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Extracellular Loose-Patch Recording of Purkinje Cell Activity in Awake Zebrafish and Emergence of Functional Cerebellar Circuit

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Extracellular Recording Approaches

Part of the book series: Neuromethods ((NM,volume 134))

Abstract

The cerebellum calibrates movements for accuracy and precision and is critical for motor learning. Purkinje cells, which are the sole output neurons of the cerebellar cortex, play an essential role in fine-tuning motor behavior. Determining how sensory stimuli, after processing by higher brain centers, modulate Purkinje cell activity and how changes in Purkinje cell firing improve the accuracy of movement is crucial for understanding cerebellar function. Due to its rapid development, small size, and transparency, the zebrafish is a promising system for functional brain mapping and for investigating the neural control of behavior in a vertebrate animal. To aid in understanding the role of the cerebellum in behavior, we have developed an approach for recording the electrical activity of cerebellar Purkinje cells in live, awake zebrafish using extracellular loose-patch electrodes. Using this method, we have found that zebrafish Purkinje cells fire simple and complex spikes similar to those seen in mammalian Purkinje cells. Spontaneous firing and a functional cerebellar circuit emerge early in zebrafish development in parallel with complex, visually guided behaviors. This preparation has significant advantages for investigating the effects of sensory stimuli on Purkinje cell firing, mapping the functional effects of afferent inputs to the cerebellum, and exploring how the cerebellum fine-tunes motor behavior.

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References

  1. Lang EJ, Apps R, Bengtsson F, Cerminara NL, De Zeeuw CI, Ebner TJ, Heck DH, Jaeger D, Jörntell H, Kawato M, Otis TS, Ozyildirim O, Popa LS, Reeves AMB, Schweighofer N, Sugihara I, Xiao J (2017) The roles of the olivocerebellar pathway in motor learning and motor control. A consensus paper. Cerebellum 16:230–252

    Article  PubMed  Google Scholar 

  2. Raman IM, Bean BP (1999) Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons. J Neurosci 19:1663–1674

    CAS  PubMed  Google Scholar 

  3. Raman IM, Sprunger LK, Meisler MH, Bean BP (1997) Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron 19:881–891

    Article  CAS  PubMed  Google Scholar 

  4. Khaliq ZM, Gouwens NW, Raman IM (2003) The contribution of resurgent sodium current to high-frequency firing in Purkinje neurons: an experimental and modeling study. J Neurosci 23:4899–4912

    CAS  PubMed  Google Scholar 

  5. Martina M, Yao GL, Bean BP (2003) Properties and functional role of voltage-dependent potassium channels in dendrites of rat cerebellar Purkinje neurons. J Neurosci 23:5698–5707

    CAS  PubMed  Google Scholar 

  6. Akemann W, Knöpfel T (2006) Interaction of Kv3 potassium channels and resurgent sodium current influences the rate of spontaneous firing of Purkinje neurons. J Neurosci 26:4602–4612

    Article  CAS  PubMed  Google Scholar 

  7. D’Angelo E, Mazzarello P, Prestori F, Mapelli J, Solinas S, Lombardo P, Cesana E, Gandolfi D, Congi L (2011) The cerebellar network: from structure to function and dynamics. Brain Res Rev 66:5–15

    Article  PubMed  Google Scholar 

  8. Schmolesky MT, Weber JT, De Zeeuw CI, Hansel C (2002) The making of a complex spike: ionic composition and plasticity. Ann N Y Acad Sci 978:359–390

    Article  PubMed  Google Scholar 

  9. Lee KH, Mathews PJ, Reeves AMB, Choe KY, Jami SA, Serrano RE, Otis TS (2015) Circuit mechanisms underlying motor memory formation in the cerebellum. Neuron 86:529–540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Woodruff-Pak DS (2006) Stereological estimation of Purkinje neuron number in C57BL/6 mice and its relation to associative learning. Neuroscience 141:233–243

    Article  CAS  PubMed  Google Scholar 

  11. Wittmann W, McLennan IS (2011) The male bias in the number of Purkinje cells and the size of the murine cerebellum may require Müllerian inhibiting substance/anti-Müllerian hormone. J Neuroendocrinol 23:831–838

    Article  CAS  PubMed  Google Scholar 

  12. Joshua M, Lisberger SG (2015) A tale of two species: neural integration in zebrafish and monkeys. Neuroscience 296:80–91

