Rối loạn chức năng vận động kéo dài mặc dù có sự cứu cánh ổn định của quá trình hình thành cấu trúc tiểu não ở chuột đột biến Car8 waddles

Neural Development - Tập 14 - Trang 1-22 - 2019
Lauren N. Miterko1,2,3, Joshua J. White1,4,3, Tao Lin1,3, Amanda M. Brown1,4,3, Kevin J. O’Donovan5,6, Roy V. Sillitoe1,4,2,3
1Department of Pathology and Immunology, Dan Duncan Neurological Research Institute of Texas Children’s Hospital, Houston, USA
2Program in Developmental Biology, Baylor College of Medicine, Dan Duncan Neurological Research Institute of Texas Children’s Hospital, Houston, USA
3Jan and Dan Duncan Neurological Research Institute of Texas Children’s Hospital, Houston, USA
4Department of Neuroscience, Dan Duncan Neurological Research Institute of Texas Children’s Hospital, Houston, USA
5Department of Chemistry and Life Science, United States Military Academy, New York, USA
6Burke Neurological Institute, Weill Cornell Medicine, White Plains, USA

Tóm tắt

Các tế bào Purkinje đóng vai trò trung tâm trong việc thiết lập mạch tiểu não. Do đó, việc làm gián đoạn sự phát triển của các tế bào Purkinje gây ra sự suy giảm trong hình thành cấu trúc tiểu não và chức năng vận động. Trong mô hình chuột Car8wdl mắc chứng mất điều hòa di truyền, những khuyết tật vận động nghiêm trọng xuất hiện mặc dù tiểu não đã vượt qua những khiếm khuyết ban đầu về kích thước và hình thái. Để hiểu rõ cách thức bù đắp này xảy ra, chúng tôi đã xem xét cách mà sự mất đi carbonic anhydrase 8 (CAR8), một yếu tố điều hòa tín hiệu Ca2+ qua IP3R1 trong các tế bào Purkinje, làm thay đổi sự phát triển tiểu não ở chuột Car8wdl. Bằng cách sử dụng tổ hợp phân tích mô học, sinh lý và hành vi, chúng tôi đã xác định mức độ mà sự mất CAR8 ảnh hưởng đến cấu trúc tiểu nao, hoạt động của nơron và phối hợp vận động trong quá trình phát triển. Kết quả của chúng tôi cho thấy sự tăng sinh của tế bào hạt giảm trong những đột biến thời kỳ sau sinh sớm, mặc dù đến tuần thứ ba sau sinh có sự tăng cường và kéo dài sự tăng sinh, cùng với sự tăng cường biểu hiện Sox2 ở lớp ngoài tế bào hạt. Việc thay đổi kiểu mạch của các tế bào Purkinje và tế bào glia Bergmann diễn ra kèm theo các điều chỉnh của tế bào hạt này. Chúng tôi cũng nhận thấy rằng mặc dù cấu trúc cuối cùng trở về bình thường, nhưng hoạt động bất thường của các nơron và cơ bắp vẫn tồn tại. Dữ liệu của chúng tôi cho thấy rằng việc mất CAR8 chỉ hạn chế sự phát triển của tiểu não tạm thời, nhưng gây thiệt hại vĩnh viễn cho chức năng của nó. Những dữ liệu này hỗ trợ hai giả thuyết hiện tại về sự phát triển và bệnh của tiểu não: (1) Biểu hiện Sox2 có thể được tăng cường tại các vị trí bị thương và góp phần vào việc cứu vãn cấu trúc tiểu não và (2) Sự chậm trễ tạm thời trong các quá trình phát triển có thể xảy ra trước khi có chức năng vận động vĩnh viễn. Hơn nữa, chúng tôi đã mô tả hình thái và hành vi của chuột đột biến waddles trong quá trình phát triển và đề xuất vai trò do tế bào trung gian Sox2 dương tính trong việc cứu cánh trong một mô hình chuột của các bệnh vận động ở người.

