair cells in vivo. This is a significant advantage compared to mammalian systems in which hair cells are surrounded by bony structures of the inner ear. This lack of access has made it very difficult to acquire functional in vivo measurements of mammalian hair cells.
The protocol outlined here describes how to monitor MET channel- and presynapse-dependent changes in calcium within individual hair cells and among cells within neuromasts in larval zebrafish. This protocol utilizes an established transgenic zebrafish line that expresses a membrane-localized GCaMP6s under the control of the hair-cell specific myosin6b promoter28. This membrane localization positions GCaMP6s to detect calcium influx through ion channels located in the plasma membrane that are critical for hair-cell function. For example, membrane-localized GCaMP6s can detect calcium influx through MET channels in apical hair bundles and through CaV1.3 channels near synaptic ribbons at the base of the cell. This contrasts with using GECIs localized in the cytosol, as cytosolic GECIs detect calcium signals that are a combination of MET and CaV1.3 channel activity as well as calcium contributions from other sources (e.g., store release). This protocol outlines how to immobilize and paralyze GCaMP6s transgenic larvae prior to imaging. It then describes how to prepare and use a fluid-jet to deflect the hair bundles to stimulate lateral-line hair cells in a controlled and reproducible manner. Representative data that can be achieved using this protocol are presented. Examples of data that represent movement artifacts are also presented. Control experiments that are used to verify results and exclude artifacts are described. Lastly, a method to visualize spatial calcium signals in the Fiji software is described. This Fiji analysis is adapted from previously established visualization methods developed using MATLAB5. Overall, this protocol outlines a powerful preparation technique that uses GECIs in larval zebrafish to measure and visualize hair-cell calcium dynamics in vivo.

