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>
	<li>Russell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+signals+in+vivo:+a+powerful+tool+in+physiology+and+pharmacology.">Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology.</a> British Journal of Pharmacology. 163 (8), 1605-1625 (2011).</li><li>P&#233;rez Koldenkova, V., Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+Properties+and+evaluation.">Genetically encoded Ca2+ indicators: Properties and evaluation.</a> Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1833 (7), 1787-1797 (2013).</li><li>Lin, M. Z., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+indicators+of+neuronal+activity.">Genetically encoded indicators of neuronal activity.</a> Nature Neuroscience. 19 (9), 1142-1153 (2016).</li><li>Tian, L., Hires, S. A., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Activity+with+Genetically+Encoded+Calcium+Indicators.">Imaging Neuronal Activity with Genetically Encoded Calcium Indicators.</a> Cold Spring Harbor Protocols. 2012 (6), (2012).</li><li>Zhang, Q., 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=Synaptically+silent+sensory+hair+cells+in+zebrafish+are+recruited+after+damage.">Synaptically silent sensory hair cells in zebrafish are recruited after damage.</a> Nature Communications. 9 (1), 1388 (2018).</li><li>Harris, G. G., Frishkopf, L. S., Flock, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+potentials+from+hair+cells+of+the+lateral+line.">Receptor potentials from hair cells of the lateral line.</a> Science. 167 (3914), 76-79 (1970).</li><li>Eatock, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+Hair+Cells:+Modern+and+Historic+Perspectives.">Vertebrate Hair Cells: Modern and Historic Perspectives.</a> Vertebrate Hair Cells. , 1-19 (2006).</li><li>Tang, L. S., Montemayor, C., Pereira, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorineural+hearing+loss:+potential+therapies+and+gene+targets+for+drug+development.">Sensorineural hearing loss: potential therapies and gene targets for drug development.</a> IUBMB Life. 58 (9), 525-530 (2006).</li><li>Assad, J. A., Shepherd, G. M. G., Corey, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip-link+integrity+and+mechanical+transduction+in+vertebrate+hair+cells.">Tip-link integrity and mechanical transduction in vertebrate hair cells.</a> Neuron. 7 (6), 985-994 (1991).</li><li>Lumpkin, E. A., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Ca2++entry+through+mechanosensitive+channels+localizes+the+site+of+mechanoelectrical+transduction+in+hair+cells.">Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.</a> Proceedings of the National Academy of Sciences. 92 (22), 10297-10301 (1995).</li><li>Ricci, A. J., Wu, Y. -. C., Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endogenous+Calcium+Buffer+and+the+Time+Course+of+Transducer+Adaptation+in+Auditory+Hair+Cells.">The Endogenous Calcium Buffer and the Time Course of Transducer Adaptation in Auditory Hair Cells.</a> Journal of Neuroscience. 18 (20), 8261-8277 (1998).</li><li>Moser, T., Beutner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+exocytosis+and+endocytosis+at+the+cochlear+inner+hair+cell+afferent+synapse+of+the+mouse.">Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (2), 883-888 (2000).</li><li>Brandt, A., Khimich, D., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Few+CaV1.3+channels+regulate+the+exocytosis+of+a+synaptic+vesicle+at+the+hair+cell+ribbon+synapse.">Few CaV1.3 channels regulate the exocytosis of a synaptic vesicle at the hair cell ribbon synapse.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 25 (50), 11577-11585 (2005).</li><li>Olt, J., Allen, C. E., Marcotti, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+physiological+recording+from+the+lateral+line+of+juvenile+zebrafish.">In vivo physiological recording from the lateral line of juvenile zebrafish.</a> The Journal of Physiology. 594 (19), 5427-5438 (2016).</li><li>Olt, J., Johnson, S. L., Marcotti, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+biophysical+properties+of+hair+cells+from+the+lateral+line+and+inner+ear+of+developing+and+adult+zebrafish.">In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.</a> The Journal of Physiology. 592 (10), 2041-2058 (2014).</li><li>Griguer, C., 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=Voltage-dependent+potassium+currents+in+cochlear+hair+cells+of+the+embryonic+chick.">Voltage-dependent potassium currents in cochlear hair cells of the embryonic chick.</a> Journal of Neurophysiology. 75 (1), 508-513 (1996).</li><li>Art, J. J., Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+membrane+properties+in+hair+cells+isolated+from+the+turtle+cochlea.">Variation of membrane properties in hair cells isolated from the turtle cochlea.</a> The Journal of Physiology. 385, 207-242 (1987).</li><li>Goutman, J. D., Pyott, S. 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. G., Nicolson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8+-+Physiological+Recordings+from+Zebrafish+Lateral-Line+Hair+Cells+and+Afferent+Neurons.">Chapter 8 - Physiological Recordings from Zebrafish Lateral-Line Hair Cells and Afferent Neurons.</a> Methods in Cell Biology. 100, 219-231 (2010).</li><li>Olt, J., Ordoobadi, A. J., Marcotti, W., Trapani, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+recordings+from+the+zebrafish+lateral+line.">Physiological recordings from the zebrafish lateral line.</a> Methods in Cell Biology. 133, 253-279 (2016).</li><li>Stawicki, T. M., Esterberg, R., Hailey, D. W., Raible, D. W., Rubel, E. 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. M., 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 236 (11), 3088-3099 (2007).</li><li>Zhang, Q. X., He, X. J., Wong, H. C., Kindt, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10+-+Functional+calcium+imaging+in+zebrafish+lateral-line+hair+cells.">Chapter 10 - Functional calcium imaging in zebrafish lateral-line hair cells.</a> Methods in Cell Biology. 133, 229-252 (2016).</li><li>Sheets, L., 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=Enlargement+of+Ribbons+in+Zebrafish+Hair+Cells+Increases+Calcium+Currents+But+Disrupts+Afferent+Spontaneous+Activity+and+Timing+of+Stimulus+Onset.">Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 37 (26), 6299-6313 (2017).</li><li>Chou, S. -. W., 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=A+molecular+basis+for+water+motion+detection+by+the+mechanosensory+lateral+line+of+zebrafish.">A molecular basis for water motion detection by the mechanosensory lateral line of zebrafish.</a> Nature Communications. 8 (1), 2234 (2017).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. 7 (1), 27-41 (1998).</li><li>. Fiji is just ImageJ Available from: <a target="_blank" href="https://fiji.sc/">https://fiji.sc/</a> (2018)</li><li>Esterberg, R., Hailey, D. W., Coffin, A. B., Raible, D. W., Rubel, E. 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. C., 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=LITE+microscopy:+Tilted+light-sheet+excitation+of+model+organisms+offers+high+resolution+and+low+photobleaching.">LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching.</a> Journal of Cell Biology. , (2018).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:1155px;" width="1155">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:224px;">Section 1</td>
