ONTOGENETIC VARIATION AND THE PLASTICITY OF NEUROBIOLOGICAL AND BEHAVIORAL PHENOTYPES IN THE SOCIALLY MONOGAMOUS PRAIRIE VOLE (MICROTUS OCHROGASTER)

A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

by Lisa Christine Hiura August 2020

© 2020 Lisa Christine Hiura

ONTOGENETIC VARIATION AND THE PLASTICITY OF NEUROBIOLOGICAL AND BEHAVIORAL PHENOTYPES IN THE SOCIALLY MONOGAMOUS PRAIRIE VOLE (MICROTUS OCHROGASTER)

Lisa Christine Hiura, Ph. D. Cornell University 2020

With everchanging environmental contexts and dynamic social partners, the ability to continuously integrate information and make adaptations is essential for an animal’s survival and fitness. Early experiences powerfully shape the phenotypic trajectories of offspring, such that conspecifics can exhibit wildly divergent behaviors in the same contexts. The causes and consequences of social behavioral diversity are rooted in the plasticity of neuroendocrine signaling systems, which also serve fundamental roles in the functional and structural development of the brain. Critically, we still know little of the mechanisms by which complex experiences are encoded in the brains of developing offspring to pattern behavioral variation in latter stages of life. This thesis investigates the impact(s) of multifactorial early life experiences on the ontogeny of neural phenotypes in the biparental prairie vole system. In chapter 2, I describe the ages at which distinct subregions of the medial extended amygdala begin to functionally respond to changes in social context. In chapters 3 and 4 I follow with a characterization of how multiple dimensions of the rearing environment independently and synergistically shape the profiles of oxytocin and vasopressin receptors, their peptide-producing cell groups, and age-specific offspring behaviors.

The data reveal that region-specific receptor densities are contingent upon an animal’s sex, a father’s presence or absence within the natal nest, the complexity of the juvenile weaning environment, and the higher-order interactions thereof. Furthermore, the absence of a father during the rearing period predicts a more socially permissive offspring phenotype, and lower numbers of oxytocinergic cells in the adult hypothalamus. However, these outcomes are contingent upon family handling conditions, indicating that alternate early life factors can offset or exacerbate the influence of paternal care. Finally, in chapter 5 I describe the reciprocal effect of variation in pup rearing experiences on parental phenotypes. Parental behaviors are surprisingly consistent across parenting contexts, but the associations between behavior and vasopressin cell groups display experience dependent plasticity.

Altogether this body of work exhibits the extensive plasticity of behavior and underlying nonapeptide mechanisms, emphasizing the importance of examining environmental interactions within and across the lifespan of the prairie vole.

BIOGRAPHICAL SKETCH

Lisa Christine Hiura was born in Hamamatsu, Japan, and moved to the United States at four years old. She grew up in Eugene, Oregon, where she spent much of her time exploring the evergreen forest trails of the Pacific Northwest and trying to get as close as possible to every animal that came across her path. She graduated from North Eugene High School after completing an International Baccalaureate independent research project on racism and stereotypic depictions of marginalized characters in children’s media. Wanting to understand why people do the things that they do, she became engrossed in the study of behavior and pursued her Bachelor of Arts in Psychology at Reed College in Portland, Oregon. At Reed, she began her research career in comparative cognition and behavioral neuroscience and conducted her senior thesis on the reinforcement value of social contact in the laboratory rat. She became deeply interested in the study of social behavior, in particular the neurobiological mechanisms that give rise to individual and species differences in social phenotypes. This interest led her to pursue her PhD in Psychology with a focus on Behavioral and Evolutionary Neuroscience in the laboratory of Dr. Alexander Ophir at Cornell University. There, she began working with the socially monogamous prairie vole, sealing her lifelong obsession with rodents. Funded by a National Science Foundation Graduate Research Fellowship, she investigated the role of early social experiences on social behavioral and neurobiological development, with a focus on plasticity of the neuropeptides oxytocin and vasopressin.

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To my mother, whose support is unconditional and fierce And to my husband, the epitome of joy.

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ACKNOWLEDGMENTS

I owe everything that I have to my community. There are so many people that deserve recognition for the ways, big and small, in which they have helped me. First and foremost I need to thank my mother, Kazumi Hiura, who is the hardest working person that I have ever had the honor of knowing. She is a veritable fountain of love and support and has spent her life setting an example of resilience for myself and for others. I am where I stand today because of her bravery. I am profoundly lucky to have the privilege of calling her my mother. I am all the more so to get to call her my friend. I have so much gratitude for my whole family in Japan, who beyond a literal ocean and language barriers, have been a source of love and laughter.

I would like to thank my lab mates, my compatriots, past and present. Marissa

Rice, Caitlyn Finton, Santiago Forero, Angela Freeman, Wen-Yi Wu, Lindsay Sailer,

Jesus Madrid, Eileen Chun, George Prounis, Danielle Lee. They have each brought so much joy to my day to day life and inspired me in ways that have influenced my work and my spirit. There is no doubt in my mind that each will continue to do so for the other fortunate friends in their lives.

I am eternally grateful for my research assistants for their endless enthusiasm and dedication. They came into the lab to learn about science but were the ones to teach me what it truly means to be a scientist. Practicing mentorship has been among the most fulfilling components of my graduate training. It has been an honor to work with each and every one of the students that have joined my projects. I would especially like to thank Vanessa Lazaro and Mandy Chan for their devotion to the

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voles, their brilliance in their work, and their patience with me.

Aubrey Kelly has done more for my personal and professional development than most people I have met in my lifetime. She has been a fierce advocate, a steadfast friend, and an inspirational mentor. She taught me about the perfect pairings between wine and food, tissue and antibody. I race behind her in the trail that she has blazed for myself and for other women in science.

My committee members, Elizabeth Adkins-Regan, Katie Kinzler and Mike

Goldstein, have been constant sources of intellectual stimulation and wisdom. My conversations with them have radically changed my perspectives on biology and psychology, and these teachings have forever altered my developmental trajectory.

I profusely thank Alex Ophir for taking a chance on me. For pushing me and teaching me and believing in my capacity to succeed. Even when he ceases to be my formal advisor, he will be a lifelong mentor to myself and my colleagues. I have changed more under his tutelage than that of any other mentor that I have ever had.

I could write an entire separate dissertation about the love of my life, Alan

Baur. He followed me across the country with 100% confidence in our path, even when I was unsure of my own plans. He’s been a daily source of encouragement, both to keep pushing and to rest. This feat is as much his as it is my own. I aspire to be even a fraction as diligent and creative and fearless and kind as he demonstrates himself to be every day. He has changed my life in innumerable ways, and I wake up every single day excited to see what our next adventure entails.

I won the lottery when I joined the Marshall / Baur family. Sue and Rob have treated me as one of their own long before the wedding. Kelly, Will, and Ali have

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shared their love and kindness and inventiveness with me, and have become the family that I never knew that I could have. Wesley and Sienna are the most delightful niblings whom I have the honor of watching grow, even if from afar.

My community of friends, too many wonderful people to name here. Just a few of my board game buddies and fiction co-enthusiasts - Kristina Smiley, Sam Carouso-

Peck, Katerina Faust, Mary Elson, Erin Isbelin, Joel Tripp, Hannah Chapman Tripp,

Raj Anderson. My trivia team, for making 4th place always feel like 1st, and for teaching me to double down. My kickball team, Ballz to the Falls, for reminding me that even when you’re not the best, you can certainly have the most fun. For those times when I had to force myself to think about something other than rodents, I leaned on these friends. Venting and dancing and laughing with them feels like coming home.

I would be remiss if I were to ignore my beloved cat, Henry, who brings me endless laughter and amusement. At both of our expenses. And of course, godmother to our cat, Phoebe Young, for being our first and forever Ithaca friend.

The faculty of the Psychology department and the BEN program have taught me innumerable lessons, academic and beyond. The staff have made this work possible. Without Pam Cunningham, Cindy Durbin, and Lisa Proper, my experience in the program would have been much less joyful. I will miss walking the halls and seeing all of their faces and hearing their lively discussions. The CARE staff deserve so much recognition for their daily contributions to our work. I was extremely fortunate to have so many individuals go above and beyond to support our projects.

I would also like to thank my funding sources - my progress was made possible in large part due to the National Science Foundation Graduate Research

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Fellowship (2016196111) and by the Eunice Kennedy Shriver National Institute of

Child Health & Human Development of the National Institute of Health (HD079573).

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TABLE OF CONTENTS

BIOGRAPHICAL SKETCH ...... V

ACKNOWLEDGMENTS ...... VII

LIST OF FIGURES ...... XIV

LIST OF TABLES ...... XVII

CHAPTER 1 - GENERAL INTRODUCTION: EARLY EXPERIENCES AND THE

DEVELOPMENTAL PLASTICITY OF NEUROBIOLOGY AND

BEHAVIOR IN THE PRAIRIE VOLE (MICROTUS OCHROGASTER) ...... 1

REFERENCES ...... 15

CHAPTER 2 - AGE-SPECIFIC AND CONTEXT-SPECIFIC RESPONSES OF THE

MEDIAL EXTENDED AMYGDALA IN THE DEVELOPING PRAIRIE

VOLE ...... 21

ABSTRACT ...... 21

INTRODUCTION ...... 22

MATERIALS AND METHODS ...... 27

RESULTS ...... 34

DISCUSSION ...... 37

CONCLUSIONS ...... 47

REFERENCES ...... 49

FIGURES ...... 57

CHAPTER 3 - INTERACTIONS OF SEX AND EARLY LIFE SOCIAL

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EXPERIENCES AT TWO DEVELOPMENTAL STAGES SHAPE

NONAPEPTIDE RECEPTOR PROFILES ...... 66

ABSTRACT ...... 66

INTRODUCTION ...... 67

MATERIALS AND METHODS ...... 70

RESULTS ...... 73

DISCUSSION ...... 76

CONCLUSIONS ...... 88

REFERENCES ...... 90

FIGURES ...... 101

TABLES ...... 109

CHAPTER 4 - PATERNAL ABSENCE, FAMILIAL HANDLING, AND

OFFSPRING AGE INDEPENDENTLY AND INTERACTIVELY SHAPE

THE DEVELOPMENT OF PRAIRIE VOLES (MICROTUS

OCHROGASTER) ...... 112

ABSTRACT ...... 112

INTRODUCTION ...... 113

MATERIALS AND METHODS ...... 116

RESULTS ...... 124

DISCUSSION ...... 130

CONCLUSIONS ...... 140

REFERENCES ...... 142

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FIGURES ...... 151

TABLES ...... 158

CHAPTER 5 - PRAIRIE VOLE (MICROTUS OCHROGASTER) PARENTAL

BEHAVIORS AND VASOPRESSIN CELL GROUPS EXHIBIT

PLASTICITY IN RESPONSE TO EXPERIENCES AS A PARENT ...... 160

ABSTRACT ...... 160

INTRODUCTION ...... 161

MATERIALS AND METHODS ...... 164

RESULTS ...... 169

DISCUSSION ...... 179

CONCLUSIONS ...... 192

REFERENCES ...... 194

FIGURES ...... 202

TABLES ...... 215

CHAPTER 6 - GENERAL DISCUSSION ...... 223

REFERENCES ...... 236

FIGURES ...... 240

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LIST OF FIGURES

CHAPTER 2

Figure 2.1 Top-to-bottom flowchart depicting experimental treatment conditions. .... 57

Figure 2.2 Diagram of target medial extended amygdala brain regions...... 58

Figure 2.3 Tyrosine hydroxylase cells counts within the medial extended amygdala over pup ages...... 59

Figure 2.4 cFos-ir counts as a function of pup age and experimental condition within the medial division of the bed nucleus of the stria terminalis (BSTm)...... 60

Figure 2.5 cFos-ir counts as a function of pup age and experimental condition within the principal nucleus of the bed nucleus of the stria terminalis (pBST) ...... 61

Figure 2.cFos-ir counts as a function of pup age and experimental condition within the medial amygdala (MeA)...... 62

Figure 2.7 Proportion of tyrosine hydroxylase neurons co-labeled with cFos-ir as a function of pup age and experimental condition within the BSTm ...... 63

