UNIVERSITY of CALIFORNIA, SAN DIEGO Neural

UNIVERSITY OF CALIFORNIA, SAN DIEGO

Neural substrates of impaired behavioral inhibition in Williams syndrome, a disorder of social cognition

A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy

Anthropology with a Specialization in Anthropogeny

Kari L. Hanson

Committee in charge:

Professor Katerina Semendeferi, Chair Professor Eric Halgren Professor Janis Jenkins Professor Mary Ann Raghanti Professor Shirley Strum

2017

Kari L. Hanson, 2017

The Dissertation of Kari L. Hanson is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

______

______Chair

University of California, San Diego

2017

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DEDICATION

This dissertation is dedicated to the memory of Dr. Lisa Stefanacci, whose insatiable curiosity, fierce heart, perseverance, and genuine kindness served as an inspiring model for women in science generally, and the amazing sort of person I will continue to aspire to be.

TABLE OF CONTENTS

SIGNATURE PAGE…………………………………………………………... iii DEDICATION…………………………………………………………………. iv TABLE OF CONTENTS……………………………………………………… v LIST OF FIGURES …………………………………………………………… vii LIST OF TABLES ………………………………………………………….… viii ACKNOWLEDGMENTS …………………………………………..………… ix CURRICULUM VITAE …………………………………………………….... xiii ABSTRACT OF THE DISSERTATION ………………………………..……. xvi CHAPTER 1: INTRODUCTION …………………………………..…………. 1 References……………………………………………………………… 8 CHAPTER 2: A DUAL COMPARATIVE APPROACH: INTEGRATING LINES OF EVIDENCE FROM HUMAN EVOLUTIONARY NEUROANATOMY AND NEURODEVELOPMENTAL DISORDERS ………………………………….. 14 Abstract ………………………………………………………………. 14 Introduction…………………………………………………………….. 15 Inhibitory Neurons in the Cortex……………………………………….. 19 Beyond the Cortex: Subcortical Systems for Behavioral Control…………………………………….……………. 24 Modeling Human Evolution through Genetic Disorders: the Case for Williams Syndrome…………………………………….………… 29 Conclusions ……………………………………………………………. 33 Acknowledgements………………………………………….…………. 35 References…………………………………………………………...…. 36 CHAPTER 3: INCREASED GLIA DENSITY IN THE CAUDATE NUCLEUS IN WILLIAMS SYNDROME: IMPLICATIONS FOR FRONTOSTRIATAL DYSFUNCTION……………………………………………………………….. 49 Abstract………………………………………………………………… 49 Introduction……………………………………………………………. 50 Methods ………………………………………….……………………. 52 Materials………………………………..……………………… 52 Tissue Processing……………………………………………… 53 Anatomical Regions of Interest……………………………….. 53 Statistical Analyses……………………………………………. 58 Results……………….. …………………………………………………. 58 Dorsal Caudate Nucleus …………………………………………. 58 Medial Caudate Nucleus…………………………………………. 59 Associative Putamen …………………………………………….. 59

Nucleus Accumbens………………………….……………………… 60 Discussion…………..…………………………………………………….. 60 Summary…………….……………………………………………. 60 Comparisons of the Striatum in TD, ASD, and WS ……….……. 62 Frontostriatal Dysfunction in ASD and WS …………………..… 63 Conclusion ………………………………………………………………. 65 Acknowledgements ………………………………………………………... 65 References………………………………………………………………….. 67

CHAPTER 4: DECREASED DENSITY OF CHOLINERGIC INTERNEURONS IN THE MEDIAL CAUDATE NUCLEUS IN WILLIAMS SYNDROME ...... 80 Abstract…………………………………………………………..….…... 80 Introduction……………………………………………….……………….. 81 Methods………………………… ………………………………………… 84 Materials……..…………………………………………………… 84 Regions of Interest…………………………………...…………… 86 Stereological Quantification ……………...…………………….... 87 Results…………….. ……………………………………………………… 88 Dorsal Caudate …………………………………………………… 88 Medial Caudate …………………………………………………… 88 Putamen ………………………………………………………….. 88 Nucleus Accumbens ………………………………………….…… 89 Discussion ………………………………………………………………… 89 Comparisons to other disorders of social and emotional disinhibition………………………………………………. 90 SCIN depletion and disorders of behavioral control………………………………………………….…. 92 Conclusions ……………………………………………………………… 95 References………………………………………………………………... 97

CHAPTER 5: CONCLUSIONS……………………...………………………….. 113 References………………………………………………………………... 117

LIST OF FIGURES

Figure 1.1. Regions of Interest…………………………………………………….. 13 Figure 3.1. Anatomical regions of interest in the associative territories of the dorsal (dC) and medial caudate nucleus (mC), associative putamen (aP), and nucleus accumbens (NA) regions………………………………………………..…..75 Figure 3.2. Neurons and glia in the striatum…………………………………....… 76 Figure 3.3. Results of stereological analyses in the four ROIs in WS and TD…... 77 Figure 3.4. Density of oligodendrocytes in the medial caudate nucleus………….. 79 Figure 4.1. Cholinergic interneurons of the striatum ………………………..…... 106 Figure 4.2. Schematic representation of the striatum and regions of interest targeted …………………………………………….…………………….. 107 Figure 4.3. Cholinergic interneuron density in four striatal ROIs ………………. 109

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LIST OF TABLES

Table 3.1. Subject Background Information……………………………………… 74 Table 4.1. Specimen Information…………………………..……..……………… 105 Table 4.2. Mean density of striatal cholinergic interneurons…………………….. 108 Table 4.3. Summary of evidence for striatal and cholinergic dysfunction in several neurological disorders……………………………………………. 110

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ACKNOWLEDGEMENTS

Though we must stand on the shoulders of giants, it is by my side that I have always found inspiration and strength, and my greatest debt is principally to the women who have carried me forward. My chair and primary mentor, Prof. Katerina Semendeferi, revolutionized the field of comparative neuroscience through an integration with anthropological perspectives, and in so doing, paved the way for my own and others’ grasping at a better understanding of the human brain’s diversity and complexity.

Another giant in the field, Prof. Ursula Bellugi, not only recognized the potential of a rare disorder to transform our understanding of the human social brain, but entrusted our lab with the rare collection of materials described in these pages. Dr. Lisa Stefanacci, whose journey ended far too soon, provided me with much of the rigorous training, theoretical knowledge, and emotional fortitude required to carry on in this journey, and is sorely missed. Prof. Mary Ann Raghanti, whose work inspired a great deal of my interest in neurotransmitter systems and interneurons in evolutionary context, has transformed from academic idol to close mentor, providing unwavering support (material and otherwise), and vital training and mentorship, throughout these many years.

Drs. Kate Teffer and Natalie Schenker, who came before me, helped to set a standard of excellence for research in our lab, but it is of course Dr. Nicole Barger who, through hard work and a great deal of patience, transformed me from a kid from Alaska who had barely touched a microscope into a finely-tuned dissecting, cryosectioning, brain-staining, and cell-counting machine. I have had the fortune to share these skills and innumerable hours in labs near and far with the great Branka Hrvoj-Mihic, who has shared this journey with me practically from the beginning. In much the same vein,

Caroline Lew has served as a constant source of strength, wisdom, laughter, and grace.

The many volunteers in our lab over the years, but most especially Chelsea Brown,

Hailey Orfant, Valerie Judd, Deion Cuevas, and Kimberly Groeniger, have provided me with the help and support to get things done, for which I am eternally grateful.

All of the biological anthropologists at UCSD, professors and students, have contributed to my professional and personal development in unique ways, and in particular I would like to thank Prof. Shirley Strum for contributing so much to my understanding of primate sociality, and Prof. Margaret Schoeninger for challenging me to always consider the role of ecology in human evolution. Both of these brilliant women contributed unique perspectives that will have a lasting impact on my theoretical grounding as a scientist, in addition to providing outstanding role models as pioneering women in science. Their intellectual largesse is clearly reflected in their amazing graduate students, and particularly Corinna Most, Andrew Somerville, and Melanie

Beasley, who have been among my closest allies and friends. Melanie, above and beyond all others, has been there for me in times of pain and joy, challenge and triumph, grant rejections and awards, knifepoint robberies, sushi dates, and everything in between. I am so thrilled that we began this program together and now, even though we will be starting our postdocs hundreds of miles away from one another, our paths will remain intertwined, and I will always love you. Jim Moore, Pascal Gagneux, Alyssa Crittenden and everyone at CARTA must also be acknowledged for their years of support and encouragement and a few death marches across the African savannah that were in no way a metaphor for the arduousness of completing a dissertation. My external committee members, Prof. Eric Halgren and Prof. Janis Jenkins, have provided insightful comments

x from widely varying perspectives during the planning of this research that have helped shape it thus far, for which I am also grateful.

Finally, to my family and my partner for their support, encouragement, and insight, I thank you for your patience and for everything you have helped me to become.

My mother’s insatiable curiosity and communication skills, combined with my father’s critical thinking and dogged work ethic, have been the values I have tried the hardest to carry with me—in other words, I was probably always doomed to a life of science. I don’t know whether to blame you or thank you, but I guess I’ll choose the latter since it never would have happened without your encouragement and support. My brother Robert

Hanson and his wife Tiffany have always kept me humble and laughing, and their kids,

Abby and Jeffery, have given me much joy. Last but not least, to my partner and best friend Robert Miller IV, thanks for enduring the many highs and lows of my career thus far by my side, and building a happy, loving home with me, and our many tiny, furry friends.

Funding has been provided in part by a Wenner Gren Foundation Dissertation

Improvement Grant to the author of this dissertation, in addition to fellowship support from the Institute for Neural Computation (INC) and the Center for Academic Research and Training in Anthropogeny (CARTA). Human control brain materials have been provided in collaboration with the University of Maryland Brain and Tissue Bank, a repository of the NIH NeuroBioBank. We are grateful to members of the Williams

Syndrome Association and the community, particularly Terry Monkaba, for assistance in facilitating brain donation. We additionally thank Yvonne Searcy, Michelle DeWitt,

Andy Arnold, Martin Rosas, and members of the team at the Laboratory for Cognitive

Neuroscience at Salk for meticulous record keeping and impressive research efforts on the behavioral side of things. Most of all, we are forever grateful for the individuals with

Williams syndrome and their families who have participated in all research efforts, past and present, aimed at characterizing the phenotype in Williams syndrome.

Chapter 2 is an abridged version of a review previously published by the author of this dissertation for an invited submission. Citation: Hanson KL, Hrvoj-Mihic B,

Semendeferi K (2014). A Dual Comparative Approach: Integrating Lines of Evidence from Human Evolutionary Neuroanatomy and Neurodevelopmental Disorders. Brain,

Behavior, and Evolution, 84(2), 135-155. The author of this dissertation was the primary investigator and author.

Chapter 3 is adapted from a manuscript currently under review and submitted as an invited paper to the journal Developmental Neurobiology for a special edition on autism spectrum disorder (ASD) and related disorders: Hanson KL, Groeniger K, Lew C,

Hrvoj-Mihic B, Bellugi U, Halgren E, Semendeferi K. Increased glia density in the caudate nucleus in Williams syndrome: implications for frontostriatal dysfunction in autism. Manuscript Under Review, Developmental Neurobiology (Invited Submission).

The author of this dissertation was the primary investigator and author.

Chapter 4 is a manuscript currently in preparation for submission for publication:

Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Raghanti MA,

Semendeferi K. Decreased density of cholinergic interneurons in the medial caudate nucleus in Williams syndrome. Manuscript in prep. The author of this dissertation was the primary investigator and author.

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CURRICULUM VITAE

EDUCATION 2017 Ph.D., Anthropology, Specialization in Anthropogeny, University of California San Diego 2011 M. A., Anthropology, University of California San Diego 2008 B. S., Anthropology, University of Alaska Anchorage 2007 B. S., Psychology, University of Alaska Anchorage

PUBLICATIONS Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Raghanti MA, Semendeferi K. Decreased density of cholinergic interneurons in the medial caudate nucleus in Williams syndrome. Manuscript in prep. Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Semendeferi K. Increased glia density in the caudate nucleus in Williams syndrome: implications for frontostriatal dysfunction in autism. Manuscript Under Review, Developmental Neurobiology (Invited Submission). Hrvoj-Mihic B, Hanson KL, Lew CH, Stefanacci L, Bellugi U, Jacobs B, Semendeferi K (2017). The prefrontal cortex in individuals with Williams syndrome: dendritic branching across cortical areas. Manuscript Under Review, Frontiers in Neuroscience (Invited Submission). Chailangkarn T, Trujillo CF, Freitas B, Hrvoj-Mihic B, Yu DX, Brown TT, Marchetto MCM, Bardy C, McHenry L, Stefanacci L, Jarvinen A, Searcy YM, DeWitt M, Wong W, Lai P, Ard MC, Herai RC, Hanson KL, Jacobs B, Dale AM, Freitas B, Korenberg JR, Halgren E, Bellugi U, Gage FH, Semendeferi K, Muotri AR (2016). A human neurodevelopmental model for Williams syndrome. Nature, 536(7616), 338-343. Semendeferi K, Hanson KL (2016). Plastic and heterogenous: postnatal developmental changes in the human brain. In Trevathan W and Rosenberg K (Eds.) Cute and Costly: How Helpless Infants Made Us Human. SAR Press. Hanson KL, Hrvoj-Mihic B, Semendeferi K (2014). A Dual Comparative Approach: Integrating Lines of Evidence from Human Evolutionary Neuroanatomy and Neurodevelopmental Disorders. Brain, Behavior, and Evolution, 84(2), 135-155. Barger N, Hanson KL, Schenker NM, Teffer K, Semendeferi K (2014). Evidence for evolutionary specialization in human limbic structures. Frontiers in Human Neuroscience 8 doi: 10.3389. Herai R, Stefanacci L, Hrvoj-Mihic B, Chailangkarn T, Hanson KL, Semendeferi, K, & Muotri AR (2014). Micro RNA detection in long-term fixed tissue of cortical glutamatergic pyramidal neurons after targeted laser-capture neuroanatomical microdissection. Journal of Neuroscience Methods 235:768-82. Lupfer-Johnson G, Hanson KL, Edwards LE, Elder RE, Evans SL (2009). Social cues influence foraging in dwarf hamsters (Phodopus campbelli). Journal of Comparative Psychology 123(2):226-9.

FELLOWSHIPS, GRANTS & AWARDS 2016 Institute for Neural Computation, UCSD, NIMH T32 Training Fellowship in Computational Neuroscience 2016 Williams Syndrome Association, Scholarship to attend annual convention 2016 Dean of Social Sciences Travel Award 2015 Summer Graduate Teaching Scholars Fellowship, UCSD

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2011-2014 CARTA Anthropogeny Specialization Student Fellowship 2013 Wenner Gren Foundation Dissertation Fieldwork Grant 2013 Freddie G. Bailey Student Research Fund, UCSD Anthropology 2013 Dean of Social Sciences Travel Award 2011 Dean of Social Sciences Travel Award 2010-11 Melford Spiro Award, Anthropology Department, UCSD 2009-10 Graduate Student Research Fellowship, Anthropology Department, UCSD 2008 Discovery Award, Psychology Department, University of Alaska Anchorage

PUBLISHED ABSTRACTS & PRESENTATIONS Hanson KL, Horton CF, Bellugi U, Semendeferi K (2017). Understanding human brain evolution through neuropathology: the case for Williams Syndrome. Podium presentation: Annual Meetings of the American Association of Physical Anthropologists, New Orleans, LA. Hanson KL, Groeninger K, Bellugi U, Semendeferi K (2017). Increased glia density in the associative and limbic striatum in Williams syndrome, a disorder of social cognition. Poster Presentation: Society for Social Neuroscience Annual Meetings, San Diego, CA. Hanson KL, Semendeferi K (2016). New approaches in modeling human brain evolution using neurodevelopmental disorders: the case for Williams syndrome. Poster Presentation: Annual Meetings of the Human Biology Association, Atlanta, GA. Hanson KL, Brown CN, Judd VA, Orfant H, Bellugi U, Semendeferi K (2016). Defining the phenotype: increases in neuronal density and glia-to-neuron ratio in the pre-commissural caudate head in Williams syndrome. Poster Presentation: Society for Neuroscience Annual Meetings, Chicago, IL. Semendeferi K & Hanson KL (2015). Plastic and Fantastic: Postnatal developmental changes and the evolution of the human brain. Podium Presentation: Annual Meetings of the American Association of Physical Anthropologists, St. Louis, MO. Hanson KL, Horton CF, Hrvoj-Mihic, B, Semendeferi K (2014). New approaches in modeling human brain evolution using neurodevelopmental disorders: the case for Williams syndrome. Podium Presentation: Annual Meetings of the Southwestern Association of Biological Anthropologists, Tucson, AZ. Hanson KL, Semendeferi K (2014). Evidence for derived cellular organization in the ventral striatum of the human brain. Poster Presentation: American Association of Physical Anthropologists, Calgary, AB, CA. Horton CF, Stefanacci L, Hanson KL, Wilder L, Yzurdiaga L, Brown, C, Bellugi U, Semendeferi K (2013). Williams Syndrome: a histological study of cortical neuronal density in the social brain. Poster Presentation: Society for Neuroscience Annual Meetings, San Diego, CA. Hrvoj-Mihic B, Stefanacci L, Hanson KL, Bellugi U, Jacobs B, Semendeferi K (2013). Williams syndrome: a preliminary investigation of the morphology of cortical pyramidal neurons. Poster Presentation: Society for Neuroscience Annual Meetings, San Diego, CA. Stefanacci L, Hanson KL, Horton C, Bienvenu T, Wattenberg A, Bellugi U, Semendeferi K (2012). Cortical layer distribution in the frontal cortex of humans: A histological study in typically developing adults and in Williams Syndrome. Poster Presentation: Society for Neuroscience Annual Meetings, New Orleans, LA. Barger N, Hanson KL, Semendeferi K (2012). Limbic Structures in Human Evolution: New Data and Meta-Analysis. Poster Presentation: American Association of Physical Anthropologists, Portland, OR.

