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Overexpression of neuronal K+–Cl co-transporter enhances dendritic spine plasticity and motor learning

  • Kayo Nakamura
  • Andrew John Moorhouse
  • Dennis Lawrence Cheung
  • Kei Eto
  • Ikuko Takeda
  • Paul Wiers Rozenbroek
  • Junichi NabekuraEmail author
Original Paper

Abstract

The neuronal K+–Cl cotransporter KCC2 maintains a low intracellular Cl concentration and facilitates hyperpolarizing GABAA receptor responses. KCC2 also plays a separate role in stabilizing and enhancing dendritic spines in the developing nervous system. Using a conditional transgenic mouse strategy, we examined whether overexpression of KCC2 enhances dendritic spines in the adult nervous system and characterized the effects on spine dynamics in the motor cortex in vivo during rotarod training. Mice overexpressing KCC2 showed significantly increased spine density in the apical dendrites of layer V pyramidal neurons, measured in vivo using two-photon imaging. During modest accelerated rotarod training, mice overexpressing KCC2 displayed enhanced spine formation rates, greater balancing skill at higher rotarod speeds and a faster rate of learning in this ability. Our results demonstrate that KCC2 enhances spine density and dynamics in the adult nervous system and suggest that KCC2 may play a role in experience-dependent synaptic plasticity.

Keywords

Two-photon microscopy In vivo Motor learning KCC2 Synaptic plasticity 

Notes

Acknowledgements

We thank Ms. Tatsuko Oba for the support of animal maintenance and preparation and Dr. Miho Watanabe at Hamamatsu Medical University for a useful suggestion for the analysis of KCC2 expression and staining.

Author contributions

KN, DLC, PWR, KE and IT conducted experiments; KN, AJM and JN wrote the paper; AJM and JN conceived the study. All authors approved the final version of the manuscript.

Funding

This study was supported by JSPS KAKENHI grant no. JP17H01530 and JP25253017 to JN. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants. All animal experiments were approved by the Okazaki Institutional Animal Care and Use Committee or by the UNSW Sydney Animal Care and Ethics Committee.

