摘要 5
Abstract 6
Introduction 7
Zebrafish model 7
Ammonia excretion in freshwater fish 8
Ammonia toxicity 8
Lateral line system in fish 10
Ototoxic drugs and number of hair cells 11
SIET 12
Purpose 14
Experimental Designs 15
Materials and Methods 16
Experimental animals 16
Preparation of solutions 16
Zebrafish larvae acclimation 17
Rheotaxis behavior assay 18
Swimming activity assay 18
Immunostaining of lateral line hair cells 18
FM1-43 staining of lateral line hair cells 19
Fluorescent imaging and hair cell number counting 19
Measurement of Ca2+ and NH4+ flux on L1 neuromast 19
Statistical analysis 20
Results 21
Establishment of rheotaxis assay and swimming activity assay 21
Effects of NH4+ on rheotaxis time and swimming activity of zebrafish larvae 21
Effects of NH4+ on the number of lateral line hair cells in zebrafish larvae 22
Effects of NH4+ on Ca2+ influx at hair cells in L1 neuromast of zebrafish larvae 22
Effects of environmental high Ca2+ concentration on Ca2+ and NH4+ influx of NH4+-exposed hair cells 23
Protective effect of environmental high Ca2+ on rheotaxis behavior of NH4+-exposed zebrafish larvae 24
Effects of long-term exposure of NH4+ on the number of lateral line hair cells 24
Effects of long-term exposure of NH4+ on Ca2+ influx and NH4+ influx of L1 neuromast 24
Discussion 26
Ammonia toxicity in fish 26
Deleterious effects of NH4+ on rheotaxis behavior 26
Effects of NH4+ on the number of hair cell per neuromast 28
Effects of NH4+ on Ion flux at L1 lateral line hair cells 28
MET channel blockers 29
Protection of environmental high Ca2+ on Ca2+ influx of lateral line hair cells and rheotaxis behavior in zebrafish larvae 30
Long-term effects of NH4+ on lateral line hair cells 30
Sensitivity of SIET and FM1-43 in describing functional changes in hair cells 31
References 33
Figures 39
Fig. 1. Device for analyzing rheotaxis behavior. 39
Fig. 2. Effects of drugs on the rheotaxis behavior of zebrafish larvae. 40
Fig. 3. Effects of neomycin on the swimming velocity of zebrafish larvae. 41
Fig. 4. Effects of NH4+ on rheotaxis time and swimming velocity of zebrafish larvae. 42
Fig. 5. Effects of NH4+ on the number of lateral line hair cells in zebrafish larvae. 43
Fig. 6. Effects of NH4+ on Ca2+ and NH4+ influx at hair cells in L1 neuromast of zebrafish larvae. 45
Fig. 7. Effects of MET channel blockers (neomycin and La3+) on NH4+ influx at hair cells in L1 neuromast of zebrafish larvae. 46
Fig. 8. Effects of environmental high Ca2+ on Ca2+ influx and NH4+ influxes at hair cells in L1 neuromast of zebrafish larvae. 47
Fig. 9. Effect of environmental high Ca2+ on rheotaxis time of NH4+-exposed zebrafish larvae. 48
Fig. 10. Effects of long-term exposure of NH4+ on the number of lateral line hair cell. 49
Fig. 11. Long-term effects of NH4+ on Ca2+ influx at L1 neuromast. 50

Allert, N., H. Koller, and M. Siebler. 1998. Ammonia-induced depolarization of cultured rat cortical astrocytes. Brain Res. 782:261-270.
Baldisserotto, B., J.A. Martos-Sitcha, C.C. Menezes, C. Toni, R.L. Prati, O. Garcia Lde, J. Salbego, J.M. Mancera, and G. Martinez-Rodriguez. 2014. The effects of ammonia and water hardness on the hormonal, osmoregulatory and metabolic responses of the freshwater silver catfish Rhamdia quelen. Aquat Toxicol. 152:341-352.
Becker, T., M.F. Wullimann, C.G. Becker, R.R. Bernhardt, and M. Schachner. 1997. Axonal regrowth after spinal cord transection in adult zebrafish. J Comp Neurol. 377:577-595.
Binstock, L., and H. Lecar. 1969. Ammonium ion currents in the squid giant axon. J Gen Physiol. 53:342-361.