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Lupfer-Johnson G, Hanson KL. Social foraging in P. campbelli: a laboratory investigation of the information centre hypothesis (2008). Oral Presentation: Behavioral Sciences Conference of the North (BSCN), Anchorage, AK. Hanson KL (2008). Juvenile-directed aggressive and agonistic interactions in Alouatta paliatta, mantled howler monkeys. Poster Presentation: American Association of Physical Anthropologists, Chicago, IL. Hanson KL (2008). Evidence for confirmation bias in perceptions of family resemblance. Oral Presentation: Behavioral Sciences Conference of the North (BSCN), Anchorage, AK.

TEACHING EXPERIENCE 2009-2010, 2014-2016 Teaching Assistant, Anthropology Department, UCSD

2015 Instructor of Record/Summer Graduate Teaching Scholar, ANTH 2: Human Origins, Summer Session II, University of California San Diego.

2008-2009 Substitute Teacher, Anchorage School District, STEM and Spanish Language Focus

LAB & FIELD EXPERIENCE 2009-present Graduate Research Assistant, Anthropology Department, UCSD Dr. Katerina Semendeferi (Principal Investigator), Laboratory of Human Comparative Neuroanatomy. Ongoing investigations include comparative histological analyses of the striatum in typically-developing humans and apes, and in humans with Williams syndrome, a neurodevelopmental disorder with a known genetic etiology, to target functional implications of neuroanatomical phenotypes.

2006-8 Research Assistant, Psychology Department, University of Alaska Anchorage Dr. Gwen Lupfer-Johnson (Principal Investigator). Designed and conducted experiments investigating dwarf hamster social foraging behavior.

2008 Student Excavator, The Rudabanya Hominid Origins Project Dr. David Begun (Principal Investigator), Miocene Ape Project

2007 Student Researcher, La Suerte Biological Field Station, Costa Rica Dr. Michelle Bezanson (field advisor), Primate Behavior & Ecology Field School

SPECIALIZED PROFESSIONAL TRAINING 2017 Certification in Clinical Trials Design and Management, UCSD Extension 2015 Participant, “The College Classroom” and “Preparing to Teach” with Dr. Peter Newbury Center for Teaching Development at UCSD 2014 Participant, Dr. Daniel Peterson’s Stereology Workshop for Biological Quantification Co-hosted by NeuroRenew and MBF Bioscience 2011 Graduate Student Researcher, Workshop in Light, Confocal, & Electron Microscopy National Center for Microscopy and Imaging Research (NCMIR), UCSD

ABSTRACT OF THE DISSERTATION

Neural substrates of impaired behavioral inhibition in Williams syndrome, a disorder of social cognition

Kari L. Hanson

Doctor of Philosophy in Anthropology with a Specialization in Anthropogeny

University of California, San Diego, 2017

Professor Katerina Semendeferi, Chair

The evolution of the human brain has yielded advanced cognitive capacities supporting the development of language, technologically advanced material culture, and highly complex social behavior that has allowed for the development of the rich diversity of human cultures. Comparative neuroanatomy in evolutionary perspective continues to make great strides in characterizing and defining unique elements of the human

xvi neuroanatomical phenotype at the gross and microscopic level that underlie these key behavioral adaptations. In conjunction with these studies, an understanding of the functional implications of derived anatomical traits is gained through analyses of neurodevelopmental disorders, which help to define a spectrum of variation in the diversity of human brain phenotypes. Williams syndrome (WS) is a rare neurodevelopmental disorder caused by a hemideletion of ~1.6 Mb (25-28 genes) on human chromosome 7q11.23, a highly dynamic region associated with recent adaptive selection in hominoid lineages. Analyzing the neuroanatomical phenotype in WS provides the unique opportunity to study correlates of a distinctive cognitive and behavioral phenotype in a neurodevelopmental disorder of known genetic cause. Among the most notable behavioral phenotypes observed in WS is a generalized disinhibition of social behavior, likely rooted in the dysfunction of frontostriatal circuitry. We targeted the rostral territories of the striatum in that share important connectivity with the prefrontal cortex in reward circuitry. We provide new evidence for variation in neuroanatomy in WS underlying its unusual social and cognitive phenotype. Specifically, we found increased glial cell density in the caudate nucleus of the striatum, as well as a significant increase in the density of oligodendrocytes in the medial caudate nucleus, likely related to dysfunctional connectivity with the prefrontal cortex. We additionally found a decrease in the density of cholinergic interneurons in the medial caudate nucleus, which may serve important functions in the regulation of social interaction. These data suggest that deficits in behavioral control may be linked to dysfunction of local circuitry within the striatum in WS, mediated by imbalance between neuronal and glial cell density and interneuron function, which may underlie important differences in social behavior.

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Chapter 1

INTRODUCTION

The human brain is comprised of more than one hundred billion neurons, interconnected in vast networks enabling specialized cognitive features including extremely sophisticated social systems (Dunbar, 1998; Kappeler and Van Schaik, 2002), habit, tradition, and ritual, introspection and mentalizing (Premack and Woodruff, 1978), advanced systems of material culture (Davidson and Mcgrew, 2005), and language

(Seyfarth et al., 2005; Arbib et al., 2008), that in many ways set us apart from our closest living relatives. Some have argued that brain size alone accounts for these derived abilities, and that the human brain represents a ‘scaled-up’ primate brain (Herculano-

Houzel, 2009). Still others have suggested that the brain’s macrostructural reorganization

(e.g., Holloway et al., 2003; Barger et al., 2007, 2014) and microstructural differences in the distribution of its neurons (Semendeferi et al., 1998, 2001, 2011; Barger et al., 2012;

Teffer et al., 2013) and glia (Sherwood et al., 2006) account for vast differences in cognition between hominoid taxa. Examinations of neurotransmitter systems additionally reveal differences in the brain’s innervation that may provide the substrates for important differences in synaptic plasticity and processing (Raghanti et al., 2008a, 2008b, 2008c,

2016). Varying morphology of individual neurons may further indicate specializations that contribute to differences in neurotransmission between anthropoid taxa (Bianchi et al., 2013a, 2013b; Stephenson et al., 2017). Ongoing investigations seek to parse these important differences between species with a focus on the functional significance of derived patterns of anatomical specializations.

1 2

In conjunction with these important studies, understanding the human brain’s evolution and function is aided by examining extreme variants of uniquely derived human features of cognition, such as complex social behavior and language. A variety of neurodevelopmental disorders differentially affect these domains of function, and patterns of differences observed between typically-developing individuals and those with such disorders bears consideration in the context of the considerable diversity of the human neurological phenotype. Many disorders, such as autism (Badcock and Crespi,

2006; Baron-Cohen, 2006; Crespi, 2016) and schizophrenia (Crow and T.J., 1995;

Randall, 1998; Brune and Brune-Cohrs, 2006; Crespi et al., 2007; Khaitovich et al., 2008;

Srinivasan et al., 2015), have been the focus of attempts to frame neurological disorders in evolutionary context. However, given the overwhelming heterogeneity of these disorders’ genetic underpinnings and phenotypic manifestations, such theories can offer interesting insights into the evolution of human-specific cognitive abilities and associated vulnerabilities, but may be highly speculative and rely on broad generalizations with regard to the mechanisms and timelines for the evolution of these traits.

Williams syndrome (WS) is a rare neurodevelopmental disorder caused by the hemideletion of ~25-28 genes at 7q11.23 on the long arm of the seventh chromosome that typically occurs during meiosis. It is characterized by multiple somatic effects including cardiac abnormalities, distinctive facial morphology, dental abnormalities, slowed growth, and short stature. Cognitively, individuals may present with an unusually hypersocial, gregarious, and disinhibited personality. Among the most interesting observations regarding WS is that its phenotype seems to be, in many ways, antithetical to the phenotype observed in Autism Spectrum Disorders (ASD), given the proclivity of 3 individuals with WS to engage in social interactions with strangers, maintain strong eye contact, and be quite talkative. By contrast, features of ASD can include social avoidance and language delays. This observation is all the more remarkable given that research has shown that a duplication of the region deleted in WS is associated almost universally with a classically ‘autistic’ behavioral phenotype (Mervis et al., 1993; Berg et al., 2007;

Velleman and Mervis, 2011; Sanders et al., 2012). Therefore, a spectrum of variation exists at this locus defined by hypersociality and relatively preserved language function in WS at one extreme, and pronounced speech and language deficits and social avoidance in duplication 7q11.23 syndrome (Velleman and Mervis, 2011) on the other. Notably, the this region has been analyzed across a range of nonhuman primate species, which has shown evidence for recent and rapid evolution at this locus, indicated by high numbers of segmental duplications. Alu sequence transposable elements located at the edges of the deleted region may have served as a source of very important interspecific variation in the anthropoid primate lineage that may predispose this region of the genome to genetic disruption. In other words, the region implicated in WS and Dup7 syndrome represents a

“hot spot” for recent genomic change.

WILLIAMS SYNDROME: A HISTORY OF THE COLLABORATION

Williams syndrome (WS) was first described in the early 1960’s by physicians

(Williams et al., 1961; Beuren et al., 1962) who independently noted the presentation of supravalvular aortic stenosis (SVAS, a congenital narrowing of the aorta) in a subset of patients who also had distinctive facial characteristics in common, coupled with mild to moderate mental retardation. Early reports (e.g., von Arnim and Engel, 1964) suggested that the disorder may include some unusual behavioral traits including a ‘friendly and 4 loquacious’ disposition with a tendency toward broad vocabulary in individuals that outpaced others (particularly children) of similar age and intellectual disability.

Diagnostic criteria were established in partnership with Dr. Kenneth Lyon Jones (Jones and Smith, 1975) at the University of California San Diego. Prof. Ursula Bellugi, then a specialist in language disorders at the Salk Institute for Biological Studies, began studying patients with WS in the most comprehensive, transdisciplinary long-term effort yet undertaken to understand the disorder’s unique cognitive and psychosocial profile

(Bellugi et al., 1994). Prof. Bellugi has recruited scientists from diverse fields including genetics, neuroimaging, and postmortem histology to help characterize the disorder’s unique neurological profile (e.g., NICHD-5P01HD033113-16).

Alongside these efforts, Prof. Bellugi has worked in close partnership with the

Williams Syndrome Association (Terry Monkaba, president) to facilitate postmortem brain donation between families who have lost loved ones living with the disorder. A collection of some 14 brains (11 adults and 3 infants) from individuals with WS are currently housed in the Laboratory for Human Comparative Neuroanatomy (PI: Prof.

Katerina Semendeferi) at UCSD. This collection constitutes the largest known repository of brain materials from individuals with WS. Among these, 7 individuals have been added to the collection within the last 6 years under the care of Prof. Semendeferi. Brain samples from 5 adults with WS have been utilized for the purposes of this dissertation, chosen for the completeness of the targeted regions of interest and their preservation allowing for their successful use in immunohistochemical staining protocols.

REGIONS OF INTEREST TARGETED

One way to conceptualize the hypersociability characteristic of WS may be to view it as a disorder of behavioral control, marked by the disinhibition of social behavior and the heightened saliency of specifically social stimuli. This would heavily implicate structures of reward circuitry including the prefrontal cortex and dorsal and ventral striatum. There is evidence to support this idea, given that performance in go/no-go tasks is characterized by slower reaction times and reduced activation of the anterior caudate nucleus in WS (Mobbs et al., 2007), in territories that receive important projections from the prefrontal cortex. Further evidence for this hypothesis was gained through structural imaging studies undertaken by Prof. Eric Halgren’s Multimodal Imaging Laboratory (Fan et al., 2017), which has shown that orbitofrontal cortex regions and the striatum display some of the most profound reductions in total volume observed in living subjects with

Williams syndrome, which may underlie the deficits in behavioral control. Research from our lab (Lew et al., 2017) has shown evidence for decreased neuronal density in

Brodmann area (BA) 11 of the orbitofrontal cortex, which likely contributes to this important frontostriatal dysfunction. Notably, the reduction in neuronal density in prefrontal cortex is most marked in the infragranular layers V/VI, which implicates deficits in functional connectivity with subcortical regions like the striatum, as opposed to corticocortical connectivity characteristic of upper layers 2/3. The prefrontal cortex shares important functional connectivity that defines a broad reward-related territory in the striatum, which has been the focus of the investigations outlined in this dissertation.

The striatum comprises the principle input nuclei of the basal ganglia, which receive diverse projections from areas of the prefrontal cortex (Haber, 2003; Haber et al., 6

2006) involved in reward-based learning and behavioral control. Frontostriatal connectivity (see Figure 1) is thought to underlie many complex processes associated with behavioral control, including behavioral response inhibition, particularly in the right hemisphere (Casey et al., 1997; Aron et al., 2014). Haber and colleagues (2006) have utilized tract tracing methods in the primate brain to define overlapping regions of interest within the striatum that receive projections from key prefrontal areas, defined as orbital prefrontal cortex (BAs 11, 13, 14, and 12), dorsolateral prefrontal cortex (BAs 9 and 46), and anterior cingulate cortex (BAs 24, 25, and 32). These rostral striatal territories were the focus of our investigations in WS, with the goal of understanding differences in certain cellular populations in these regions of interest that may contribute to frontostriatal deficits, behavioral disinhibition, and emotional dysregulation in WS.

OUTLINE OF THE DISSERTATION

Three manuscripts comprise the bulk of this dissertation, and are meant to serve as stand-alone publications. Chapter 2 serves as a theoretical introduction to dissertation, with the central assertion that neurodevelopmental disorders are of great value in conceptualizing the functional significance of derived morphological traits in the evolution of the human brain. It was originally published in part as an invited submission to the journal Brain, Behavior, and Evolution in conjunction with a symposium presentation for the annual Karger Workshop in Evolutionary Neuroscience, which sought to bring together diverse perspectives for studying the evolution of the human brain. The symposium was organized by Prof. Chet C. Sherwood (The George

Washington University), and Branka Hrvoj-Mihic and Prof. Katerina Semendeferi served as co-authors. The author of this dissertation was the primary author of the publication. 7

In Chapter 3, we targeted neurons and glia using Nissl-stained sections from our sample compared to typically-developing human control subjects. Previous research from our collection using Nissl-stained tissue in the prefrontal cortex (Lew et al., 2017) found a reduction in neuronal density in BA 11. We chose to quantify the density of neurons in the striatum, as well as glia, to better conceptualize the role that both cell types may play in characterizing the disorder’s neurological phenotype.

In Chapter 4, immunohistochemical methods were implemented in the WS collection for the very first time, owed to the specialized expertise of Prof. Mary Ann

Raghanti (Kent State University) who served as an important theoretical, technical, and methodological advisor. Prof. Raghanti developed the protocol we used, and provided extensive material and technical support during the entire course of the project, which would not have been possible without her expert support and guidance. Here, we targeted cholinergic interneurons in the striatum for the purpose of understanding these unique cells’ contribution to the WS phenotype.

Chapter 5 provides a general discussion and conclusions for the dissertation, and includes future directions for defining the phenotype in WS that contributes to its unusual behavioral and cognitive phenotype. Future investigations will include extended study of subtypes of interneurons and glia in WS, as well as in non-human primates, with the goal of understanding trends in the diversity and evolution of the human social brain.

