THE EFFECTS OF KETAMINE ON DOPAMINERGIC FUNCTION Michelle Kokkinou Psychiatric Imaging Group London Institute of Medical Sciences (LMS), Medical Research Council (MRC) Faculty of Medicine, Imperial College London Thesis submitted for the degree of Doctor of Philosophy 1 Declaration of originality The experiments described and data analysis were part of my own work, unless otherwise stated. The work was conducted at the London Institute of Medical Sciences (LMS) in collaboration with Imanova Ltd. Specialist pre-clinical imaging analysis was conducted in collaboration with Mattia Veronese (King’s College London). Specialist meta-analysis was conducted in collaboration with Abhishekh Ashok. Peer-reviewed accepted paper Michelle Kokkinou, Abhishekh H Ashok, Oliver D Howes. The effects of ketamine on dopaminergic function: meta-analysis and review of the implications for neuropsychiatric disorders. Molecular Psychiatry. Oct 3. doi: 10.1038/mp.2017.190. Published conference abstracts arising from this thesis Chapters 3&4: Howes OD., Kokkinou M. The Effects of Repeated NMDA Blockade with Ketamine on Dopamine and Glutamate Function. Biological Psychiatry. 72nd Annual Scientific Convention and Meeting. https://doi.org/10.1016/j.biopsych.2017.02.091 Chapters 4&5: Kokkinou M, Irvine EE, Bonsall DR Ungless MA, Withers DJ, Howes OD. Modulation of sub-chronic ketamine-induced locomotor sensitisation by midbrain dopamine neuron firing. European Neuropsychopharmacology (ENP) Chapters 3 & 4 are in preparation for publication. Presentation regarding the experiments in this thesis British Association for Psychopharmacology Meeting July 2017. Midbrain dopamine neuron firing mediates the effects of ketamine treatment on locomotor activity: A DREADD experiment 2 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives Licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 3 One of the most remarkable experiences I have had throughout my PhD was to take part in a BBC Horizon programme along with my supervisor Professor Oliver Howes and discuss how an excess of the brain chemical dopamine can lead to paranoia and delusions. Therefore I would like to start with a photo I took on the day of the BBC2 film interview which was set in a Funfair. 4 Dedication I would like to dedicate this thesis to my grandmother Maro. 5 Acknowledgments Most of all I would like to thank my supervisor Professor Oliver Howes for his support, encouragement and expert advice throughout the PhD project. Oliver has given me the opportunity and resources to perform exciting experiments using state in the art technologies and has trained me to learn about the world of research. He has a great impact in my career development as a neuroscientist. I do not have the words to express how grateful I am to him. For all their help, support and professional ideas about experiments I would like to thank my co-supervisors Dr Mark Ungless and Professor Dominic Withers. Many thanks also to Elaine Irvine for help and advice regarding the behavioural experiments presented and her time to train me to perform the experimental techniques, such as the stereotaxic surgeries. Importantly I would also like to thank many people who helped to make this research possible Dr David Bonsall, Dr Abhishekh Ashok, Justyna Glegola, Maria Paiva Pessoa, Dr Paulius Viskaitis, Dr Kyoko Tossell and Ele Paul. I would also like to thank my academic mentors Professor Thomas Barnes and Dr Alex Sardini who kept me on track with my assessments. Many thanks to all my lovely colleagues in the Psychiatric Imaging group, Metabolic Signalling group and Neurophysiology group at the London Institute of Medical Sciences (LMS), Susan Watts and Deborah Oakley from the science communications team at the LMS, Central Biomedical Services Imperial College London, the PET modeller Mattia Veronese and all my collaborators at the Institute of Psychiatry King’s College London, the biology and radiochemistry teams at Imanova Ltd. I would also like to thank Dr Paula Moran for her expert advice on the latent inhibition experiment. 6 I would also like to thank my family, especially Mum, Dad, Andrea, Michalis, Marisa, Poly, Chloe, my grandparents, my godparents and my friends especially Antonia, Elli, Maria, Irene, Georgia, Stephanos and Andrienne for supporting me throughout this journey. The research was financially supported by Medical Research Council (UK) Grant (MC- A656-5QD30) to Professor Howes. 