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Title マウス小脳パンソー構造の分子解剖学的研究 : パンソーはプルキンエ細胞の軸索初節に対してGABA作動性の化学的抑制を行うための分子解剖学的基盤を欠く

Author(s) 岩倉, 淳

Citation 北海道大学. 博士(医学) 甲第11203号

Issue Date 2014-03-25

DOI 10.14943/doctoral.k11203

Doc URL http://hdl.handle.net/2115/55714

Type theses (doctoral)

Note 配架番号：2075

File Information Atsushi_Iwakura.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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i�s�

1. Ogawa, Y. & Rasband, M. N. The functional organization and assembly of the axon

initial segment. Curr Opin Neurobiol 18, 307-13 (2008). 2. Debanne, D. & Rama, S. Astrocytes shape axonal signaling. Sci Signal 4, pe11

(2011). 3. Clark, B. D., Goldberg, E. M. & Rudy, B. Electrogenic tuning of the axon initial

segment. Neuroscientist 15, 651-68 (2009). 4. Kordeli, E., Lambert, S. & Bennett, V. AnkyrinG. A new ankyrin gene with

neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J Biol Chem 270, 2352-9 (1995).

5. Berghs, S. et al. betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system. J Cell Biol 151, 985-1002 (2000).

6. Kole, M. H. & Stuart, G. J. Signal processing in the axon initial segment. Neuron 73, 235-47.

7. Somogyi, P. A specific 'axo-axonal' interneuron in the visual cortex of the rat. Brain Res 136, 345-50 (1977).

8. Peters, A., Proskauer, C. C. & Ribak, C. E. Chandelier cells in rat visual cortex. J Comp Neurol 206, 397-416 (1982).

9. Freund, T. F., Martin, K. A., Smith, A. D. & Somogyi, P. Glutamate decarboxylase-immunoreactive terminals of Golgi-impregnated axoaxonic cells and of presumed basket cells in synaptic contact with pyramidal neurons of the cat's visual cortex. J Comp Neurol 221, 263-78 (1983).

10. Somogyi, P. et al. Identified axo-axonic cells are immunoreactive for GABA in the hippocampus and visual cortex of the cat. Brain Res 332, 143-9 (1985).

11. DeFelipe, J. & Gonzalez-Albo, M. C. Chandelier cell axons are immunoreactive for GAT-1 in the human neocortex. Neuroreport 9, 467-70 (1998).

12. Nusser, Z., Sieghart, W., Benke, D., Fritschy, J. M. & Somogyi, P. Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells. Proc Natl Acad Sci U S A 93, 11939-44 (1996).

13. Howard, A., Tamas, G. & Soltesz, I. Lighting the chandelier: new vistas for axo-axonic cells. Trends Neurosci 28, 310-6 (2005).

14. Roman y Cajal, S. Histolory of the nervous system of man and vertebrates. Oxford University Press (1911).

44

15. Gobel, S. Axo-axonic septate junctions in the basket formations of the cat cerebellar cortex. J Cell Biol 51, 328-33 (1971).

16. Sotelo, C. & Llinas, R. Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J Cell Biol 53, 271-89 (1972).

17. Palay, S. L., Billings-Gagliardi, S. & Chan-Palay, V. Neuronal perikarya with dispersed, single ribosomes in the visual cortex of Macaca mulatta. J Cell Biol 63, 1074-89 (1974).

18. Rosina, A., Morara, S. & Provini, L. GAT-1 developmental expression in the rat cerebellar cortex: basket and pinceau formation. Neuroreport 10, 1613-8 (1999).

19. Gianola, S., Savio, T., Schwab, M. E. & Rossi, F. Cell-autonomous mechanisms and myelin-associated factors contribute to the development of Purkinje axon intracortical plexus in the rat cerebellum. J Neurosci 23, 4613-24 (2003).

20. Ango, F. et al. Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at purkinje axon initial segment. Cell 119, 257-72 (2004).

21. Sotelo, C. Development of "Pinceaux" formations and dendritic translocation of climbing fibers during the acquisition of the balance between glutamatergic and gamma-aminobutyric acidergic inputs in developing Purkinje cells. J Comp Neurol 506, 240-62 (2008).