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BA 9, 46

BA 11,12,13,14 IC Dorsolateral Caudate

Medial Caudate

Putamen Nucleus Accumbens BA 24b, 25, 32

Figure 1.1. Regions of Interest. Striatal territories investigated are defined by connectivity with prefrontal cortical areas. Adapted from Haber et al., 2006. Sampling took place in a consistent manner well within the

regions of interest with care to avoid transitional areas and white matter inclusions.

Chapter 2

A Dual Comparative Approach: Integrating Lines of Evidence from Human Evolutionary Neuroanatomy and Neurodevelopmental Disorders1

ABSTRACT

The evolution of the human brain has been marked by a nearly three-fold increase in size since our divergence from the last common ancestor shared with chimpanzees and bonobos. Despite increased interest in comparative neuroanatomy and phylogenetic methods, relatively little is known regarding the effects that this enlargement has had on its internal organization, and how certain areas of the brain have differentially expanded over evolutionary time. Analyses of the microstructure of several regions of the human cortex and subcortical structures have demonstrated subtle changes at the cellular and molecular level, suggesting that the human brain is more than simply a ‘scaled-up’ primate brain. Ongoing research in comparative neuroanatomy has much to offer our understanding of human brain evolution.

Through analysis of the neuroanatomical phenotype at the level of reorganization in cytoarchitecture and cellular morphology, new data continue to highlight changes in cell density and organization associated with volumetric changes in discrete regions.

An understanding of the functional significance of variation in neural circuitry can further be approached through studies of atypical human development. Many

1 Chapter 2 is an abridged version of a review previously published by the author of this dissertation for an invited submission. Citation: Hanson KL, Hrvoj-Mihic B, Semendeferi K (2014). A Dual Comparative Approach: Integrating Lines of Evidence from Human Evolutionary Neuroanatomy and Neurodevelopmental Disorders. Brain, Behavior, and Evolution, 84(2), 135-155. The author of this dissertation was the primary investigator and author.

14 15 neurodevelopmental disorders cause disruption in systems associated with uniquely human features of cognition, including language and social cognition. Understanding the genetic and developmental mechanisms that underlie variation in the human cognitive phenotype can help to clarify the functional significance of interspecific variation. By uniting approaches from comparative neuroanatomy and neuropathology, insights can be gained that clarify trends in human evolution. Here, we explore these lines of evidence, and their significance for understanding functional variation between species, and within neuropathological variation in the human brain.

INTRODUCTION

For primates, as particularly social mammals, the complexity of the social environment has been suggested as a principal driving force in the evolution of advanced cognitive faculties (Humphrey, 1976; Byrne and Whiten, 1989; Dunbar, 1998), encouraging the evolution of increased size and greater complexity in brain areas associated with social cognition, and the understanding of interindividual relationships between conspecifics. The challenges that a dynamic and hierarchical social environment present have fostered the development of advanced capacities for behavioral flexibility

(Strier, 2006), inhibitory control of behavior (Amici et al., 2008), and associative learning

(Haber et al., 2006, Williams et al., 2006) across primate lineages. Precipitous gains in object-focused intelligence and tool use (Byrne, 2004; Whiten and van Schaik, 2007), as well as language use (Tomasello et al., 1997; Deacon, 1997), and increased capacity for social learning through imitation (e.g., Call and Tomasello, 1995; Subiaul, 2007) and theory of mind (Povinelli and Eddy, 1996; Hare, 2007) have further characterized uniquely human features of human cognition and behavior. 16

By utilizing a comparative approach, we can better understand the differences in the brain’s anatomy that underlie these key cognitive specializations. In addition to overall changes in brain size (Falk et al., 2000; Holloway, 2004) and the expansion of the cerebral cortex (Rilling and Insel, 1999), volumetric studies at the gross level have shown modifications that suggest reorganization in certain regions of the human brain (Barton and Harvey, 2000; Aldridge, 2011). While humans and apes share a large frontal cortex

(Semendeferi et al., 2002; Bush and Allman, 2004; Barton and Venditti, 2013), volumetric differences in certain areas suggest reorganization in areas of the prefrontal cortex (Semendeferi et al., 2001; Smaers, 2013; and Passingham, 2014). Reduction in the size of the primary visual cortex (Holloway et al., 2003), coupled with the expansion of parietal cortices (Bruner, 2010), additionally seem to suggest a trade-off in relative sizes of these regions. Furthermore, major bilateral expansion of the temporal lobes has been observed (Semendeferi and Damasio, 2000; Rilling and Seligman, 2002). Increases in white matter volume have been noted in areas of the prefrontal cortex (Smaers et al.,

2010; but see also Sherwood et al., 2005) and broadly in the gyral regions of the cortex as compared to the core (Schenker et al., 2005) suggesting important differences in local connectivity between cortical regions. With regard to long-range connectivity between regions, increases in fractional anisotropy (FA) have been noted in the arcuate fasciculus

(Rilling et al., 2008; Rilling et al., 2011), associated with increased density of fiber tracts connecting areas involved in language use and production, along with important differences in fiber terminations in expanded associative territories. Associations between frontal white matter volume and basal ganglia volume (Smaers et al., 2010) further show evidence for human-specific increases in fronto-striatal connectivity underlying key 17 differences in executive control.

Differences in volume and connectivity in functional areas are likely reflected in subtle, yet important changes at the level of cellular organization. Indeed, differences in cytoarchitecture have been observed in certain areas of the prefrontal cortex, including increased neuropil spacing in human frontopolar (Brodmann’s area; BA 10) and frontoinsular cortices as compared to chimpanzees (Spocter et al., 2012). Increased spacing between cortical minicolumns in the human frontal pole (Semendeferi et al.,

2011) provides further evidence for reorganization in this region, suggesting differences in cellular organization and morphology related to its relative increase in size

(Semendeferi et al., 2001). These species differences in BA 10 in adults seem to reflect important changes in development: whereas horizontal spacing distance (HSD) between cortical minicolumns increased with age pre- vs. post-weaning in human subjects, similar increases were not observed in chimpanzees (Teffer et al., 2013). Increased dendritic branching of pyramidal neurons in BA 10 (Bianchi et al., 2013) seems to correlate with this trend toward increased space, presumably to accommodate increased connections between cells. Wider minicolumnar spacing in development may necessarily correspond to expanded surface area (Rakic and Kornack, 2001). Indeed, differences in white matter density seem to reflect regionally-specific differences in connectivity (Schenker et al.,

2005), and disruption in cortical layering patterns is also reflected in gyral convolutions

(Van Essen 1997, Hilgetag and Barbas, 2006). Therefore, differences observed at the level of individual cortical layers and neurons across several primate species can provide insights into evolutionary changes underlying gross-anatomical variations across taxa.

Increasingly, studies targeting neurodevelopmental disorders characterized by 18 perturbations of gross morphology of the brain also find differences in its intrinsic microstructure. For example, narrower minicolumnar spacing has been noted in the prefrontal cortex of individuals with autism (Casanova et al., 2002; Casanova et al., 2006;

Buxhoeveden et al., 2006), which may contribute to selective deficits in executive function and social engagement characteristic of the disorder. A developmental shift to wider minicolumnar spacing in the frontal pole post-weaning is a feature of human, but not chimpanzee, cortical development (Teffer et al., 2013). We can therefore expect that this important developmental event affects cellular morphology in a way that, in turn, influences cellular function, affecting important features of cognition and behavior that are impaired in autism (Courchesne et al., 2011). Thus, variation in development observed within the human species could be of significance for understanding the functional implications of differences in cognition between species.

Uniting approaches from the study of neurodevelopmental disorders and comparative evolutionary neuroanatomy offers powerful tools for understanding the genetic, developmental, and neuroanatomical influences on the human cognitive and behavioral phenotype. Here, we will discuss variation in cortical and subcortical microstructure both between hominoid species (i.e., apes and humans), and among pathological variations in the human neuroanatomical phenotype, drawing parallels in morphology and function to elucidate mechanisms that underlie inter- and intraspecific variability in cognition and behavior. Some neurodevelopmental disorders may provide better models than others for understanding functional implications of neurodevelopmental abnormalities, such as autism and schizophrenia, due to their specific influences on uniquely derived human cognitive traits and social behaviors. 19

Given its well-characterized genetic etiology and consistent social and cognitive phenotype, we will further argue that Williams syndrome can provide an ideal model for understanding variation in the human neuroanatomical phenotype underlying important behavioral specializations.

Inhibitory Interneurons in the Cortex

While pyramidal neurons are the most ubiquitous cell type in the cerebral cortex and may be a primary target of evolutionary specialization (see Hanson, Hrvoj-Mihic, and Semendeferi 2014 for review), a diverse array of interneuron subtypes act locally to modulate the activity of these pyramidal cells. In the primate cerebral cortex, approximately 15-30% of neurons are inhibitory, GABAergic interneurons (DeFelipe,

1997; Sherwood et al., 2010). Inhibitory interneurons of the cerebral cortex serve an important role in modulating excitatory neuronal function, including control of rhythmic oscillations across assemblages of pyramidal neurons (DeFelipe, 1997) important for the regulation of spike-timing dependent synaptic plasticity. GABAergic interneurons additionally regulate subcortical input (Hendry et al., 1987; Porter et al., 2001) to the cortex.

In the cortex, the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin co-localize with GABA in 90-95% of interneurons. These interneurons display a variety of morphological types, including parvalbumin-reactive (PV) chandelier and large basket cells, calbindin-reactive (CB) double bouquet cells, calretinin-reactive

(CR) bipolar, double bouquet cells, and some Cajal-Retzius cells. The exact morphology and pattern of synaptic formation of each class of GABAergic inhibitory interneurons varies relative to the cortical region and layer in which it resides, but can be distinguished 20 according to general patterns (Ascoli et al., 2008; DeFelipe et al., 2013). Functionally- distinct types of inhibitory interneurons, characterized by differences in immunochemical markers and postsynaptic morphology, owe their distinctive traits to differences in their origin and regulation (Wonders and Anderson, 2006) during fetal development.

Maintaining the delicate balance between excitatory and inhibitory neuronal activity is essential for proper brain function. Many neurodevelopmental disorders have been shown to involve disruption in the balance between excitatory and inhibitory cortical neurons, as revealed through postmortem anatomical studies, and differential activity, as measured in vivo in animal models of these disorders (Marín, 2012). Many of these disorders differentially affect certain cognitive faculties that are peculiar to the human behavioral phenotype, including language use and complex aspects of social cognition. Schizophrenia, for example, is a severe psychiatric disorder characterized by delusions and hallucinations, in addition to cognitive deficits in attention and working memory, as well as major deficits in social behavior and recognition of affect (Green and

Horan, 2010). Recent research suggests that schizophrenia’s unique symptoms are the result of vulnerabilities associated with recent adaptive genomic changes associated with the human neurocognitive phenotype (Crespi et al., 2007; Crow, 2000; Khaitovich et al.,

2008). Specifically, the rapid evolution of certain traits, including linguistic capacity

(Crow, 1998; Crow, 2000) and advanced aspects of social cognition, such as empathy and mentalizing (Burns, 2006; Brüne, 2005) and executive function, which are severely affected in schizophrenia, point to derived features of the human neuroanatomical phenotype supporting these unique faculties. Cognitive effects of schizophrenia seem to point to the involvement of a breakdown in synchronization of gamma oscillations in the 21 dorsolateral prefrontal cortex. These electrophysiological data implicate dysfunction in fast-spiking, parvalbumin-positive inhibitory interneurons, which are reportedly decreased in number (Benes, 1991). More nuanced mechanisms are likely also involved: in parvalbumin-positive basket cells (PVBCs), decreased expression of GAD67, an enzyme associated with GABA transmission, underlies inefficacy in the inhibitory control of pyramidal neurons (Lewis et al., 2012). A variety of other genes are likely involved in the dysfunction of PVBCs, and animal models of the disorder have highlighted the involvement of several promising candidate genes in their dysfunction.

Autism spectrum disorder (ASD) additionally affects aspects of social cognition that merit study in a comparative framework, and associated pathophysiologies point to the involvement of interneuron dysfunction in these disorders’ neuroanatomical phenotype. Functional studies (Orekhova et al., 2007; Dinstein et al., 2011) have shown disruptions in oscillatory frequencies, particularly in response inhibition tasks

(Rubenstein and Merzenich, 2003; Yizhar et al., 2011) that implicate dysfunction in the balance of inhibitory and excitatory neuronal activity. Reductions in GABAergic receptors in the anterior (ACC) and posterior (PCC) cingulate cortices, as well as the fusiform gyrus, have also been demonstrated in autism (Oblak et al., 2009, 2010, 2011).

Postmortem histological investigations have found reductions in the number of PV interneurons in the dorsolateral prefrontal cortex—specifically BA 9—in a small sample of autistic patients (Zikopoulos and Barbas, 2013). Additionally, this study found that the overall ratio of PV vs. CB interneurons showed a decrease by nearly one third, suggesting decreased inhibitory efficacy in local networks. A relative increase of PV interneuron ratios is a derived trait in the primate cortex, as compared to other mammalian species 22

(Dombrowski, 2001). This could have interesting implications for derived features of inhibitory control that underlie social cognition, such that cellular mechanisms contributing to inhibitory control of behavior could have been a target of selection, of particular importance for animals living in hierarchical social groups (Amici et al., 2008).

Many other cortical areas, including the fusiform face area (FFA), show evidence for disrupted synchrony between alpha and gamma oscillatory frequencies (Buard et al.,

2013), which further implicate PV interneuron dysfunction. It is likely that the downstream effects of the decrease in PV interneurons in BA 9 are systemic in nature

(Zikopoulos and Barbas, 2013), and contribute to both local and global dysfunction in mechanisms of attention and cognition.

Whereas multiple molecular and environmental mechanisms have been shown to contribute to autism and schizophrenia, other disorders with a more clear-cut genetic etiology show evidence for pathologies of interneuronal function that affect social cognition. In Fragile X syndrome, for example, reduction in numbers of interneurons have been noted, as well as a reduction in numbers of GABA receptors, likely mediated by a global reduction in the expression of GAD65 and GAD67 (Mulle et al., 2007; Curia et al., 2009). Similar mechanisms seem to underlie deficits in learning and memory in

RTT, where decreased levels of cortical GABAergic transmission cause impairments in inhibitory function (Chao et al., 2010). Further study of the neuroanatomical phenotype and associated perturbations of inhibitory function in disorders characterized by known genetic deletions will help to elucidate mechanisms of interneuronal function that underlie differences in cognition.

Studying the developmental and genetic mechanisms that underlie disorders 23 affecting uniquely human aspects of cognition can provide an essential window into mechanisms guiding normative social and cognitive development and its evolution.

Through comparative analysis of the neuroanatomical phenotype, we can begin to further understand how the function of specialized cells contributes to interspecific variation in cognitive abilities. Previous research, however, has not found differences in inhibitory interneuron numbers or their distribution in cortical areas, including BA 9, BA 4, BA 32, and BA 44, between humans and anthropoid primates (Sherwood et al., 2010). The conserved phenotype displayed by inhibitory interneurons in the cortex suggests instead that other genetic and regulatory mechanisms contribute to differences in functional regulation of pyramidal cells by inhibitory interneurons. These may be particularly vulnerable to the effects of cognitive dysfunction manifested in neuropsychiatric disorders.

The contribution of neurotransmitter systems to cortical function likely implicates key species-level differences in the distribution of neurotransmitter fibers and varicosities

(Raghanti et al., 2008a; Raghanti et al., 2008b; Raghanti et al., 2008c; Raghanti et al.,

2010) which contribute to differences in the function of assemblages of cortical neurons.

Specifically, loss of cholinergic innervation in schizophrenia (Sarter et al., 2005) and

ASD (Lam et al., 2006) plays an important role in imbalances in calcium regulation in

GABAergic interneurons, particularly CB-reactive double bouquet cells, which are major sites of cholinergic inputs (Xiang, 1998). Given that distinctive clusters of cholinergic fibers were found in human and chimpanzee prefrontal cortex, but not in macaques, fundamental differences in cholinergic innervation likely play a role in cognitive differences (Raghanti et al., 2008c). This is likely related to species-level differences in 24 the organization of the nucleus basalis of Meynert (Raghanti et al., 2011), where cholinergic projections originate. Additionally, differences in serotonergic innervation seem to play a role in cognitive symptomatology in schizophrenia via regulation of oscillatory frequencies (Puig and Gulledge, 2011). Serotonergic innervation shows some evidence of species-specific patterning, with greater innervation in BAs 9 and 32 in layers V/VI of the human and chimpanzee cortex as compared to macaques (Raghanti et al., 2008b). Further, differences in dopaminergic innervation in layers III and V/VI among humans and chimpanzees, but not macaques, have been noted. This is of particular interest, given that layer VI shows a pattern of decreased dopaminergic innervation in schizophrenic subjects as opposed to controls in BA 9 of the prefrontal cortex (Akil et al., 1999). Given the involvement of BA 9 in theory-of-mind tasks

(Gallagher and Frith, 2003), variation in neurotransmitter innervation in this area likely contributes to inter- and intraspecific variation in mentalizing and social cognition.