References

  1. 1.
    Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999) The K+/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255CrossRefPubMedGoogle Scholar
  2. 2.
    Blaesse P, Airaksinen MS, Rivera C, Kaila K (2009) Cation-chloride cotransporters and neuronal function. Neuron 61:820–838CrossRefPubMedGoogle Scholar
  3. 3.
    Kaila K, Price TJ, Payne JA, Puskarjov M, Voipio J (2014) Cation-chloride cotransporters in neuronal development, plasticity and disease. Nat Rev Neurosci 15:637–654CrossRefPubMedGoogle Scholar
  4. 4.
    Watanabe M, Fukuda A (2015) Development and regulation of chloride homeostasis in the central nervous system. Cell Neurosci Front.  https://doi.org/10.3389/fncel.2015.00371 Google Scholar
  5. 5.
    Kahle KT, Delpire E (2016) Kinase-KCC2 coupling: Cl rheostasis, disease susceptibility, therapeutic target. J Neurophysiol 115:8–18CrossRefPubMedGoogle Scholar
  6. 6.
    Li H, Khirug S, Cai C, Ludwig A, Blaesse P, Kolikova J, Afzalov R, Coleman SK, Lauri S, Airaksinen MS, Keinänen K, Khiroug L, Saarma M, Kaila K, Rivera C (2007) KCC2 interacts with the dendritic cytoskeleton to promote spine development. Neuron 56:1019–1033CrossRefPubMedGoogle Scholar
  7. 7.
    Fiumelli H, Briner A, Puskarjov M, Blaesse P, Belem BJT, Dayer AG, Kaila K, Martin J-L, Vutskits L (2013) An ion transport-independent role for the cation-chloride cotransporter KCC2 in dendritic spinogenesis in vivo. Cereb Cortex 23:378–388CrossRefPubMedGoogle Scholar
  8. 8.
    Llano O, Smirnov S, Soni S, Golubtsov A, Guillemin I, Hotulainen P, Medina I, Nothwang HG, Rivera C, Ludwig A (2015) KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX. J Cell Biol 209:671–686CrossRefPubMedGoogle Scholar
  9. 9.
    Chevy Q, Heubl M, Goutierre M, Backer S, Moutkine I, Eugène E, Bloch-Gallego E, Lévi S, Poncer JC (2015) KCC2 gates activity-driven AMPA receptor traffic through cofilin phosphorylation. J Neurosci 35:15772–15786CrossRefPubMedGoogle Scholar
  10. 10.
    Chamma I, Heubl M, Chevy Q, Renner M, Moutkine I, Eugene E, Poncer JC, Levi S (2013) Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons. J Neurosci 33:15488–15503CrossRefPubMedGoogle Scholar
  11. 11.
    Gulyas AI, Sik A, Payne JA, Kaila K, Freund TF (2001) The KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampus. Eur J Neurosci 13:2205–2217CrossRefPubMedGoogle Scholar
  12. 12.
    Gauvain G, Chamma I, Chevy Q, Cabezas C, Irinopoulou T, Bodrug N, Carnaud M, Lévi S, Poncer JC (2011) The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines. Proc Natl Acad Sci USA 108:15474–15479CrossRefPubMedGoogle Scholar
  13. 13.
    Fu M, Yu X, Lu J, Zuo Y (2012) Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo. Nature 483:92–95CrossRefPubMedGoogle Scholar
  14. 14.
    Yu X, Zuo Y (2011) Spine plasticity in the motor cortex. Curr Opin Neurobiol 21:169–174CrossRefPubMedGoogle Scholar
  15. 15.
    Holtmaat A, Svoboda K (2009) Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci 10:647–658CrossRefPubMedGoogle Scholar
  16. 16.
    Kasai H, Fukuda M, Watanabe S, Hayashi-Takagi A, Noguchi J (2010) Structural dynamics of dendritic spines in memory and cognition. Trends Neurosci 33:121–129CrossRefPubMedGoogle Scholar
  17. 17.
    Xu T, Yu X, Perlik AJ, Tobin WF, Zweig JA, Tennant K, Jones T, Zuo Y (2009) Rapid formation and selective stabilization of synapses for enduring motor memories. Nature 462:915–919CrossRefPubMedGoogle Scholar
  18. 18.
    Yang G, Pan F, Gan WB (2009) Stably maintained dendritic spines are associated with lifelong memories. Nature 462:920–924CrossRefPubMedGoogle Scholar
  19. 19.
    Bosch M, Hayashi Y (2012) Structural plasticity of dendritic spines. Curr Opin Neurobiol 22:383–388CrossRefPubMedGoogle Scholar
  20. 20.
    Goulton CS, Watanabe M, Cheung DL, Wang KW, Oba T, Khoshaba A, Lai D, Inada H, Eto K, Nakamura K, Power JM, Lewis TM, Housley GD, Wake H, Nabekura J, Moorhouse AJ (2018) Conditional upregulation of KCC2 selectively enhances neuronal inhibition during seizures. bioRxiv.  https://doi.org/10.1101/253831 Google Scholar
  21. 21.
    Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551CrossRefPubMedGoogle Scholar
  22. 22.
    Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER (1996) Control of memory formation through regulated expression of a CaMKII transgene. Science 274:1678–1683CrossRefPubMedGoogle Scholar
