UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Comparative compositional analysis of the gut microbiome in animal models of addiction and stress Author(s) Peterson, Veronica L. Publication date 2018 Original citation Peterson, V. L. 2018. Comparative compositional analysis of the gut microbiome in animal models of addiction and stress. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2018, Veronica L. Peterson. http://creativecommons.org/licenses/by-nc-nd/3.0/ Embargo information Not applicable Item downloaded http://hdl.handle.net/10468/7941 from Downloaded on 2021-10-09T07:41:39Z Ollscoil na hEireann National University of Ireland Colaiste na hOllscoile, Corcaigh University College Cork Department of Anatomy & Neuroscience Department Head: Prof. John F. Cryan Comparative Compositional Analysis of the Gut Microbiome in Animal Models of Addiction and Stress Thesis presented by Veronica L. Peterson under the supervision of Prof. John F. Cryan Prof. Timothy G. Dinan for the degree of Doctor of Philosophy November 2018 1-1 Table of Contents Declaration……………………………………………………………….…1-6 Acknowledgements…………………………….……………………….…1-8 Publications……….…………………………….……………………….…1-10 1 General Introduction .......................................... 1-15 1.1 The Gut Microbiome .................................................................... 1-15 1.1.1 Bacteriome ............................................................................ 1-16 Fig. 1.1.1: Factors influencing microbiome composition ................. 1-17 1.1.2 Virome - Bacteriophage and Other Viruses ........................... 1-18 1.2 The Microbiome-Gut-Brain Axis (MGBA) ..................................... 1-21 1.2.1 Overview: Bacteria & MGB Axis ............................................ 1-22 Fig. 1.2.1: Anatomy of the Microbiome-Gut-Brain Axis (MGBA) and pathways of communication ........................................................... 1-24 1.2.2 Overview: Viruses & Microbiome-Gut-Brain Axis (MGBA) .... 1-25 1.2.3 Methods of Communication ................................................... 1-25 1.3 Psychological Stress .................................................................... 1-29 1.3.1 Animal Models of Stress ........................................................ 1-29 1.4 The Addicted Microbiome: Role of Gut Microbiota in Addiction ... 1-31 1.4.1 Drug Addiction and Substance Abuse Disorders .................. 1-31 Figure 1.4.1: Gut Microbiota in the cycle of addiction: .................... 1-33 1.4.2 Psychological Comorbidities ................................................. 1-34 1.4.3 Genetics in Addiction ............................................................. 1-35 1.4.4 Neurodevelopment and Addiction ......................................... 1-36 1.4.5 Inflammation and Addiction ................................................... 1-37 1.4.6 Substance Abuse and the Gut Microbiota ............................. 1-37 Figure 1.4.6: Recurrent Changes in Gut Microbiota in Human Studies Related to Addiction ....................................................................... 1-40 1.4.7 Animal Models of Addiction ................................................... 1-41 1.5 Mining the Microbiome – Next-Generation Sequencing, Bioinformatics, and Biostatistics ......................................................... 1-45 1.5.1 “The Poop Phase” ................................................................. 1-45 1.5.2 Sequencing ........................................................................... 1-45 1.5.3 Bioinformatic Pipelines .......................................................... 1-50 1.5.4 Alpha and Beta Diversity ....................................................... 1-51 1.5.5 Biostatistical Analysis ............................................................ 1-53 Fig. 1.5.5: Ways to measure the microbiome. ................................ 1-55 1.6 Primary hypothesis and aims of thesis......................................... 1-56 2 Chapter .............................................................. 2-58 Drunk Bugs: Chronic Vapour Alcohol Exposure Induces Marked Changes in the Gut Microbiome in Mice ............................................. 2-58 2.1 Abstract 2-59 2.2 Introduction 2-60 2.3 Methods 2-61 2.4 Results 2-63 1-2 Figure 2.3: Effects of CIE vapour exposure on the microbiome ..... 2-65 Figure 2.4: Effects of CIE vapour exposure on bacterial composition 2- 67 2.5 Discussion 2-66 3 Chapter: ............................................................. 3-70 Gut microbiome correlates with altered striatal dopamine receptor expression in a model of compulsive alcohol seeking ........................ 