Overall, the dysfunction of cortical interneurons in a variety of neurodevelopmental disorders involving deficits in cognition points to their crucial role in supporting normative behavior. Similarly to pyramidal neurons, their distribution and morphology bear further investigation in comparative studies. Aberrations of neuronal circuitry contributing to selected deficits in cognition are not restricted to the cortex, however, and we must also consider the functional role that differences cellular organization may play in subcortical systems supporting behavior as well.

Beyond the Cortex: Subcortical Systems for Behavioral Control

Given that subcortical regions share diverse connections with the cerebral cortex, and in light of the phylogenetically dynamic nature of cortical modification (Semendeferi 25 et al., 2011; Bauernfeind et al., 2013; Semendeferi et al., 1998), it should be expected that subcortical structures reflect the changes in the cortical areas with which they share connections (Vilensky et al., 1982). Our recent analyses of subcortical structures in a broad sample of primate species have shown that some have not kept pace with the expanding cerebral cortex, while others are disproportionately enlarged (Barger et al.,

2014). Whereas two limbic structures, the amygdala and hippocampus, scale larger in humans than would be expected in a hominoid brain of its size, the striatum falls below expected values for total volume among anthropoids. This has interesting implications for the intrinsic organization of these structures, such that relative size of structures may affect cell density and number, and the distribution of different cell types within interconnected regions of the brain.

The amygdala, for example, has been shown to be an important site of reorganization in the evolution of the human brain. Volumetric reorganization of the basolateral division has been marked by differential expansion of discrete nuclei, with the lateral nucleus seeing disproportionate increases, compared to the basal nucleus, which constitutes the largest subdivision in nonhuman apes (Barger et al., 2007). These important volumetric differences are reflected in reorganization at the cellular level: the lateral nucleus in humans shows disproportionate increases in total cellular number when compared to nonhuman apes (Barger et al., 2012). The shift in emphasis on the lateral nucleus in human evolution, which shares dense connections with the expanded temporal cortex, likely underlies functional differences in emotional processing and social cognition which helps to account for behavioral differences between humans and our closest living relatives. 26

Total volume of the hippocampus has not been shown to stand out as differentially enlarged in primate species (Barton, 2000), though its reorganization may underlie key differences. In a small sample of insectivores, rodents, and three species of primates, discrete regions of the hippocampus were found to have increased in relative size among primate species (West, 1990). Therefore, comparative data may show important differences in the distribution of neuronal types and their morphology in the human hippocampus related to its relative increase in size in the human lineage (Barger et al., 2014). This suggests the potential for modification at the cellular level underlying specializations in learning and memory.

Other subcortical structures have been shown to be sites of reorganization in the human brain. The hominoid thalamus has seen differential changes in the size of several of its nuclei, with evidence for major modifications at the cellular level. Among motor components of the thalamus, the ventrolateral complex has about 1.5 times as many neurons in humans as are found in the same region in great apes (Armstrong, 1980a). In its associative territories, the human pulvinar nucleus scales larger than expected as compared to other hominoids, and contains nearly twice as many neurons (Armstrong,

1981). Among the anterior nuclei, most closely associated with limbic connectivity and function, major differences in the relative size of these nuclei have not been found. While a general decrease in cell density is a feature common to most thalamic nuclei in humans, this trend is less pronounced in the anterior nuclei, suggesting differential migration of greater numbers of neurons to limbic associated territories in the thalamus during development (Armstrong, 1980b; Armstrong, 1986). Indeed, differences in migration have been noted in regions of the human thalamus, which attract migrating streams of 27

GABAergic interneurons from the telencephalic ganglionic eminence, as compared to the diencephalic origins of these interneurons (Letenic and Rakic, 2001) observed in the development of the macaque brain.

Many other subcortical structures of particular potential interest for human cognitive evolution remain unexplored with respect to how changes in relative size has affected their intrinsic organization at the cellular level. Elements of the basal ganglia are of particular interest, owing to their important role in cognition and behavior. Evidence from cognitive studies has increasingly elucidated the importance of the striatum, particularly the caudate nucleus, as a site of integration for higher-order processes, including action selection, decision-making, reward-based and associative learning

(Grahn et al., 2009; Balleine and O’Doherty, 2010), as well as inhibitory control of behavioral responses to stimuli (Aron and Poldrack, 2005). Recent findings have shown that inhibitory control of behavior varies in species-specific patterns based on ecological and behavioral factors (Shumaker et al., 2001; Amici et al., 2008; Wobber et al., 2010).

The involvement of the caudate in planning and execution of motor sequences (Graybiel,

1998; van den Heuvel et al., 2003), essential for tool use and production, further hints at its important role in human cognition. Further, insights from Huntington’s disease, which causes volumetric loss and cell death in the caudate, have highlighted its functional importance in recursive elements of language, such as syntax and hierarchical structure

(Longworth et al., 2005; Teichmann et al., 2006), that are defining features of human language (Pinker and Jackendoff, 2005; Read, 2008).

Analyzing potentially aberrant neuronal distributions in neurodevelopmental disorders that seem to implicate subcortical structures as sites of dysfunction also helps to 28 elucidate mechanisms underlying uniquely human features of cognition. In autism, for example, the amygdala seems to be particularly affected by developmental processes inherent to the disorder. While Schumann and Amaral (2006) found no volumetric differences in the size of the whole amygdala or its main constituent nuclei, substantial reductions in cell numbers in the lateral nucleus were observed, which may contribute to selective deficits in social behavior. Increases in cell number in the lateral nucleus were found to be a unique feature of the human amygdala as compared to apes, which likely underlies functional differences contributing to uniquely human features of emotional regulation and cognition (Barger et al., 2012).

Just as differences in the organization of the cortex have been noted between humans and non-human hominoids, reorganization of subcortical structures, including limbic regions, seems to be an important feature of the human brain’s evolution.

Targeting disruptions in their intrinsic microstructure in neuropathological conditions, and how this affects cognition and behavior, is of key importance for inferring functional consequences of evolutionary reorganization. Disorders that affect language use, social cognition, object-focused intelligence, and other features of human cognition that may be scaled up in our lineage are of particular interest for understanding anatomical specializations and their function. Particularly where the genetic mechanisms guiding variations in neural circuitry may be discernable, we can begin to examine the developmental processes underlying functional variations in neural systems.

Modeling Human Evolution through Genetic Disorders: The Case for Williams

Syndrome

A variety of neurodevelopmental disorders affect important features of human cognition and behavior. The processes underlying pathological variation are often complex, and involve diverse genetic mechanisms, as is the case in schizophrenia

(Eisenberg and Berman, 2010) and ASD (Freitag, 2007), which seems to involve a high rate of de novo mutations (Sebat et al., 2007; Sanders et al., 2012). Other neurodevelopmental disorders, particularly those with a more well-defined genetic etiology, may provide better models for studying links between neuropathology, function, and variation in human evolution. Disorders, such as Williams syndrome, that are caused by a known deletion can provide a “living knock-out model” for studying associations between atypical genotypes and neuropathological phenotypes (Bellugi et al., 1999;

Järvinen-Pasley et al., 2008). Williams syndrome is a rare genetic disorder caused by a hemizygous deletion of about 26-28 genes on chromosome band 7q11.23. This region has undergone a number of sweeps of purifying selection in the hominoid lineage

(Antonell et al., 2005) suggesting it represents a “hot spot” for genomic change in recent human evolution.

Williams syndrome is characterized by multiple systemic effects, including distinctive facial morphology, slowed growth, and cardiac abnormalities (Williams et al.,

1961; Beuren et al., 1962), in part related to the deletion of the gene for elastin, which is used as the definitive marker for the disorder. Individuals with Williams syndrome additionally display a relatively predictable and consistent cognitive and behavioral phenotype, including global reductions in total IQ, particularly with respect to 30 visuospatial intelligence (Meyer-Lindenberg et al., 2006). Curiously, individuals with

Williams syndrome have been shown to demonstrate linguistic capacity characterized by elaborated vocabulary and unusually expressive use of phrases (Udwin and Yule, 2005;

Reilly et al., 2004), but struggle with elements of syntax, grammatical structure, and embedded meaning (Karmiloff-Smith et al., 1997; Brock, 2007). Among the most salient features of Williams syndrome, affected individuals tend to show an abnormally high drive to engage in social and affiliative behavior, and exhibit an unusual willingness to approach strangers (Doyle et al., 2004; Järvinen-Pasley et al., 2008).

Specific cognitive impairments in Williams syndrome have been linked to a variety of structural and functional abnormalities noted in patients diagnosed with the disorder. Brains of individuals with Williams syndrome tend to be smaller overall than in typically-developing individuals (Jernigan and Bellugi, 1990), with notable size reductions found in the parietal lobule (Eckert et al., 2005), occipital grey matter (Reiss et al., 2000), intraparietal sulcus, and orbitofrontal cortex (Meyer-Lindenberg et al., 2006).

Functional abnormalities have been demonstrated with respect to primary auditory

(Levitin et al., 2003) and visual perception (Atkinson et al., 2007). Additionally, deficits in object-focused and spatial cognition and memory have implicated the dorsal visual stream (Atkinson et al., 2003) and hippocampal formation (Meyer-Lindenberg et al.,

2005a).

To date, histological studies in Williams syndrome have targeted primarily cortical areas. Primary visual cortex in a postmortem sample of Williams syndrome patients has shown abnormalities in cell size and packing density (Galaburda and Bellugi,

2000). Additionally, primary auditory cortex shows no significant difference in cell 31 density, despite lacking differences in asymmetry in this region as compared to normal controls. A greater proportion of larger neurons in layer II of the primary auditory cortex, as well as enlarged neuronal size in layer IV (Holinger et al., 2005), suggests differences in connectivity with limbic regions that reflect differences in emotional reactivity to sound, particularly music.

Subcortical structures merit further study in Williams syndrome to determine microstructural abnormalities that underlie tradeoffs between cognitive deficits and conserved or enhanced processes that manifest in the disorder. Given structural abnormalities present in the hippocampal formation, and its importance in spatial cognition, cytoarchitectural study of this region is warranted (Meyer-Lindenberg et al.,

2005a; Meyer-Lindenberg et al., 2006). Additionally, the amygdala has shown decreased reactivity to threatening stimuli and fearful faces in individuals with Williams syndrome as compared to typically-developing controls (Meyer-Lindenberg et al., 2005b).

Investigations of cellular distributions and morphology in the amygdala will help to elucidate the limbic correlates of emotional processing differences (Doyle et al., 2004;

Bellugi et al., 1999) displayed in Williams syndrome.

Evidence from functional imaging studies has also suggested that the striatum may be differentially affected by developmental abnormalities characteristic of the disorder. Hypersociality and increased affiliative drive in Williams syndrome patients represent selected deficits in the ability to suppress behavioral responses (Frigerio et al.,

2006). Attention deficit hyperactivity disorder (ADHD) is a frequent comorbid diagnosis in these individuals (Carrasco et al., 2005). Mobbs and colleagues (2007) showed slower reaction times among Williams syndrome patients in a go/no-go task as compared with 32 typically-developing control participants, and substantially reduced BOLD signal activity in the striatum was observed. Given the role of frontostriatal circuits in controlling behavioral responses, microstructural abnormalities in the striatum, and particularly the caudate (Reiss et al., 2004) may underlie behavioral deficits in response inhibition and the control of behavioral responses.

Features of language use in Williams syndrome further point to a role for the striatum in the disorder’s neurogenetic phenotype. Deficits in sentence morphology and syntax (Karmiloff-Smith et al., 1997) indicate difficulty in encoding rules of proper language use and grammatical structure (Thomas and Karmiloff-Smith, 2005; Clahsen,

1998). Given the role of the striatum in recursive elements and rule-encoding in language use and acquisition (Teichmann et al., 2006) these processing abnormalities might be related to differences in striatal microstructure.

Perhaps the best-described feature of individuals with Williams syndrome includes what has been described as hypersociability, and the intense drive to engage in social interactions with strangers. Given this exaggerated proclivity to engage in affiliative behavior, as well as heightened reactivity to emotional stimuli, such as music,

Dai and colleagues (2012) expected to find an increase in basal levels of the social neuropeptide oxytocin in adult individuals with Williams syndrome as compared to typically-developed control participants. Indeed, individuals with Williams syndrome had higher baseline levels of oxytocin, and these results correlated positively with social approach, and negatively with adaptive social behavior. Given the relationship between oxytocin receptors and dopaminergic input to the nucleus accumbens (Aragona et al.,

2006; Insel, 2003; Carter, 1998) differences in dopaminergic innervation in the ventral 33 striatum, as well as the morphology of the nucleus accumbens, hint at a functional role of the subcortical systems in modulating these behavioral features.

Preliminary analysis of pyramidal neurons in Williams syndrome has revealed anomalies in the organization of dendritic trees specific to a particular cortical layer. In layers II/III the neurons displayed aberrations from typical cells across cortical areas as seen in controls (Hrvoj-Mihic et al., 2013a), whereas in the layers V/VI the neurons displayed more elaborated dendritic branching compared to controls (Chailangkarn et al.,

2013). Interestingly, the deletion that causes Williams syndrome, at the 7q11.23 chromosomal region (Osborne et al., 1996), is duplicated in some cases of ASD (Sanders et al., 2012). At the same time, the two disorders are characterized by different social, cognitive, and verbal phenotypes. A comparison between Williams syndrome and ASD at different levels, encompassing macroanatomical and microstructural changes, cognitive phenotypes, and genetic underpinnings of the each disorder, can offer an exciting new paradigm for evolutionary analyses encompassing several levels of inquiry across several scientific fields.

Conclusions

Ecological pressures, including the increased complexity of the social milieu, have driven the development of highly specialized morphology in primate taxa. Despite the importance of the human brain’s anatomical specializations for providing the material substrate to support extraordinary behavioral flexibility, complex cognition, and language as hallmark features of human evolution, relatively little is known about the reorganization of the brain that supports these unique faculties. While studying the evolution of the brain through fossil endocasts offers the only direct evidence of its 34 expansion and potential reorganization, these investigations are limited to examining changes in size and external gross morphology of its structural features. Targeting indirect evidence through comparative approaches allows for inferences to be made about human brain evolution from a phylogenetic perspective. A variety of noninvasive, non- destructive neuroimaging techniques, including magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI), allow for the comparative study of brain structure in extant taxa, and can provide great insights into structure and connectivity of structures, respectively.

Changes in the volume and connectivity of brain structures often entail an increase in total neuron number and processing capacity, and evidence at the microstructural level continues to reveal unique patterns of cellular organization, neurotransmitter distribution, and development that show that the human brain is not simply an enlarged primate brain.

Patterns of reorganization in the human brain remain enigmatic, and the effects that the expanding cerebral cortex has had on cellular distributions in cortical and subcortical regions as a result bear further study. Investigations of hominoid cellular neuroanatomy in comparative perspective have been limited by the practical considerations involved in securing rare brain materials from critically endangered great ape taxa. Analyzing potentially aberrant neuronal distributions in neurodevelopmental disorders, in addition to phylogenetic comparative approaches, will help to elucidate functional mechanisms underlying complex cognition. Given the dynamic and rapid changes observed in the region of the human genome deleted in Williams syndrome, and its unusual and consistent behavioral phenotype, understanding the effects of this deletion may further help us to better conceptualize the dynamic interplay of genetic influences and 35 development on the uniquely human neuroanatomical phenotype.

ACKNOWLEDGEMENTS

We would like to thank Dr. Chet Sherwood and the participants of the 25th annual

Karger Workshop in Evolutionary Neuroscience for the opportunity to share diverse, transdisciplinary perspectives with the common goal of understanding and describing the evolution of brain and mind. This paper is dedicated to the memory of Drs. Lisa

Stefanacci and Gary van Hoesen, who continue to inspire ongoing research and scholarship in comparative neuroanatomy from an evolutionary perspective. Support has been provided by the National Institute for Health (P01NICHD033113), and the Wenner

Gren Foundation (Dissertation Fieldwork Grant).

