Functional and Anatomical study of the Inferior Olive: From slice physiology to in vivo recordings Sebastián Loyola Arroyo Functional and Anatomical study of the Inferior Olive: From slice physiology to in vivo recordings Sebastián Loyola Arroyo Functional and anatomical study of the inferior olive: From slice physiology to in vivo recordings The author’s work described herein was done at: Netherlands Institute for Neuroscience (NIN), Amsterdam & Department of Neuroscience, Erasmus MC, Rotterdam Cover art: “The inferior olive is like an ingenious guitar” by Sebastián Loyola. It was inspired by that statement which was said by my supervisor, Chris De Zeeuw, during a data discussion meeting. it was also inspired for the love I have for the guitar, the music, and the inferior olive. Copyright © 2021 Sebastián Loyola Arroyo All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without permission of the author. Printed by Gildeprint || www.gildeprint.nl ISBN: 978-94-6419-126-4 Functional and Anatomical study of the Inferior Olive: From slice physiology to in vivo recordings Functionele en anatomische studie van de onderste olijf: Van in vitro fysiologie tot in vivo opnames Thesis to obtain the degree of Doctor from the Erasmus University Rotterdam by command of the Rector Magnificus Prof. dr. F.A. van der Duijn Schouten, and in accordance with the decision of the Doctorate board. The public defence shall be held on March 23rd, 2021 at 13:00 hrs by Sebastián Loyola Arroyo born in Santiago, Chile Doctoral committee Promotor: Prof.dr. C.I. de Zeeuw Other members: Dr. T.J.H. Ruigrok Prof. dr. Y. Yarom Dr. P. Isope Copromotor: Dr. T.M. Hoogland For all the people who care about me and for the ones who care about our old and well conserved friend across evolution, the inferior olive…. Table of contents 1 General Introduction………………………………………………………………………… 1 1.1 Abstract………………………………………………………………………………………… 2 1.2 Development of the inferior olive and climbing fibers 1.2.1 The origin of inferior olivary neurons………………………………………………. 3 1.2.2 Migration of inferior olivary neurons…………………………………………………6 1.2.3 Inferior olivary subdivisions and cell types………………………………………… 7 1.2.4 Climbing fiber outgrowth and elimination…………………………………............. 8 1.3 Ultrastructure of the inferior olivary neuropil 1.3.1 Glomeruli and gap junctions…………………………...………………………….. 12 1.3.2 Inputs and origin……………………………………………………………………... 15 1.3.3 Neurotransmitters and receptors…………………………………………………... 16 1.4 Cell physiology of inferior olivary neurons 1.4.1 Subthreshold oscillations and spike Timing………………………………………. 18 1.4.2 Electrical synapses in the inferior Olive…………………………………………… 20 1.4.3 Synaptic modification of oscillations and coupling………………………………. 21 1.4.4 Action potential waveforms…………………………………………………………. 24 1.5 Models of the olivary Neurons 1.5.1 Single-cell models…………………………………………………………………… 25 1.5.2 Network models……………………………………………………………………… 27 1.6 Climbing fibers patterns and behavioral consequences 1.6.1 Spatiotemporal patterms……………………………………………………………. 28 1.6.2 Behavioral consequences………………………………………………................. 30 1.7 Functional models of the olivocerebellar system 1.7.1 Marr-Albus-Ito learning models…………………………………………………….. 34 1.7.2 Motor timing models………………………………………………………………….37 1.8 Pathology of the inferior olive…………………………………………………………………39 1.9 Inferior olive and sleep……………………………………………………………………… 40 1.10 Conclusions……………………………………………………………………………………. 42 1.11 Scope of the thesis………………………………………………………….………………… 42 1.13 References……………………………………………………………………………………. 43 2 Variability and directionality of inferior olive neuron dendrites revealed by detailed 3D characterization of an extensive morphological library Abstract………………………………………………………………………………………… 74 Introduction…………………………………………………………………………………….. 75 Materials and methods……………………………………………………………………….. 76 Results…………………………………………………………………………………………. 81 Discussion………………………………………………………………………………………94 References……………………………………………………………………………………..100 Supplementary figures………………………………………………………………………. 105 3 Impact of temporal interplay of excitatory and inhibitory inputs on sub- and suprathreshold activity of inferior olivary neurons and motor learning Abstract ……………………………………………………………………………………….. 108 Introduction……………………………………………………………………………………. 109 Results Physiological properties of IO neurons…………………………………………………...111 Impact of subthreshold CN input on IO neurons……………………………………….. 113 Impact of suprathreshold CN input on IO neurons……………………………………... 116 Impact of subthreshold MDJ input on IO neurons………………………………………. 118 Impact of suprathreshold MDJ input on IO neurons……………………………………. 