Figure 2.8 Proportion of tyrosine hydroxylase neurons co-labeled with cFos-ir as a function of pup age and experimental condition within the pBST ...... 64

Figure 2.9 Proportion of tyrosine hydroxylase neurons co-labeled with cFos-ir as a function of pup age and experimental condition within the MeA ...... 65

CHAPTER 3

Figure 3.1. Experimental design ...... 101

Figure 3.2 Main effects of Sex on mean vasopressin 1a receptor (V1aR) binding densities ...... 102

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Figure 3.3 Main effect of Pre-wean condition on mean V1aR binding densities ...... 103

Figure 3.4 Main effect of Pre-wean condition on mean oxytocin receptor (OTR) binding densities ...... 104

Figure 3.5 Main effect of Post-wean condition on mean V1aR binding density for the anterior hypothalamus...... 105

Figure 3.6 Interaction effect of Pre-wean condition and Sex on mean V1aR binding density for the ventromedial hypothalamus ...... 106

Figure 3.7 Interaction effect of Pre-wean condition and Post-wean condition on OTR binding density for the septohippocampal nucleus ...... 107

Figure 3.8 Interaction effect of Pre-wean condition, Post-wean condition and Sex on

OTR binding density for the lateral septum ...... 108

CHAPTER 4

Figure 4.1 Timeline of experimental treatments ...... 151

Figure 4.2 Offspring performance in the Open Field test ...... 152

Figure 4.3 Offspring performance in the Social Interaction test ...... 153

Figure 4.4 Offspring performance in the Resident-Intruder test ...... 154

Figure 4.5 Offspring performance in the social dominance Tube test ...... 155

Figure 4.6 Offspring performance in the Partner Preference test ...... 156

Figure 4.7 OT-ir cell counts by experimental conditions ...... 157

CHAPTER 5

Figure 5.1 Parental pup grooming duration ...... 202

Figure 5.2 Combined parental pup retrievals ...... 203

Figure 5.3 Pup retrievals across conditions ...... 204

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Figure 5.4 Mother’s behavior across conditions ...... 205

Figure 5.5 Pup grooming durations in biparental families...... 206

Figure 5.6 Pup retrievals in biparental families ...... 207

Figure 5.7 Nonparental behaviors in biparental families ...... 208

Figure 5.8 Father’s pup directed care over conditions ...... 209

Figure 5.9 Changes in nonparental behaviors in fathers ...... 210

Figure 5.10 OFT performance in mothers ...... 211

Figure 5.11 OFT performance in biparental families ...... 212

Figure 5.12 Relationships between hypothalamic cell counts and behavioral principal components in mothers ...... 213

Figure 5.13 Relationships between hypothalamic cell counts and behavioral principal components in fathers ...... 214

CHAPTER 6

Figure 6.1 Extension of social behavior network schematic ...... 240

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LIST OF TABLES

CHAPTER 3

Table 3.1 Vasopressin 1a receptor densities across structures ...... 109

Table 3.2 Oxytocin receptor densities across structures ...... 111

CHAPTER 4

Table 4.1 Final parameters of selected GLMM for behavioral and neural analyses. . 158

Table 4.2 Effects of experimental factors on hypothalamic cell groups...... 159

CHAPTER 5

Table 5.1 Ethogram of scored behaviors with operationalized definitions...... 215

Table 5.2 Effects of conditions on parental behavior of mothers ...... 216

Table 5.3 Post-hoc contrasts between pup ages on maternal behaviors ...... 217

Table 5.4 Effects of conditions on non-parental behavior of mothers ...... 218

Table 5.5 Female behavior PCA ...... 219

Table 5.6 Male behavior PCA ...... 220

Table 5.7 GLMM results for predictors of cell counts in mothers ...... 221

Table 5.8 GLMM results for predictors of cell counts in fathers ...... 222

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CHAPTER 1

GENERAL INTRODUCTION: EARLY EXPERIENCES AND THE

DEVELOPMENTAL PLASTICITY OF NEUROBIOLOGY AND BEHAVIOR IN

THE PRAIRIE VOLE (MICROTUS OCHROGASTER)

Our earliest experiences have profoundly enduring effects on mental illness, physiology, social wellbeing, and behavior. Experiences that take place within hours of parturition can have lasting consequences for an animal’s phenotype, even well into adulthood. Life-threatening environmental conditions, such as the availability of food, or the presence of predators, can exert strong influences on the development of an organism (Taborsky, 2017). But natural variation exists along many dimensions of the early environment; temperature, humidity, nutrition, season, and social interactions all change over the course of animal development. Why do fluctuations within the tolerable range of these seemingly innocuous factors lead to sustained alterations in developmental trajectories? And what mechanisms translate perinatal experiences into adult phenotypes?

Developmental plasticity is the ability of an animal to respond to biotic and abiotic ontogenetic experiences by altering its developmental phenotype (West-

Eberhard, 2003). For example, dung beetles (Onthophagus taurus) alter their morphological phenotypes based on their early nutritional experiences; if males are provisioned with enough high-quality food as larvae, they develop large horns as adults. Otherwise, they mature as hornless morphs (Moczek, 1998). Developmentally contingent outcomes in morphological characteristics (color, size, physical traits) are

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commonly studied, but the phenotypic flexibility enabled by variation in behavioral development is becoming increasingly appreciated in the field of evolutionary developmental biology (Bertossa, 2011; Hoke et al., 2019).

Stimuli throughout the environment provide context clues about the future environment in which organisms may find themselves (Bateson, 2001).

Developmental plasticity is adaptive when the use of such early cues enables the organism to make accurate predictions about future environments, such that their altered phenotype is more competitive under the subsequent environmental demands

(Bateson, 2001). For instance, when adolescent male guinea pigs (Cavia porcellus) were either housed in mixed-sex pairs or in large mixed-sex colonies, then later cohoused with two sexually-mature females, males from the paired background were more aggressive and sired significantly more offspring than males from the colony background (Zimmermann et al., 2017). Conversely, when such males were moved into a large mixed-sex colony, males from paired backgrounds exhibited heightened physiological stress responses, lost more weight, and engaged in more fights than males from colony backgrounds (Sachser et al., 2011). Together these results demonstrate that animals performed optimally under adult conditions that better approximated their early-life housing conditions. This example of developmental plasticity highlights how altering a developing phenotype can increase reproductive fitness, producing a condition under which developmental plasticity should be under positive selection. However, this example also demonstrates how the adaptive value of phenotypic plasticity is dependent upon the degree to which cues from the early environment accurately predict the following environmental conditions. For many

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developing animals, social interactions provide a rich source of contextual information. As such, the influence of the early social environment on the development of offspring phenotypes has become a fruitful area of research. In the following section I describe the role that caregivers play in mammalian offspring development.

Parental influences and offspring development

For many species, one of the greatest influences that offspring experience in early development is the quality and quantity of parental care they receive. This is particularly true among mammalian species, for which maternal care is ubiquitous and offspring depend on milk provisioning by mothers during perinatal development

(Kleiman and Malcolm, 1981). Interactions with mothers make up the majority of a neonate’s social world, and environmental conditions are mediated through a mother’s behavior towards her offspring (Wells, 2019). For the 5-10% of mammalian species that exhibit biparental care, paternal interactions also comprise the earliest social relationships for offspring (Kleiman and Malcolm, 1981). Early maternal (or paternal) behaviors such as huddling, grooming, and retrieving function to regulate body temperatures and provide intensive bodily stimulation, exposing neonates to a complex set of sensory experiences (Ronca et al., 1993). In this way, caregivers construct the offspring’s earliest postnatal ontogenetic niche (Alberts, 2008). As offspring mature, their interactions with their mothers (or other caregivers, such as fathers or older siblings) and their physical environments change in tandem, enabling access to additional cues afforded by ongoing changes in their niche. Substantial

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empirical evidence has demonstrated that such early liminal social interactions hold the capacity to push an individual down alternate paths of a species-typical trajectory

(Champagne and Curley, 2005).

A valuable case study of socially-mediated developmental plasticity comes from the rich literature describing the functions of early-life adversity on the neurobiology of stress (Levine, 2005). Decades of work studying rats have leveraged natural and experimentally induced variation in maternal caregiving to demonstrate that alterations in mother-infant interactions can impart life-long changes in the behavior and physiology of offspring. More specifically, paradigms that modify pup- directed maternal behaviors can produce offspring that are either more resilient to, or more vulnerable to, the effects of later life stressors. Experience-induced modification of hypothalamic-pituitary-adrenal (HPA) axis regulation provides a likely mechanism by which this kind of developmental plasticity in behavior is possible. For example, higher levels of maternal care during the rearing period produce offspring with attenuated corticosterone signaling and potentiated negative feedback in response to stressors (Champagne and Curley, 2009). This work demonstrates the incredibly powerful role that mothers play in offspring phenotypic development and has provided the field with some of the most comprehensive evidence of the molecular mechanisms that facilitate developmental plasticity.

Nonapeptide cells, receptors, and social experiences

The mammalian nonapeptide homologues oxytocin (OT) and arginine vasopressin (VP) are two closely related nine amino acid peptides that function as

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neuromodulators in the central nervous system and as hormones in the periphery

(Stoop, 2012). Their functions have been extensively tied to the regulation of various social behaviors such as parental care, courtship, mating, social recognition, and pair bonding (Goodson 2008; Dumais & Veenema 2016). The nonapeptides are deeply conserved across vertebrate evolution, but show considerable interspecific and intraspecific plasticity in the distribution of their receptor densities and the activity patterns of their peptide producing cell groups (Goodson and Kabelik, 2009). This diversity is believed to be a key mechanism underlying the variation in social behavior evident across the animal kingdom.

OT and VP exert their actions by binding to their G-protein coupled receptors, which initiate intracellular signaling cascades that participate in processes including the regulation of gene expression, mobilization of intracellular calcium, or alteration of cell excitability (Stoop, 2012). The particular molecular consequences of a binding event are contingent upon which class of G-proteins the activated receptor is bound to, contributing to the pleiotropic effects of nonapeptide signaling systems. OT has one receptor subtype (OTR) and VP has three (V1aR, V1bR, V2R), although OTR and

V1aR are the most intensively studied in relation to social behavior (Jard et al., 1987;

Young, 1999). Activation of these receptors regulates broad physiological and behavioral processes (Landgraf and Neumann, 2004). Furthermore, each receptor is capable of binding both OT and VP, increasing the degrees of freedom by which the nonapeptide systems may enact their central effects (Chini and Manning, 2007; Smith et al., 2019; Song and Albers, 2018).

Patterns of peptide-producing cells and receptor densities are specific to

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species, sex, and developmental age (Dumais and Veenema, 2016; Insel and Young,

2000). Furthermore, these nonapeptide profiles are shaped by variation in early-life experiences, suggesting that developmental processes alter phenotypic trajectories by differentially organizing related nonapeptide systems (Vaidyanathan and Hammock,

2017). The developmentally-contingent calibration of the brain, specifically with respect to the nonapeptide system, may enhance an individual’s ability to adapt to social and asocial challenges (Knudsen, 2004). Unfortunately, the majority of work that describes the impact of early experiences on the development of nonapeptide systems focuses on a singular, isolated dimension of the early environment (paternal absence, or variation in maternal care, or isolation, etc.). As such, the ways in which nonapeptide systems are shaped by interactions amongst multiple early experiences remain unclear.

Prairie voles as a model for understanding the impact of parental care on adult behavioral and neural phenotype.

Prairie voles (Microtus ochrogaster) have been especially amenable to laboratory research focused on the impacts of early-life parental care on offspring development. Prairie voles are socially monogamous and bi-parental and offer great opportunity to disentangle the contributions of maternally and paternally mediated impacts on offspring behavioral and neurobiological outcomes. Free-living families consist of three types of social groups; male-female pairs with pups, single mothers with a litter, and communal groups made up of a mother or both parents with multiple generations of offspring (Getz and Carter, 1996). These social groups are found in

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roughly equal frequencies, and thus variation in familial composition is a natural occurrence in this species. Unlike sires of uniparental mammals, prairie vole fathers exhibit robust paternal behaviors and provide all forms of offspring care that females display with the exception of nursing (Thomas and Birney, 1979).