Chapter 2 is an abridged version of a review previously published by the author of this dissertation for an invited submission. Citation: Hanson KL, Hrvoj-Mihic B,

Semendeferi K (2014). A Dual Comparative Approach: Integrating Lines of Evidence from Human Evolutionary Neuroanatomy and Neurodevelopmental Disorders. Brain,

Behavior, and Evolution, 84(2), 135-155. The author of this dissertation was the primary investigator and author. 36

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Chapter 3

Increased glia density in the caudate nucleus in Williams syndrome: implications for frontostriatal dysfunction2

Abstract

Williams syndrome (WS) is a rare neurodevelopmental disorder with a well- described, known genetic etiology. In contrast to Autism Spectrum Disorders (ASD), WS has a unique phenotype characterized by global reductions in IQ and visuospatial ability, with relatively preserved function, enhanced reactivity to social stimuli and music, and an unusual eagerness to interact socially with strangers. A duplication of the deleted region in WS has been implicated in a subset of ASD cases, defining a spectrum of genetic and behavioral variation at this locus defined by these opposite extremes in social behavior.

The hypersociability characteristic of WS may be linked to abnormalities of frontostriatal circuitry that manifest as deficits in inhibitory control of behavior. Here, we examined the density of neurons and glia in associative and limbic territories of the striatum including the caudate, putamen, and nucleus accumbens regions in Nissl stained sections in five pairs of age, sex, and hemisphere-matched WS and typically-developing control (TD) subjects. In contrast to what is reported in ASD, no significant increase in overall neuron density was observed in this study. However, we found a significant increase in the density of glia in the dorsal caudate nucleus, and in the ratio of glia to neurons in the dorsal and medial caudate nucleus in WS, accompanied by a significant increase

2 Chapter 3 is adapted from a manuscript currently under review and submitted as an invited paper to the journal Developmental Neurobiology for a special edition on autism spectrum disorder (ASD) and related disorders: Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Semendeferi K. Increased glia density in the caudate nucleus in Williams syndrome: implications for frontostriatal dysfunction in autism. Manuscript Under Review. The author of this dissertation was the primary investigator and author.

in density of oligodendrocytes in the medial caudate nucleus. These cellular abnormalities may underlie reduced frontostriatal activity observed in WS, with implications for understanding altered connectivity and function in ASD.

Introduction

Autism Spectrum Disorders (ASD) are characterized by an extremely complex and heterogenous genetic (Sebat et al., 2007; Geschwind, 2011; Miles, 2011; Persico and

Napolioni, 2013; Jeste and Geschwind, 2014) and phenotypic (Tager-Flusberg and

Joseph, 2003; Ring et al., 2008; Lombroso et al., 2009) profile. By contrast, Williams syndrome (WS) is known to be caused by a hemizygous microdeletion of 25-28 genes on chromosome band 7q11.23. It is a rare disorder (<1 in 7500, Strømme et al., 2002) characterized by multiple somatic effects, including distinctive facial morphology, slowed growth, and cardiac abnormalities (Williams et al., 1961; Beuren et al., 1962).

Individuals with WS additionally display a relatively predictable and consistent cognitive and behavioral phenotype, including global reductions in total IQ, particularly with respect to visuospatial abilities (Meyer-Lindenberg et al., 2006). Curiously, individuals with WS have been shown to demonstrate linguistic capacity characterized by relatively broad vocabulary and unusually expressive use of phrases (Karmiloff-Smith et al., 1997;

Reilly et al., 2004; Udwin and Yule, 2005), but may struggle with temporal, spatial, and relational elements (Thomas et al., 2001; Phillips et al., 2004; Brock, 2007; Mervis and

Becerra, 2007) of language use.

In stark contrast to ASD, perhaps the best-described feature of individuals with

WS includes what has been described as hypersociability, and the intense drive to engage in social interactions with strangers (Jones et al., 2000; Doyle et al., 2004; Järvinen-

Pasley et al., 2008, 2010; Järvinen et al., 2013). This unique trait may be best viewed as a deficit in inhibitory control of socially-directed behavior (Little et al., 2013). Evidence from functional imaging studies has suggested that frontostriatal circuitry may be differentially affected by developmental abnormalities characteristic of the disorder

(Mobbs et al., 2007). Hypersociality and increased affiliative drive in Williams syndrome patients may represent selected deficits in the ability to suppress behavioral responses

(Frigerio et al., 2006), and attention deficit hyperactivity disorder is a frequent comorbid diagnosis in these individuals (Carrasco et al., 2005). Mobbs and colleagues (2007) showed slower reaction times among WS patients in a go/no-go task as compared with typically-developing control participants, and substantially reduced BOLD signal activity in the striatum was observed. Given the role of frontostriatal circuits in controlling behavioral responses, microstructural abnormalities in the striatum (Fan et al., 2017), and particularly the caudate (Reiss et al., 2004) may underlie behavioral deficits in response inhibition and the control of behavioral responses.

Brains of individuals with WS tend to be smaller overall than in typically- developing individuals (Jernigan and Bellugi, 1990), including reductions in cortical and subcortical grey matter. Reductions in grey matter volume have been noted in the caudate in individuals with Williams syndrome (Reiss et al., 2004) and recent structural imaging studies have found the greatest volumetric reductions occur in the orbitofrontal cortex and striatum in WS (Fan et al., 2017). Microstructural analyses of Brodmann areas 10 and 11 of the orbitofrontal cortex have demonstrated a reduction in neuronal density in these regions (Lew et al., 2016), further supporting the assertion that neural systems implicated in emotional processes are affected in WS at the cellular level. No histological

studies have yet aimed to identify the microstructural changes underlying volume differences in the striatum in WS.

Here we examined the caudate, putamen, and nucleus accumbens regions corresponding to the associative and limbic territories within the striatum, a key part of the reward-processing system in the primate striatum associated with learning, flexibility, and behavioral control (Fudge and Haber, 2002; Haber et al., 2006) to examine microstructural features of the striatum that may underlie cognitive and behavioral features of the disorder. In these regions in ASD, the caudate has been shown to involve structural increases in size (Langen et al., 2007, 2012) and reductions of total neuronal density (Wegiel et al., 2014). We utilized postmortem tissue from ten adult individuals including a unique sample of five WS and five age, sex, and hemisphere-matched controls, to measure the density of neurons and glia in the rostral striatum.

Methods

Materials. Subjects were adults, ranging in age from 18 to 45 years at time of death and included two males and two females with genetically confirmed diagnoses of WS, and one male (WS1) with a behaviorally confirmed diagnosis of WS with significant cardiac history consistent with the genetic disorder’s biomedical profile for which no genetic information was available (Table 1). These five specimens in the Ursula Bellugi Williams

Syndrome Brain Collection met the criteria for inclusion here based on completeness of the targeted ROIs. Another set of five age, sex, and right hemisphere-matched specimens from five adult TD cases were provided by University of Maryland Brain and Tissue

Bank (UMBTB), a repository of the NIH NeuroBioBank.

Tissue Processing. Blocks including the rostral portion of the striatum were used in our analyses owed to the close association of these territories with functional connectivity with regions of the prefrontal cortex (Fudge and Haber, 2002; Haber, 2003; Haber et al.,

2006; Delmonte et al., 2013; Averbeck et al., 2014; Jarbo and Verstynen, 2015). For each

WS brain, 3-4 cm thick blocks of tissue from the right hemisphere were removed and cryoprotected using successive concentrations of 10, 20, and 30% buffered sucrose solutions, and sectioned at 40m on a Leica SM2010 freezing microtome. TD subjects were dissected following standard procedures at the UMBTB, which include blocking of hemispheres into 1 cm thick slabs. These blocks were also cryoprotected using sucrose solutions and sectioned at 40m. Following cryosectioning procedures, a 1-in-10 series of tissue sections was stored for 48 hours in a neutral phosphate buffer solution to rehydrate the tissue. Sections were then mounted on gelatin-coated slides, dried at room temperature for 48 hours, and stained using a 0.25% thionine stain for Nissl substance to visualize cytoarchitecture and quantify neuron and glia densities in WS and TD subjects.

Remaining sections were stored in cryoprotectant at -20°C for use in later immunohistochemical and other analyses.

Anatomical Regions of Interest (ROIs). The striatum is organized topographically by the overlapping projections it receives from cortical territories, which have been categorized on the basis of functionally significant loops (Holt et al., 1997; Nakano et al., 2000;

Fudge and Haber, 2002; Haber, 2003; Choi et al., 2012). The regions of interest targeted were in the associative and limbic territories in the dorsal (dC) and medial caudate nucleus (mC), associative putamen (aP), and nucleus accumbens (NA) regions (Figure 1), comprised of areas corresponding to cognitive and limbic loops (Holt et al., 1997;

Nakano et al., 2000; Haber, 2003) and reward systems (Balleine et al., 2007; Delgado,

2007; Tanaka et al., 2015). Stereological quantification took place in a consistent manner between sections and cases, within these nuclei as described below.

Dorsal caudate nucleus (dC). The caudate head emerges anterior to the appearance of the lateral ventricle and caudal to the genu of the corpus callosum in coronal sections. It is bounded laterally by the emerging fibers of the internal capsule, which separates it from the putamen. Medially, the caudate is bounded by the lateral ventricle, and functional territories receiving projections primarily from prefrontal cortical regions are targeted rostral to the anterior commissure (Haber et al., 2006; Choi et al., 2012). The dorsal territories of the caudate nucleus share overlapping projections from premotor areas and the dorsal prefrontal cortex (Brodmann area 9/ 46) forming part of a frontal executive loop (Haber, 2003).

Medial caudate nucleus (mC). To target morphology associated with areas connecting to orbitofrontal cortices, we specifically targeted the medial regions of the caudate nucleus (Haber, 2003; Stephenson et al., 2017) which shares connectivity with areas 11, 12, 13 and 14 of the orbitofrontal and BA 24, 25, and 32 of the anterior cingulate cortex (Haber, 2003; Haber et al., 2006). These regions form an important site of convergence for inputs related to cognitive processes including decision-making, reward processing, and cognitive control (Averbeck et al., 2014).

Associative putamen (P). The rostral associative territory of the putamen is bounded laterally by the external capsule, which separates it from the claustrum, and medially by the fibers of the internal capsule in coronal sections anterior to the emergence of the globus pallidus. Posterior boundaries of the rostral putamen were

delimitated by the emergence of the anterior commissure rostral to the external medullary lamina of the globus pallidus (Choi et al., 2012). We targeted the medioventral region of the rostral putamen that also receives projections from the dorsal prefrontal (PFC) and anterior cingulate cortices (Haber et al., 2006) as part of an associative network defined by its connectivity with the PFC. In each coronal section we sampled within the associative putamen found in the lower two thirds of the nucleus of the putamen, to the exclusion of its dorsal territory (Haber, 2003; Haber et al., 2006), which receives primarily sensorimotor projections.

Nucleus accumbens (NA). Previous studies have noted that core and shell regions of the human NA show a difference in the distribution of calbindin D28k (Meredith et al.,

1996); however, consistent with our own observations in our sample, these differences in staining represent a gradient of CB distribution that do not allow for absolute delineations of boundaries between these regions (Selden et al., 1994, Parent et al., 1996). Since there is cytoarchitectonic continuity of the dorsal striatum and NA region, we have defined our region of interest consistent with previous investigations in TD and ASD subjects

(Wegiel et al., 2014), superiorly bounded by the inferior limit of the internal capsule, by drawing a line perpendicular to its orientation. The most rostral limit of the NA is its emergence inferior to the internal capsule. Posteriorly, the region is bounded by the termination of the anterior commissure. The NA is bounded by white matter along the inferior extent of the NA, separating it from cortical regions and the olfactory tubercle.

Stereological Analyses. Unbiased stereological methods were utilized to quantify the density of neurons and glia and neuronal soma area in each ROI, consistently defined by cytoarchitectonic and anatomical criteria across specimens as outlined above, using

StereoInvestigator software (v.10, MBF Bioscience, Williston, VT). The rater (KLH) was blinded to diagnosis and subject ID at the time of data collection and no obvious qualitative pathological changes were noted between subject groups that distinguished

WS from TD subjects. Intrarater reliability was insured through repeated measures of neuron and glia counting probes in 3 out of the 10 subjects in each ROI with less than 2% difference in estimated counts.

In each region of interest, 8-10 sections were selected in a 1-in-20 series (800 m apart) spanning its rostral to caudal extent anteriorly from the emergence of the lateral medullary lamina of the globus pallidus in the coronal plane, with care taken to sample consistently across subjects from the region of interest. Stereological analyses were performed on a Dell workstation receiving live video feed from an Optronics MicroFire color video camera (East Muskogee, OK) attached to a Nikon Eclipse 80i microscope equipped with a Ludl MAC5000 stage (Hawthorn, NY) and a Heidenhain z-axis encoder

(Plymouth, MN). Regions of interest were hand-traced at 2x magnification, and a square grid was superimposed over the tracing of the region of interest. Grid size was adjusted to optimize counting based on the size of the regions of interest, and measured 2000m 2 for the nucleus accumbens and dorsal caudate regions, and 3000m2 for the caudate and putamen regions. The height of the disector measured 9m, with a 1m top guard zone. For the quantification of neurons and glia, cells were counted by placing a software marker on each neuron visible in the given counting frame. As white matter inclusions are common within the striatum, care was taken to avoid sampling within white matter by drawing boundaries of the region of interest well outside the boundaries of the internal capsule. Additionally, no markers for glia were counted where neurons were not observed

within the field of view, though not necessarily in the counting frame itself, to ensure that counts were not performed within white matter inclusions in striatal territories.

Neurons and glia were counted at 100x magnification using an oil lens (NA 1.4) using the Optical Fractionator probe in StereoInvestigator. Neurons and glial cells were counted using different software markers within the same frame, and distinguished on the basis of their morphology (Figure 2). Briefly, neurons are characterized by their large size, distinct nucleus, and the presence of a darkly-stained nucleolus (Barger et al., 2012;

Wegiel et al., 2014). Soma area was also measured in one out of every three neurons counted using the Nucleator probe in StereoInvestigator, using a 4-point array.

Glia were distinguished from neurons and non-cellular artifacts by the presence of darkly stained cytoplasm, smaller size, and the lack of a distinctive nucleolus (Sherwood et al., 2006; Kreczmanski et al., 2007). Following initial glia counts, we also chose to focus specifically on oligodendrocytes due to their relative abundance within the caudate nucleus, and their distinctive morphology. Oligodendrocytes were identified using criteria described in the literature; as smaller and rounder than astrocytes and microglia, and more darkly-stained, with a more compact nucleus and less granular-appearing cytoplasm, and lacking a distinctive nucleolus in contrast to immature neurons (Baumann and Pham-Dinh, 2001; Hamidi et al., 2004; Pelvig et al., 2008; Karlsen and Pakkenberg,

2011; Morgan et al., 2014) (Figure 2). Given the heterogeneity in morphotypes of activated microglia (Morgan et al., 2014) and the difficulty in distinguishing these from astrocytes in Nissl-stained tissue, only oligodendrocytes were quantified. Additional immunostaining will be implemented in future series to quantify these elements

independently. Endothelial cells were sparsely present within the tissue, but were not quantified.

Neuron and glia density for the regions measured was estimated by dividing the number of cells by the projected volume of the sampled region in each ROI using planimetric sampling in StereoInvestigator. A target of 100-200 cells of each type was counted per region of interest with a coefficient of error (Gundersen m=1) less than 0.1.

Statistical Analysis. Standard 2-tailed t-tests (P< 0.05) were calculated using GraphPad

Prism software to compare means for neuron density, glia density, glia-to-neuron ratio, and soma volume. Grubb’s Outlier test was applied to all data to determine if any single value was a statistically significant outlier, but no outliers were detected and no data points were removed.

Results

Results for all regions of interest are summarized in Figure 3. A significantly higher density of glia was found in the dC, as well as an increase in the ratio of glia to neurons. In the mC, an increased ratio of glia to neurons, and a significant increase in oligodendrocyte density was found. Overall, neuron density did not vary significantly between WS and TD for any regions sampled. Total glia density was elevated significantly in the dC, and mean density of glia was also higher in the mC and aP, but these values did not reach statistical significance.

Dorsal Caudate Nucleus

A significant increase in the total density of glia was found in the dC

(58,304±3,595 WS vs. 47,156±2,482 glia/mm3; P=0.038). The ratio of glia to neurons was also significantly elevated (2.92 WS vs. 2.24 TD glia/neuron, P=0.028).

Oligodendrocyte density was higher on average in WS (33,810±3,742 cells/mm3, Figure

4) than in TD subjects (25,281±1,343 cells/mm3), though these results did not reach statistical significance (P=0.085). Though no significant difference in neuron density was found (19,496±1,471 WS vs. 21,061±776 cells/mm3 TD), mean neuronal soma area was smaller in WS (128±8 μm2) than in TD subjects (154±4 μm2, P=0.026).