122 Interaction of CN and MDJ inputs and their impact on IO neurons…………………… 124 Interaction of CN and MDJ inputs and their impact on motor learning……………….. 126 Discussion…………………………………………………………………………………….. 129 References……………………………………………………………………………………. 134 Supplementary figures………………………………………………………………………. 138 Supplementary tables………………………………………………………………………... 147 Appendix………………………………………………………………………………………. 153 Materials and Methods………………………………………………………………………. 153 4 Sleep stage dependent modularion of complex spike activity ensemble activity Abstract………………………………………………………………………………………... 160 Introduction……………………………………………………………………………………. 161 Results………………………………………………………………………………………… 162 Discussion……………………………………………………………………………………...170 Materials and methods………………………………………………………………………..175 References……………………………………………………………………………………. 178 5 General Discussion 5.1 A morphological map of IO neuron morphology 5.1.1 IO dendritic morphology………………………………………………………………. 185 5.1.2 Anamotical clustering of IO neurons………………………………………………... 185 5.2 The role of cerebellar nuclei and midbrain afferents in shaping the STOs and output of IO neurons 5.2.1 Impact of CN and MDJ afferents on conditional oscillators……………………….. 186 5.2.2 Impact of CN and MDJ afferents on oscillating neurons…………………………...187 5.2.3 Temporal interactions of CN and MDJ afferents and their impact on STOs and output …………………………………………………………………………………………………. 189 5.2.4 Temporal interactions of CN and MDJ afferents and their impact on motor learning…………………………………………………………………………………………190 5.3 Modulation of complex spike activity during sleep………………………………………... 191 5.4 Future perspectives………………………………………………………………………….. 194 5.5 References……………………………………………………………………………………. 195 Summary …………………………………………………………………………………………… 201 Samenvatting………………………………………………………………………………………. 203 Curriculum Vitae…………………………………………………………………………………… 205 Acknowledgments…………………………………………………………………………………211 Chapter 1 General Introduction This chapter is the thoroughly revised and updated second edition of the book chapter: Inferior Olive: All Ins and Outs. Handbook of the Cerebellum and Cerebellar disorders. Springer, Cham. Authors of the first edition: De Gruijl JR, Bosman LWJ, De Zeeuw CI, De Jeu MTG Year of publication of the first edition of the book chapter: 2013 Year of publication of the second edition of the book chapter: 2019 Sebastián Loyola, Laurens Bosman, Jornt De Gruijl, Marcel De Jeu, Mario Negrello, Tycho Hoogland, Chris De Zeeuw General introduction 1.1 Abstract The inferior (IO) is a structure located in the ventrolateral part of the brainstem that plays a fundamental role in motor learning and motor coordination by providing one of the two major excitatory inputs to the cerebellar cortex: the climbing fibers. These climbing fibers elicit an all or none response in Purkinje cells named the “complex spike”. In addition, the olivary axons provide collaterals to the cerebellar nuclei, further modulating the cerebellar output. IO neurons have several distinguishing features one of them is exhibiting complex dendritic arbor morphologies with particular spatial orientations. Another striking feature is the presence of dendro-dendritic gap junctions in which electrotonically coupled dendritic spines receive both excitatory and inhibitory inputs. Due to this unique synaptic arrangement of the glomerulus, which forms the core of the hallmark of the olivary neuropil (De Zeeuw, 1990), temporal interaction of both inputs would have a big impact on synaptic integration and spatiotemporal activity patterns (Segev and Parnas, 1983; De Zeeuw et al., 1995). Furthermore, IO neurons also show subthreshold oscillations (STOs) that may well play an important role in motor learning and motor coordination by controlling spike timing (Van der Giessen et al., 2008; Yarden-Rabinowitz and Yarom, 2019). Due the low spike frequency of individual IO neurons (~1Hz), coordination of complex behaviors, such as eating, grooming or digging, is mediated by ensembles of IO neurons that generate dynamic spatiotemporal patterns of complex spikes in the cerebellar cortex (Welsh et al., 1995). Despite the multiple studies focused on olivary function, there is little knowledge about the morphology of IO neurons and the anatomical organization of their inputs. Likewise, the impact of the temporal interaction of excitatory and inhibitory inputs on STOs, spike output and motor learning remains to be elucidated. In this thesis I will address those topics using in vitro and in vivo techniques that will allow us to have a better understanding of this structure. Furthermore, I will provide new insights in the engagement of olivo-cerebellar activity during sleep. 2 | Chapter 1 1.2 Development of the inferior olive and climbing fibers 1.2.1