The amount of care that prairie vole parents provide to their offspring is naturally variable. Adult offspring of high-contact parents and low-contact parents differ in their intrasexual aggression, and in their subsequent pup-directed behaviors

(Perkeybile et al., 2013; Perkeybile and Bales, 2015a). Furthermore, the maternal care that rodent mothers provide to their offspring can be experimentally upregulated by handling either the mother or her pups (Levine, 2002; Perkeybile et al., 2019). When prairie vole parents are handled to induce an increase in their pup directed care behaviors, their adult offspring show less anxiety-like behavior (Bales et al., 2007), and male offspring demonstrate more alloparental care (Bales et al., 2011, 2007).

Female offspring from control handling conditions (that experienced putatively less parental care as a neonate) showed impairments in their ability to form pair bonds

(Bales et al., 2007), indicating that experimental manipulation of maternal care can induce robust behavioral effects on species-typical social behavior. Moreover, paternal effects on prairie vole offspring development have been assessed by rearing pups with and without fathers present in the natal nest. Such studies have demonstrated that paternal presence influences behaviors including the onset of consuming solid foods

(Wang and Novak, 1992), the age of first foray out of the natal nest (Wang and Novak,

1992), anxiety-like behaviors (Ahern and Young, 2009), alloparental care behaviors

(Ahern and Young, 2009), social affiliation (Tabbaa et al., 2017), and the latency to

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develop a pair bond (Ahern and Young, 2009). Collectively, these studies provide considerable evidence for the impact that natural and experimentally induced variation in parental care can have on the diversity of offspring social development. Diversity in behavior is facilitated by variation in the underlying neural substrates. Therefore, behavioral group-differences in vole offspring that were produced by parental effects must be functionally related to diversity in their neural phenotypes.

Prairie voles have served as a valuable system in which to investigate the impacts of developmental experiences on the nonapeptide system. Variation in the familial composition of the natal nest has been found to alter OT mRNA expression,

OTR densities, and V1aR densities in regions of the forebrain in later life (Ahern and

Young, 2009; Prounis et al., 2015). The amount of postnatal care that pups receive impacts the densities of VP-producing cells in the hypothalamus (Perkeybile and

Bales, 2015b), and early handling-induced alterations in pup-directed parental behaviors impact the counts of hypothalamic OT-producing cells (Carter et al., 2008).

Reduced levels of maternal care leads to increases in the methylation of the OTR gene, and lower levels of OTR mRNA and protein expression, indicating that early care modifies the development of prairie vole OT systems through experience-dependent epigenetic regulation (Perkeybile et al., 2019). These studies demonstrate that the profiles of both nonpeptide-producing cells and the densities of their receptors are susceptible to alterations by early experiences. Considering the roles that nonapeptides have in modulating social behavior, and that such developmental experiences change nonapeptide phenotypes that persist into adulthood, it stands to reason that perturbations in the social environmental can and should impact adult social behavior.

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Critically, laboratory studies of developmental experiences have primarily implemented the manipulation of single dimensions of the early environment (such as paternal presence, social isolation after weaning, or altered parental care). However, the real world is a complex, dynamic physical and social environment. Developing organisms receive environmental information from numerous sources of their postnatal contexts, and also integrate experiences across multiple points across their lifespan (Taborsky, 2017). Early experiences that differentially impact brain regions at one stage of life could bias the functionality of those brain regions during subsequent life experiences by patterning distinct nonapeptide phenotypes.

Prounis et al., (2015) demonstrated that the preweaning social environment of a vole pup (specifically, the presence or absence of a father) interacts with the postweaning social environment (isolate or pair housing) to impact social discrimination and socio-spatial memory performance and OTR density in the lateral septum (a structure heavily implicated in social recognition). Furthermore, a study in male mice revealed that a forced-swimming acute stressor in young adulthood induced an increase in hippocampal OTR mRNA when those mice experienced chronic social isolation in early-life, but not if they were reared in standard housing conditions

(Lesse et al., 2017). These studies emphasize the idea that variation across multiple dimensions of early experience has the potential to synergistically shape offspring phenotypes across animals’ life-history.

Aims and objectives

The goal of this dissertation is to analyze how interactions between distinct

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factors within the early social environment contribute to social and neurochemical phenotypes in prairie voles. This is accomplished by characterizing the concordant changes in the developmental plasticity of behavioral phenotypes and the nonapeptide system. More specifically, we systematically vary multiple dimensions of the ontogenetic environment during the generally sensitive period of development and measure the trajectories of nonapeptide producing cells, receptor distributions, and behaviors.

Chapter 2: How does the perinatal vole brain’s response to the social environment change over the course of development?

The ability of the organism to incorporate information from the social environment requires that the brain can first recognize and encode social environmental changes at a cellular level. The nervous systems of perinatal pups are immature at birth, and it is not clear to what extent they are sensitive to social stimuli.

The behavioral repertoires of infants rapidly change with physical maturation, and the development of neural systems must change in parallel. Therefore, it would be prudent to assess when during development the brains of preweanling prairie voles become sensitive to their social environments.

In chapter 2, I present a reproduction of a published paper (Hiura et al., 2018,

Developmental Neurobiology) that describes the patterns in which immature brains respond to features of the social environment. We measured induction of the immediate early gene cFos following an acute isolation paradigm to identify the ages at which prairie vole pup brains begin discriminating between social contexts. We

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reveal that distinct brain regions begin to functionally respond at separate developmental stages, and to either isolation or social contact, demonstrating that the course of prairie vole social brain development is heterogenous in nature.

Chapter 3: How does variation within, and interactions across, multiple dimensions of a prairie vole’s early environment shape patterns of OTR and V1aR densities?

Receptors represent the capacity for cells to respond to incoming signals.

Modulating receptor density within a specific brain-region dynamically regulates the sensitivity of that structure to neurochemical inputs. Changes in receptor densities have downstream consequences for cell signaling networks, and ultimately, for behavior. As brains begin to integrate social information, these experiences shape the trajectories of neuromodulatory systems critical for social behaviors - notably the nonapeptides oxytocin and vasopressin. The nonapeptide receptors exert pleiotropic effects on physiology and social behavior based upon their site of expression.

Therefore, characterizing how OTR and V1aR densities are impacted by early experiences will inform our understanding of the mechanisms by which developmental inputs tune the social brain.

In chapter 3, I present a reproduction of a publication (Hiura and Ophir, 2018,

Integrative Zoology) in which we characterized the effects of perinatal and postnatal social contexts, sex, and their interactions on nonapeptide receptor densities across the forebrain. We ascertained that variation in distinct dimensions of the early environment leads to group differences in nonapeptide receptor densities in a region- specific, sex-specific, and rearing condition-specific manner. These data demonstrate

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that nonapeptide receptor profiles are a product of complex interactions among developmental contexts and biology. Our results exemplify the necessity of implementing research designs that better reflect the variability of the natural world if we wish to fully understand the causes and consequences of social developmental plasticity.

Chapter 4: How are behavioral profiles, as well as the nonapeptide-producing cell groups critical for sociality, shaped by independent and interacting early-life experiences?

Beyond experience-dependent variation in receptor densities, diversity in nonapeptide-producing cells can also drive behavioral flexibility. In chapter 4 (in prep for submission), I manipulated paternal presence and experimental handling conditions to investigate how distinct dimensions of the early environment may interact to mediate hypothalamic cell counts and age-specific behaviors. I found that rearing conditions impact behavioral phenotypes in an age-specific manner, where variation in social behavior arises or dissipates over separate life stages. Furthermore, distinct early experiences do not impart homogenous effects on offspring development, indicating that particular developing systems may be differentially susceptible to the influences of qualitatively distinct experiences. Oxytocin-producing, but not vasopressin-producing, cell groups varied as a function of interactions among multiple early-life factors, demonstrating that the developmentally contingent diversity in neuroendocrine profiles may underlie behavioral variation.

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Chapter 5: How do early postnatal environments and diversity in rearing experiences drive the subsequent behavioral and neurochemical profiles of parents themselves?

It is critical to acknowledge that growing pups are not passive vessels into which environmental information is poured by parents, but rather a rich dynamic stimulus that responds to and elicits interactions (Schwab and Moczek, 2017). Early- life social experiences not only shape the development of pups, but they also fundamentally modify the social world of the caregiving parents. Adult animals exhibit neurophysiological and behavioral phenotypic plasticity in response to social environments, despite falling outside of the range of developmental sensitive periods

(Dufty et al., 2002). The evolution of developmental plasticity is intrinsically tied to the behavior of parents. Thus, an investigation of how experiences as a parent shape the ensuing phenotypes of mothers and fathers will provide insight into the reciprocal relationship between offspring and parental plasticity.

In chapter 5 (in prep for submission), I manipulated the presence or absence of a parenting partner and applied an acute handling stressor to systematically vary the parenting experiences of adults. I demonstrated that parents are exquisitely sensitive to the maturation of offspring, but mothers and fathers differ in how their behaviors shift with pup age. Parents did not exhibit dramatic behavioral changes as a function of rearing conditions. However, the associations between vasopressin-producing cell groups and behavioral suites were altered by rearing experience, demonstrating that brain-behavior relationships are plastic in adulthood. These results suggest that experience-dependent programming of nonapeptide systems is not a phenomenon exclusive to developing young, but rather may serve as an ongoing mechanism to

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facilitate behavioral plasticity throughout the lifespan.

This dissertation attempts to integrate information on the plasticity of developmental systems across the levels of cellular activity, receptor densities, cell group profiles, and behavior. In chapter 6, I synthesize the empirical data and fit these results into an existing multiple-hits framework of phenotypic development inspired by the clinical literature. I conclude by describing future directions of research that could provide causal evidence linking transient neural phenotypes to adult outcomes, with the aim of moving towards a more complete picture of the mechanisms and functions of developmental plasticity.

14

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CHAPTER 2

AGE-SPECIFIC AND CONTEXT-SPECIFIC RESPONSES OF THE

MEDIAL EXTENDED AMYGDALA IN THE

DEVELOPING PRAIRIE VOLE

As published in: Hiura, L.C., Kelly, A.M. & Ophir, A.G. (2018) Age-specific and context-specific responses of the medial extended amygdala in the developing prairie vole. Dev. Neurobiol. 78, 1231–1245.

ABSTRACT

The social needs of organisms change as they mature. Yet, little is known about the mechanisms that subserve processing social interactions or how these systems develop. The medial extended amygdala (meEA) is comprised of the medial bed nucleus of the stria terminalis (BSTm) and the medial amygdala (MeA). This neural complex holds great promise for understanding how the social brain processes information. We assessed expression of the immediate early gene cFos and the enzyme tyrosine hydroxylase (TH) at three developmental time-points (postnatal day

[PND] 2, 9, and 21) to determine how developing prairie voles process familial social contact, separation, and reunion. We demonstrate that (1) BSTm cFos responses were sensitive to separation from family units at PND 9 and PND 21, but not at PND 2; (2)

MeA cFos responses were sensitive to reunion with the family, but only in PND 21 pups; (3) BSTm TH neurons did not exhibit differential responses to social condition

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at any age; and (4) MeA TH neurons responded strongly to social contact (remaining with family or following reunion), but only at PND 21. Our results suggest that the sub-units of the meEA become functionally responsive at different developmental time points, and are differentially activated in response to distinct social contexts.

Overall, our results support the notion that interconnected regions of the meEA follow divergent developmental timelines and are sensitive to distinct properties of social contexts.

INTRODUCTION

Much of what we understand about the mechanisms of social behavior is derived almost entirely from research on adult animals. From studies of social motivation and pair bonding to cooperation and emotional processing, the neural mechanisms that underlie social behaviors have been primarily explored in mature brains (Happé and Frith, 2014). The social (and asocial) needs of perinatal and juvenile offspring differ from those of adults, and these needs change substantially as young mature and become less vulnerable (Nelson et al., 2016). For instance, social exploration outside of the nest might be rewarding for sexually mature individuals, but stressful for defenseless perinatal young. Thus, as young develop, the ways in which their brains processes social cues are likely to transform throughout ontogeny (Rehling et al., 2012). Despite the potential for age-dependent differences in sociality, we know very little about when the neural systems that support social functioning develop, or where in the brain these processes functionally emerge.