Brignull, H.R., D.W. Raible, and J.S. Stone. 2009. Feathers and fins: non-mammalian models for hair cell regeneration. Brain Res. 1277:12-23.
Chablais, F., and A. Jazwinska. 2010. IGF signaling between blastema and wound epidermis is required for fin regeneration. Development. 137:871-879.
Ching, B., S.F. Chew, W.P. Wong, and Y.K. Ip. 2009. Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddarti (mudskipper). Aquat Toxicol. 95:203-212.
Chitnis, A.B., D.D. Nogare, and M. Matsuda. 2012. Building the posterior lateral line system in zebrafish. Dev Neurobiol. 72:234-255.
Dambly-Chaudiere, C., D. Sapede, F. Soubiran, K. Decorde, N. Gompel, and A. Ghysen. 2003. The lateral line of zebrafish: a model system for the analysis of morphogenesis and neural development in vertebrates. Biol Cell. 95:579-587.
Engeszer, R.E., L.B. Patterson, A.A. Rao, and D.M. Parichy. 2007. Zebrafish in the wild: a review of natural history and new notes from the field. Zebrafish. 4:21-40.
Esterberg, R., D.W. Hailey, A.B. Coffin, D.W. Raible, and E.W. Rubel. 2013. Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death. J Neurosci. 33:7513-7525.
Evans, D.H., P.M. Piermarini, and K.P. Choe. 2005. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev. 85:97-177.
Fettiplace, R. 2009. Defining features of the hair cell mechanoelectrical transducer channel. Pflugers Arch. 458:1115-1123.
Gale, J.E., W. Marcotti, H.J. Kennedy, C.J. Kros, and G.P. Richardson. 2001. FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel. J Neurosci. 21:7013-7025.
Gemberling, M., T.J. Bailey, D.R. Hyde, and K.D. Poss. 2013. The zebrafish as a model for complex tissue regeneration. Trends Genet. 29:611-620.
Gillespie, P.G., and U. Muller. 2009. Mechanotransduction by hair cells: models, molecules, and mechanisms. Cell. 139:33-44.
Goldshmit, Y., T.E. Sztal, P.R. Jusuf, T.E. Hall, M. Nguyen-Chi, and P.D. Currie. 2012. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci. 32:7477-7492.
Goodrich, L.V. 2005. Hear, hear for the zebrafish. Neuron. 45:3-5.
Harris, J.A., A.G. Cheng, L.L. Cunningham, G. MacDonald, D.W. Raible, and E.W. Rubel. 2003. Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio). J Assoc Res Otolaryngol. 4:219-234.
Hernandez, P.P., V. Moreno, F.A. Olivari, and M.L. Allende. 2006. Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio). Hear Res. 213:1-10.
Howe, D.G., Y.M. Bradford, T. Conlin, A.E. Eagle, D. Fashena, K. Frazer, J. Knight, P. Mani, R. Martin, S.A. Moxon, H. Paddock, C. Pich, S. Ramachandran, B.J. Ruef, L. Ruzicka, K. Schaper, X. Shao, A. Singer, B. Sprunger, C.E. Van Slyke, and M. Westerfield. 2013a. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics. Nucleic Acids Res. 41:D854-860.
Howe, K., M.D. Clark, C.F. Torroja, J. Torrance, C. Berthelot, M. Muffato, J.E. Collins, S. Humphray, K. McLaren, L. Matthews, S. McLaren, I. Sealy, M. Caccamo, C. Churcher, C. Scott, J.C. Barrett, R. Koch, G.J. Rauch, S. White, W. Chow, B. Kilian, L.T. Quintais, J.A. Guerra-Assuncao, Y. Zhou, Y. Gu, J. Yen, J.H. Vogel, T. Eyre, S. Redmond, R. Banerjee, J. Chi, B. Fu, E. Langley, S.F. Maguire, G.K. Laird, D. Lloyd, E. Kenyon, S. Donaldson, H. Sehra, J. Almeida-King, J. Loveland, S. Trevanion, M. Jones, M. Quail, D. Willey, A. Hunt, J. Burton, S. Sims, K. McLay, B. Plumb, J. Davis, C. Clee, K. Oliver, R. Clark, C. Riddle, D. Elliot, G. Threadgold, G. Harden, D. Ware, S. Begum, B. Mortimore, G. Kerry, P. Heath, B. Phillimore, A. Tracey, N. Corby, M. Dunn, C. Johnson, J. Wood, S. Clark, S. Pelan, G. Griffiths, M. Smith, R. Glithero, P. Howden, N. Barker, C. Lloyd, C. Stevens, J. Harley, K. Holt, G. Panagiotidis, J. Lovell, H. Beasley, C. Henderson, D. Gordon, K. Auger, D. Wright, J. Collins, C. Raisen, L. Dyer, K. Leung, L. Robertson, K. Ambridge, D. Leongamornlert, S. McGuire, R. Gilderthorp, C. Griffiths, D. Manthravadi, S. Nichol, G. Barker, et al. 2013b. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496:498-503.