7 Glossary of abbreviations 3Rs- Replacement, Reduction and Refinement 3-MT- 3-Methoxytyramine 6-OHDA- 6-hydroxydopamine [(18)F]-DOPA- 3,4-dihydroxy-6-[(18)F]-fluoro-L-phenylalanine [18]F-fluorodeoxyglucose (18F-FDG) ([11C] β-CFT)- [11C]2-β- carbomethocy3β-(4-fluorophenyl) tropane [123I]-iodobenzamide AADC- Aromatic L-amino acid decarboxylase AC- adenylate cyclase Ach- acetylcholine AMPAR- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ANCOVA- Analysis of covariance ANOVA- Analysis of variance AAV- adeno-associated virus BBB- Blood brain barrier Carbon- C Ca+2 - Calcium ion CaMKII- calmodulin-dependent protein kinase II cAMP- cyclic adenosine monophosphate 8 CNO- Clozapine N-oxide COMT-Catechol O-methyltransferase CS- Conditioned stimulus Ct region– cortex Ct value- Cycle threshold CT- computed tomography DA – Dopamine D1, D2, D3 – Dopamine receptor sub-type 1, 2, and 3 respectively DAT - Dopamine transporter dB- Decibel df- degrees of freedom dlPFC – dorsolateral prefrontal cortex DNA- Deoxyribonucleic acid dNTPs – deoxyribonucleotides DOPA- L-3,4-dihydroxyphenylalanine DOPAC- 3,4-Dihydroxyphenylacetic acid DREADD – Designer Receptors Exclusively Activated by Designer Drugs EDTA - ethylenediaminetetraacetic acid FDA - Food and Drug administration 9 FOV- Field of view GABA- γ-Aminobutyric acid GIRK- G protein-coupled inwardly-rectifying potassium Hipp- Hippocampus HNK- Hydroxynorketamine Hprt1- Hypoxanthine phosphoribosyltransferase 1 HVA-homovanillic acid hM3- human muscarinic acetylcholine receptor IAW- Inveon Acquisition Workplace i.p- intraperitoneal i.v- intravenous ITG – Inferior temporal gyrus K+ - Potassium ion Ket- Ketamine Ki – Influx rate constant mod Ki – Influx rate constant relative to the cerebellum corrected for kloss std Ki – Influx rate constant relative to the cerebellum kloss – rate constant for the loss of radioactive metabolites from the striatum KS- Kolmogorov-Smirnov LAT-1- Large-neutral Amino Acid Transporter 1 10 L-DOPA- L-3,4-dihydroxyphenylalanine LSO- Lutetium Oxyorthosilicate LI- Latent inhibition µPET µ positron emission tomography MAM- methylazoxymethanol acetate MAO-B-monoamine oxidase B Mg+2 - Magnesium ion Min- minutes MK-801- Dizocilpine mRNA- Messenger RNA MRS - Magnetic Resonance Spectroscopy n- sample size N- Nitrogen Na+- Sodium ion n/a- not available NAc – Nucleus accumbens NaOH- Sodium hydroxide NDS- Normal donkey serum NMDA - N-methyl-D-aspartate NMDAR - N-methyl-D-aspartate receptor 11 NPE- Non pre-exposed ns - not significant p- probability Parvalbumin- PV PBS- phosphate buffered saline PCP - Phencyclidine PE- Pre-exposed PET - Positron Emission Tomography PFA- paraformaldehyde PFC- Prefrontal cortex PMT- Photomultiplier tubes PR- Progressive ratio QPCR- quantitative polymerase chain reaction RNA- ribonucleic acid RT- reverse transcriptase Sal- saline s.c- subcutaneous s.d.- standard deviation SEM- standard error of the mean SD- Sprague Dawley 12 SNc - Substantia nigra pars compacta Str – striatum SUV- standardized uptake value TAC- Time activity curve TAE- Tris acetate EDTA TH- Tyrosine hydroxylase Thal- Thalamus TM3- transmembrane domains 3 US- Unconditioned stimulus VHipp- ventral hippocampus VSub- ventral subiculum VS – ventral striatum VTA - Ventral tegmental area Zn+2- Zinc ion 13 Abstract Schizophrenia is a chronic debilitating disorder which affects about 21 million people worldwide. One of the robust neurochemical abnormalities in schizophrenia is significantly elevated striatal dopamine synthesis capacity in patients compared to controls. Ketamine is a club drug of abuse and induces psychotomimetic effects at sub-anaesthetic doses in healthy human and exacerbates psychotic symptoms in patients with schizophrenia. Therefore it has been used to model neurochemical alterations seen in schizophrenia such as dopaminergic overactivity. However, the effect of sub-chronic ketamine on dopamine synthesis capacity in vivo is not known. This thesis synthesizes the evidence of sub-chronic ketamine’s action on the dopaminergic function and behavioural measures previously associated with the dopaminergic system in the male mouse and it can be divided into four key findings. Firstly, sub-chronic ketamine administration significantly increases presynaptic striatal dopamine synthesis capacity as measured by 3,4-dihydroxy-6-[(18)F]-fluoro-l- phenylalanine ([18F]-DOPA) positron emission tomography (PET) imaging and induces locomotor sensitization. Secondly, inhibiting midbrain dopamine neuron firing using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in vivo prevents the effects of sub-chronic ketamine administration on the presynaptic striatal dopaminergic function and locomotor sensitization. Thirdly,