The few studies that have examined the development of nonsocial and social

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neural functioning have demonstrated age-dependent neural responses. For example, in rats, physical restraint induces dramatically different expression patterns of cFos (an immediate early gene (IEG) and marker of neuronal activation) in the anterior olfactory nucleus, piriform cortex, tenia tecta, and amygdala of postnatal day (PND)

28 juveniles compared to adult PND 60 animals (Kellogg et al., 1998). Critically, neural responses to social stimuli are also refined with age. For example, compared to

PND 28 juvenile hamsters, PND 58-66 adult males show divergent patterns of cFos expression in the ventral tegmental area (VTA), nucleus accumbens (NAcc) and medial prefrontal cortex after the presentation of female vaginal secretions, a biologically relevant cue for the sexually mature male (Bell et al., 2013). Furthermore, patterns of cFos responses differ across the brains of PND 7, PND 14, and PND 21 rat pups following exposure to a novel adult male rat (an important event for preweanling pups that are susceptible to infanticide), specifically within the paraventricular nucleus of the hypothalamus, the amygdala, the periaqueductal gray, and the locus ceruleus

(Wiedenmayer and Barr, 2001). PND 7 rats do not mount discriminative neural responses to the adult stimulus, whereas older age groups show distinct, age-specific patterns of cFos expression following exposure to the social stimulus.

Together, these data demonstrate that the neural processing of postnatal experiences (stressful and/or social) changes across development, and within particular subregions of the brain. However, these few studies on developing brains are conducted within the second week of postnatal life, and thus we still know extremely little about the neural functioning of neonatal offspring. Furthermore, the majority of these data characterize brain responses to novel stimuli, and therefore the

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development of neural responses to the most common social partners of a young helpless animal, namely the parents and siblings, remains unclear. In the present study, we examine the development of social neural function beginning at a neonatal age

(PND 2) in a neural circuit specifically involved in social behavior.

The extended amygdala represents a promising region of interest to investigate the ontogeny of social functioning. The extended amygdala is an important node of the social behavior network (Newman, 1999) that encompasses the reciprocally connected amygdaloid complex and bed nucleus of the stria terminalis (BST). The amygdaloid complex is composed of cytoarchitecturally and functionally distinct subregions

(Johnston, 1923), which notably includes the medial amygdala (MeA) - a subregion of the amygdaloid complex that has been repeatedly implicated in fearful and emotional responses, olfactory information processing, and socio-sexual reward (Bergan et al.,

2014; Davis, 1992; Newman, 1999). Indeed, disruptions to the MeA impair a variety of rodent social behaviors, including male sexual behavior, aggression, and parental care (Kirkpatrick et al., 1994; Vochteloo and Koolhaas, 1987), highlighting the importance of the MeA in social functioning.

Like the MeA, the BST can be anatomically and functionally divided into subcomponents, which each have sets of distinct and overlapping efferent and afferent projections to various amygdaloid nuclei (Coolen and Wood, 1998). Of particular interest here is the medial division (BSTm), which shares dense reciprocal projections with the MeA (Coolen and Wood, 1998; Pardo-Bellver et al., 2012). Traditionally, the

BSTm and MeA comprise the medial extended amygdala (meEA), and share functional characteristics in response to social stimuli (Alheid, 2003). In rodents, these

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regions exhibit elevated cFos levels after agonistic encounters (Kollack-Walker and

Newman, 1995), exposure to alarm pheromones (Kiyokawa et al., 2005), and predator odor (Dielenberg et al., 2001).

Notably, in some species, the meEA contains tyrosine hydroxylase (TH) neurons (Northcutt et al., 2007). TH is the rate-limiting enzyme in the biosynthesis of

L-DOPA, the precursor molecule to catecholamines such as adrenaline, noradrenaline

(NA), and dopamine (DA) (Kobayashi and Nagatsu, 2012). The largest population of

TH cells, and most commonly studied, originates from the ventral tegmental area, and has been linked to rodent play behavior and social-dominance (Filipenko,

Alekseyenko, Beilina, Kamynina, & Kudryavtseva, 2001; Northcutt & Nguyen, 2014).

TH cell groups are not found within the rat meEA (Northcutt, Wang, & Lonstein,

2007), however, dense populations of TH neurons are found in the MeA and BST of the socially monogamous prairie vole (Microtus ochrogaster), and in adult animals these TH cell groups exhibit functional plasticity in response to the presentation of various conspecifics (Northcutt and Lonstein, 2009). Together, these data suggest that meEA TH cells may contribute to the expression of species-typical social behavior.

The molecular consequences of activating MeA and BSTm TH cells is unclear.

In adult prairie voles, TH cells in the MeA and principal BST (pBST; dorsomedial to the BSTm) are not immunoreactive for aromatic L-amino acid decarboxylase (AADC; necessary for the conversion of L-DOPA to DA) or dopamine-beta-hydroxlyase

(DBH; necessary for the conversion of L-DOPA to NA) (Ahmed et al., 2012).

However, studies in rats have demonstrated that nearby AADC-producing cells can coordinate with TH cells to synthesize DA (Ugrumov et al., 2004). Furthermore, the

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multifaceted role of L-DOPA in neural modulation is relatively underappreciated (De

Deurwaerdère et al., 2017), highlighting the importance of investigating TH neural function more broadly.

The species-specific expression of meEA TH cell groups and their sensitivity to the social environment in adult prairie voles, combined with the multiple molecular pathways by which TH can contribute to downstream peptide production, makes these cell groups promising regions to analyze in the context of encoding dynamic social environments. Furthermore, because these TH cell groups were discovered only in the last decade, and have only been analyzed in adult animals, there is still much to learn about their involvement in social behavior and development. Understanding the ontogeny of neural activity within these cells, and the conditions under which these cell groups become responsive will help elucidate their contributions to the social behavior of the developing animal. Furthermore, prairie voles have been touted as a model for the neurobiology of human social attachment behavior because they exhibit pair bonding behaviors and biparental care like humans (McGraw and Young, 2010).

Thus, examining the functional development of TH systems within the meEA of the prairie vole could have substantial translational implications for social neuroscience.

We aimed to investigate how neuronal function within the BSTm and the MeA changes across developmental time. To do this we conducted an IEG study in prairie vole pups at three stages of early development (PND 2, 9, and 21). Utilizing an acute social isolation and reunion paradigm designed to induce stressful and/or emotional states, we examined meEA neural activity (assessed by cFos expression) and meEA

TH neural activity (assessed by TH-cFos colocalization) after social environmental

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manipulation. To our knowledge, this study is the first to examine the ontogeny of meEA function and the development of TH neural function within this circuit. The interconnectedness of the BSTm and the MeA led us to hypothesize that the responsiveness of these regions may emerge simultaneously, and that they may exhibit similar neural response profiles. Either or both of these structures could be involved in the processing of social contexts, and we therefore did not predict a particular direction in their expected responses to each social context.

MATERIALS AND METHODS

Subjects

All prairie vole pups used in this study were the first litter of the F2 generation of breeding pairs derived from wild-caught animals housed in our animal colony. We trapped all wild animals in Champagne County, Illinois, USA. Breeding pairs were established, and the first litter of each pair was culled to three subjects to control for variation in early social experience. Families were assigned unique Litter IDs to control for pup-relatedness in the statistical analyses (see below). Animals were housed under a 14L:10D light cycle in standard polycarbonate rodent cages (29 x 18 x

13cm) lined with Sani-chip bedding. Animals were provided nestlets, water, and standard rodent chow (Laboratory Rodent Diet 5001, LabDiet, St. Louis, MO, USA) ad libitum. All procedures were approved by the Institutional Animal Care and Use

Committee of Cornell University (2013-0102).

Design

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To assess whether meEA function changes across development, we conducted an IEG experiment utilizing a 3 x 3 design (see Kelly et al., 2018). We tested animals that were at three different stages of early development: PND 2, PND 9, and PND 21.

These ages were chosen because they reflect specific behavioral and physiological milestones of prairie vole development. At PND 2, pups are relatively immobile, incapable of thermoregulation, and entirely dependent on parental care for survival. At

PND 9, pups open their eyes and are physically capable of environmental and social exploration. By PND 21, pups are weaned and can survive independently outside of the nest (McGuire & Novak, 1984).

Subjects underwent testing in one of three social conditions in which we manipulated contact with parents and siblings (see Fig. 2.1). Subjects were isolated from their family (Isolate, PND 2 - M:11, F:11; PND 9 - M:10, F:8; PND 21 - M:7,

F:7), reunited with their family after isolation (Reunite, PND 2 - M:9, F:9; PND 9 -

M:11, F:12; PND 21 - M:9, F:9), or interacted with their family under normal conditions (Together, PND 2 - M:10, F:10; PND 9 - M:7, F:14; PND 21 - M:10, F:9).

This design was utilized to assess neural activity in response to emotionally salient and stressful social experiences. Social isolation from siblings and parents presumably represents a life-threatening context for young neonates, and thus is likely to produce a neural response.

Regions of Interest

Because previous studies implicate the MeA and BSTm in stress response and affiliation (Choi et al., 2007; Lim and Young, 2004; Pardo-Bellver et al., 2012), we

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sought to determine the age at which these brain regions become responsive to variation in social context. To this end, we quantified the immediate early gene product cFos within the meEA, because this protein is an established marker of neural activity that can be time-locked to experimental manipulations (Hoffman et al., 1993).

Given that adult prairie voles exhibit changes in cFos activity within regions of the meEA as a function of social context (Northcutt and Lonstein, 2009), we also examined neural activity in TH cell groups within the meEA of developing pups by assessing cFos colocalization with TH neurons. Earlier work from Northcutt et al.

(2007) identified the principal nucleus of the bed nucleus of the stria terminalis

(pBST) as a TH neuron rich region within the extended amygdala of prairie voles.

Although the pBST is not classically considered a component of the medial extended amygdala, it contributes to rodent sociosexual olfactory processing (Dong and

Swanson, 2004) and responds to the presentation of various social stimuli (Northcutt and Lonstein, 2009). As such, we also measured pBST cFos activation and TH-cFos colocalization in relation to the experimental manipulations as described for the MeA and BSTm neural data.

Experimental Manipulations

Breeding pairs (n = 92) were formed and closely monitored until parturition.

Litters were culled to three pups at birth. Families were then randomly assigned to one of nine experimental groups that spanned all possible combinations across age (PND

2/9/21) and social condition (Together, Isolate, and Reunite). All three pups in each litter remained with their families under normal colony conditions until they reached

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test-age.

The social manipulations were designed to assess how the neural profiles of the pups differed when they were removed from, or reunited with, their families at specific ages. The Together condition was intended to serve as a baseline (control) for neural activity when pups coexist with their family groups under normal conditions.

Each manipulation consisted of a 30-min pre-test phase, followed by a 90-min IEG- test phase. The novel cages in all phases were lined with clean bedding, did not contain food or water, and were outfitted with transparent, perforated Plexiglas lids to allow for overhead video recording.

In the Together and Isolate conditions, parents and pups were transferred from their home cages to neutral, clean polycarbonate cages for the pre-test phase. After 30 mins, the Together families were moved as a group to a novel test-cage. In the Isolate condition, the pups were moved into individual novel test-cages. For the Reunite condition, pups were moved into individual novel cages for the 30 min pretest, after which they were reunited with each other and their parents in a novel cage for the 90 min IEG-test phase. All parents and pups experienced the same number of standardized handling experiences, and the same number of cage changes, regardless of experimental group. All subjects were sacrificed immediately following the IEG- test. Thus, the measured neural activity reflects the neurochemical changes that occurred when animals transitioned from pre-test conditions to the IEG-test phase.

We controlled for thermoregulatory and temperature differences across ages in phases where pups were isolated in a neutral cage. An infrared thermometer was used to take an average temperature reading under normal home cage pre-test conditions across

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five families at each age group. Electric heating pads were placed underneath the cage and heated to 36°C for PND 2 pups, 34°C for PND 9 pups, and 30°C for PND 21 pups.