Medial Caudate Nucleus

Mean density of glia was slightly elevated in the WS as compared to TD subjects

(65,990±4,266 vs. 55,046±4,2103 glia/mm3) though this did not reach significance

(P=0.105). However, the ratio of glia to neurons in the medial caudate was significantly elevated in WS as compared to TD subjects (2.81 vs. 2.18 glia/neuron, P=0.004).

Additionally, a significant increase of oligodendrocytes was found in the caudate in WS subjects (40,605±3,161 cells/mm3) as compared to TD controls (27,375±2,566 cells/mm3;

P=0.012). No significant difference in overall neuron density was observed between TD and WS subjects in the mC (22,935±843 WS vs. 24,931±1,210 TD neurons/mm3), though average soma area was significantly smaller in WS (121±12 μm2) as compared to TD

(158±8 μm2, P=0.034).

Associative Putamen

Average glia density was higher on average in WS as compared to TD cases

(73,106±8,075 vs. 59,100±4,754glia/mm3), but this did not reach statistical significance

(P=0.182). The ratio of glia to neurons in the associative putamen was slightly elevated in

WS as compared to TD subjects (2.70 vs. 2.22 glia per neuron), though this also did not reach statistical significance (P=0.074). Mean soma volume was also slightly decreased in the associative putamen, but did not reach statistical significance (P=0.077).

Nucleus Accumbens

No significant differences were observed in the nucleus accumbens region for neuron density (P=0.824), glia density (P=0.843), or glia-to-neuron ratio (P=0.330) between WS and TD subjects. Average soma area was modestly smaller in WS (112±11

μm2) than in TD (143±12 μm2) subjects, but this did not reach significance (P=0.087).

Discussion

Summary. Mean glia density was significantly elevated in the dC, and increased glia density was observed across all ROIs of the caudate and putamen, but not in the NA. The glia-to-neuron ratio was significantly elevated in subjects with WS in the medial caudate

(P=0.003), and although average glia-to-neuron ratio was higher in the associative putamen, this did not reach significance (P=0.068). (Figure 3C). Significant differences in the density of neurons in territories of the striatum were not observed between WS and

TD subjects. Mean values of glia density were greater in WS as compared to TD controls in the associative territories of the caudate and putamen but not in the limbic nucleus accumbens region. Average neuronal soma area was consistently smaller across all regions of interest in WS (Figure 3D).

Specific cognitive and neurological impairments in Williams Syndrome have been noted in individuals diagnosed with the disorder. Brains of individuals with

Williams Syndrome tend to be smaller overall than in typically-developing individuals

(Jernigan and Bellugi, 1990), with notable size reductions found in the parietal lobule

(Eckert et al., 2005), occipital grey matter (Reiss et al., 2000), intraparietal sulcus and orbitofrontal cortex (Meyer-Lindenberg et al., 2005; Fan et al., 2017). Functional abnormalities have been demonstrated with respect to primary auditory (Levitin et al.,

2003) and visual perception (Galaburda et al., 2002; Atkinson et al., 2007). Additionally, deficits in object-focused and spatial cognition and memory have implicated the dorsal visual stream (Atkinson et al., 1997, 2003) and the hippocampal formation (Meyer-

Lindenberg et al., 2005).

Previous histological studies in Williams Syndrome have targeted cellular density and soma size in cortical areas (Galaburda and Bellugi, 2000), with a specific focus on sensory territories. Primary visual cortex in a postmortem sample of Williams Syndrome patients has shown abnormalities in cell size and packing density (Galaburda et al.,

2002). Additionally, primary auditory cortex shows no overall significant difference in cell density compared to controls, but enlarged neuronal size in layer IV suggests that there may be differences in connectivity with limbic regions that reflect differences in emotional reactivity to sound (Holinger et al., 2005), particularly music. Lew and colleagues (2016) found a decrease in neuron density specific to BA 11 of the prefrontal cortex, specifically in the infragranular layers, that was not found in somatosensory (BA

3), motor (BA 4), or visual cortex (BA 18), further implicating dysfunction of subcortical connectivity.

Analyzing specific neural networks that are affected in WS serves the dual purpose of identifying structures and systems that contribute to selected behavioral and cognitive deficits in WS, and informing the spectrum of neuroanatomical variation that could be compromised in various other neurodevelopmental disorders, including ASD.

Given that a portion of the deleted region at chromosome band 7q11.23 is frequently seen as a duplication in the closely-related Dup7 syndrome, which presents universally with an

ASD-like social and cognitive phenotype (Berg et al., 2007; Sanders et al., 2012)

examining the neuroanatomical phenotype in WS offers the unique opportunity to contrast phenotypic features underlying this unique disorder as a counterpoint to pathological processes in ASD.

Comparisons of the Striatum in TD, WS and ASD. Our results for neuron density in TD subjects fell within expected ranges based on previously reported data (Kreczmanski et al., 2007; Khundakar et al., 2011; Wegiel et al., 2014). Average glia density in TD controls measured was somewhat higher than previously reported for the entire caudate nucleus (Khundakar et al., 2011), but within the range of variation defined by previous studies (Karlsen and Pakkenberg, 2011). This likely results from the specificity of our region of interest as a high-integration area, or perhaps due to the average age of our subjects being somewhat higher than in the sample reported by Khundakar (2011), as older age may be a factor in increased glia densities (Pelvig et al., 2008).

Significant volumetric (Langen et al., 2009, 2014) and microstructural (Wegiel et al., 2014) differences have been noted in the striatum in ASD. Specifically, reductions in total neuronal density were observed in the caudate and nucleus accumbens in ASD as compared to TD controls, with ASD subjects displaying overall increases in the volume of the caudate (Wegiel et al., 2014). In contrast, we found no significant differences in the density of neurons in four striatal regions of interest in WS. We found an increase in the density of glia in the dorsal caudate nucleus, in addition to increased glia-to-neuron ratio in the medial caudate nucleus, which our results suggest is driven by a significant overall increase in oligodendrocytes. Similar quantitative data for the striatum in ASD are not published, but glial pathologies have been noted in several cortical areas (Edmonson et al., 2014) and in the amygdala (Morgan et al., 2014). Here, we targeted

oligodendrocytes due to their consistent morphology and abundant appearance in WS cases. This likely has important implications for understanding hypoactivity in frontostriatal circuitry that underlies deficits in behavioral control seen in WS.

Specifically, the functional role of oligodendrocytes in myelination may point to mechanisms underlying dysfunction of frontostriatal connectivity related to hypermyelination and associated white matter abnormalities.

Frontostriatal Dysfunction in ASD and WS. Targeting psychiatric endophenotypes common to both disorders may help to elucidate patters of striatal neuroanatomy, particularly with reference to territories that share extensive connectivity with the PFC, in a way that contributes to deficits of executive function. This may be particularly true with respect to features of behavioral control that are impaired in both ASD (Christ et al.,

2007; Kana et al., 2007; Langen et al., 2012) and WS (Carrasco et al., 2005; Mobbs et al.,

2007) as well.

Previous research has shown anatomical abnormalities of the PFC in both ASD and WS. Significant overgrowth in the PFC in early development is reflected in increased neuron number in dorsal and medial prefrontal cortical regions in ASD (Courchesne et al., 2011), which corresponds to narrower spacing of cortical minicolumns in the PFC of

ASD (Buxhoeveden et al., 2006; Casanova et al., 2006). This may relate to local overconnectivity in the PFC to the detriment of long-range connectivity (Courchesne and

Pierce, 2005) with subcortical structures. Conversely, reduced neuronal density in lower layers V/VI of BA 11 was observed in WS (Lew et al., 2016), which implicates a differential decrease in PFC neurons that share connectivity with subcortical structures.

Additionally, layer III pyramidal neurons of BA 10 and 11 in WS may not display the

pattern of longer and more branched basal dendrites as observed in TDs (Hrvoj-Mihic et al., 2017) relatively to primary processing areas, suggesting compromised circuitry in high-integration PFC areas.

Important connectivity between prefrontal cortical areas and territories of the striatum observed in primates (Haber, 2003; Haber et al., 2006; Averbeck et al., 2014) investigated here has been established in human subjects (Choi et al., 2012; Delmonte et al., 2013; Jarbo and Verstynen, 2015) suggesting that abnormalities of this network may account for downstream dysfunction of striatal territories. Alterations in striatal morphology and function have been noted as a characteristic of both disorders. Structural findings consistently report alterations of striatal morphology in ASD, including increased volume of the caudate (Hollander et al., 2005; Langen et al., 2007; 2009).

Functionally, reduced frontostriatal activity in ASD subjects as compared to controls has been observed, particularly in response to social stimuli (DelMonte et al., 2012; Kohls et al., 2013). Deficits of behavioral control in response inhibition tasks have also been attributed to frontostriatal hypoactivity in WS generally (Mobbs et al., 2007) though specifically social stimuli were not utilized. Repetitive behaviors are also a common feature of ASD related to deficits in behavioral control thought to relate to striatal abnormalities (Hollander et al., 2005), though it is not a common behavioral feature associated with WS (Rodgers et al., 2012). These differences may be rooted in the different patterns of functional connectivity in frontostriatal territories in WS and ASD: whereas connectivity with associative regions of the caudate may be reflected in reduced activity in response inhibition tasks, connectivity between frontal regions and the putamen has shown significant impairment (Balsters et al., 2013) in ASD.

Overall, dysfunction in frontostriatal territories in WS contributes to deficits in the ability to inhibit the drive for social interaction (Mobbs et al., 2007) ultimately characterizing WS as a disorder of inhibitory behavioral control in distinctively social domains. Here, we reported abnormalities at the cellular level that may underlie frontostriatal dysfunction in the caudate nucleus that complements previous evidence for reduced neuron density in prefrontal cortical regions (Lew et al., 2016). It is likely that dysfunction in other systems in WS, including impaired frontolimbic connectivity between the prefrontal cortex and amygdala (Ng et al., 2016), is similarly reflected in pathologies within the amygdala in WS, which is an additional target of ongoing investigations given known aberrations in neuron numbers in ASD (Schumann et al.,

2006) in discrete amygdala nuclei. Frontostriatal (Fan et al., 2016) and frontolimbic (Ng et al., 2016) systems contribute to distinctive functional deficits in WS in different patterns that affect the cognitive and emotional profiles of this unique disorder in characteristic ways providing an interesting contrast to patterns seen in ASD.

Acknowledgements

This research was supported by the National Institutes of Health P01

NICHD033113 and 5R03MH103697. We are grateful to the individuals with WS and their families, past and present, who have participated in research characterizing this disorder’s unique cognitive and social phenotype. In particular, we thank the families who have graciously donated WS human tissue, which was obtained under the Ursula

Bellugi WS Brain Collection, curated by KS at UCSD. Human TD tissue was obtained from the NIH Neurobiobank at the University of Maryland, Baltimore, MD. We

additionally thank Chelsea Brown, Deion Cuevas, Valerie Judd, Hailee Orfant, and

Linnea Wilder for assistance with tissue processing and feedback in the development of this manuscript.

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Table 3.1: Subject Background Information. PMI = postmortem interval.

Subject Age Sex Diagnosis Hemisphere PMI Cause of death at (hours) Death WS 10 18y M WS R 24 WS/Cardiac complications 4916 19y M TD R 5 Drowning WS 1 31y M WS R 26 WS/Cardiac complications 5539 31y M TD R 24 Acute Drug Intoxication WS 14 42y F WS R 12 WS/Cardiac complications 5445 42y F TD R 10 Pulmonary Thromboembolism WS 9 43y F WS R 12 WS/Cardiac complications 4636 43y F TD R 19 Pulmonary Thromboembolism WS 12 45y M WS R 24 WS/Cardiac complications 5875 45y M TD R 19 Aortic Dissection

Figure 3.1. Anatomical regions of interest in the associative territories of the dorsal (dC) and medial caudate nucleus (mC), associative putamen (aP), and nucleus accumbens (NA) regions. The dorsolateral portion of the putamen, which receives primarily sensorimotor projections, was not measured. Scale bar = 5mm.

Figure 3.2. Neurons and Glia in the Striatum. Neurons (black arrow) were distinguished from oligodendrocytes (solid white arrows) and other glia (dashed white arrow) by their large size

and distinctively stained nucleolus. Image taken at 100x (1.4 NA). Scale bar = 10 μm.

A Dorsal Caudate Medial Caudate

40000 40000

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0 0 D S D S T W T W Figure 3.3. Results of stereological analyses in the four ROIs in WS and TD A. Neuron density. B. Density of glia. C. Glia-to-neuron ratio. D. Average soma area.

C Dorsal Caudate Medial Caudate 4 4 * **

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0 0 D S D S T W T W Figure 3.3 (continued). Results of stereological analyses in the four ROIs in WS and TD A. Neuron density. B. Density of glia. C. Glia-to-neuron ratio. D. Average soma area. 79

Figure 3.4. Density of oligodendrocytes in the medial caudate nucleus.

Chapter 4

Decreased density of cholinergic interneurons in the medial caudate nucleus in Williams syndrome3

ABSTRACT

Williams syndrome (WS) is a rare neurodevelopmental disorder caused by the deletion of approximately 25-28 genes at 7q11.23. Its unusual social and cognitive phenotype is most strikingly characterized by the disinhibition of social behavior, in addition to reduced global IQ, with a relative sparing of language ability. Hypersociality and increased social approach behavior in WS may represent a unique inability to inhibit responses to specifically social stimuli, and is likely rooted in part in abnormalities of frontostriatal circuitry. Recent research has demonstrated that selective ablation of striatal cholinergic interneurons (SCINs) results in significant increases in compulsive conspecific-directed, but not object-focused, exploratory behavior in mice, indicating a role for SCINs in social approach behavior. Here, we examined the density of SCINs in the striatum of five subjects with WS and four typically-developing (TD) controls and found a significant reduction in their density in the medial caudate nucleus, an important region receiving cortical afferents from the orbitofrontal and ventromedial prefrontal cortex in circuitry involved in language and reward systems. This pattern of decreased density in WS is contrasted by anatomical findings in other disorders, including Tourette syndrome and schizophrenia, where differing patterns of SCIN depletion are found.

3 Chapter 4 is a manuscript currently in preparation for submission for publication: Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Raghanti MA, Semendeferi K. Decreased density of cholinergic interneurons in the medial caudate nucleus in Williams syndrome. Manuscript in prep. The author of this dissertation was the primary investigator and author.

INTRODUCTION

Though traditionally best understood with reference to motor function, a growing body of literature has highlighted the importance of the basal ganglia in cognitive processes. The striatum constitutes the principle input nuclei for the basal ganglia, and receives important overlapping projections from cortical areas that topographically organize its territories into functional loops. In the cognitive loop, shared connectivity with the prefrontal cortex subserves important circuits associated with behavioral control, executive function, and emotional regulation. In primates, projections from prefrontal cortex, Brodmann area (BA) 9/46 sends projections to the dorsal head of the caudate nucleus, while BA 11, 13, 14 and 12 send projections to the medial caudate nucleus

(Haber et al., 2006), comprising important connectivity critical to action selection, decision making, reward-based and associative learning (Grahn et al., 2008; Balleine and

O’Doherty, 2010). The rostral putamen additionally receives projections from the orbitofrontal and ventromedial cortex in its associative territories (Haber et al., 2003;

2006). The limbic loop sends projections from the anterior cingulate cortex (BAs 24, 25, and 32) to the medial aspects of the caudate and putamen and the nucleus accumbens, which sends projections to subcortical areas important for the regulation of affective states (Fudge and Haber, 2002; Haber et al., 2006). The nucleus accumbens shares reciprocal connectivity with the anterior cingulate cortex, (Masterman & Cummings,

1997) important for integrating emotional states with cognitive inputs.

The striatum is characterized by a diversity of inhibitory interneurons and excitatory cholinergic interneurons that modulate the function of inputs from regions sharing connectivity with the striatal nuclei and output from striatal medium spiny

neurons. Among these, diverse populations of inhibitory GABAergic interneurons express calbindin, calretinin, parvalbumin, somatostatin, neuropeptide Y, and NADPH- diaphorase, and combined constitute more than 20% of total striatal neuron populations in the typically-developing human brain (Wu and Parent, 2000; Bernácer et al., 2012).

Striatal cholinergic interneurons (SCINs), by contrast, are excitatory neurons that comprise around 1% of total neuron populations in the human striatum (Bernacer et al.,

2007, 2012; Stephenson et al., 2017). Despite their sparse distribution, these extremely large, heavily branched neurons share extensive connections with cortical and subcortical projections, producing some 500,000 axonal varicosities (Bolam et al., 1984; Contant et al., 1996) and receiving direct projections from the substantia nigra (Kubota et al., 1987), thalamus, and cortex (Lapper & Bolam, 1992), modulating inputs to local inhibitory interneurons and medium spiny projection neurons (Tepper & Bolam, 2004; Pakhoti &

Bracci, 2007) within the striatum.