Từ khóa

#tế bào Purkinje #carbonic anhydrase 8 #phát triển tiểu não #rối loạn vận động #chuột Car8wdl #Sox2 #sự tăng sinh tế bào hạt

Tài liệu tham khảo

Orr HT. SCA1-phosphorylation, a regulator of Ataxin-1 function and pathogenesis. Prog Neurobiol. 2012;99:179–85. Ledoux MS, Lorden JF. Abnormal spontaneous and harmaline-stimulated Purkinje cell activity in the awake genetically dystonic rat. Exp Brain Res. 2002;145:457–67. Wilson BK, Hess EJ. Animal models for dystonia. Mov Disord. 2013;28:982–9. Louis ED, Faust PL, J-PG V. Purkinje cell loss is a characteristic of essential tremor. Park Relat Disord. 2011;17:406–9. Gennarino VA, Singh RK, White JJ, De Maio A, Han K, Kim JY, et al. Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels. Cell. 2015;160:1087–98. Fremont R, Tewari A, Angueyra C, Khodakhah K. A role for cerebellum in the hereditary dystonia DYT1. Elife. 2017;6:e22775. White JJ, Sillitoe RV. Genetic silencing of olivocerebellar synapses causes dystonia-like behaviour in mice. Nat Commun. 2017;8:1–16. Reeber SL, Loeschel C, Franklin A, Sillitoe R. Establishment of topographic circuit zones in the cerebellum of scrambler mutant mice. Front Neural Circuits. 2013;7:122. Becker EBE, Oliver PL, Glitsch MD, Banks GT, Achilli F, Hardy A, et al. A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci. 2009;106:6706–11. Lim J, Hao T, Shaw C, Patel AJ, Szabó G, Rual J-F, et al. A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell. 2006;125:801–14. He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD. Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci. 2006;26:9975–82. Perkins EM, Clarkson YL, Sabatier N, Longhurst DM, Millward CP, Jack J, et al. Loss of beta-III spectrin leads to Purkinje cell dysfunction recapitulating the behavior and neuropathology of spinocerebellar ataxia type 5 in humans. J Neurosci. 2010;30:4857–67. Ramani B, Harris GM, Huang R, Seki T, Murphy GG, Do Carmo Costa M, et al. A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript. Hum Mol Genet. 2015;26:3232–3. White JJ, Sillitoe R. Development of the cerebellum: from gene expression patterns to circuit maps. Wiley Interdiscip Rev Dev Biol. 2013;2:149–64. Sillitoe RV, Joyner A. Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum. Annu Rev Cell Dev Biol. 2007;23:549–77. Corrales JD, Blaess S, Mahoney EM, Joyner AL. The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation. Development. 2006;133:1811–21. Sudarov A, Joyner AL. Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers. Neural Dev. 2007;2:26. Yu T, Yaguchi Y, Echevarria D, Martinez S, Basson MA. Sprouty genes prevent excessive FGF signalling in multiple cell types throughout development of the cerebellum. Development. 2011;138:2957–68. Leto K, Arancillo M, Becker EBE, Buffo A, Chiang C, Ding B, et al. Consensus paper: Cerebellar development. Cerebellum. 2015;15:789–828. Takeo YH, Kakegawa W, Miura E, Yuzaki M. RORα regulates multiple aspects of dendrite development in cerebellar Purkinje cells in vivo. J Neurosci. 2015;35:12518–34. Hashizume M, Miyazaki T, Sakimura K, Watanabe M, Kitamura K, Kano M. Disruption of cerebellar microzonal organization in GluD2 (GluRδ2) knockout mouse. Front Neural Circuits. 2013;7:130. Letellier M, Wehrlé R, Mariani J, Lohof AM. Synapse elimination in olivo-cerebellar explants occurs during a critical period and leaves an indelible trace in Purkinje cells. Proc Natl Acad Sci U S A. 2009;106:14102–7. Miyazaki T, Yamasaki M, Hashimoto K, Yamazaki M, Abe M, Usui H, et al. Cav2.1 in cerebellar Purkinje cells regulates competitive excitatory synaptic wiring, cell survival, and cerebellar biochemical compartmentalization. J Neurosci. 2012;32:1311–28. Shih EK, Sekerková G, Ohtsuki G, Aldinger KA, Chizhikov VV, Hansel C, et al. The spontaneous ataxic mouse mutant tippy is characterized by a novel Purkinje cell morphogenesis and degeneration phenotype. Cerebellum. 2015;14:292–307. White JJ, Arancillo M, Stay TL, George-Jones NA, Levy SL, Heck DH, et al. Cerebellar zonal patterning relies on Purkinje cell neurotransmission. J Neurosci. 2014;34:8231–45. Sarna JR, Hawkes R. Patterned Purkinje cell death in the cerebellum. Prog Neurobiol. 