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Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously [1][2][3][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. Video LinkThe video component of this article can be found at https://www.jove.com/video/4281/ Protocol 1. Pre-experimental Preparations 1. Prepare six solutions (see Table 1 for compositions): (a) 100 ml NZR (normal zebrafish Ringer's), (b) 50 ml NZR + Tricaine (MS222), (c) 10 ml NZR +BSA; (d) 100 ml LoCaS (low Ca 2+ solution), (e) 10 ml LoCaS + papain, (f) 100 ml K + -internal solution. Solutions (a), (b), (d) and (f) can be stored for up to one month at 4 °C. Solutions (c) and (e) should be prepared the day of experimentation. All solutions should be at room temperature before beginning experiments. 2. Label and fill four 35 mm Petri dishes approximately halfway with solutions (a), (c), (d) and (e). 3. Make a dissecting dish by filling a 60 mm Petri dish with Sylgard (Dow Corning, Midland, MI) and then cutting a cavity into it that will allow a fish to sit upright without tipping over. 4. Prepare at least two cell isolation tools by gluing the follicle end of a dog hair to the end of a glass Pasteur pipette with superglue, allowing the hair to extend past the end of the pipette by approximately 0.5 cm. The hairs from soft-furred dogs like Labrador Retrievers and Weimaraners work well. Isolation of Auditory and Vestibular Labyrinth1. Sacrifice one adult zebrafish by immersion in a beaker containing NZR + Tricaine. Visually monitor the gills until all opercular movements have ceased. Wait an additional ten minutes before removing fish and rinsing with fresh NZR. These procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) of Pepperdine University but approval should be obtained from your own institution. 2. Pin the fish to the dissecting dish dorsal side up by inserting one standard sewing straight pin (approximately 2.5 cm long) through each eye socket and a third pin through the dorsal-ventral axis about one-third to one-half of the distance from head to tail as illustrated in Figure 1A. 3. Under a zoom (0.62-5x) stereo dissecting microscope fitted with 10x eyepieces, use spring scissor...

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