Animal models support the hypothesis that SCINs contribute importantly to the modulation of social behavior. Martos and colleagues (2017) showed that selective ablation of SCINs led to a significant increase in compulsive, socially-directed (but not object-focused) exploratory behavior in mice. Rapanelli and colleagues (2017) conversely demonstrated that the ablation of both SCINs and fast-spiking parvalbumin- positive interneurons (FSIs) in the caudate and putamen may result in ‘autistic’ behaviors including repetitive and stereotyped behavior, and reduced social interaction, particularly in male (but not female) mice. The behavioral pattern was observed only when both types of neurons were targeted. These deficits may relate to the relationship between interneurons and medium spiny projecting neurons (MSNs) in the striatum. Previous

research has suggested that specific deficits of a PDE10A, phosphodiesterase involved in the hydrolysis of CAMP and cGMP at intercellular sites, was sufficient to produce a profound increase in social interaction in mice (Sano et al., 2008). Together, these findings suggest that SCINs, along with FSIs, contribute to a mechanism that selectively inhibits exploratory social behavior.

Differences in the distribution of SCINs have been identified that likely contribute to a variety of neuropsychiatric disorders in humans. For example, Tourette syndrome is characterized by a decrease in SCIN density in associative and sensorimotor but not limbic regions, suggesting that impaired function of corticostriatal-thalamic circuitry contributes to the emergence of motor/language disinhibition and tics observed in

Tourette syndrome. A decrease in SCINs in the ventral portions of the caudate, as well as a profound depletion of SCINs in the nucleus accumbens region, has also been noted in schizophrenia (Holt et al., 1999, 2005), which may contribute to deficits in prefrontal cortex function and relate to abnormalities in emotional responses charactersitc of the disorder.

Williams syndrome (WS) is a rare genetic disorder with an unusual cognitive phenotype characterized most saliently by the profound disinhibition of social behavior

(Järvinen-Pasley et al., 2008; Järvinen et al., 2013), as well as a cognitive profile defined by impaired visuospatial function (Atkinson et al., 2003) with relatively spared language ability (Mervis and Klein-Tasman, 2000; Mervis and Becerra, 2007), expressive use of phrases (Reilly et al., 2004; Udwin and Yule, 2005) and a proclivity for music (Levitin and Bellugi, 1998; Don et al., 1999). Individuals with WS are known to have an unusually high drive to engage in social interactions, which may be attributed to a deficit

in the inhibitory control of social behavior (Little et al., 2013). Deficits in response inhibition have been demonstrated in WS in go/No-go tasks, associated with hypoactivity of the anterior caudate nucleus (Mobbs et al., 2007). Further evidence for pathology of striatal morphology and its contribution to functional deficits has been identified from structural imaging, where decreases in grey matter in the caudate (Reiss et al., 2000,

2004) have been noted. Additionally, disproportionate decreases in the size of the putamen and nucleus accumbens, in addition to reductions in orbitofrontal cortical volumes, are highly distinctive features of the disorder (Fan et al., 2017). Taken together, these findings suggest that features of the unique cognitive profile in WS may be owed to abnormalities found in frontostriatal circuitry.

In our previous research, we examined neuronal density in WS (Hanson et al.,

2017) and found no significant difference in total neuronal density between WS and TDs.

However, we noted significant increases in the density of glia in WS, specifically in the caudate nucleus, suggesting that specific cellular populations may be uniquely affected.

Here, we extended our analysis of the striatum in WS and sought to examine the density of SCINs. Our aim was to determine if there are differences in the density of SCINs, and how the pattern of their differential distribution may relate to differences observed in other neuropsychiatric disorders. Given the unusual social phenotype observed in WS, it is expected that pathologies in the density of SCINs could in part underlie the lack of behavioral inhibition seen in WS.

Methods

Materials. Table 4.1 lists information on all subjects used in the current study. Five subjects with behaviorally-confirmed diagnoses of WS (three males and two females) in

the Ursula Bellugi Williams Syndrome Brain Collection were cryosectioned at 40μm and a 1-in-10 series was Nissl-stained using methods previously described (Hanson et al.,

2017). Genetic confirmation of WS, based on FISH for elastin, was confirmed in four out of five subjects. Medical history for one subject, for whom genetic deletion data was not available (WS1) indicated many key phenotypic traits, including distinctive facies and significant history of supravalvular aortic stenosis (SVAS) consistent with the genetic disorder’s biomedical profile (Morris and Mervis, 2000; Pober, 2010). Three TD subjects

(two male and one female) were provided in collaboration with the University of

Maryland Brain and Tissue Bank, a repository of the NIH NeuroBiobank. These subjects were also cryosectioned at 40 μm and a 1-in-10 series stained with 0.25% thionine solution for Nissl. A fourth control subject (male) was provided by the Northwestern

University Alzheimer’s Disease Center Brain Bank, which was additionally processed and sectioned at 40 μm and stained using 0.5% cresyl violet for Nissl. No significant difference in age or postmortem interval was observed between subjects.

Immunohistochemical Staining. A 1-in-20 series was selected from remaining adjacent sections stored at -20°C in cryoprotective solution. Sections were stained for choline acetyltransferase using an avidin-biotin peroxidase method previously described

(Raghanti et al., 2008) developed for optimal use in heavily fixed tissues. Briefly, floating tissue sections were rinsed in PBS for 50 minutes, and pre-treated for antigen retrieval utilizing a 0.05% citraconic acid solution at 86°C for 30 minutes. Endogenous peroxidases were quenched by incubation in a methanol and hydrogen peroxide solution.

Sections were pre-blocked in a solution of normal sera and Triton-X detergent prior to incubation in a purified anti-ChAT polyclonal antibody raised in goat (Millipore AB-

144P) at a 1:500 dilution, continuously shaken for 24h at room temperature, followed by

24h at 4°C. The primary antibody selected labels a band at approximately 68 kDa in humans and non-human primates, and has been validated for immunohistochemical use in non-human primate and human tissue (Bernacer et al., 2007; Raghanti et al., 2008).

Sections were rinsed and incubated in a biotinylated secondary antibody (dilution 1:200) and an avidin-peroxidase complex (ABC kit; PK-6100, Vector Laboratories, Burlingame,

CA). A DAB chromogen (SK-4100, Vector Laboratories) was utilized to visualize immunoreactive cells and fibers with robust staining of cell bodies and their processes

(Figure 1). Stained sections were mounted on gelatin-coated slides that were blinded to the investigator for diagnosis prior to quantification. No qualitative differences in staining or in the distribution of positively-stained cells was observable between TD and WS subjects that would distinguish them for diagnosis during counting procedures.

Regions of Interest (ROIs). We investigated four ROIs including the dorsal (dC) and medial (mC) territories of the caudate nucleus, ventral territories corresponding to the nucleus accumbens region (NA), and associative regions of the putamen (aP) using Nissl- stained sections as previously described (Hanson et al., 2017), (Figure 2). Utilizing cytoarchitectural landmarks, a line was drawn in vertical orientation through the center of the internal capsule, and a line perpendicular to this line beneath the inferior termination of the internal capsule was drawn to demarcate the superior border of the ventral striatum corresponding to the NA region (Selden et al., 1994; Wegiel et al., 2014). The dC was sampled separately from the mC, with a line drawn dividing the superior one-third of the caudate nucleus from inferior medial territories. Care was taken to sample within these

regions of interest consistently, avoiding transitional areas, and the fibers of the internal capsule.

Stereological Quantification. Quantitative analyses were performed with computer- assisted stereology using a Dell workstation receiving live video feed from an Optronics

MicroFire color video camera (East Muskogee, OK) attached to a Nikon 80i light microscope, equipped with a Ludl MAC5000 motorized stage and a Heidenhain z-axis encoder. The optical disector probe was used in conjunction with fractionator sampling using StereoInvestigator software (v. 10, MBF Bioscience, Williston, VT). Regions of interest were hand-traced at 4x magnification (N.A. 0.75), and a square grid no larger than 1000μm2 was superimposed in random orientation over the region of interest. Six sections through all regions of interest, counting rostrally from the emergence of the anterior commissure in the coronal plane, were sampled for density of cholinergic interneurons. Cholinergic interneurons were counted at 20x magnification (N.A. 0.75) in a 350μm2 counting frame, with a coefficient of error (Gundersen m=1) less than or equal to 0.1. Soma area was measured in one out of every five neurons counted using the

Nucleator probe in StereoInvestigator with a 4-point array. Cholinergic interneurons were counted only if they had a well-defined, darkly staining soma and at least one visibly stained process. Total density of cholinergic interneurons was calculated as the projected number for the region of interest divided by the planimetric volume of the region sampled. We additionally compared the density of SCINs relative to the total density of neurons for all regions of interest, as previously determined from Nissl stained sections

(Hanson et al., 2017).

Statistical Analyses. Statistical analyses were performed on Prism v. 7 (GraphPad

Software, Inc.). Standard 2-tailed t tests with Welch’s correction were calculated (P <

0.05) to compare the density of SCINs in WS and TD for all regions of interest. All data were subject to Grubb’s test for outliers (GraphPad test for outliers) to determine if any single value represented a significant outlier from others by diagnostic category (WS or

TD), but no outliers were observed. The percent of SCINs relative to the total density of neurons was additionally compared across all ROIs from previously established values

(Hanson et al., 2017).

Results

Results for the density of SCINs in all ROIs are summarized in Figure 3. A significant decrease in the density of SCINs in WS was observed in the medial caudate nucleus, but not in the dorsal caudate or putamen. A modest decrease in SCIN density was observed in the nucleus accumbens, but did not reach statistical significance. Table 2 summarizes SCIN density and percent of total neurons for all areas.

Dorsal Caudate. Consistent with previous reports for the associative territory of the head of the caudate nucleus (Bernácer et al., 2007), the highest average values for SCIN density in the dorsal striatum (caudate and putamen) were found in the dC. No difference in the density of SCINs (mean±SEM) was observed in the dC between WS

(146.8±8.1/mm3) and TD (163.3±11.3/mm3) subjects. Neuronal soma area also did not differ significantly (837.3±58.5 μm2 WS vs. 800.5±52.8 μm2 TD).

Medial Caudate. A significant decrease in the density of SCINs was observed in the medial caudate nucleus (94.7±6.5/mm3 in WS vs. 144.5±11.2/mm3 TD; P=0.0128).

Average soma area did not differ significantly between TD (731.0±78.2 μm2) and WS

(767.8±57.0 μm2). Overall density of SCINs was lower in the mC than in all other territories examined in WS, in contrast to TD subjects, which showed the lowest values for SCIN density in the putamen.

Putamen. The lowest density of SCINs in TD subjects was observed in the putamen, consistent with previously reported average values for its precommissural territories

(Bernácer et al., 2007). Average values for SCIN density were nearly identical between

TD and WS subjects (132.9±4.0/mm3 TD vs. 134.6±11.2/mm3 WS). No difference in soma area was observed (746.3±76.3 μm2 TD vs. 751.1±61.4 μm2 WS).

Nucleus Accumbens. Qualitative observations included greater variability in the size of neurons in the NA in all TD and WS subjects as compared to other regions of interest, but these differences in morphotypes were not quantified separately. Average density of

SCINs was nearly 41% lower in WS subjects (157.2±10.7/mm3) than in TDs

(220.2±24.8) though this did not reach statistical significance (P = 0.0783). Though soma area was slightly larger in WS (757.1±57.0 μm2) as compared to TD subjects (686.1±51.5

μm2), this difference was not significant.

Discussion

In the present work, our aim was to analyze density of SCINs in the rostral striatum, including the dorsal and medial caudate, putamen and nucleus accumbens in

WS. The regions of interest targeted here comprise a significant portion of the striatum rostral to the anterior commissure, and were selected for their connections with several regions of the prefrontal cortex: orbitofrontal (BAs 11, 13, 14, and 12), anterior cingulate

and ventromedial (BAs 24b, 25, and 32) and dorsal (BA 9/46) regions of the prefrontal cortex (Haber et al., 2003, 2006). SCIN densities for all regions of interest reported here lie within the range of variation previously reported for normal controls (Kataoka et al.,

2010). Our consistent sampling schema constrained to these rostral territories and strict criteria for the inclusion of intact neurons may account for slightly lower densities than previously reported (Bernácer et al., 2007) for a broader region of interest.

Our previous analyses (Hanson et al., 2017) have indicated that the caudate nucleus in WS is characterized by an excess of glia, and particularly oligodendrocytes, which may relate to local differences in connectivity with regions of the prefrontal cortex.

These findings are particularly interesting given that previous research (Lew et al., 2017) has also noted a decrease in neuron density in the orbital prefrontal cortex (specifically

BA 11) that was not observed in primary somatosensory, motor, or visual cortex (BAs 3,

4, and 18). Taken together, these findings suggest differences between WS and TDs found in PFC, as well as in a prefrontal area that sends projections to the striatum.

Therefore, significant changes are found in WS in both sub-cortical and cortical structures guiding social responses and inhibitory behavioral control, suggesting that the circuits subserving these functions are compromised in WS.

Comparisons to other disorders of social and emotional disinhibition

Taken together, these interesting findings in the striatum in WS can be considered in the context of other neuropsychiatric disorders (See Table 3). For example, SCINs are found to be decreased in density in Tourette syndrome (TS), schizophrenia, and

Alzheimer’s disease (AD). Depletions in SCIN density in these disorders follow distinctive patterns with respect to the striatal territories where decreased density is

found. The topographical organization of the striatum by the cortical projections it receives suggests these differences may be of significance for understanding how differences in SCIN density may present with different behavioral symptomatology.

Schizophrenia, for example, is characterized by disruptions in emotional regulation, deficits in reward prediction, and generalized anhedonia, attributed in part to abnormalities of the midbrain dopaminergic system affecting innervation of the basal ganglia (Perez-Costas et al., 2010). Interestingly, a decrease in SCIN density was observed in schizophrenia, most notably in the ventral striatum, inclusive of the most ventral territories of the caudate and nucleus accumbens regions (Holt et al., 1997, 2005).

These areas also may be affected in our sample in WS, though decreased SCIN density was predominately in the mC and did not reach significance in the NA region.

Studies of emotional processing in WS have reported abnormalities including increased responsiveness to emotional contagion and empathy (Järvinen et al., 2013) as well as what has been described as a fearlessness in approaching strangers (Doyle et al.,

2004). In contrast, schizophrenia is characterized by social anxiety (Pallanti et al., 2004) and pronounced deficits in empathy (Shamay-Tsoory et al., 2007; Derntl et al., 2009).

Thus, the distinctively pro-social phenotype observed in WS and contrasting social- avoidant phenotype characteristic of schizophrenia may share the common trait of altered reward perception and risk rooted in circuitry in the ventral striatum (Juckel et al., 2006;

Barch and Dowd, 2010) as well as the amygdala (Haber and Fudge, 1997) which remains the subject of ongoing investigations in WS.

Alzheimer’s disease (AD), by contrast, is best known to be a disorder of impaired memory function, though deficits in emotional control of behavior have been noted as a

feature in some studies of the disorder (e.g., Vasterling et al., 1997). Specific deficits of

SCINs were noted in early immunohistochemical studies (Selden et al., 1994a) in the ventral striatum, where a dramatic (70%+) decrease in their numbers was observed in a two-dimensional study of sections stained for acetylcholinesterase. Caution is warranted in the interpretation of these results, given that early characterizations of AD were inclusive of what is now known to be a heterogeneous group of forms of dementia and cognitive decline in the aging brain, such as frontotemporal dementia (McKhann, 2001;

Dubois et al., 2007). However, early hypotheses regarding the progression of AD suggest the involvement of the cholinergic system in the disease’s neurodegenerative processes

(Siegfried, 1993), though cholinergic systems may primarily be affected in AD in limbic, and specifically septal nucleus-hippocampal circuitry (Craig et al., 2011). Nevertheless, it may be the case that SCINs are differentially depleted in the striatum in AD, which may relate to the high density of neurofibrillary tangles observed in the nucleus accumbens

(Selden et al., 1994b) but not in the head of the caudate nucleus.