<table><tbody><tr><td>&lt;strong&gt;Section 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin</td><td>R&amp;amp;D Systems</td><td>2133</td><td>For paralyzing larvae prior to imaging</td></tr><tr><td>Phenol red Solution 0.5%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P0290</td><td>For visualization of &amp;alpha;-bungarotoxin solution during heart injection</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (MESAB, MS-222, tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td>For anesthetizing larvae</td></tr><tr><td>&lt;strong&gt;Section 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imaging chamber</td><td>Siskiyou</td><td>PC-R</td><td>Platform to mount larvae for imaging</td></tr><tr><td>No. 1.5 Coverslips square, 22*22 mm</td><td>VWR</td><td>48366-227</td><td>To seal imaging chamber&amp;nbsp;</td></tr><tr><td>High vacuum silicone grease</td><td>Fischer Scientific</td><td>14-635-5D&amp;nbsp;</td><td>For affixing of coverslip to imaging chamber</td></tr><tr><td>Silicone encapsulant clear 0.5 g kit</td><td>Ellsworth Adhesives</td><td>Dow Corning Sylgard, 184 SIL ELAST KIT 0.5KG</td><td>To fill imaging chamber to create a surface to pin fish&amp;nbsp;</td></tr><tr><td>Oven</td><td>Techne</td><td>HB-1D</td><td>For drying silicone encapsulant</td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss Microscopy</td><td>Stemi 2000 with transmitted light illumination</td><td>For illuminating wire, forceps and scissors to make pins</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>For making pins&amp;nbsp;</td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Cole-Palmer</td><td>5.5&quot;, EW-10818-00</td><td>For cutting tunsgten wire to make pins</td></tr><tr><td>Tungsten wire, 0.035 mm</td><td>Goodfellow</td><td>W005131</td><td>For head pins to immobilize larvae</td></tr><tr><td>Tungsten wire, 0.025 mm</td><td>ThermoFischer Scientific</td><td>AA10405-H4</td><td>For tail pins to immobilize larvae</td></tr><tr><td>&lt;strong&gt;Section 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micropipette guller</td><td>Sutter Instrument Company</td><td>P-97</td><td>For pulling capillary glass for fluid-jet and heart injection needles</td></tr><tr><td>Borosilicate glass capillaries w/ filament</td><td>Sutter Instrument Company</td><td>BF 150-86-10</td><td>Glass to be pulled into &amp;alpha;-bungarotoxin injection needles for heart injection</td></tr><tr><td>Borosilicate glass capillaries w/o filament</td><td>Sutter Instrument Company</td><td>B 150-86-10</td><td>Glass to be pulled into fluid-jet needles to stimulate hair cells</td></tr><tr><td>Pipette polisher</td><td>Narishige</td><td>MF-830 microforge</td><td>To polish fluid-jet pipette tips to smooth jagged breaks</td></tr><tr><td>Ceramic tile</td><td>Sutter Instrument Company</td><td>NC9569052</td><td>For scoring and evening breaking fluid-jet needles&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Sections 4 &amp;amp; 5&lt;/strong&gt;</td><td></td><td></td><td></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>For pinning larvae</td></tr><tr><td>Gel loading tips</td><td>Eppendorf</td><td>5242956003</td><td>For backfilling heart injection needles</td></tr><tr><td>Stereomicroscope</td><td>Carl Zeiss Microscopy</td><td>Stemi 2000 with transmitted light illumination</td><td>For illuminating larvae during pinning and heart injection</td></tr><tr><td>Glass capillary/needle holder</td><td>WPI</td><td>MPH315</td><td>To hold fluid-jet needles</td></tr><tr><td>Manual micromanipulator</td><td>Narishige</td><td>M-152</td><td>For holding and positioning of needle holder to inject &amp;alpha;-bungarotoxin&amp;nbsp;</td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1</td><td>For holding manual micromanipulator for &amp;alpha;-bungarotoxin injection</td></tr><tr><td>Pressure injector</td><td>Eppendorf</td><td>Femtojet 4x</td><td>To deliver &amp;alpha;-bungarotoxin</td></tr><tr><td>&lt;strong&gt;Sections 6, 7 &amp;amp; 8&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>Bruker</td><td>Swept field/Opterra confocal microscope</td><td>Fixed, upright microscope with DIC optics and 488 nm laser with appropriate filters</td></tr><tr><td>Microscope software</td><td>Bruker</td><td>Prairieview 5.3</td><td>To coordinate and control the microscope, lasers, stage, piezo-z, cameras and fluid jet</td></tr><tr><td>10x air objective</td><td>Nikon</td><td>MRH00101</td><td>Low magnification for positioning of larvae and fluid jet</td></tr><tr><td>60x water objective</td><td>Nikon</td><td>MRF07620</td><td>Water immerison objective with high NA (1.0) and adequate working distance (2.0 mm)</td></tr><tr><td>Piezo-Z objective scanner with controller/driver</td><td>Physik Intruments instruments/Bruker</td><td>01144210/UM-Z-PZ</td><td>High-speed z-stack acquisition with 0.025um accuracy</td></tr><tr><td>EMCCD camera&amp;nbsp;</td><td>QImaging</td><td>Rolera EM-C2 EMCCD camera&amp;nbsp;</td><td>Camera with small pixel size that can acquire up to 100 frames per s</td></tr><tr><td>Circular chamber adapter</td><td>Siskiyou</td><td>PC-A</td><td>For holding and rotating imaging chamber on microscope stage</td></tr><tr><td>Motorized Z-deck stage</td><td>Prior Scientific</td><td>ZDN12MP</td><td>Microscope stage that can hold and move sample and micromanipulator with fluid-jet needle together</td></tr><tr><td>Z-deck stage insert adaptor</td><td>NIH Machine shop</td><td>custom</td><td>To fit the circular chamber adaptor onto the z-deck stage</td></tr><tr><td>Fluid-jet apparatus</td><td>ALA scientific instruments</td><td>HSPC-1 High-speed pressure Clamp with PV-PUMP</td><td>For controlling and delivering the fluid-jet stimulus</td></tr><tr><td>Masterflex Peroxide-cured silicone tubing (1 feet)</td><td>Cole-Palmer</td><td>Masterflex L/S 13, 96400-13</td><td>For connecting the fluid-jet needle holder to pressure pump</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter Instrument Company</td><td>MP-225</td><td>For holding and positioning of needle holder for fluid jet&amp;nbsp;</td></tr><tr><td>Micromanipulator controller</td><td>Sutter Instrument Company</td><td>MPC-200</td><td>For controlling fluid-jet needle manipulator</td></tr><tr><td>Gel loading tips</td><td>Eppendorf</td><td>5242956003</td><td>For backfilling fluid-jet needles</td></tr><tr><td>Glass capillary/needle holder</td><td>WPI</td><td>MPH315</td><td>To hold fluid-jet needles</td></tr><tr><td>PSI manometer</td><td>Sper Scientific</td><td>840081</td><td>For measuring pressure clamp output</td></tr><tr><td>&lt;strong&gt;Section 9&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BAPTA, Tetrapotassium Salt, cell impermeant</td><td>ThermoFischer Scientific</td><td>B1204</td><td>To cleave tips links, uncouple MET channels and block all evoked GCaMP6s signals</td></tr><tr><td>Isradipine</td><td>Sigma-Aldrich</td><td>I6658</td><td>To block signals dependent on the L-type calcium channels (CaV1.3) at the presynapse</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>Solvent for pharmacological compounds</td></tr><tr><td>Section 10</td><td></td><td></td><td></td></tr><tr><td>Prism7</td><td>Graphpad</td><td>Prism7</td><td>Software to plot GCaMP6 intensity changes</td></tr><tr><td>FIJI</td><td>Schindelin, et., al.&lt;sup&gt;34&lt;/sup&gt;</td><td>https://fiji.sc/</td><td>Software to process images and create spatio-temporal signal maps</td></tr><tr><td>Turboreg Plugin</td><td>Th&amp;eacute;venaz et., al.&lt;sup&gt;29&lt;/sup&gt;&amp;nbsp;</td><td>http://bigwww.epfl.ch/thevenaz/turboreg/</td><td>Plugin to register GCaMP6 image sequences in FIJI</td></tr><tr><td>StackReg Plugin</td><td>Th&amp;eacute;venaz et., al.&lt;sup&gt;29&lt;/sup&gt;</td><td>http://bigwww.epfl.ch/thevenaz/stackreg/</td><td>Plugin to register GCaMP6 image sequences in FIJI</td></tr><tr><td>Times Series Analyzer V3 Plugin</td><td>Balaji J 2007, Dept. of Neurobiology, UCLA</td><td>https://imagej.nih.gov/ij/plugins/time-series.html</td><td>Plugin to create multiple ROIs to measure GCaMP6 intensity changes</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish at JoVE.com

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

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 ...

in-vivo-calcium-imaging-of-lateral-line-hair-cells-in-larval-zebrafish

Research

JoVE Journal

Neuroscience

11.3K Views.  NIDCD/National Institutes of Health. 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.

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

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Developmental Biology

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

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 ...

Biology

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

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.

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 ...

In Vivo Whole-Brain Imaging of Zebrafish Larvae

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

Author Spotlight: In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy  

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

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 ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://app.jove.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...