22. Ichikawa, R. et al. Developmental switching of perisomatic innervation from climbing fibers to basket cell fibers in cerebellar Purkinje cells. J Neurosci 31, 16916-27 (2011).

23. Wang, H., Kunkel, D. D., Schwartzkroin, P. A. & Tempel, B. L. Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. J Neurosci 14, 4588-99 (1994).

24. Laube, G. et al. Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux. Brain Res Mol Brain Res 42, 51-61 (1996).

25. Bobik, M., Ellisman, M. H., Rudy, B. & Martone, M. E. Potassium channel subunit Kv3.2 and the water channel aquaporin-4 are selectively localized to cerebellar pinceau. Brain Res 1026, 168-78 (2004).

26. Zhou, D. et al. AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing. J Cell Biol 143, 1295-304 (1998).

27. Jenkins, S. M. & Bennett, V. Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje

45

neuron initial segments. J Cell Biol 155, 739-46 (2001). 28. Foust, A., Popovic, M., Zecevic, D. & McCormick, D. A. Action potentials initiate in

the axon initial segment and propagate through axon collaterals reliably in cerebellar Purkinje neurons. J Neurosci 30, 6891-902 (2010).

29. Somogyi, P. & Hamori, J. A quantitative electron microscopic study of the Purkinje cell axon initial segment. Neuroscience 1, 361-5 (1976).

30. Korn, H. & Axelrad, H. Electrical inhibition of Purkinje cells in the cerebellum of the rat. Proc Natl Acad Sci U S A 77, 6244-7 (1980).

31. Gabbott, P. L., Somogyi, J., Stewart, M. G. & Hamori, J. GABA-immunoreactive neurons in the rat cerebellum: a light and electron microscope study. J Comp Neurol 251, 474-90 (1986).

32. Fukaya, M. & Watanabe, M. Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: a study in the adult mouse brain. J Comp Neurol 426, 572-86 (2000).

33. Watanabe, M. et al. Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur J Neurosci 10, 478-87 (1998).

34. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75 (1951).

35. Palay, S. L., Sotelo, C., Peters, A. & Orkand, P. M. The axon hillock and the initial segment. J Cell Biol 38, 193-201 (1968).

36. Peters, A., Feldman, M. & Saldanha, J. The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. II. Terminations upon neuronal perikarya and dendritic shafts. J Neurocytol 5, 85-107 (1976).

37. Chaudhry, F. A. et al. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 15, 711-20 (1995).

38. Jiao, Y. et al. Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice. Genetics 171, 1239-46 (2005).

39. Patrizi, A. et al. Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors. Proc Natl Acad Sci U S A 105, 13151-6 (2008).

40. Kosaka, T., Kosaka, K., Nakayama, T., Hunziker, W. & Heizmann, C. W. Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling. Exp Brain Res 93, 483-91 (1993).

41. Itouji, A., Sakai, N., Tanaka, C. & Saito, N. Neuronal and glial localization of two

46

GABA transporters (GAT1 and GAT3) in the rat cerebellum. Brain Res Mol Brain Res 37, 309-16 (1996).

42. Chiu, C. S. et al. Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions. J Neurosci 22, 10251-66 (2002).

43. Kaufman, D. L., McGinnis, J. F., Krieger, N. R. & Tobin, A. J. Brain glutamate decarboxylase cloned in lambda gt-11: fusion protein produces gamma-aminobutyric acid. Science 232, 1138-40 (1986).

44. McIntire, S. L., Reimer, R. J., Schuske, K., Edwards, R. H. & Jorgensen, E. M. Identification and characterization of the vesicular GABA transporter. Nature 389, 870-6 (1997).

45. tom Dieck, S. et al. Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 142, 499-509 (1998).

46. Oertel, W. H., Schmechel, D. E., Mugnaini, E., Tappaz, M. L. & Kopin, I. J. Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6, 2715-35 (1981).