			<td style="width:229px;"></td>
			<td style="width:239px;"></td>
			<td style="width:463px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Bungarotoxin</td>
			<td>R&#38;D Systems</td>
			<td>2133</td>
			<td>For paralyzing larvae prior to imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red Solution 0.5%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td>For visualization of &#945;-bungarotoxin solution during heart injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate (MESAB, MS-222, tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>For anesthetizing larvae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Section 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging chamber</td>
			<td>Siskiyou</td>
			<td>PC-R</td>
			<td>Platform to mount larvae for imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No. 1.5 Coverslips square, 22*22 mm</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td>To seal imaging chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum silicone grease</td>
			<td>Fischer Scientific</td>
			<td>14-635-5D&#160;</td>
			<td>For affixing of coverslip to imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven</td>
			<td>Techne</td>
			<td>HB-1D</td>
			<td>For drying silicone encapsulant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors&#160;</td>
			<td>Cole-Palmer</td>
			<td>5.5&#34;, EW-10818-00</td>
			<td>For cutting tunsgten wire to make pins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten wire, 0.035 mm</td>
			<td>Goodfellow</td>
			<td>W005131</td>
			<td>For head pins to immobilize larvae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten wire, 0.025 mm</td>
			<td>ThermoFischer Scientific</td>
			<td>AA10405-H4</td>
			<td>For tail pins to immobilize larvae</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Section 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillaries w/ filament</td>
			<td>Sutter Instrument Company</td>
			<td>BF 150-86-10</td>
			<td>Glass to be pulled into &#945;-bungarotoxin injection needles for heart injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Ceramic tile</td>
			<td>Sutter Instrument Company</td>
			<td>NC9569052</td>
			<td>For scoring and evening breaking fluid-jet needles&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sections 4 &#38; 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Gel loading tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For backfilling heart injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Glass capillary/needle holder</td>
			<td>WPI</td>
			<td>MPH315</td>
			<td>To hold fluid-jet needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td>For holding and positioning of needle holder to inject &#945;-bungarotoxin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td>For holding manual micromanipulator for &#945;-bungarotoxin injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure injector</td>
			<td>Eppendorf</td>
			<td>Femtojet 4x</td>
			<td>To deliver &#945;-bungarotoxin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sections 6, 7 &#38; 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Bruker</td>
			<td>Swept field/Opterra confocal microscope</td>
			<td>Fixed, upright microscope with DIC optics and 488nm laser with appropriate filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">10X air objective</td>
			<td>Nikon</td>
			<td>MRH00101</td>
			<td>Low magnification for positioning of larvae and fluid jet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">EMCCD camera&#160;</td>
			<td>QImaging</td>
			<td>Rolera EM-C2 EMCCD camera&#160;</td>
			<td>Camera with small pixel size that can acquire up to 100 frames per s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Circular chamber adapter</td>
			<td>Siskiyou</td>
			<td>PC-A</td>
			<td>For holding and rotating imaging chamber on microscope stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Masterflex Peroxide-cured silicone tubing (1 ft)</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 height="21">
			<td height="21" style="height:21px;">Motorized micromanipulator</td>
			<td>Sutter Instrument Company</td>
			<td>MP-225</td>
			<td>For holding and positioning of needle holder for fluid jet&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator controller</td>
			<td>Sutter Instrument Company</td>
			<td>MPC-200</td>
			<td>For controlling fluid-jet needle manipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel loading tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For backfilling fluid-jet needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillary/needle holder</td>
			<td>WPI</td>
			<td>MPH315</td>
			<td>To hold fluid-jet needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PSI manometer</td>
			<td>Sper Scientific</td>
			<td>840081</td>
			<td>For measuring pressure clamp output</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Section 9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>Solvent for pharmacological compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Section 10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism7</td>
			<td>Graphpad</td>
			<td>Prism7</td>
			<td>Software to plot GCaMP6 intensity changes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FIJI</td>
			<td>Schindelin, et., al.34</td>
			<td>https://fiji.sc/</td>
			<td>Software to process images and create spatio-temporal signal maps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Turboreg Plugin</td>
			<td>Th&#233;venaz et., al.29&#160;</td>
			<td>http://bigwww.epfl.ch/thevenaz/turboreg/</td>
			<td>Plugin to register GCaMP6 image sequences in FIJI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StackReg Plugin</td>
			<td>Th&#233;venaz et., al.29</td>
			<td>http://bigwww.epfl.ch/thevenaz/stackreg/</td>
			<td>Plugin to register GCaMP6 image sequences in FIJI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

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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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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 ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...