3-70 3.1 Abstract 3-71 3.2 Introduction 3-72 3.3 Material and methods .................................................................. 3-75 3.3.1 Animals ................................................................................. 3-75 3.3.2 Behavioural phenotyping of alcohol use disorder .................. 3-75 3.3.3 Ceacal microbiome collection and sequencing ..................... 3-76 3.3.4 Microbiome Sequence Processing ........................................ 3-77 3.3.5 Gene expression analysis ..................................................... 3-77 3.3.6 Statistical Analysis ................................................................. 3-78 3.4 Results 3-79 Figure 3.3: Identification of rats at risk of alcohol use disorder. ...... 3-81 3.4.2 Behavioural profiling of selected resilient and vulnerable rats ... 3- 83 Figure 3.3.2.2: ................................................................................ 3-86 3.4.3 16S microbiome analyses in caecal contents of resilient and vulnerable rats ................................................................................ 3-87 3.5 Discussion 3-93 3.6 Supplementary information .......................................................... 3-98 3.6.1 Material and methods ............................................................ 3-98 3.6.2 Apparatus: ............................................................................. 3-98 3.6.3 Impulsivity- 5 Choice serial reaction time task ....................... 3-99 3.6.4 Anxiety-related behaviors .................................................... 3-101 3.6.5 Animal’s training for alcohol self-administration .................. 3-102 3.6.6 Screening for addiction-like behaviour ................................ 3-102 3.6.7 Partial grid paradigm ........................................................... 3-104 3.6.8 Reinstatement paradigm ..................................................... 3-104 3.6.9 Results ................................................................................ 3-104 4 Chapter: ........................................................... 4-113 Sex, Drugs, and the Microbiome: Goal/Sign-Tracking Phenotype Reveals Associations between Behavior and Microbiome in a Sex- Dependent Manner in the Rat .......................................................... 4-113 4.1 Abstract 4-114 4.2 Introduction 4-115 4.3 Methods 4-117 4.3.1 Animals ............................................................................... 4-117 4.3.2 Behavioral tests ................................................................... 4-118 4.3.3 Cecal microbiome collection and sequencing ..................... 4-123 4.3.4 Statistical Analysis ............................................................... 4-124 4.4 Results 4-128 1-3 4.4.1 Behavioral cluster analysis .................................................. 4-128 4.4.2 Behavioral differences by behavioral cluster within sex ...... 4-128 4.4.3 Group differences in Pavlovian Conditioned Reinforcement (CRF) ........................................................................................... 4-129 4.4.4 Group differences in Cocaine Cue Preference (CCP) ......... 4-129 4.4.5 Group differences in Choice Reaction Time Task (RT) ....... 4-129 4.4.6 Group differences in Locomotor Response to Novelty (Loco) ... 4- 130 4.4.7 Group differences in Light Reinforcement (LR) ................... 4-130 4.4.8 Group differences in Delay Discounting (DD) ...................... 4-130 Figure 2 – Male Behavioral Phenotypes and Behavioral Comparisons 4-131 Figure 3 – Female Behavioral Phenotypes and Behavioral Comparisons ................................................................................ 4-132 4.4.9 Microbiome diversity ............................................................ 4-133 Figure 4 – Male Alpha Diversity by Phenotype Group .................. 4-134 Figure 5 – Female Alpha Diversity by Phenotype Group ............. 4-135 4.4.10 Bacterial differences by groups within sex .................... 4-136 4.4.11 OTU level bacterial correlations to behavior ................. 4-136 Figure 6 – Correlation Analysis in Male and Female Goal/Sign- Tracking Phenotype. .................................................................... 4-138 Figure 7 – Correlation Analysis in Male and Female Behavioral Clusters ........................................................................................ 4-139 Figure 8 – Correlation Analysis by sex ......................................... 4-140 4.5 Discussion 4-141 4.6 Conclusion 4-146 4.7 Supplementary documentation .................................................. 4-147 4.7.1 4.7.1 Preliminary results of Sex Differences (Male vs Female) . 4- 147 5 Chapter: ..........................................................