SCIN depletion and disorders of behavioral control

Tourette syndrome (TS) is characterized by the persistence of multiple motor and at least one vocal tic, or unprovoked, non-rhythmic movement or utterance that is involuntary. Depletion of SCINs is also found in TS, primarily in associative and sensorimotor territories of the dorsal striatum (Kataoka et al., 2010). By contrast, we found a decrease of SCINs specific to the medial caudate nucleus, with no significant differences observed in other territories. The medial caudate nucleus corresponds to territories that receive afferents from the orbitofrontal and ventromedial prefrontal cortices in primates (Haber, 2003; Haber et al., 2006; Averbeck et al., 2014) forming

networks important for decision-making, motivation, and reward (Haber and Behrens,

2014). This region is additionally important for language function and vocalizations, and corresponds to territories where humans with dysfunctional FOXP2 genes display hyperactivation relative to TD participants in fMRI and PET language tasks (Vargha-

Khadem et al., 1998; Liégeois et al., 2003).

The medial caudate nucleus may be particularly important for social interaction and language, and has been the site of important neuroanatomical changes during human evolution. While the whole striatum has undergone a substantial reduction in relative size in the human brain in recent primate evolution (Barger et al., 2014), significant increases in dopaminergic innervation of the mC have been observed in humans as compared to anthropoid primates, including apes (Raghanti et al., 2016). Though cholinergic innervation did not differ across taxa in the mC, humans and apes display a significant predominance of multipolar SCINs as compared to unipolar and bipolar morphotypes, which displayed a higher frequency in monkeys (Stephenson et al., 2017). Though basal ganglia components are frequently regarded as being highly conserved, these differences may indicate enhanced synaptic plasticity and neurotransmission, which may relate to the evolution of language.

Though language function in WS is typically referred to as preserved relative to other cognitive faculties, such as visuospatial cognition (Nakamura et al., 2001; Atkinson et al., 2003, 2007; Farran and Jarrold, 2003; Meyer-Lindenberg et al., 2004), global IQ

(Searcy et al., 2004), and working memory (Morris and Mervis, 2000; Robinson et al.,

2003; Sampaio et al., 2008, 2010), abnormalities of language use and function are observed in the disorder. Individuals with WS may have unusually expressive vocabulary

relative to age-matched peers (Reilly et al., 2004; Mervis and Becerra, 2007) but display deficiencies in syntax, phonology, and lexical semantics (Brock, 2007) as well as spatial and temporal elements of language (Karmiloff-Smith et al., 1997; Phillips et al., 2004).

Features of TS are not typical to WS, though one subject, WS12, had a history of echolalia in noted in his physician’s medical records in adulthood. Echolalia may occasionally be a feature of WS related to anxiety in the disorder (Mervis and Klein-

Tasman, 2000), as imitative behavior may serve similar adaptive functions in neurotypical individuals in early childhood, and in TS (Ganos et al., 2012) rooted in altered neural circuitry.

A thread of commonality may be shared in WS and TS in the high rate of comorbid diagnoses of Attention Deficit Hyperactivity Disorder (ADHD) present in both disorders (Spencer et al., 1998; Carrasco et al., 2005), as well as in Autism Spectrum

Disorder (ASD) (Geurts et al., 2008). Studies of ADHD have also revealed altered frontostriatal connectivity (Durston et al., 2003) as well as a strong pattern of volumetric reductions in the caudate nucleus and reductions in structural asymmetry (Hynd et al.,

1993; Singer et al., 1993; Filipek et al., 1997). Interestingly, structural abnormalities present in ADHD may respond to treatment with stimulant medication in early childhood, yielding better outcomes in adulthood (Frodl and Skokauskas, 2012) but stimulant medication is not typically prescribed to individuals with WS due to the high potential for cardiac side effects.

Patterns of morphological differences in ASD serve as an interesting counterpoint to those observed in WS, particularly in light of their contrasting behavioral phenotypes and genetic profiles. Duplications in the deleted region in WS have been shown to result

in a classically autistic behavioral phenotype (Berg et al., 2007; Velleman and Mervis,

2011; Sanders et al., 2012) suggesting disparate effects of the gene deletion or duplication in WS and ASD respectively, with implications for the underlying neuroanatomy. Structural imaging studies have revealed increased caudate volume (Sears et al., 1999; Langen et al., 2007) and early overgrowth of the caudate (Langen et al.,

2012) in ASD, which is strongly associated with repetitive behaviors (Estes et al., 2011) in the disorder. Decreased frontostriatal connectivity in the caudate nucleus (Di Martino et al., 2011) has also been observed. Functional imaging studies have also noted reduced frontostriatal activation (Shafritz et al., 2008), as well as aberrant processing of reward and reduced connectivity in the nucleus accumbens (Delmonte et al., 2013), particularly related to social stimuli (Kohls et al., 2013).

Whereas reduced neuron density is observed in the caudate and nucleus accumbens in ASD (Wegiel et al., 2014), no differences in total neuron density were observed in WS, but an excess of glia, and particularly oligodendrocytes, was found

(Hanson et al., 2017). Complementary data for SCINs do not exist in the literature for

ASD, but reduced acetylcholine receptor binding has been found in the cerebellar cortex

(Perry et al., 2001), as well as a loss of nicotinic acetylcholine receptors in the thalamus

(Ray et al., 2005). Based on these findings, investigations of SCIN and other interneuron distributions in the striatum in ASD are warranted.

Conclusions

Multiple neurological disorders involve decreases in the density of SCINs, which are likely important for social behavior, emotional regulation, and behavioral control.

Decreases in SCIN density in neurological disorders follows distinctive patterns that

seem to relate to shared connectivity with cortical regions. The topographical organization of the striatum, and particularly the caudate nucleus, by projections from regions of the prefrontal cortex and other areas suggests that a more nuanced parcellation of its territories may help to elucidate frontostriatal circuits contributing to pathophysiology of select neurological disorders. Future research will target additional interneuron subtypes in the striatum, particularly FSIs, to better understand how these may be impaired in WS, contributing to the disorder’s unique behavioral phenotype.

Acknowledgements

Chapter 4 is a manuscript currently in preparation for submission for publication:

Hanson KL, Groeniger K, Lew C, Hrvoj-Mihic B, Bellugi U, Halgren E, Raghanti MA,

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Table 4.1. Specimen Information. All WS subjects met diagnostic criteria for Williams syndrome on the basis of established behavioral criteria. All subjects with the exception of WS1 were genetically confirmed by fluorescence in-situ hybridization to have the deletion for the gene ELN; WS1 was not tested but had significant history of supravalvular aortic stenosis (SVAS) consistent with the disorder’s biomedical profile.

Subject Age at Sex Hemi PMI Cause of Psychiatric ID Death (hours) death Medication (years) History WS 10 18 M R 24 WS/Cardiac Cardiac/anti- hypertensives only WS 1 31 M R 26 WS/Cardiac Dilantin, beta blockers WS 14 42 F R 12 WS/Cardiac Unknown WS 9 43 F R 12 WS/Cardiac Lorazepam, quetiapine, sertraline WS 12 45 M R 24 WS/Cardiac Cardiac/anti- hypertensives only TD 4916 19 M R 5 Drowning TD 5875 45 M R 10 Aortic Dissection TD 5445 42 F R 10 Pulmonary Embolism 09A611 45 M L 24 Myocardial Infarction

A B C D

E F G H

Figure 4.1. Cholinergic interneurons of the striatum in TD (top) and WS (bottom) in the dorsolateral caudate (A, E), medial caudate (B, F), putamen (C, G), and nucleus accumbens (D, H). Scale bar = 50 μm.

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Dorsolateral Caudate IC Medial Caudate

Putamen Nucleus Accumbens

Figure 4. 2. Schematic representation of the striatum and regions of interest targeted.

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Table 4.2. Mean density of striatal cholinergic interneurons

Region of Interest dC mC aP NA

TD ChAT+ Density 163.3±11.3 144.5±11.2 133±8 220.2±24.8

Percent of total neuron density 0.72% 0.59% 0.51% 0.71%

WS ChAT+ Density 146.8±8.1 94.7±6.5 135±19 157.2±10.7

Percent of total neuron density 0.75% 0.41% 0.53% 0.58%

% difference in ChAT+ -11.64% -51.58%* +0.01% -40.13%

density

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Dorsolateral Caudate Medial Caudate

300 300 *

n o

200 o 200

100 100

C C 0 0 D S D S T W T W

P=0.2615 ns *P=0.0128

Associative Putamen Nucleus Accumbens Region

300 300

3 3

m m

/ /

s s

n n o

o 200 200

r r

u u

e e

n n

r r

e e

t t

n n

I I

100 100

+ +

T T

A A

h h

C C 0 0 D S D S T W T W

P=0.0783 ns P=0.8656 ns

Figure 4.3. Cholinergic interneurons density in four striatal ROIs. 110

Table 4.3. Summary of evidence for striatal and cholinergic dysfunction in several neurological disorders.

Disorder Study Type Key Finding Citation

Structural MRI Reduced caudate grey Reiss et al., 2000, matter volume 2004 fMRI Reduced putamen and Fan et al., 2017 nucleus accumbens volume Hypoactivation of Mobbs et al., 2007 Williams anterior caudate in syndrome (WS) go/No-go tasks

Postmortem Increased glia density in Hanson et al., anatomy caudate (Nissl) 2017 Decreased neuron density in prefrontal Lew et al., 2017 cortex (Nissl) Decreased density of present study ChAT+ neurons in medial caudate Structural MRI Increased caudate Sears et al., 1999; volume, caudate Langen et al., overgrowth associated 2007, 2012; Estes with repetitive behaviors et al., 2011 Autism Spectrum fMRI Disorders (ASD) Abnormal processing of Delmonte et al., social reward 2013; Kohls et al., Reduced frontostriatal 2013 Functional activation Shafritz et al., Connectivity 2008 MRI/DTI Enhanced connectivity Turner et al., with associative/limbic 2006; Di Martino cortices, decreased et al., 2011 Postmortem connectivity of caudate anatomy Decreased neuron Wegiel et al., 2014 density in caudate and nucleus accumbens Perry et al., 2001, Reduced acetylcholine Ray et al., 2005 receptors (AChR)

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Table 4.3 (continued). Summary of evidence for striatal and cholinergic dysfunction in several neurological disorders.

Disorder Study Type Key Finding Citation

Structural MRI Reduced structural Singer et al., 1993; asymmetry of striatal Forde et al., 2017 volume Tourette’s syndrome (TS) fMRI Increased frontostriatal Mazzone et al., activation in blink 2010 inhibition

Functional Altered cortico-striato- Worbe et al., 2015 Connectivity pallido-thalamocortical MRI/DTI connectivity Kalanithi et al., Postmortem Reduced density of 2005; Kataoka et striatal parvalbumin and al., 2010 cholinergic interneurons Reduced expression of Lennington et al., genes associated with 2014 interneurons in the caudate, activated microglia

Structural MRI Smaller left caudate, Hynd et al., 1993; reduced asymmetry Filipek et al., 1997 Medication affects Frodl and striatal morphology in Skokauskas, 2012 Attention Deficit ADHD Hyperactivity Disorder (ADHD) fMRI Decreased frontostriatal Durston et al., activation 2003

Functional Decreased fractional Connectivity anisotropy of De Zeeuw et al., MRI/DTI frontostriatal tracts 2012

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Table 4.3 (continued). Summary of evidence for striatal and cholinergic dysfunction in several neurological disorders.

Disorder Study Type Key Finding Citation

Structural MRI Reduced putamen De Jong et al., volume, thalamus 2008; Roh et al., Alzheimer’s disease volume associated with 2011 (AD) Postmortem degree of impairment

Decreased density of Lehéricy et al., cholinergic interneurons 1989; Selden et in ventral striatum al., 1994a High density of Selden et al., neurofibrillary tangles in 1994b accumbens and caudate tail

Chapter 5

Conclusions

This dissertation has sought to identify cellular mechanisms underlying frontostriatal dysfunction that may contribute to behavioral disinhibition and heightened emotional reactivity to social stimuli in Williams syndrome (WS), a genetic disorder with a highly unique social, behavioral, and cognitive profile. We targeted rostral territories of the striatum involved in reward networks in a postmortem sample from brains of individuals with WS and, when available, used age, sex, and hemisphere matched controls to perform stereological analyses to quantify densities of neurons, glia, and cholinergic interneurons. Variations in the density of select cell types in the striatum were observed that likely contribute importantly to the disorder’s neurological phenotype.

Ongoing work will seek to target differences in populations of glia and interneurons in the striatum and cerebral cortex in WS, as well as in non-human primate species, with the goal of characterizing specializations in the human brain important for derived variation in social behavior and cognition.

Summary of key findings

Neuron and glia density. No significant difference in the density of total neurons was observed in any of the three striatal nuclei in WS from Nissl-stained sections.

However, increased density of glia was observed in the dorsal caudate nucleus, and the ratio of glia to neurons was also significantly higher in the medial caudate nucleus.

Oligodendrocytes were additionally quantified, and their density was found to be significantly higher in the medial caudate nucleus and elevated in the dorsal caudate, though this did not reach significance. Oligodendrocytes function to produce myelin and

113 114 enhance nerve conduction along axons, and an excess of oligodendrocytes in grey matter of the caudate nucleus may reflect hyperproliferation of oligodendrocyte progenitor cells, impairments in their migration into white matter, and/or the failure of programmed cell death. This difference is thought in part to contribute to differences in connectivity with regions of the prefrontal cortex, as hypoactivity in the caudate nucleus is observed in WS as compared to TD subjects (Mobbs et al., 2007) in tasks designed to test behavioral response inhibition rooted in frontostriatal circuits.

Cholinergic interneuron density. We observed a significant decrease in the density of striatal cholinergic interneurons (SCINs) in the medial caudate nucleus, but not in the dorsal caudate nucleus, suggesting specificity in patterning of cholinergic interneuron depletion in neurological disorders that reflects important functional connectivity and specialization. In other words, previous investigations that have parsed the striatum into associative, motor, and limbic territories broadly may overlook important anatomical differences based on functional connectivity. In light of recent research that has suggested that the depletion of striatal cholinergic interneurons may result in compulsive social exploratory behavior in mice (Martos et al., 2017), this may have exciting implications for understanding the social disinhibition and gregariousness that constitutes the most well-described and notable feature associated with WS.

Future directions

Postdoctoral funding from the Institute for Neural Computation (UCSD) has been obtained by the author of this dissertation to continue pursuing questions related to defining the neuroanatomical phenotype in WS. These projects will be undertaken in conjunction with continuing efforts to quantify interneuron densities in the non-human 115 primate brain with a focus on differences between humans and apes in hominoid evolution. An additional goal will be to extend the use of immunohistochemical techniques in the WS brain collection, particularly in the cerebral cortex. This dissertation represent the first use of immunohistochemical staining in postmortem brain tissue in WS, which will extend the range of research questions that can be addressed with this extremely rare collection.

Quantification of glia types and morphology. In this dissertation, we quantified the average density of glia in our regions of interest, with a focus on oligodendrocytes, which could be easily recognized due to their distinctive morphology. However, additional types of glia, including astrocytes and microglia, will require more advanced staining techniques to examine their distribution and morphology. Microglial activation is a pathological feature of many neurological disorders (Dheen et al., 2007; Kierdorf and

Prinz, 2013). I expect to find an increase in microglial numbers as well as the presence of microglial activation consistent with other disorders affecting cognition, including autism

(Morgan et al., 2010; Tetreault et al., 2012; Suzuki et al., 2013). Increases in numbers of microglia and microglial activation may represent a compensatory mechanism for balancing the overproliferation of dendritic segments and synapses in PFC in WS mediated by FZD9 in a stem cell model (Chailangkarn et al., 2016) of the disorder.

Quantification of additional interneuron subtypes in the striatum and cerebral cortex. Analyses of parvalbumin and calretinin positive inhibitory interneurons in the striatum and the prefrontal cortex in WS may yield additional patterns of aberrant distribution that merit comparison to other neurobehavioral disorders. Similar staining methods will be applied in select regions of the prefrontal cortex to examine the 116 distribution of inhibitory interneurons in these regions. Previous research (Lew et al.,

2017) found a decrease in neuronal density in BA10 and BA11 in Nissl stained sections.

Applying methods in these regions and other areas in the prefrontal cortex to quantify inhibitory interneurons will highlight which populations of neurons may be differentially affected in WS.

Examining myelination and white matter distribution in the striatum and prefrontal cortex. The research in this dissertation found that myelin-producing oligodendrocytes are differentially increased in density in the caudate nucleus. This has important implications for examining local connectivity and associated white matter microstructure in our postmortem sample of adult brains. I plan to utilize methods for myelin staining in neuroanatomically identified striatal and prefrontal cortical areas. I expect to see an overall increase in the density of myelinated fibers in adult subjects consistent with the overabundance of myelin-producing oligodendrocytes.

Rigorous scientific research often fails to provide answers, but will always generate more questions. Given the fascinating social and behavioral phenotype present in WS, a wealth of future research is likely to yield surprising findings that support a better understanding of functional neuroanatomy in the human social brain.

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