2003;70:473–507. Bichelmeier U, Schmidt T, Hübener J, Boy J, Rüttiger L, Häbig K, et al. Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3: in vivo evidence. J Neurosci. 2007;27:7418–28. Cemal CK, Carroll CJ, Lawrence L, Lowrie MB, Ruddle P, Al-Mahdawi S, et al. YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit. Hum Mol Genet. 2002;11:1075–94. Gould VFC. Mouse models of spinocerebellar ataxia type 3 (Machado-Joseph disease). Neurotherapeutics. 2012;9:285–96. White JJ, Arancillo M, King A, Lin T, Miterko LN, Gebre SA, et al. Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration. Neurobiol Dis. 2016;86:86–98. Kato K. Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in purkinje cells. FEBS Lett. 1990;271:137–40. Taniuchi K, Nishimori I, Takeuchi T, Ohtsuki Y, Onishi S. cDNA cloning and developmental expression of murine carbonic anhydrase-related proteins VIII, X, and XI. Mol Brain Res. 2002;109:207–15. Tripp BC, Smith K, Ferry JG. Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem. 2001;276:48615–8. Hirota J, Ando H, Hamada K, Mikoshiba K. Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1. Biochem J. 2003;372:435–41. Hirasawa M, Xu X, Trask RB, Maddatu TP, Johnson BA, Naggert JK, et al. Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum. Mol Cell Neurosci. 2005;35:161–70. Jiao Y, Yan J, Zhao Y, Donahue LR, Beamer WG, Li X, et al. Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice. Genetics. 2005;171:1239–46. Ali BR, Silhavy JL, Gleeson JG, Al-Gazali L. A missense founder mutation in VLDLR is associated with Dysequilibrium syndrome without quadrupedal locomotion. BMC Med Genet. 2012;13:80. Türkmen S, Guo G, Garshasbi M, Hoffmann K, Alshalah AJ, Mischung C, et al. CA8 mutations cause a novel syndrome characterized by ataxia and mild mental retardation with predisposition to quadrupedal gait. PLoS Genet. 2009;5:e10000487. Reeber SL, Sillitoe RV. Patterned expression of a cocaine- and amphetamine-regulated transcript peptide reveals complex circuit topography in the rodent cerebellar cortex. J Comp Neurol. 2011;519:1781–96. Sillitoe RV, Benson MA, Blake DJ, Hawkes R. Abnormal dysbindin expression in cerebellar mossy fiber synapses in the mdx mouse model of Duchenne muscular dystrophy. J Neurosci. 2003;23:6576–85. Sillitoe RV, Vogel MW, Joyner AL. Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map. J Neurosci. 2010;30:10015–24. White JJ, Sillitoe RV. Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression. Front Neuroanat. 2013;7:9. Cardiff RD, Miller CH, Munn RJ. Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harb Protoc. 2014;2014:655–8. Sillitoe RV, Chung S-H, Fritschy J-M, Hoy M, Hawkes R. Golgi cell dendrites are restricted by Purkinje cell stripe boundaries in the adult mouse cerebellar cortex. J Neurosci. 2008;28:2820–6. Mead TJ, Lefebvre V. Proliferation assays (BrdU and EdU) on skeletal tissue sections. Methods Mol Biol. 2014;1130:233–43. Paxinos G, Franklin K. The mouse brain in stereotaxic coordinates: Elsevier Academic Press; Amsterdam. 2001. Chambers WW, Sprague JM. Functional localization in the cerebellum: Somatotopic organization in cortex and nuclei. Arch Neurol Psychiatr. 1955;74:653–80. Udo M, Matsukawa K, Kamei H, Oda Y. Cerebellar control of locomotion: effects of cooling cerebellar intermediate cortex in high decerebrate and awake walking cats. J Neurophysiol. 1980;44:119–34. Coffman KA, Dum RP, Strick PL. Cerebellar vermis is a target of projections from the motor areas in the cerebral cortex. Proc Natl Acad Sci. 2011;108:16068–73. Sauerbrei BA, Lubenov EV, Siapas AG. Structured variability in Purkinje cell activity during locomotion. Neuron. 2015;87:840–52. Eccles JC, Provini L, Strata P, Taborikova H. Topographical investigations on the climbing fiber inputs from forelimb and hindlimb afferents to the cerebellar anterior lobe. Exp Brain Res. 1968;6(3):195–215. Arancillo M, White JJ, Lin T, Stay TL, Sillitoe RV. In vivo analysis of Purkinje cell firing properties during postnatal mouse development. J Neurophysiol. 2015;113:578–91. White JJ, Lin T, Brown AM, Arancillo M, Lackey EP, Stay TL, et al. An optimized surgical approach for obtaining stable extracellular single-unit recordings from the cerebellum of head-fixed behaving mice. J Neurosci Methods. 