    Article  CAS  PubMed  Google Scholar 

  13. Rinkwitz S, Mourrain P, Becker TS (2011) Zebrafish: an integrative system for neurogenomics and neurosciences. Prog Neurobiol 93:231–243

    Article  PubMed  Google Scholar 

  14. Wyatt C, Bartoszek EM, Yaksi E (2015) Methods for studying the zebrafish brain: past, present and future. Eur J Neurosci 42:1746–1763

    Article  PubMed  Google Scholar 

  15. Feierstein CE, Portugues R, Orger MB (2015) See the whole picture: a comprehensive imaging approach to functional mapping of circuits in behaving zebrafish. Neuroscience 296:26–38

    Article  CAS  PubMed  Google Scholar 

  16. Orger MB, de Polavieja GG (2017) Zebrafish behavior: opportunities and challenges. Annu Rev Neurosci., epub ahead of print. https://doi.org/10.1146/annurev-neuro-071714-033857

  17. Nusslein-Volhard C, Dahm R (eds) (2002) Zebrafish: a practical approach. Oxford University Press, Oxford

    Google Scholar 

  18. Westerfield M (2007) The Zebrafish Book, 5th edition: A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene

    Google Scholar 

  19. Brustein E, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Drapeau P (2003) Steps during the development of the zebrafish locomotor network. J Physiol Paris 97:77–86

    Article  PubMed  Google Scholar 

  20. Kikuta H, Kawakami K (2009) Transient and stable transgenesis using Tol2 transposon vectors. Methods Mol Biol 546:69–84

    Article  CAS  PubMed  Google Scholar 

  21. Arrenberg AB, Driever W (2013) Integrating anatomy and function for zebrafish circuit analysis. Front Neural Circuits 7:74

    Article  PubMed  PubMed Central  Google Scholar 

  22. Dunn TW, Mu Y, Narayan S, Randlett O, Naumann EA, Yang CT, Schier AF, Freeman J, Engert F, Ahrens MB (2016) Brain-wide mapping of neural activity controlling zebrafish exploratory locomotion. Elife 5:e12741

    Article  PubMed  PubMed Central  Google Scholar 

  23. Ahrens MB, Orger MB, Robson DN, Li JM, Keller PJ (2013) Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods 10:413–420

    Article  CAS  PubMed  Google Scholar 

  24. Naumann EA, Fitzgerald JE, Dunn TW, Rihel J, Sompolinsky H, Engert F (2016) From whole-brain data to functional circuit models: the zebrafish optomotor response. Cell 167:947–960

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Scalise K, Shimizu T, Hibi M, Sawtell NB (2016) Responses of cerebellar Purkinje cells during fictive optomotor behavior in larval zebrafish. J Neurophysiol 116:2067–2080

    Article  PubMed  PubMed Central  Google Scholar 

  26. Berkefeld H, Fakler B, Schulte U (2010) Ca2+-activated K+ channels: from protein complexes to function. Physiol Rev 90:1437–1459

    Article  CAS  PubMed  Google Scholar 

  27. Bae Y-K, Kani S, Shimizu T, Tanabe K, Hojima H, Kimura Y, Higashijima S, Hibi M (2009) Anatomy of zebrafish cerebellum and screen for mutations affecting its development. Dev Biol 330:406–426

    Article  CAS  PubMed  Google Scholar 

  28. Hibi M, Shimizu T (2012) Development of the cerebellum and cerebellar neural circuits. Dev Neurobiol 72:282–301

    Article  PubMed  Google Scholar 

  29. Hashimoto M, Hibi M (2012) Development and evolution of cerebellar neural circuits. Dev Growth Differ 54:373–389

    Article  CAS  PubMed  Google Scholar 

  30. Hamling KR, Tobias ZJC, Weissman TA (2015) Mapping the development of cerebellar Purkinje cells in zebrafish. Dev Neurobiol 75:1174–1188

    Article  CAS  PubMed  Google Scholar 

  31. Hsieh J-Y, Ulrich B, Wan J, Papazian DM (2014) Rapid development of Purkinje cell excitability, functional cerebellar circuit, and afferent sensory input to cerebellum in zebrafish. Front Neural Circuits 8:147