Following perfusions, all pups were visually sexed. PCR amplification was used to detect male-specific SRY genes to confirm the sexes of the PND 2 and PND 9 animals, because at these ages it can be difficult to assess sex visually. Genomic DNA was extracted from tissue punches of lung and spleen samples using the DNEasy

Blood and Tissue Kit and protocol (Qiagen, USA). SRY primer sequences were forward 5’- TTATGCTGTGGTCTCGTGGTC-3’; and reverse 5’-

GCAGTCTCTGTGCCTCTTGG-3’. DNA segments were amplified in a thermocycler

(Eppendorf realplex4) in 25 µL for 35 cycles (94 °C 30 s, 55 °C 1 min, and 72 °C

30 s). The amplified PCR products were separated on 2% agarose gels, and visualized under a UV transilluminator after staining (GelRed, Biotium, USA).

Histology and Immunocytochemistry

Immediately after the 90 min IEG-test, pups were deeply anesthetized by isoflurane and intracardially perfused with 0.1M phosphate-buffered saline (PBS, pH

= 7.4), followed by 4% paraformaldehyde in PBS. Brains were extracted and post- fixed in 4% paraformaldehyde for 24 h, sunk in 30% sucrose for 48 h, then stored at -

80°C until sectioning. Brains were mounted and coronally cryosectioned at 40 μm thickness into three series for PND 21 pups, and into two series for PND 2 and PND 9 pups (to account for smaller brain sizes). Tissue was stored at -80°C in cryoprotectant until immunocytochemical processing.

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Antibody reporting

One series of tissue from each subject was immunofluorescently double- labeled for TH and cFos. Free-floating sections were rinsed twice for 30 min in PBS, and blocked for 1 h (PBS + 10% normal donkey serum + 0.03% Triton-X-100). The primary antibodies used were monoclonal mouse anti-TH (1:1000 μL, Millipore Corp,

USA, Cat. # MAB318) and polyclonal rabbit anti-Fos (4:1000 μL, Millipore Corp,

USA, Cat. # ABE457). Tissue was incubated in primary antibodies for 24 h, rinsed in

PBS for 40 min, and incubated in secondary antibodies for 2 h. The secondary antibodies were donkey anti-mouse conjugated to Alexa Fluor 680 (6:1000 μL) and donkey anti-rabbit conjugated to Alexa Fluor 594 (4:1000 μL), with 5% donkey serum

+ 0.03% Triton-X-100 in PBS. Sections were rinsed in PBS overnight, mounted onto microscope slides, and cover-slipped with Prolong Gold antifade + DAPI nuclear stain

(ThermoFisher Scientific).

Visualization and Quantification

Bilateral photomicrographs were taken at 10X on a Zeiss Axioimager II scope with an AxioCam MRm attachment, z-drive, and Apotome optical dissector (Carl

Zeiss Inc., Gottingen, Germany) at two consecutive levels of the BSTm and MeA.

Areas of interest were localized using neuroanatomical landmarks - the ventral side of the anterior commissure for the BSTm, and optic tract for the MeA (Fig. 2.2). Images were manually-counted for cFos and TH using the GNU Image Manipulation Program

(GIMP, 2.8.22) and ImageJ (National Institutes of Health, Bethesda, MD).

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Monochromatic images were visually quantified for TH-immunoreactivity (-ir), cFos- ir, and colocalization of TH-ir and cFos-ir (TH-ir cells expressing Fos-ir). Inherently, brain sizes varied across age groups, and to a much lesser extent across individuals within age groups. To avoid unfairly biasing the number of cells per structure we could capture, we did not use a use a standard sized box to score a region of interest across all brains. Instead, we outlined and counted immunoreactive cells for the entire region of interest to account for brain size variation. This method had the advantage of providing an exhaustive metric that scales for age-dependent brain size differences across groups.

Statistical Analysis

Cell counts were combined and averaged across rostral-caudal sections within each region of interest. To analyze the colocalization of TH-ir and cFos-ir neurons, the proportions of TH-ir neurons expressing cFos over the total number of TH-ir cells in each region were arcsine transformed. Cell count data were analyzed via Linear Mixed

Models (LMM) in R v.3.2.1 (R Core Team, 2013), using the R package lme4 (Bates et al., 2015). LMMs are robust against slight departures from normality; model residuals were graphically assessed and either log-transformed or square-root-transformed to satisfy assumptions of normality when necessary. Social condition, Sex, and Age were included as fixed effects, while Litter ID was included as a random effect to control for genetic variation. p-values were derived from a likelihood ratio test within the R package lmerTest (Kuznetsova et al., 2017).

We hypothesized that isolation and reunion with families would induce unique

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neural profiles in pups as a function of their age. Our a priori interaction of interest was between the Age and Condition factors. When an Age x Condition interaction was significant, we conducted planned contrasts of social condition within age groups using the R package lsmeans (Lenth, 2016). All pairwise post-hoc tests were

Bonferroni corrected in R to control for multiple comparisons, with a 0.05 α-level threshold for statistical significance.

We examined neural responses to variation in social context. We, therefore, did not explore the main effects of Age because the results lack context. As a result, we conducted analyses to specifically examine main effects and significant interactions involving Condition. We observed no three-way interactions of Age x

Condition x Sex in any of the brain regions analyzed (all p’s > 0.05). Furthermore, we did not find any interactions of Sex x Age (all p’s > 0.05), and collapsed sex for the results presented below.

RESULTS

TH-ir neurons.

In order to characterize the neuroanatomical development of TH-ir neurons in the prairie vole, we analyzed the raw number of TH-ir cells present in each region of interest.

The medial bed nucleus of the stria terminalis (BSTm). We observed a main effect of

Age (F(2, 155) = 23.93, p < 0.001) in the BSTm, where PND 21 animals had significantly more TH-ir neurons compared to both the PND 9 (t(155) = 5.21, p <

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0.0001) and PND 2 (t(155) = 6.65, p < 0.0001) groups (Fig. 2.3A).

The principle bed nucleus of the stria terminalis (pBST). Analyses revealed a main effect of Age (F(2, 62.251) =32.125, p < 0.001) in the pBST. PND 21 animals had significantly more TH-ir neurons compared to both PND 9 (t(65.46) = 5.34, p < 0.0001) and PND 2 (t(60.59) = 7.86, p < 0.0001) groups. In addition, PND 9 animals has significantly more TH-ir cells than PND 2 pups (t(60.69) = 2.82, p = 0.019) (Fig. 2.3B).

The medial amygdala (MeA). We found a main effect of Age (F(2, 73.07) = 14.81, p <

0.001) in the MeA, for which both PND 21 (t(72.51) = 5.28, p < 0.0001) and PND 9 pups (t(65.64) = 3.81, p = 0.0009) had significantly more TH-ir neurons than the PND 2 pups (Fig. 2.3C).

BSTm cFos responses.

We observed a significant main effect of condition in the BSTm (F(2,78.52) =

6.08; p = 0.004), with subjects in the Isolate condition exhibiting significantly higher levels of cFos-ir compared to pups in the Together condition (p = 0.003). However, a significant Age x Condition interaction in the BSTm (F(4,78.39) = 2.49; p = 0.05; Fig.

2.4) suggests that neural responses in the older animals likely drives this main effect:

Post-hoc comparisons revealed that Isolated PND 9 (t(90.79) = 2.71 ; p = 0.024) and

PND 21 (t(97.6) = 3.38 ; p = 0.003) pups exhibited significantly more cFos-ir induction compared to pups in the Together condition. Isolated PND 9 pups also exhibited greater cFos-ir compared to pups in the Reunite condition (t(84.42) = 2.61 ; p = 0.032).

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We found that PND 2 pups did not show differences in cFos-ir across conditions

(Together x Reunite t(59.81) = 0.192, Together x Isolate t(71.95) = 0.31; Reunite x Isolate t(64.3) = 0.5; all p’s > 0.05).

pBST cFos responses.

We did not observe a significant main effect of Condition (F(2, 74.17 ; p = 0.37 ) or an interaction of Age x Condition for cFos-ir in the pBST (F(4, 73.68 ) = 0.55 ; p = 0.7;

Fig. 2.5), suggesting that this subdivision of the BST was not differentially sensitive to our experimental conditions at any stage of development.

MeA cFos responses.

We observed a significant Age x Condition interaction for cFos-ir in the MeA

(F(4, 51.86) = 3.36 ; p = 0.016; Fig. 2.6). Post-hoc comparisons revealed that PND 21 pups in the Reunite condition exhibited significantly more cFos-ir compared to PND

21 pups in the Together (t(56.98) = 2.51 ; p = 0.045) and Isolate (t(67.78) = 3.73 ; p =

0.001) conditions. Interestingly, neither the PND 2 nor PND 9 animals showed significantly different levels of cFos-ir as a function of social condition (Isolate x

Reunite – PND 2 t(39.29) = 0.39 , PND 9 t(52.74) = 1.14; Isolate x Together – PND 2 t(45.43) = 0.085, PND 9 t(57.22) = 1.79; Reunite x Together – PND 2 t(35.45) = 0.452, PND

9 t(49.47) = 0.72; all p’s > 0.05).

TH-cFos colocalization in the BSTm.

Analysis of the proportion of TH neurons expressing cFos in the BSTm

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yielded null results. We found no significant Age x Condition interaction for TH-cFos colocalization in the BSTm (F(4, 70.13) = 2.159; p = 0.083; Fig. 2.7).

TH-cFos colocalization in the pBST.

We did not find any significant main effect of Condition (F(2,75.81) = 2.13; p =

0.126), nor any significant Age x Condition interaction for TH-cFos colocalization in the pBST (F(4,75.28) = 0.96; p = 0.44; Fig. 2.8).

TH-cFos colocalization in the MeA.

We observed a significant Age x Condition interaction for TH-cFos colocalization in the MeA (F(4,59.69) = 2.8275; p = 0.032; Fig. 2.9). Post-hoc comparisons revealed that in PND 21 pups, TH-cFos colocalization was significantly greater in pups in the Together (t(84.08) = 2.51; p = 0.042) and Reunite (t(75.91) = 3.127; p = 0.0075) conditions compared to those in the Isolate condition. Neither the PND 2 nor PND 9 pups showed differences in MeA TH-cFos colocalization as a function of social condition (Isolate x Reunite – PND 2 t(46.09) = 0.94, PND 9 t(60.53) = 0.98; Isolate x Together – PND 2 t(52.74) = 0.55, PND 9 t(65.37) = 0.89; Reunite x Together – PND 2 t(41.84) = 1.42, PND 9 t(57.14) = 0.043; all p’s > 0.05).

DISCUSSION

In the present study, we found that subregions within the meEA differentially responded to social context in an age-dependent manner. For both PND 9 and PND 21 animals, the BSTm demonstrated more general neuronal responsiveness (i.e., cFos

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expression) in animals experiencing the Isolate condition compared to those in the

Together condition. These results indicate that the BSTm may be particularly sensitive to the presumably stressful experience of social isolation. Moreover, cFos responses within the MeA of PND 21 subjects were greatest in the Reunite condition compared to either the Together or Isolate conditions, suggesting that this region is sensitive to the emergence of social stimuli and/or potentially the rewarding context of being reunited with familial individuals after social isolation. However, the youngest age we examined (PND 2) did not show BSTm or MeA cFos response profiles that discriminated between social conditions, suggesting that the meEA becomes selectively responsive to changes in the social environment later in postnatal development.

Interestingly, the patterns we observed for cFos activity within the meEA were not identical to those we observed for TH neural activity within the meEA.

Specifically, TH-cFos colocalization within the MeA was higher in both the Together and Reunite groups compared to the Isolate group (only in PND 21 animals, discussed below). These data suggest that there might be a specialized function for this sub- population of TH positive cells relative to other cell types in the MeA. However, additional data on the sensitivity of types of MeA cells to various environmental stimuli would be helpful to support this interpretation. Finally, although neurons of the

BSTm were particularly responsive to social isolation (as indicated by cFos expression), TH neurons were not exclusively responsible for BSTm neuronal activation because we did not observe group differences for TH-cFos colocalization in the BSTm.

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Altogether, these data provide evidence that 1) cell groups in the MeA and the

BSTm differ in their sensitivities to aspects of the social environment, and 2) the social responsiveness of the BSTm appears to emerge earlier in prairie vole development (by PND 9) compared to that of the MeA (by PND 21). Below we discuss the ontogenetic differences in the timing and location of emerging functional cell groups across the meEA. Because significantly more developmental neurobiology has been characterized in rat pups, and rats share key developmental features with prairie voles (altriciality and a gestation period of 21 days; Smotherman and Robinson,

1987) we draw from the rat literature to provide a useful basis for understanding the ontogeny of prairie vole social development.