47. Chaudhry, F. A. et al. The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons. J Neurosci 18, 9733-50 (1998).

48. Fritschy, J. M. & Panzanelli, P. Molecular and synaptic organization of GABAA receptors in the cerebellum: Effects of targeted subunit gene deletions. Cerebellum 5, 275-85 (2006).

49. Varoqueaux, F., Jamain, S. & Brose, N. Neuroligin 2 is exclusively localized to inhibitory synapses. Eur J Cell Biol 83, 449-56 (2004).

50. Richter, K. et al. Presynaptic cytomatrix protein bassoon is localized at both excitatory and inhibitory synapses of rat brain. J Comp Neurol 408, 437-48 (1999).

51. Fritschy, J. M., Panzanelli, P., Kralic, J. E., Vogt, K. E. & Sassoe-Pognetto, M. Differential dependence of axo-dendritic and axo-somatic GABAergic synapses on GABAA receptors containing the alpha1 subunit in Purkinje cells. J Neurosci 26, 3245-55 (2006).

52. Somogyi, P., Freund, T. F. & Cowey, A. The axo-axonic interneuron in the cerebral cortex of the rat, cat and monkey. Neuroscience 7, 2577-607 (1982).

53. Kistner, U. et al. SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J Biol Chem 268, 4580-3 (1993).

54. Yamada, K., Fukaya, M., Shimizu, H., Sakimura, K. & Watanabe, M. NMDA

47

receptor subunits GluRepsilon1, GluRepsilon3 and GluRzeta1 are enriched at the mossy fibre-granule cell synapse in the adult mouse cerebellum. Eur J Neurosci 13, 2025-36 (2001).

55. Yamada, K. et al. Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression. J Neurosci 18, 5706-13 (1998).

56. Nakamura, M. et al. Signaling complex formation of phospholipase Cbeta4 with metabotropic glutamate receptor type 1alpha and 1,4,5-trisphosphate receptor at the perisynapse and endoplasmic reticulum in the mouse brain. Eur J Neurosci 20, 2929-44 (2004).

57. Miyazaki, T., Fukaya, M., Shimizu, H. & Watanabe, M. Subtype switching of vesicular glutamate transporters at parallel fibre-Purkinje cell synapses in developing mouse cerebellum. Eur J Neurosci 17, 2563-72 (2003).

58. Li, X. G., Somogyi, P., Tepper, J. M. & Buzsaki, G. Axonal and dendritic arborization of an intracellularly labeled chandelier cell in the CA1 region of rat hippocampus. Exp Brain Res 90, 519-25 (1992).

59. Fritschy, J. M. & Mohler, H. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359, 154-94 (1995).

60. Kralic, J. E. et al. Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice. J Clin Invest 115, 774-9 (2005).

61. Kasugai, Y. et al. Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling. Eur J Neurosci 32, 1868-88.

62. Morara, S., Brecha, N. C., Marcotti, W., Provini, L. & Rosina, A. Neuronal and glial localization of the GABA transporter GAT-1 in the cerebellar cortex. Neuroreport 7, 2993-6 (1996).

63. Attwell, D., Barbour, B. & Szatkowski, M. Nonvesicular release of neurotransmitter. Neuron 11, 401-7 (1993).

64. Nakajima Y & K, K. Fine structure of the Mauthner cell:synaptic topography and comparative study. In: Neurobiology of the Mauthner cell (Faber D, Korn H, eds). New York: Raven (1978).

65. Triller, A. & Korn, H. Glio-axonic junctional like complexes at the Mauthner cell's axon cap of teleosts: a possible morphological basis for field effect inhibitions. Neurosci Lett 18, 275-81 (1980).

66. Furukawa, T. & Furshpan, E. J. Two inhibitory mechanisms in the Mauthner

48

neurons of goldfish. J Neurophysiol 26, 140-76 (1963). 67. Weiss, S. A. & Faber, D. S. Field effects in the CNS play functional roles. Front

Neural Circuits 4, 15. 68. Faber, D. S. & Korn, H. Electrical field effects: their relevance in central neural

networks. Physiol Rev 69, 821-63 (1989).