    Article  PubMed  PubMed Central  Google Scholar 

  32. Tanabe K, Kani S, Shimizu T, Bae Y-K, Abe T, Hibi M (2010) Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus. J Neurosci 30:16983–16992

    Article  CAS  PubMed  Google Scholar 

  33. Brochu G, Maler L, Hawkes R (1990) Zebrin II: a polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J Comp Neurol 291:538–552

    Article  CAS  PubMed  Google Scholar 

  34. Hurd MW, Cahill GM (2002) Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish. J Biol Rhythms 17:307–314

    Article  PubMed  Google Scholar 

  35. Chhetri J, Jacobson G, Gueven N (2014) Zebrafish—on the move towards ophthalmological research. Eye (Lond) 28:367–380

    Article  CAS  Google Scholar 

  36. Kano M, Hashimoto K (2012) Activity-dependent maturation of climbing fiber to Purkinje cell synapses during postnatal cerebellar development. Cerebellum 11:449–450

    Article  PubMed  Google Scholar 

  37. Hashimoto K, Kano M (2013) Synapse elimination in the developing cerebellum. Cell Mol Life Sci 70:4667–4680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Westphal RE, O’Malley DM (2013) Fusion of locomotor maneuvers and improving sensory capabilities give rise to the flexible homing strikes of juvenile zebrafish. Front Neural Circuits 7:108

    Article  PubMed  PubMed Central  Google Scholar 

  39. Bianco IH, Kampff AR, Engert F (2011) Prey capture behavior evoked by simple visual stimuli in larval zebrafish. Front Syst Neurosci 5:101

    Article  PubMed  PubMed Central  Google Scholar 

  40. Aizenberg M, Schuman EM (2011) Cerebellar-dependent learning in larval zebrafish. J Neurosci 31:8708–8712

    Article  CAS  PubMed  Google Scholar 

  41. Matsui H, Namikawa K, Babaryka A, Köster RW (2014) Functional regionalization of the teleost cerebellum analyzed in vivo. Proc Natl Acad Sci U S A 111:11846–11851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sengupta M, Thirumalai V (2015) AMPA receptor mediated synaptic excitation drives state-dependent bursting in Purkinje neurons of zebrafish larvae. Elife 4:e09158

    Article  PubMed Central  Google Scholar 

  43. Mundell NA, Beier KT, Pan YA, Lapan SW, Göz Aytürk D, Berezovskii VK, Wark AR, Drokhlyansky E, Bielecki J, Born RT, Schier AF, Cepko CL (2015) Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms. J Comp Neurol 523:1639–1663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Beier KT, Mundell NA, Pan YA, Cepko CL (2016) Anterograde or retrograde transsynaptic circuit tracing in vertebrates with vesicular stomatitis virus vectors. Curr Protoc Neurosci 74:1.26–1.27

    Google Scholar 

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Acknowledgments

With the exception of Fig. 5a, c, the data in this paper were originally published by Hsieh et al. (2014) (Ref. 31). This work was supported by NIH grant R01NS058500 and a UCLA Stein-Oppenheimer Seed Grant to DMP. JYH was partially supported by the Jennifer S. Buchwald Graduate Fellowship in Physiology at UCLA. We are grateful to Drs. Thomas Otis, Joanna Jen, Fadi Issa, and Jijun Wan for advice and helpful discussions. We thank Dr. Masahiko Hibi for the gift of a pT2K aldoca:gap43-Venus plasmid.

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Authors and Affiliations

  1. Department of Physiology and the Interdepartmental Program in Molecular, Cellular, and Integrative Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Jui-Yi Hsieh & Diane M. Papazian

  2. Circuit Therapeutics, Inc., Menlo Park, CA, USA

    Jui-Yi Hsieh

Authors
  1. Jui-Yi Hsieh
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  2. Diane M. Papazian
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Correspondence to Diane M. Papazian .

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Editors and Affiliations

  1. Department of Pathology & Immunology and Neuroscience, Baylor College of Medicine, Houston, Texas, USA

    Roy V. Sillitoe

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Hsieh, JY., Papazian, D.M. (2018). Extracellular Loose-Patch Recording of Purkinje Cell Activity in Awake Zebrafish and Emergence of Functional Cerebellar Circuit. In: Sillitoe, R. (eds) Extracellular Recording Approaches. Neuromethods, vol 134. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7549-5_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7549-5_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7548-8

  • Online ISBN: 978-1-4939-7549-5

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