The duality of the meEA and the processing of social context.

Our results showed that the highest level of neuronal activation within the

BSTm was detected under social isolation. In contrast, the neurons of the MeA only showed differential heightened activation in conditions in which the pups were in contact with caregivers. The implication of these results is that, within the meEA, the

BSTm and the MeA appear to respond to distinct properties of the social environment.

Current understanding of the BST suggests that it acts as a relay center, receiving inputs from sensory and cortical structures and modulating steroid hormone and nonapeptide release via projections to the paraventricular nucleus of the hypothalamus (Choi et al., 2007). Thus, the BST could play a critical role in facilitating appropriate responses to different stressful contexts. Stressful novel experiences increase BST cFos counts in rat dams (Smith and Lonstein, 2008),

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whereas BST stimulation produces behavioral responses similar to those elicited by restraint stress (Casada and Dafny, 1991). Pharmacological synaptic blockade of the rat BST attenuates behavioral and autonomic responses to fear cues, indicating the importance of the BST in processing threatening contexts and subsequently producing appropriate behavioral and endocrine stress responses (Resstel et al., 2008). We found neurons of the BSTm are functionally sensitive to the absence of social contact (at

PND 9 and PND 21). This outcome is consistent with documented functional roles of the BSTm in stress and anxiety-like responses, and suggests a broader role for the

BSTm in processing stressful social contexts.

In contrast, the MeA was active particularly after the transition from brief isolation to reunion with family. This result provides ostensible evidence that the MeA plays a role in processing the exposure to social conspecifics. Supporting this interpretation is the longstanding association between MeA function and social affiliation and aggression in prairie voles (Curtis and Wang, 2003; Wang et al., 1997;

Wang and De Vries, 1993). In male voles, MeA cFos expression is heightened after exposure to a sexually-receptive female, compared to cFos levels after exposure to a familiar sibling, or to isolation (Lim and Young, 2004). This suggests that the MeA appears to be sensitive to the presentation of social stimuli, and to the biological relevance of the conspecific. This interpretation is supported by the known functional role of the MeA in social recognition and social interest in rats (Gur et al., 2014).

Furthermore, lesioning the MeA of male prairie voles decreases their levels of paternal care, and time spent in contact with a familiar female (Kirkpatrick et al., 1994). These results suggest that MeA function is necessary in facilitating adaptive behavioral

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responses to conspecifics, which is congruent with evidence that demonstrates selective representations of social stimuli within mouse MeA sub-circuitry (Bergan et al., 2014).

Alternatively, the neural activity we observed in the MeA might have resulted from manipulating the availability of general sensory information (olfactory, visual, or auditory cues). We think this is unlikely because if the mere presence of sensory cues were driving these neural responses, we would expect to see heightened levels of cFos expression in the Together condition in conjunction with the Reunite condition.

Moreover, in female prairie voles, both the dorsal medial and the anterior medial nuclei of the amygdala show heightened cFos expression after exposure to male urine, compared to either saline or milk stimuli (Hairston et al., 2003). This result suggests that the prairie vole MeA is capable of responding selectively to the presentation of social odors, relative to odors more generally. Furthermore, rats and mice as young as

PND 2 can be conditioned under an odor-aversion learning paradigm (Rudy and

Cheatle, 1977) or odor-conditioned for food reinforcement (Armstrong, DeVito, &

Cleland, 2006), suggesting that we should have seen similar neural responses in the

PND 2 and PND 9 groups if the capacity for olfactory processing was driving the

MeA cFos responses.

It is possible that simple exposure to any stimulus (regardless of its social or nonsocial properties) could explain the induction of cFos in the MeA in the Reunite condition. However, the MeA cFos responses of male hamsters and mice discriminate between the presentation of conspecific and heterospecific odors, and both social odors induce significantly greater amount of cFos relative to control unscented and

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peppermint stimuli (Meredith & Westbury, 2004; Samuelson & Meredith, 2009).

These studies of MeA function suggest that the rodent MeA is highly sensitive to distinct social stimuli compared to nonsocial stimuli. Without including a non-social stimulus in our design, we cannot definitively conclude that our results are uniquely due to reunion with family, per se. Nonetheless, the ability to assess the social environment is inextricably tied to the mechanisms underlying sensory processing.

Investigating how sensory modalities serve as potential routes by which the social environment is encoded is beyond the scope of the present work, but would be an important and valuable avenue of research that would elucidate how social contexts are processed in the developing brain.

Developmental timing of meEA response to social context.

It is interesting to consider the differential neural activation of the BSTm and

MeA in response to social conditions with respect to some notable developmental benchmarks. We did not find discriminative patterns of cFos activation in either the

BSTm or MeA in PND 2 animals, which was surprising given that separation from parents is likely to leave PND 2 pups at their greatest risk for predation, thermoregulatory vulnerability, or starvation. It is plausible that systems beyond the functional sensitivity of the extended amygdala are online to address these life- threatening challenges. For example, from the day of parturition until the weaning period, prairie vole pups emit calls in response to cooling (a consequence of isolation), which is believed to elicit retrieval responses from parents (Blake, 2002). Whether these calls are a physiological byproduct of the respiratory response to cold exposure

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(Blumberg and Alberts, 1990) or an evolutionarily adaptive communicative signal

(Hofer and Shair, 1993), pup vocalizations may serve as a compensatory mechanism that mediates infant survival during a period in which isolation is not yet encoded by the meEA.

Importantly, our data do not suggest that the brains of neonatal pups are entirely incapable of reacting to context. Work in rats has shown that pups transiently upregulate cFos mRNA expression in the neocortex and midbrain 30 mins following parturition (Ringstedt et al., 1995), and PND 0 rats show significantly more cFos expression in the olfactory bulbs after the presentation of odors such as peppermint and propionic acid compared to clean air (Guthrie and Gall, 2003). These pieces of evidence indicate that the brains of even the youngest postnatal rat pups seem capable of responding to environmental changes. Our results depict an insensitivity of PND 2 cell groups within the meEA to social context, which is consistent with the hypothesis that the brains of perinatal pups lack the functional capacity for adult-like responses to changes in the social environment. The relatively delayed functional maturation of the

MeA has been documented in other rodents. For example, rat pups show heightened

MeA cFos expression when exposed to a threatening novel adult male at PND 21, but not at PND 7 (Wiedenmayer and Barr, 2001). One hypothesis posits that the hypo- activity of the amygdala in the neonatal rodent is not merely a result of neurobiological immaturity, but rather an evolutionarily adaptive mechanism that attenuates amygdala-dependent fear or aversion-learning during a critical sensitive period in which pups learn to form an attachment to their mothers (Landers and

Sullivan, 2012). It follows that neonatal brains should not be considered mere

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immature versions of adult brains. Instead, by considering the developmental context, we may begin to appreciate the ways in which the particular neurobiology of infants facilitates adaptations to the demands of their changing environment (Alberts, 2008).

We found that neural responsiveness of the BSTm to distinct social contexts was first observable as early as PND 9, when the pups opened their eyes and became more mobile. In contrast, the MeA demonstrated differential responses to social contexts only at PND 21, during the developmental stage of weaning. These results suggest that subregions within the meEA exhibit differential functional development, with the BSTm beginning to discriminate between social contexts earlier in development relative to the MeA. It is unclear whether our findings stem from the underdevelopment of meEA brain structures, or immaturity of the afferent projections from sensory regions. In rats, projections from both the olfactory and accessory olfactory bulbs to the medial amygdala via the stria terminalis are seen in prenatal rats

(Schwob and price 1978). Projections from the MeA to the BST are established prior to parturition, while reciprocal fibers are not seen in rat pups until PND1 (Cooke and

Simerly, 2005). However, the density of projections between these regions of the meEA increase over the postnatal period, and only approach adult-typical densities around PND15. Consequently, it is plausible that structures outside of the meEA and upstream in the olfactory pathway become functionally responsive earlier in postnatal life to facilitate behavioral responses to environmental changes in preweanling animals. The functional consequences of differences in developmental timing for the

BSTm and the MeA remain unclear. To this end, our data provide evidence that regions of the meEA are not homogenous in their functional developmental rates.

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TH function within the meEA.

TH-ir cells are present in the BSTm, pBST, and MeA as early as PND 2 in prairie voles, indicating the potential for TH-dependent action in the meEA at an early age. Despite the presence of TH-ir neurons in the BSTm, we found no effects across

Age and Social Context for TH-cFos co-localization in this region. On the other hand,

MeA TH neurons in PND 21 brains responded strongly to social context, suggesting that family interactions at weaning elicit a TH-related neural response within the MeA.

Notably, the sub-population of MeA neurons expressing TH demonstrated enhanced activation (indicated by TH-cFos co-localization) in the Reunite context (paralleling our cFos only data) and in the Together context (a departure from the cFos only data in this region). These results suggest the TH positive neurons of the MeA exhibit a general sensitivity to pro-social contexts. Other studies have shown that male prairie vole TH neural activity is greater following a variety of social interactions compared to social isolation (Northcutt & Lonstein, 2009), supporting the interpretation here that

MeA TH activation reflects sensitivity to social interactions.

It is tempting to equate the presence of TH to dopamine action, because TH serves as a precursor to DA. Given that our data showed that MeA TH activation responded most intensely to prosocial contexts, it is even more tempting to conclude that TH in the MeA facilitates the rewarding properties (so often attributed to dopamine) under social contexts. Caution should be taken with this conclusion. The activation of TH-producing cells can relate to downstream dopaminergic neurotransmission, but it can also lead to subsequent production and exocytosis of

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adrenaline or noradrenaline (De Deurwaerdère et al., 2017). Furthermore, L-DOPA can be metabolized into non-catecholaminergic products (such as melanin precursors), interact with 5-HT neurons to modify extracellular serotonin (Navailles et al., 2013) or exhibit neurotransmitter-like properties as an end product itself (De Deurwaerdère et al., 2017). As such, the multifaceted potential of TH molecules (and L-DOPA action) obscures precise functional roles of TH neurons within the meEA. It is, therefore, unclear if the sensitivity of MeA TH neurons to familial stimuli is related to dopaminergic encoding of a social reward. AADC is required for dopamine synthesis, and MeA TH cells do not immunoreactively co-express AADC in adult prairie voles

(Ahmed et al., 2012). We do not know if this is also true for pre-weaning prairie vole pups. On the other hand, AADC-expressing cells are found more generally in both the

BST and the MeA of adult prairie voles (Ahmed et al., 2012), and neighboring AADC cells can coordinate with TH cells to facilitate DA synthesis in rats (Ugrumov, 2009;

Ugrumov et al., 2004). Further studies are necessary to determine the specific products of TH neurons within the MeA of prairie voles, and how they might be involved in the encoding of social and/or rewarding contexts. Whether the TH activation we found in the MeA serves as a marker of DA activation, or the activation of other molecular pathways, the dynamic nature and multi-functional potential of TH is likely enabling

TH-positive neurons in the MeA to differentially and appropriately respond to the social environment as animals mature. A deeper appreciation of the development of social behavior will only be achieved by identifying the specific ontogenetic windows during which time these cell groups become responsive to social contexts and exhibit adult-typical response profiles, and by understanding the abilities and limitations

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imposed by the development of this neurophysiology.

CONCLUSIONS

Our data provide evidence that the meEA exhibits functional differences across early development. Interestingly, the meEA of neonatal pups was not selectively responsive to variation in the social environment. However, meEA functioning of older pups (PND 9, age of eye-opening and independent locomotion; PND 21, age of weaning) was subregion-, age- and context-specific. Although the BSTm differentially responds to isolation by the second week of postnatal life, the MeA does not differentially respond to the presence of familial individuals (i.e., parents and siblings) until weaning-age. Furthermore, our findings suggest that TH neurons in the MeA do not differentially respond to social context until later in development (PND 21). As such, our results point towards heterogeneous ontogenies of functionality across, and

TH circuitry within, the meEA. Despite the connectivity of the meEA, careful consideration of the specific subregions analyzed within the meEA should be taken given that individual nuclei have varying rates of structural, functional, and connective development (Goodson et al., 2004). Examining the development of social neural circuitry is important because it provides insight into the mechanisms that govern age- specific social phenotype. It can also identify developmental stages where changes in neuroanatomy and function occur, which might represent critical periods in development where the environment can most substantially impact behavioral profiles that persist into adulthood.

