Vagus nerve stimulation and modulation of the locus coeruleus in a preclinical model for multiple sclerosis Dinah Arys Student number: 01303288 Supervisor(s): Prof. Dr. Robrecht Raedt, Dr. Guy Laureys A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in the Biomedical Sciences Academic year: 2017-2018 Vagus nerve stimulation and modulation of the locus coeruleus in a preclinical model for multiple sclerosis Dinah Arys Student number: 01303288 Supervisor(s): Prof. Dr. Robrecht Raedt, Dr. Guy Laureys A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in the Biomedical Sciences Academic year: 2017-2018 Preface Writing this thesis has been an interesting and challenging journey. It has been an instructive experience which gave me the opportunity to expand my knowledge and skills in the field of neuroscientific research. Without the help of many people, I wouldn’t have been able to write this thesis. So, I would like to thank them all. First of all, I would like to thank my promotor Prof. Dr. Robrecht Readt, for giving me the opportunity to carry out my thesis in the Laboratory for clinical and experimental neurophysiology (LCEN) and for his guidance and many advice. Further, I would like to thank Dr. Guy Laureys for giving me the chance to carry out his project and for his guidance with designing the study protocol. I also want to thank Prof. Dr. Sarah Gerlo, for her guidance during the RT-qPCR analyzes. Furthermore, I would like to thank Latoya Stevens and Wouter van Lysebettens in particular, for teaching me everything in the past two years and for always being there for me when I had questions or problems. I would also like to thank the other doctoral students for helping me whenever I needed it. Finally, I would like to thank my fellow lab students, for making the days at the laboratory much more fun. My family and friends, for their endless support. And Jari, for his interest in my project and for his support and motivating words during the past two years. Table of contents 1. Introduction ............................................................................................................................ 3 The locus coeruleus-noradrenaline system: anatomy and physiology ............................. 3 Multiple sclerosis ............................................................................................................ 4 1.2.1 Definition and clinical subtypes ................................................................................ 4 1.2.2 Immunopathogenesis of multiple sclerosis ............................................................... 5 Role of the locus coeruleus-noradrenaline system in MS ................................................ 7 Chemogenetics ............................................................................................................... 9 1.4.1 Development of DREADDs ...................................................................................... 9 1.4.2 Classification of DREADDs .................................................................................... 10 1.4.3 Cell specific expression of DREADDs .................................................................... 12 Vagus nerve stimulation (VNS) ..................................................................................... 14 1.5.1 Principle ................................................................................................................. 14 1.5.2 Vagus nerve: anatomy ........................................................................................... 14 1.5.3 Mechanism of action (MOA) .................................................................................. 15 Objectives of the project ............................................................................................... 16 2. Materials & methods ............................................................................................................ 17 Laboratory animals ....................................................................................................... 17 Stereotactical surgery: injection of vector ...................................................................... 17 Unit recording ............................................................................................................... 18 Perfusion and immunohistochemistry (IHC) .................................................................. 19 Design of the pilot experiment ...................................................................................... 20 Validation of stereotactical coordinates of the left lateral ventricle ................................. 20 Implantation of a VNS-electrode and stereotactical placement of a cannula ................. 21 ICV injection of TNF-α or Ringers’ solution ................................................................... 21 RT-qPCR ...................................................................................................................... 21 Statistics ....................................................................................................................... 23 3. Results ................................................................................................................................ 23 Validation of stereotactical coordinates of the left lateral ventricle ................................. 23 Immunohistochemistry: validation of vector expression ................................................ 24 Unit recording ............................................................................................................... 31 RT-qPCR analysis ........................................................................................................ 33 4. Discussion ........................................................................................................................... 36 Immunohistochemistry: validation of vector expression ................................................ 36 Unit recording ............................................................................................................... 40 RT-qPCR analysis ........................................................................................................ 41 5. Conclusion ........................................................................................................................... 43 6. References .......................................................................................................................... 44 Samenvatting Achtergrond: Recente studies wijzen op een rol van gereduceerde noradrenaline (NA) levels en noradrenerge signalisatie in de pathofysiologie van multiple sclerose. Het hoofddoel van dit project is onderzoeken of een toegenomen NA release vanuit de locus coeruleus (LC), de voornaamste bron van NA in het centrale-zenuwstelsel, de immuun respons kan moduleren in het TNF-α-geïnduceerde neuro-inflammatoir-ratmodel. Twee methodes die de noradrenerge output van de LC zouden stimuleren zijn chemogenetische modulatie met de excitatoire hM3Dq designer-recepter-exclusively-activated-by-designer-drugs (DREADD) en nervus vagus stimulatie. Methoden: Een AAV2/7-PRSx8-hM3Dq-mCherry-vector werd geïnjecteerd in de LC, om de expressie van de hM3Dq DREADD te induceren. De selectiviteit van de DREADD expressie werd nagegaan a.d.h.v. een immunohistochemische dubbelkleuring tegen mCherry en tyrosine hydroxylase. De elektrofysiologische respons van LC neuronen op DREADD activatie m.b.v. clozapine werd gemeten met unit recording. Het TNF-α neuro-inflammatoir-ratmodel werd gecreëerd door intracerebroventriculaire injectie van TNF-α. Pro-inflammatoire condities werden geëvalueerd a.d.h.v. een RT-qPCR analyse voor inflammatoire target genen. Resultaten: Virale expressie was niet selectief voor noradrenerge neuronen van de LC, maar was ook aanwezig in regio’s rondom de noradrenerge nucleus. Injectie van clozapine leidt tot een stijging in vuurfrequentie van zowel controle als hM3Dq getransduceerde units. Er konden geen significante verschillen in mRNA expressie levels van target genen aangetoond worden tussen TNF-α behandelde en controle dieren. Besluiten: De chemogenetische modulatie van de LC met hM3Dq vereist verder onderzoek. Door mogelijke interacties van clozapine met endogene receptoren, kan niets besloten worden omtrent selectieve hM3Dq activatie in hM3Dq units. Het TNF-α ratmodel kon niet gevalideerd worden, vermoedelijk door een te kleine sample size en normalisatie van mRNA expressie levels na 24h. 1 Summary Background: Recent studies suggest that reduced noradrenaline (NA) levels and noradrenergic signalization may play a role in the pathophysiology of multiple sclerosis. The main goal of this project is to investigate if an increased NA release from the locus coeruleus (LC), the main source of NA in the central nervous system, can modulate the immune response in the TNF-α-induced neuroinflammatory-rat-model. Two methods that would increase the noradrenergic output of the LC are chemogenetic modulation with the excitatory hM3Dq designer-receptor-exclusively-activated-by-designer-drugs (DREADD) and vagus nerve stimulation. Methods: An AAV2/7-PRSx8-hM3Dq-mCherry vector was injected into the LC, to induce the expression of the hM3Dq DREADD. The selectivity of the DREADD expression was investigated by an immunohistochemical double-staining against mCherry and tyrosine hydroxylase.