THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE SCHOOL OF SCIENCE, ENGINEERING, AND TECHNOLOGY ALTERED RESTING-STATE BRAIN ACTIVITY IN A POSTNATALLY DEVELOPING VALPROIC ACID MOUSE MODEL OF AUTISM TREVOR LOVELL Spring 2018 A thesis submitted in partial fulfillment of the requirements for baccalaureate degree in Biology with honors in Biology Reviewed and approved* by the following: Yongsoo Kim Assistant Professor of Neural and Behavioral Sciences Thesis Supervisor Michael Chorney Professor of Biology Faculty Reader David Witwer Director of Capital College Honors Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder characterized by difficulties with social communication, coinciding with restricted or repetitive interests or behaviors. The symptoms of ASD are heterogeneous between those clinically diagnosed, however these symptoms consistently emerge early in childhood. Previous research has identified significant neuroanatomical and neurophysiological alterations in ASD brains, but the underlying neural mechanisms behind these changes are poorly understood. One of the most widely-researched theories of ASD is the imbalance of excitatory and inhibitory (E/I) neurons within the brain. Despite its significance, E/I balance in early postnatally developing brains has not been examined. In order to test the potential imbalance, we analyzed the neural activation levels in early postnatal mouse brains that were prenatally exposed to valproic acid, an environmental animal model of ASD. We measured endogenous c-Fos, a transcription factor and marker for activated neurons, in mice at postnatal (P) days 7 and 14 using serial two-photon tomography, a novel fluorescence microscopy method that enables us to achieve brain- wide imaging at cellular resolution. Furthermore, we quantified total brain-cell, neuronal and inhibitory neuron-density in the prefrontal cortex to examine altered neuronal composition at P14. The preliminary results of this study show a trend towards overall hyperactivation at P7, followed by hypoactivation at P14. Furthermore, we found the ii total number of brain cells at P14 decreased, the number of neurons decreased, and the number of inhibitory cells increased. This has led us to propose that excitatory neurons more vulnerable to VPA treatment. Conversely, the relative greater density of inhibitory neurons compared to excitatory neurons can be linked with the hypoactivation at P14. This early postnatal hypoactivation of the brain could result in developmental delays, which is one of the core symptoms of ASD in humans. iii TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................... iv LIST OF TABLES .............................................................................................................. v ACKNOWLEDGEMENTS ............................................................................................... vi Chapter 1: Introduction ....................................................................................................... 1 Chapter 2: Literature Review .............................................................................................. 5 2.1. Background of Autism Spectrum Disorder .......................................................... 5 2.2. Objective Differences of Neurotypical and Autistic Brains ................................. 6 2.3. Prenatal VPA Exposure as a Mouse Model of Autism ........................................ 8 2.4. Prenatal VPA Exposure Results in Excitatory/Inhibitory Imbalance .................. 9 2.5. Prenatal VPA Exposure Results in Neurohistological Deviations ..................... 12 2.6. Microscopy Techniques ..................................................................................... 14 2.7. Fluorescence Microscopy ................................................................................... 16 2.8. Literature Gaps and Hypotheses ........................................................................ 18 Chapter 3: Materials and Methods .................................................................................... 19 3.1. Sample ................................................................................................................ 19 3.2. Mouse Injection Method .................................................................................... 19 3.3. Mouse Brain Dissection Method ........................................................................ 20 3.4. c-Fos Automated Cell Counting ......................................................................... 21 3.5. Neurohistological Manual Cell Counting .......................................................... 21 Chapter 4: Results and Discussion .................................................................................... 23 Chapter 5: Conclusion....................................................................................................... 35 References ......................................................................................................................... 37 iv LIST OF FIGURES Figure 1: ASD Manifestations in Humans .......................................................................... 7 Figure 2: Subplate Hyperconnectivity Following Prenatal VPA Exposure...................... 11 Figure 3: Neurohistological Deviations Resultant from VPA Exposure .......................... 13 Figure 4: Neuroimaging Techniques ................................................................................ 15 Figure 5: Graphical Summary of Methods ....................................................................... 22 Figure 6: Qualitative Examination of Resting-State P7 Activation .................................. 25 Figure 7: Quantitative Examination of Resting-State P14 Activation .............................. 28 Figure 8: Qualitative Examination of Resting-State P14 Activation ................................ 29 Figure 9: Qualitative Examination of NeuroTrace in P14 Brains..................................... 32 Figure 10: Qualitative and Quantitative Examination of NeuN in P14 Brains................. 33 Figure 11: Qualitative and Quantitative Examination of GAD in P14 Brains ................. 34 v LIST OF TABLES Table 1: Resting-State Alterations at P7. .......................................................................... 24 Table 2: Resting-State Alterations at P14. ........................................................................ 27 vi ACKNOWLEDGEMENTS This thesis would not have been possible without the help of many individuals. I would first like to thank Dr. Yongsoo Kim, my thesis advisor and mentor. Dr. Kim took me when I was at my lowest. I had no applicable research experience coming into his lab, but he quickly and efficiently changed this. He taught me the value of taking ownership of my own work, and that the best way to learn something is to first take the time to try and figure it out yourself before asking for help. If you do not spend the hands-on time to try and learn something yourself, then you are never going to learn anything. Next, I want to thank Dr. Michael Chorney, my faculty reader. With his help, I was able to secure this high-caliber research position at the Penn State College of Medicine. I have also taken numerous classes with Dr. Chorney at Penn State Harrisburg and he was the first professor to make me think like a scholar. His teaching style has engaged me and encouraged me to become an independent and critical thinker. I am forever grateful for your patience and encouragement over these last four years. Third, I would like to Zachary Nolan and URee Chon. Zachary helped with mouse husbandry, perfusions, and brain dissections. He also executed the automated computational pipeline in order to analyze the brains. URee helped with genetic analysis of the mice and provided me with general guidance with mouse brain neuroanatomy. Finally, I would like to thank my mother and father. They have provided me with both financial and emotional support throughout my entire life. I would not be the young adult I am today without their guidance and wisdom. 1 Chapter 1: Introduction Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder that affects approximately one in sixty-eight children born in the United States.1 The defining characteristics used to clinically diagnose ASD include difficulties with social communication, coinciding with restricted or repetitive interests or behaviors.2 The symptoms of ASD are heterogeneous among those diagnosed, however they always emerge in early childhood. Within the first year of life, children with ASD will exhibit difficulties with interpreting and expressing social and emotional cues, such as smiling and eye contact.3 Historically, a diagnosis of ASD was made at the age of three by a psychiatric professional,4 but now, accurate diagnoses can be made by age one. Not only does this disorder cause emotional stress to the parents and affected child, but it also causes a major economic burden. The average lifetime cost for an individual with ASD is 1.4 million dollars, while the costs for a child with ASD accompanied by an intellectual disability is 2.4 million dollars.5 Although there are both environmental and genetic risk factors associated with autism, there are currently no known treatments or underlying neural mechanisms to prevent this disorder. Previous