2016;262:21–31. Holt GR, Softky WR, Koch C, Douglas RJ. Comparison of discharge variability in vitro and in vivo in cat visual cortex neurons. J Neurophysiol. 1996;75:1806–14. Alvina K, Khodakhah K. KCa channels as therapeutic targets in episodic Ataxia Type-2. J Neurosci. 2010;30:7249–57. Walter JT, Alviña K, Womack MD, Chevez C, Khodakhah K. Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia. Nat Neurosci. 2006;9:389–97. Stoodley CJ, Schmahmann JD. Evidence for topographic organization in the cerebellum of motor control versus cognitive and affective processing. Cortex. 2010;46:831–44. Habas C, Manto M. Activation of cerebellar lobules VI-VII during motor imagery but not during motor activation in unilateral cerebellar hypoplasia. Cerebellum Ataxias. 2014;1:6. Valle MS, Bosco G, Poppele RE. Cerebellar compartments for the processing of kinematic and kinetic information related to hindlimb stepping. Exp Brain Res. 2017;235:3437–48. Valle MS, Eian J, Bosco G, Poppele RE. The organization of cortical activity in the anterior lobe of the cat cerebellum during hindlimb stepping. Exp Brain Res. 2012;216:349–65. De Gruijl JR, Hoogland TM, De Zeeuw CI. Behavioral correlates of complex spike synchrony in cerebellar microzones. J Neurosci. 2014;34:8937–47. Altman J, Bayer S. Development of the cerebellar system in relation to its evolution, structure and function. Boca Rat: CRC Press; 1997. Silbereis J, Heintz T, Taylor MM, Ganat Y, Ment LR, Bordey A, et al. Astroglial cells in the external granular layer are precursors of cerebellar granule neurons in neonates. Mol Cell Neurosci. 2010;44:362–73. Wojcinski A, Lawton AK, Bayin NS, Lao Z, Stephen DN, Joyner AL. Cerebellar granule cell replenishment postinjury by adaptive reprogramming of nestin+ progenitors. Nat Neurosci. 2017;20:1361–70. Lange C, Huttner WB, Calegari F. Cdk4/CyclinD1 overexpression in neural stem cells shortens G1, delays neuorgenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell. 2009;5:320–31. Zhang RL, Zhang ZG, Lu M, Wang Y, Yang JJ, Chopp M. Reduction of the cell cycle length by decreasing G1 phase and cell cycle reentry expand neuronal progenitor cells in the subventricular zones of adult rat after stroke. J Cereb Blood Flow Metab. 2006;26:857–63. Ko L, Koestner A, Wechsler W. Characterization of cell cycle and biological parameters of transplantable glioma cell lines and clones. Acta Neuropathol. 1980;51:107–11. Cerrato V, Mercurio S, Leto K, Fucà E, Hoxha E, Bottes S, et al. Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia. Glia. 2018;66:1929–46. Neveu I, Arenas E. Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. J Cell Biol. 1996;133:631–46. Verslegers M, Van Hove I, Buyens T, Dekeyster E, Knevels E, Moons L. Identification of MMP-2 as a novel enhancer of cerebellar granule cell proliferation. Mol Cell Neurosci. 2013;57:63–72. Verslegers M, Van Hove I, Dekeyster E, Gantois I, Hu TT, D’Hooge R, et al. MMP-2 mediates Purkinje cell morphogenesis and spine development in the mouse cerebellum. Brain Struct Funct. 2015;220:1601–17. Reeber SL, Arancillo M, Sillitoe RV. Bergmann glia are patterned into topographic molecular zones in the developing and adult mouse cerebellum. Cerebellum. 2014;17:392–403. Consalez GG, Hawkes R. The compartmental restriction of cerebellar interneurons. Front Neural Circuits. 2013;6:123. Sillitoe RV. Mossy fibers terminate directly within Purkinje cell zones during mouse development. Cerebellum. 2016;15:14–7. Lordkipanidze T, Dunaevsky A. Purkinje cell dendrites grow in alignment with Bergmann glia. Glia. 2005;51:229–34. Rakic P. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electonmicroscopic study in Macacus rhesus. J Comp Neurol. 1971;141:283–312. Custer SK, Garden GA, Gill N, Rueb U, Libby RT, Schultz C, et al. Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport. Nat Neurosci. 2006;9:1302–11. Armstrong CL, Krueger-Naug AMR, Currie RW, Hawkes R. Expression of heat-shock protein Hsp25 in mouse purkinje cells during development reveals novel features of cerebellar compartmentation. J Comp Neurol. 