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Acknowledgements

We would like to express gratitude to Chang Kim for his assistance with PCR, and to the Statistical Consulting Unit at Cornell University for their guidance with our statistical analyses.

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FIGURES

Figure 2.1 A top-to-bottom flowchart depicting experimental treatment conditions. Family units were randomly assigned to one of three conditions: Together, Isolate, or Reunite. All subjects started in identical housing conditions prior to the 30-minute pre- test phase. The same procedure was used for family units with pups of each age (PND 2, PND 9, or PND 21). See text for details.

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Figure 2.2 A diagram representing the location of cFos-ir and TH-ir neurons quantified in (A) the medial bed nucleus of the stria terminalis (BSTm), and (B) the medial amygdala (MeA). Regions where cells were quantified are shaded in gray. AC, anterior commissure; f, fornix; opt, optic tract.

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Figure 2.3 Mean (±SEM) number of TH-ir neurons as a function of pup age in the (A) medial portion of the bed nucleus of the stria terminalis (BSTm), (B) principal nucleus of the bed nucleus of the stria terminalis (pBST), and (C) medial amygdala (MeA). * p < 0.05.

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Figure 2.4 Mean (±SEM) cFos-ir as a function of pup age and experimental condition within the medial division of the bed nucleus of the stria terminalis (BSTm). * p < 0.05.

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Figure 2.5 Mean (±SEM) cFos-ir as a function of pup age and experimental condition within the principal nucleus of the bed nucleus of the stria terminalis (pBST). No comparisons were statistically significant.

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Figure 2.6 Mean (±SEM) cFos-ir as a function of pup age and experimental condition within the medial amygdala (MeA). * p < 0.05.

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Figure 2.7 Mean (±SEM) proportion of tyrosine hydroxylase (TH)-ir neurons co- labeled with cFos-ir as a function of pup age and experimental condition within the medial division of the bed nucleus of the stria terminalis (BSTm). No comparisons were statistically significant.

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Figure 2.8 Mean (±SEM) proportion of TH-ir neurons co-labeled with cFos-ir as a function of pup age and experimental condition within the pBST. No comparisons were statistically significant.

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Figure 2.9 Mean (±SEM) proportion of tyrosine hydroxylase (TH)-ir neurons co- labeled with cFos-ir as a function of pup age and experimental condition within the medial amygdala (MeA). * p < 0.05.

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CHAPTER 3

INTERACTIONS OF SEX AND EARLY LIFE SOCIAL EXPERIENCES AT TWO

DEVELOPMENTAL STAGES SHAPE NONAPEPTIDE RECEPTOR PROFILES

As published in: Hiura, L.C. & Ophir, A.G. (2018) Interactions of sex and early life social experiences at two developmental stages shape nonapeptide receptor profiles.

Integr. Zool. 13, 745–760.

ABSTRACT

Early life social experiences are critical to behavioral and cognitive development, and can have a tremendous influence on developing social phenotypes.

Most work has focused on outcomes of experiences at a single stage of development

(e.g. perinatal or post-weaning). Few studies have assessed the impact of social experience at multiple developmental stages and across sex. Oxytocin and vasopressin are profoundly important for modulating social behavior and these nonapeptide systems are highly sensitive to developmental social experience, particularly in brain areas important for social behavior. We investigated whether oxytocin receptor (OTR) and vasopressin receptor (V1aR) distributions of prairie voles (Microtus ochrogaster) change as a function of parental composition within the natal nest or social composition after weaning. We raised pups either in the presence or absence of their fathers. At weaning, offspring were housed either individually or with a same-sex sibling. We also examined whether changes in receptor distributions are sexually dimorphic because the impact of the developmental environment on the nonapeptide

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system could be sex-dependent. We found that differences in nonapeptide receptor expression were region-specific, sex-specific and rearing condition-specific, indicating a high level of complexity in the ways that early life experiences shape the social brain. We found many more differences in V1aR density compared to OTR density, indicating that nonapeptide receptors demonstrate differential levels of neural plasticity and sensitivity to environmental and biological variables. Our data highlight that critical factors including biological sex and multiple experiences across the developmental continuum interact in complex ways to shape the social brain.

INTRODUCTION

An organism’s biological sex shapes and constrains the ecological and social context, and can extensively impact social behavior. Many social behaviors of interest, such as parental care or courtship, are inherently tied to the sex of an animal due to morphological or physiological requirements necessary to express specific behaviors

(i.e. nursing and secondary sexual characteristics). Interest in studying behavioral sex differences across species has logically led to studying the underlying endocrinological and neural mechanisms that give rise to sexual dimorphism in behavior (Dulac & Kimchi 2007). This focus has in part helped to reveal the functional roles that the neuropeptides oxytocin (OT) and vasopressin (VP) play in shaping behavioral sex differences. OT and VP act as neuromodulators when bound to their receptors (OTR and V1aR, respectively), which function to regulate widespread physiological and behavioral processes (Landgraf & Neumann 2004). The distributions of OTR, V1aR and nonapeptide-containing neurons are specific to

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species, age and sex, and, thus, have functional implications for the display of social behaviors, including pair-bonding, parental care, social recognition, gregariousness and courtship (Goodson 2008; Dumais & Veenema 2016).

The impact of early life social experiences, such as variation in parental caregiving, shapes the social brain. For example, the density of nonapeptide receptors across regions of the forebrain are sensitive to environmental experiences in perinatal life (Champagne et al. 2001; Curley et al. 2012; Prounis et al. 2015). Decades of research on maternal separation and caregiving in rodents has revealed the critical role that mothers play in shaping offspring brains and behaviors in relation to stress regulation, cognitive processing and sociality (Meaney 2001). Variation in the quality and quantity of maternal care given in the first few weeks of life has lasting consequences for offspring, such that lower levels of maternal care can lead to higher levels of anxiety-like behavior, elevations in glucocorticoid responses to stress, dysregulation of the hypothalamic–pituitary–adrenal axis, and even modifications to the epigenome that subsequently facilitate the intergenerational transmission of parental caregiving (Liu et al. 1997; Caldji et al. 1998; Meaney 2001).

The emphasis on the developmental impact of maternal care over paternal care has likely been driven by the rarity of paternal care in mammals; approximately 5% of mammalian species exhibit biparental care (Clutton-Brock 1991). In biparental mammals such as the socially monogamous prairie vole (Microtus orchrogaster), pups receive care from both their mothers and their fathers (Getz & Carter 1996). This characteristic makes the prairie vole a suitable species in which to ask questions regarding the role of paternal care in shaping offspring behavioral phenotypes.

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Variation in parental composition (such as manipulating the presence or absence of fathers during the rearing period) has been shown to alter the rate at which prairie vole pups develop (Wang & Novak 1992). This variation also impacts social affiliation towards conspecifics in adulthood (Tabbaa et al. 2017) and the species-typical pair- bonding behavior of adult animals (Ahern & Young 2009).

In addition to variation in social environments resulting from different experience with parental caregivers, the dynamics of post-wean social experiences influence brain development. Juvenile exposure to social experiences, such as aggressive encounters (Delville et al. 1998), opportunities to engage in social play behaviors (Van Den Berg et al. 1999) and housing conditions (Kaiser et al. 2007) all have significant consequences for adult rodent social behavior, stress physiology and neuroendocrinology (Sachser et al. 2013).

Despite ongoing interest in how early life social experiences impact the development of the social brain, a comprehensive understanding of the ways in which complex social experiences might differentially impact the development of the male and female brain remains underdeveloped. To this end, the current study asks how the distributions of nonapeptide receptors vary as a function of sex, and how interactions across postnatal social conditions subsequently impact receptor expression in both males and females. We accomplish this task by investigating prairie vole sex differences in nonapeptide receptor profiles after manipulating the presence and absence of the father during pre-weaning, and by varying social housing conditions after weaning.

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MATERIALS AND METHODS

Subjects and rearing conditions

Animals were housed in polycarbonate rodent cages (29 × 18 × 13 cm) under a

14:10 light-dark cycle (lights on at 06:00 hours). Animals had ad libitum access to water and Rodent Chow 5000 (Harlan Teklad, Madison, WI, USA). Primiparous breeding pairs were formed in opposite-sex pairs (n = 41 pairs), using colony offspring derived from wild-caught prairie voles we trapped in Champaign County, Illinois,

USA. Breeders were monitored closely for the birth of pup litters. When pups were born, the number of pups per litter was recorded (mean = 3.8 ± 1, range of 2–6).

Fathers in the Father-absent condition were then removed from the home cage. Fathers in the Father-present condition remained with the litter. All pups were weaned and sexed on post-natal day (PND) 21. Upon weaning, animals from the Father-present and Father-absent groups were each sub-divided into 2 groups: Single-housed or Pair- housed (housed with a same-sex sibling). Thus, the final design was a 2 × 2 × 2 factorial design (Fig. 3.1) with 2 levels of Pre-wean condition (Father-absent vs

Father-present), 2 levels of Post-wean condition (Single-housed vs Pair-housed) and 2 levels of Sex (Male vs Female). All procedures were approved by the Institutional

Animal Care and Use Committee of Cornell University (2013-0102).

Histology and autoradiography

Subjects were killed between PND37 and 45 by CO2 inhalation. Brains were immediately extracted, snap-frozen on powdered dry ice, and stored at −80C°. Brains were coronally cryosectioned at 20-μm thickness onto 4 sets on Superfrost Plus slides

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(Fisher Scientific, Pittsburgh, PA, USA) at 100-μm intervals. The mounted slides were stored at −80C° until autoradiographic labeling. Two sets of slides were labeled with

125I radioligands to visualize oxytocin receptor (ornithine vasotocin analogue ([125I]-

OVTA); NEX254, PerkinElmer, Waltham, MA, USA) and vasopressin 1a receptor

(vasopressin (Linear), V-1S antagonist (Phenylacetyl1,0-Me-D-Tyr2,[125I-Arg6]-);

NEX 310, PerkinElmer), as previously described (Ophir et al. 2013).

Digital imaging and analysis

The radiolabeled slides and 125I radiographic microscales (American

Radiolabeled Chemicals, St Louis, MO, USA) were stored in film cassettes and exposed to storage phosphoreimaging screens (Fujifilm, Tokyo, Japan) for 23 h. The screens were removed from the cassettes under dark light, and positioned in a

Typhoon FLA 7000 laser scanner (GE Healthcare, Marlborough, MA, USA). Screens were scanned using the Typhoon FLA 7000 control software version 1.3 (GE

Healthcare, Marlborough, MA, USA) and analyzed in ImageQuant TL Toolbox

Version 8.1 (GE Healthcare, Marlborough, MA, USA). Brain areas of interest were measured for densitometry analysis in 3 sequential slices of tissue by encircling each region of interest bilaterally. Mean values of intensity were calculated for each region and automatically adjusted for background by the ImageQuant program. 125I-labeled radiographic microscales were used to create decay formulas, which transformed mean intensity measures to standardized values of disintegrations per minute (dpm) adjusted for tissue equivalence (TE; for 1 mg in rat brain). Transformed mean non-specific binding measurements from cortex taken from the same brain sections as each region

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of interest were then subtracted from these values to calculate a final value for mean receptor density (units dpm/mg TE). V1aR was measured in the main olfactory bulb

(MOB), accessory olfactory bulb (AOB), lateral septum (LS), ventral pallidum

(VPall), lateral bed nucleus of the stria terminalis (BSTl), medial bed nucleus of the stria terminalis (BSTm), ventral bed nucleus of the stria terminalis (BSTv), anterior hypothalamus (AH), paraventricular nucleus of the hypothalamus (PVN), medio- dorsal nucleus of the thalamus (MDTh), latero-dorsal nucleus of the thalamus (LDTh), ventro-posterior nucleus of the thalamus (VPTh), central amygdala (CeA), medial amygdala (MeA), retrosplenial cortex (RSC) and ventromedial hypothalamus (VMH).