Ip, Y.K., and S.F. Chew. 2010. Ammonia production, excretion, toxicity, and defense in fish: a review. Front Physiol. 1:134.
Kishimoto, N., K. Shimizu, and K. Sawamoto. 2012. Neuronal regeneration in a zebrafish model of adult brain injury. Dis Model Mech. 5:200-209.
Kizil, C., N. Kyritsis, S. Dudczig, V. Kroehne, D. Freudenreich, J. Kaslin, and M. Brand. 2012. Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3. Dev Cell. 23:1230-1237.
Kroehne, V., D. Freudenreich, S. Hans, J. Kaslin, and M. Brand. 2011. Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors. Development. 138:4831-4841.
Langheinrich, U. 2003. Zebrafish: a new model on the pharmaceutical catwalk. Bioessays. 25:904-912.
Ledent, V. 2002. Postembryonic development of the posterior lateral line in zebrafish. Development. 129:597-604.
Lin, L.Y., W. Pang, W.M. Chuang, G.Y. Hung, Y.H. Lin, and J.L. Horng. 2013. Extracellular Ca2+ and Mg2+ modulate aminoglycoside blockade of mechanotransducer channel-mediated Ca2+ entry in zebrafish hair cells: an in vivo study with the SIET. Am J Physiol Cell Physiol. 305:C1060-1068.
Ma, E.Y., and D.W. Raible. 2009. Signaling pathways regulating zebrafish lateral line development. Curr Biol. 19:R381-386.
Marcaida, G., V. Felipo, C. Hermenegildo, M.D. Minana, and S. Grisolia. 1992. Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors. FEBS Lett. 296:67-68.
Marz, M., R. Schmidt, S. Rastegar, and U. Strahle. 2011. Regenerative response following stab injury in the adult zebrafish telencephalon. Dev Dyn. 240:2221-2231.
McNeil, P.L., D. Boyle, T.B. Henry, R.D. Handy, and K.A. Sloman. 2014. Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish (Danio rerio). Aquat Toxicol. 152:318-323.
Meyers, J.R., R.B. MacDonald, A. Duggan, D. Lenzi, D.G. Standaert, J.T. Corwin, and D.P. Corey. 2003. Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels. J Neurosci. 23:4054-4065.
Minana, M.D., C. Hermenegildo, M. Llsansola, C. Montoliu, S. Grisolia, and V. Felipo. 1996. Carnitine and choline derivatives containing a trimethylamine group prevent ammonia toxicity in mice and glutamate toxicity in primary cultures of neurons. J Pharmacol Exp Ther. 279:194-199.
Nakada, T., K. Hoshijima, M. Esaki, S. Nagayoshi, K. Kawakami, and S. Hirose. 2007. Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression. Am J Physiol Regul Integr Comp Physiol. 293:R1743-1753.
Namdaran, P., K.E. Reinhart, K.N. Owens, D.W. Raible, and E.W. Rubel. 2012. Identification of modulators of hair cell regeneration in the zebrafish lateral line. J Neurosci. 32:3516-3528.
Niihori, M., T. Platto, S. Igarashi, A. Hurbon, A.M. Dunn, P. Tran, H. Tran, J.A. Mudery, M.J. Slepian, and A. Jacob. 2015. Zebrafish swimming behavior as a biomarker for ototoxicity-induced hair cell damage: a high-throughput drug development platform targeting hearing loss. Transl Res. 166:440-450.
Nogare, D.D., M. Nikaido, K. Somers, J. Head, T. Piotrowski, and A.B. Chitnis. 2017. In toto imaging of the migrating Zebrafish lateral line primordium at single cell resolution. Dev Biol. 422:14-23.