2001;429:7–21. Luna-Cancalon K, Sikora KM, Pappas SS, Singh V, Wulff H, Paulson HL, et al. Alterations in cerebellar physiology are associated with a stiff-legged gait in Atcayji-hes mice. Neurobiol Dis. 2014;67:140–8. Miterko LN, Sillitoe RV. Climbing Fiber development is impaired in postnatal Car8wdl mice. Cerebellum. 2017;17:56–61. McKay BE, Turner RW. Physiological and morphological development of the rat cerebellar Purkinje cell. J Physiol. 2005;567:829–50. Wallace VA. Purkinje-cell-derived sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr Biol. 1999;9:445–8. Doughty ML, Delhaye-Bouchaud N, Mariani J. Quantitative analysis of cerebellar lobulation in normal and agranular rats. J Comp Neurol. 1998;399:306–20. Dussault I, Fawcett D, Matthyssen A, Bader JA, Giguère V. Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer. Mech Dev. 1998;70:147–53. Feddersen RM, Ehlenfeldt R, Yunis WS, Clark HB, Orr HT. Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice. Neuron. 1992;9:955–66. Rakic P, Sidman RL. Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J Comp Neurol. 1973;152:103–32. Cheng FY, Fleming JT, Chiang C. Bergmann glial sonic hedgehog signaling activity is required for proper cerebellar cortical expansion and architecture. Dev Biol. 2018;440:152–66. Nicholson JL, Altman J. The effects of early hypo- and hyperthyroidism on the development of the rat cerebellar cortex. II. Synaptogenesis in the molecular layer. Brain Res. 1972;44:25–36. Espinosa JS, Luo L. Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci. 2008;28:22301–12. Zhu T, Tang H, Shen Y, Tang Q, Chen L, Wang Z, et al. Transplantation of human induced cerebellar granular-like cells improves motor functions in a novel mouse model of cerebellar ataxia. Am J Transl Res. 2016;8:705–18. Ahlfeld J, Favaro R, Pagella P, Kretzschmar HA, Nicolis S, Schüller U. Sox2 requirement in sonic hedgehog-associated medulloblastoma. Cancer Res. 2013;73:3796–807. Kakizawa S, Yamasaki M, Watanabe M, Kano M. Critical period for activity-dependent synapse elimination in developing cerebellum. J Neurosci. 2000;20:4954–61. Yan J, Jiao Y, Jiao F, Stuart J, Donahue LR, Beamer WG, et al. Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the wdl mouse. Neurosci Lett. 2007;413:196–201. Kincaid AE. Spontaneous circling behavior and dopamine neuron loss in a genetically hypothyroid mouse. Neuroscience. 2001;105:891–8. Shimobayashi E, Wagner W, Kapfhammer JP. Carbonic anhydrase 8 expression in Purkinje cells is controlled by PKCγ activity and regulates Purkinje cell dendritic growth. Mol Neurobiol. 2015;53:5149–60. Altman J. Postnatal development of the cerebellar cortex in the rat II. Phase in the maturation of Purkinje cells and of the molecular layer. J Comp Neurol. 1972;145:399–463. Westerga J, Gramsbergen A. The development of locomotion in the rat. Brain Res Dev Brain Res. 1990;57:163–74. Pascual R, Hervias MC, Tohá ME, Valero A, Figueroa HR. Purkinje cell impairment induced by early movement restriction. Biol Neonate. 1998;73:47–51. Greenough WT, West RW, DeVoogd TJ. Subsynaptic plate perforations: changes with age and experience in the rat. Science. 1978;202:1096–8. Murphy J, MacKay W, Johnson F. Responses of cerebellar cortical neurons to dyanmis proprioceptive inputs from forelimb muscles. J Neurophysiol. 1973;36:711–23. Calderon DP, Fremont R, Kraenzlin F, Khodakhah K. The neural substrates of rapid-onset dystonia-parkinsonism. Nat Neurosci. 2011;14:357–65. Bauswein E, Kolb FP, Leimbeck B, Rubia FJ. Simple and complex spike activity of cerebellar Purkinje cells during active and passive movements in the awake monkey. J Physiol. 1983;339:7418–28. Andersson G, Armstrong DM. Complex spikes in Purkinje cells in the lateral vermis (b zone) of the cat cerebellum during locomotion. J Physiol. 1987;385:107–34. Horie M, Mekada K, Sano H, Kikkawa Y, Chiken S, Someya T, et al. Neurobiology of disease characterization of novel dystonia musculorum mutant mice: implications for central nervous system abnormality. Neurobiol Dis. 2016;96:271–83. Tuschi K, Clayton PT, Gospe SM, Mills PB. Dystonia/parkinsonism, Hypermanganesemia, polycythemia, and chronic liver disease. Seattle: University of Washington; 2017. Robertson LT, McCollum G. Stimulus classification by ensembles of climbing fiber receptive fields. Trends Neurosci. 1991;14:248–54.