  23. 23.
    Kakegawa W, Miyoshi Y, Hamase K, Matsuda S, Matsuda K, Kohda K, Emi K, Motohashi J, Konno R, Zaitsu K, Yuzaki M (2011) d-serine regulates cerebellar LTD and motor coordination through the delta2 glutamate receptor. Nat Neurosci 14:603–611CrossRefPubMedGoogle Scholar
  24. 24.
    Rothwell PE, Fuccillo MV, Maxeiner S, Hayton SJ, Gokce O, Lim BK, Fowler SC, Malenka RC, Sudhof TC (2014) Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors. Cell 158:198–212CrossRefPubMedGoogle Scholar
  25. 25.
    Kim SK, Hayashi H, Ishikawa T, Shibata K, Shigetomi E, Shinozaki Y, Inada H, Roh SE, Kim SJ, Lee G, Bae H, Moorhouse AJ, Mikoshiba K, Fukazawa Y, Koizumi S, Nabekura J (2016) Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain. J Clin Invest 126:1983–1997CrossRefPubMedGoogle Scholar
  26. 26.
    Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J (2009) Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci 29:3974–3980CrossRefPubMedGoogle Scholar
  27. 27.
    Kim SK, Nabekura J (2011) Rapid synaptic remodeling in the adult somatosensory cortex following peripheral nerve injury and its association with neuropathic pain. J Neurosci 31:5477–5482CrossRefPubMedGoogle Scholar
  28. 28.
    Levine ND, Rademacher DJ, Collier TJ, O’Malley JA, Kells AP, Sebastian WS, Bankiewicz KS, Steece-Collier K (2013) Advances in thin tissue Golgi-Cox impregnation: fast, reliable methods for multi-assay analyses in rodent and non-human primate brain. J Neurosci Methods 213:214–227CrossRefPubMedGoogle Scholar
  29. 29.
    Sutoo D, Akiyama K, Yabe K (2002) Comparison analysis of distributions of tyrosine hydroxylase, calmodulin and calcium/calmodulin-dependent protein kinase II in a triple stained slice of rat brain. Brain Res 933:1–11CrossRefPubMedGoogle Scholar
  30. 30.
    Meyer HS, Schwarz D, Wimmer VC, Schmitt AC, Kerr JND, Sakmann B, Helmstaedter M (2011) Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A. Proc Natl Acad Sci 108:16807–16812CrossRefPubMedGoogle Scholar
  31. 31.
    Krestel HE, Mayford M, Seeburg PH, Sprengel R (2001) A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse. Nucleic Acids Res 29:E39–E39CrossRefPubMedGoogle Scholar
  32. 32.
    Xue J, Li G, Bharucha E, Cooper NGF (2002) Developmentally regulated expression of CaMKII and iGluRs in the rat retina. Dev Brain Res 138:61–70CrossRefGoogle Scholar
  33. 33.
    Awad PN, Sanon NT, Chattopadhyaya B, Carriço JN, Ouardouz M, Gagné J, Duss S, Wolf D, Desgent S, Cancedda L, Carmant L, Di Cristo G (2016) Reducing premature KCC2 expression rescues seizure susceptibility and spine morphology in atypical febrile seizures. Neurobiol Dis 91:10–20CrossRefPubMedGoogle Scholar
  34. 34.
    Awad PN, Amegandjin CA, Szczurkowska J, Carriço JN, do Nascimento ASF, Baho E, Chattopadhyaya B, Cancedda L, Carmant L, Di Cristo G (2018) KCC2 regulates dendritic spine formation in a brain-region specific and BDNF dependent manner. Cereb Cortex 28:4049–4062CrossRefPubMedGoogle Scholar
  35. 35.
    Fu M, Zuo Y (2011) Experience-dependent structural plasticity in the cortex. Trends Neurosci 34:177–187CrossRefPubMedGoogle Scholar
  36. 36.
    Lu J, Zuo Y (2017) A local rebalancing act leads to global benefit. Neuron 96:712–713CrossRefPubMedGoogle Scholar
  37. 37.
    Tjia M, Yu X, Jammu LS, Lu J, Zuo Y (2017) Pyramidal neurons in different cortical layers exhibit distinct dynamics and plasticity of apical dendritic spines. Neural Circuits Front.  https://doi.org/10.3389/fncir.2017.00043 Google Scholar
  38. 38.
    Liston C, Cichon JM, Jeanneteau F, Jia Z, Chao MV, Gan W-B (2013) Circadian glucocorticoid oscillations promote learning-dependent synapse formation and maintenance. Nat Neurosci 16:698CrossRefPubMedGoogle Scholar
  39. 39.
    Yang G, Lai CSW, Cichon J, Ma L, Li W, Gan W-B (2014) Sleep promotes branch-specific formation of dendritic spines after learning. Science 344:1173–1178CrossRefPubMedGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Homeostatic Development, Department of Fundamental NeuroscienceNational Institutes for Physiological SciencesOkazakiJapan
  2. 2.Department of Physiological SciencesSokendaiHayamaJapan
  3. 3.Department of Physiology, School of Medical SciencesUniversity of New South WalesSydneyAustralia

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