Nunez, V.A., A.F. Sarrazin, N. Cubedo, M.L. Allende, C. Dambly-Chaudiere, and A. Ghysen. 2009. Postembryonic development of the posterior lateral line in the zebrafish. Evol Dev. 11:391-404.
Ohmori, H. 1985. Mechano-electrical transduction currents in isolated vestibular hair cells of the chick. J Physiol. 359:189-217.
Olive, R., S. Wolf, A. Dubreuil, V. Bormuth, G. Debregeas, and R. Candelier. 2016. Rheotaxis of Larval Zebrafish: Behavioral Study of a Multi-Sensory Process. Front Syst Neurosci. 10:14.
Olszewski, J., M. Haehnel, M. Taguchi, and J.C. Liao. 2012. Zebrafish larvae exhibit rheotaxis and can escape a continuous suction source using their lateral line. PLoS One. 7:e36661.
Oteiza, P., I. Odstrcil, G. Lauder, R. Portugues, and F. Engert. 2017. A novel mechanism for mechanosensory-based rheotaxis in larval zebrafish. Nature. 547:445-448.
Poss, K.D., L.G. Wilson, and M.T. Keating. 2002. Heart regeneration in zebrafish. Science. 298:2188-2190.
Raible, D.W., and G.J. Kruse. 2000. Organization of the lateral line system in embryonic zebrafish. J Comp Neurol. 421:189-198.
Randall, D.J., and T.K. Tsui. 2002. Ammonia toxicity in fish. Mar Pollut Bull. 45:17-23.
Raymond, P.A., L.K. Barthel, R.L. Bernardos, and J.J. Perkowski. 2006. Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Dev Biol. 6:36.
Sanchez, M., M.L. Ceci, D. Gutierrez, C. Anguita-Salinas, and M.L. Allende. 2016. Mechanosensory organ regeneration in zebrafish depends on a population of multipotent progenitor cells kept latent by Schwann cells. BMC Biol. 14:27.
Santoriello, C., and L.I. Zon. 2012. Hooked! Modeling human disease in zebrafish. J Clin Invest. 122:2337-2343.
Shih, T.H., J.L. Horng, P.P. Hwang, and L.Y. Lin. 2008. Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. Am J Physiol Cell Physiol. 295:C1625-1632.
Shih, T.H., J.L. Horng, Y.T. Lai, and L.Y. Lin. 2013. Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes. Am J Physiol Regul Integr Comp Physiol. 304:R1130-1138.
Shingles, A., D.J. McKenzie, E.W. Taylor, A. Moretti, P.J. Butler, and S. Ceradini. 2001. Effects of sublethal ammonia exposure on swimming performance in rainbow trout (Oncorhynchus mykiss). J Exp Biol. 204:2691-2698.
Singh, S.P., J.E. Holdway, and K.D. Poss. 2012. Regeneration of amputated zebrafish fin rays from de novo osteoblasts. Dev Cell. 22:879-886.
Stoick-Cooper, C.L., G. Weidinger, K.J. Riehle, C. Hubbert, M.B. Major, N. Fausto, and R.T. Moon. 2007. Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development. 134:479-489.
Suli, A., G.M. Watson, E.W. Rubel, and D.W. Raible. 2012. Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells. PLoS One. 7:e29727.
Thomas, A.J., D.W. Hailey, T.M. Stawicki, P. Wu, A.B. Coffin, E.W. Rubel, D.W. Raible, J.A. Simon, and H.C. Ou. 2013. Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line. J Neurosci. 33:4405-4414.
Uribe, P.M., L.H. Kawas, J.W. Harding, and A.B. Coffin. 2015. Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure. Front Cell Neurosci. 9:3.
Vihtelic, T.S., J.E. Soverly, S.C. Kassen, and D.R. Hyde. 2006. Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish. Exp Eye Res. 82:558-575.
Visek, W.J. 1984. Ammonia: its effects on biological systems, metabolic hormones, and reproduction. J Dairy Sci. 67:481-498.
Wicks, B.J., R. Joensen, Q. Tang, and D.J. Randall. 2002. Swimming and ammonia toxicity in salmonids: the effect of sub lethal ammonia exposure on the swimming performance of coho salmon and the acute toxicity of ammonia in swimming and resting rainbow trout. Aquat Toxicol. 59:55-69.