OTR was measured in the basolateral amygdala (BLA), caudate-putamen (CPu), hippocampus (HPC), anterior portion of the insular cortex (ICa), medial portion of the insular cortex (ICm), intermediodorsal thalamic nucleus (IMD), LS, nucleus accumbens (NAcc), prefrontal cortex (PFC) and septo-hippocampal nucleus (SHi).

Statistical analysis

Mean receptor density data were analyzed using linear mixed models (LMM) in R v.3.2.1 (R Core Team, 2016) using the R package lme4 (Bates et al. 2015). For each receptor type (OTR, V1aR), we included Pre-wean condition, Post-wean condition and Sex as fixed factors. Phosphoreimaging screen, Autoradiography chamber, and Litter were included as factored random effects to control for potential variation across histological processing and genetic profiles. A model was run for each brain region of interest. When the omnibus ANOVA revealed a significant effect, we conducted paired contrasts using the R package lsmeans (Lenth 2016). Post-hoc

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comparisons were adjusted using Tukey corrections, and we considered a 0.05 α-level threshold for statistical significance.

RESULTS

Our analyses revealed that there were significant effects of sex and early life experiences that shaped V1aR and OTR expression in the brains of developing prairie voles (Tables 1 and 2). Below we detail these results, first exploring main effects and then moving onto interaction effects.

Main effect of sex

We found a main effect of sex on V1aR expression in the main olfactory bulbs

(MOB; F(1, 77.5) = 8.6, P < 0.005), accessory olfactory bulbs (AOB; F(1, 80.4) = 6.1, P <

0.05), lateral septum (LS; F(1, 94.1) = 7.2, P < 0.01), bed nucleus of the stria terminalis lateral subdivision (BSTl; F(1, 100.5) = 11.0, P < 0.005), bed nucleus of the stria terminalis medial subdivision (BSTm; F(1, 98.8) = 4.6, P < 0.05), and central amygdala

(CeA; F(1, 92.2) = 3.9, P = 0.05). Post-hoc contrasts revealed that for both the MOB (P =

0.005) and the AOB (P = 0.02), males had a significantly greater density of V1aR compared to females (Fig. 3.2). Conversely, we found that females had significantly higher V1aR density compared to males in the LS (P < 0.01), BSTl (P < 0.01) and the

BSTm (P < 0.05; Fig. 3.2). There were no main effects of sex on OTR expression in any of the regions analyzed (all P > 0.05).

Main effect of pre-wean social experience

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We found a main effect of Pre-wean social experience on V1aR density in the paraventricular nucleus of the hypothalamus (PVN; F(1, 30.6) = 4.0, P = 0.05). A post- hoc contrast showed that offspring reared in the Father-present condition had significantly greater V1aR density compared to offspring reared in the Father-absent condition (P = 0.05, Fig. 3.3). No other main effects of Pre-wean on V1aR density were found for any of the other areas analyzed (all P > 0.05).

Pre-wean social experience impacted OTR density in the nucleus accumbens (NAcc;

F(1, 102.3) = 6.8, P = 0.01). A post-hoc contrast revealed that offspring in the Father- absent condition expressed significantly greater OTR density than offspring in the

Father-present condition (P = 0.01, Fig. 3.4). There were no main effects of Pre-wean condition on OTR density within any of the other regions analyzed (all P > 0.05)

Main effect of post-wean social experience

Post-wean experience affected V1aR density in the anterior hypothalamus

(AH; F(1, 97.9) = 3.9, P = 0.05). A post-hoc contrast revealed that offspring in the

Single-housed condition had significantly greater V1aR density compared to animals in the Pair-housed condition (P < 0.05, Fig. 3.5). There were no significant main effects of Post-wean experience on V1aR density in other brain areas or on OTR density in any of the OTR-expressing regions analyzed (all P > 0.05)

Two-way interaction effects

Beyond the main effects reported above, we found only 1 significant 2-way interaction for V1aR density in any brain area. Specifically, the ventromedial

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hypothalamus (VMH) demonstrated a significant Sex by Pre-wean interaction (F(1, 97.6)

= 4.6, P < 0.05). Post-hoc contrasts showed that female, but not male, offspring in the

Father-present condition had significantly higher V1aR density compared to females in the Father-absent condition (P = 0.05, Fig. 3.6). In addition, females had higher

V1aR density compared to males (P = 0.05) within the Father-present condition, but not the Father-absent condition. We did not find any significant interactions of Sex X

Pre-wean conditions, or significant interactions of Sex X Post-wean conditions on

V1aR density (all P > 0.05).

We found a significant Pre-wean by Post-wean interaction in the septohippocampal nucleus (SHi; F(1, 103.6) = 9.4, P < 0.005) for OTR density (Fig. 3.7).

Post-hoc contrasts showed that Pair-housed animals had significantly greater OTR density compared to Single-housed animals (P < 0.05) in the Father-absent condition.

Conversely, Single-housed animals had significantly greater OTR density compared to

Pair-housed animals (P < 0.05) in the Father-present condition. Finally, we found that

Single-housed offspring in the Father-present condition had higher levels of OTR density compared to Single-housed offspring in the Father-absent condition (P <

0.05). No other significant 2-way interactions were found on OTR density in any of the regions analyzed (all P > 0.05).

Three-way interaction effects

We observed only 1 significant 3-way interaction of Sex by Pre-wean by Post- wean condition. This interaction effect was found for OTR expression in the LS (F(1,

109.6) = 3.8, P = 0.05). Post-hoc contrasts revealed that males had significantly greater

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OTR density compared to females within Single-housed and Father-present animals (P

< 0.05, Fig. 3.8). No 3-way interaction effects were found for any other OTR expressing brain area or for V1aR expression in any of the regions analyzed (all P >

0.05).

DISCUSSION

Sex differences in vasopressin receptor expression

Our data revealed several areas of the brain in which there were significant sex differences in the expression of V1aR in prairie voles. These included the main and accessory olfactory bulbs (MOB and AOB), the lateral septum (LS), and the lateral and medial divisions of the bed nucleus of the stria termialis (BSTl and BSTm).

Intriguingly, these regions are structurally interconnected and work in concert to facilitate social chemosensory processing via the vasopressin system. VP activity in both the MOB and AOB is critical in rodent social recognition, which is facilitated by processing the scent signature of a conspecific. Administering a V1a receptor antagonist or using siRNA to target V1aR in the MOB impairs social recognition performance in rats (Tobin et al. 2010). Similarly, V1aR in the LS is both necessary and sufficient for facilitating social recognition in rats and mice (Bielsky et al. 2005;

Gabor et al. 2012). Although we are unaware of any portion of the BST being involved in social recognition per se, this region is known to respond to sociosexual olfactory cues in hamsters (Fiber et al. 1993), rats (Bressler and Baum, 1996), mice

(Veyrac et al. 2011) and mandarin voles (He et al. 2014). The BST also happens to be one of the densest extra-hypothalamic regions that produces central VP in rats (de

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Vries et al. 1985). Moreover, the BST is structurally connected with both the olfactory system and the LS. For example, the AOB projects to the BST, and the LS is densely innervated by VP neurons that originate in the BST (de Vries & Buijs, 1983). Sex differences in V1aR binding within these regions are not well characterized. However,

Veenema et al. (2012) found that female rats express significantly greater LS V1aR binding than males, a result that is consistent with our study. The sex differences we report are even more interesting considering that the innervation of VP fibers within the LS is sexually dimorphic; male prairie voles, meadow voles and rats possess greater VP fiber density in the LS compared to females (De Vries et al. 1981;

Bamshad et al. 1993). This distinction may partially underlie behavioral sex differences in social recognition. Female rats retain social memories for longer intervals compared to males (Bluthé & Dantzer 1990). Furthermore, peripheral blockade of V1aR impairs recognition performance in male but not in female rats

(Bluthé & Dantzer 1990). However, subsequent work has demonstrated that both adult male and female rats exhibit impaired social recognition after LS V1aR antagonism

(Veenema et al. 2012). Despite some mixed results, the sex differences in V1aR density we report support the established interpretation that VP facilitates social recognition in a sex-specific manner, and our data provide evidence of developmental- based sexual dimorphisms of receptor expression within brain areas upstream in the social chemosensory pathway.

Despite the various sex differences observed in V1aR expression, we did not find any sex differences in OTR density in any of the brain regions analyzed. This distinction falls in line with a more general trend in the rodent nonapeptide literature,

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in which cross-species comparisons have established more sex differences in the

VP/V1aR systems compared to the OT/OTR systems (Dumais & Veenema 2016). The reason for this phenomenon could be due to a general lack of comprehensive sex comparisons of OT/OTR systems in the literature (Dumais & Veenema 2016), an artifact of selecting particular OTR-expressing regions of interest, or a fundamental difference in the plasticity of OTR and V1aR expression (see Subsection 4.5).

Effect of pre-wean post-natal social experiences on nonapeptide receptor expression

An extraordinary number of studies have highlighted the ways in which early life social experiences with a mother can shape the social behaviors of offspring. By utilizing the biparental prairie vole, we demonstrated that the absence of a father yields a drastic drop in V1aR expression within the PVN. Early life social experiences, such as variation in maternal care, handling or social deprivation, have been known to impact both OT and VP immunoreactivity (ir) in the PVN. However, few studies have reported corresponding changes in PVN nonapeptide receptor expression as a function of early social experiences (Veenema 2012). Our study provides evidence that paternal presence is a significant factor for V1aR expression in the hypothalamus of a biparental mammal. Studies in rats have demonstrated that antagonism of V1aR within the PVN impairs anxiety-like and maternal behavior (Bayerl et al. 2016). In addition, levels of V1aR expression in the PVN of rat dams are highest at parturition compared to the days following parturition (Caughey et al. 2011), which suggests that V1aR in the PVN might be linked to rodent maternal behavior. The functional implications of upregulation or downregulation of V1a receptor expression in the PVN, however,

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remain unclear without corresponding behavioral measures for our subjects.

Nevertheless, these data provide a promising avenue of research for understanding how paternal presence during development may impact the mechanisms underlying maternal care.

Paternal presence at the nest shaped offspring neural phenotype, such that the absence of fathers resulted in greater OTR density in the NAcc. The NAcc is heavily involved in processing the reinforcing effects of both pharmacological and natural rewards across species, including those elicited from social stimuli (McBride et al.

1999; Young et al. 2001). Prairie voles densely express OTR within the NAcc compared to their socially promiscuous congener, the montane voles (M. montanus), and this phenotype is believed to contribute heavily to the species-specific propensity to form opposite sex pair-bonds (Insel et al. 1992). Decades of follow-up studies have contributed to this interpretation. For example, mating-induced partner preference formation is blocked in female prairie voles after NAcc infusion of an OTR antagonist

(Young et al. 2001) or RNAi knockdown of OTR (Keebaugh et al. 2015), whereas overexpressing NAcc OTR via viral vector gene transfer subsequently enhances partner preference formation in females (Ross et al. 2009; Keebaugh & Young, 2011).

Furthermore, differences in NAcc OTR are highly likely to relate to differences in natural motivation to engage in bonding behavior. For instance, of all the neural structures that laboratory studies have shown to be necessary and sufficient for establishing a pair-bond, pair-bonded male prairie voles living freely in outdoor enclosures differed only from single males in NAcc OTR density, with bonded males expressing more OTR than single males (Ophir et al. 2008; Ophir et al. 2012).

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Collectively, the aforementioned studies demonstrate a causal link between more OT/OTR activation in the NAcc translating into increased bonding. Under this context, our results demonstrating that Father-absent offspring had more NAcc OTR would indicate that single-raised animals should also be more likely to form bonds.

Unfortunately, we did not assess differences in any behavior, including general or pair-bond-specific motivated behaviors.

Interestingly, Ahern and Young (2009) found that single mother-reared prairie voles show a delayed onset of partner preference formation compared to biparentally reared offspring, a result that is the opposite of what our data would have predicted.

However, Ahern and Young (2009) also found no differences in NAcc OTR. Such inconsistencies are particularly intriguing and highlight 2 important points that must be kept in mind for any studies interested in aligning brain and behavioral data, particularly within a developmental context. First, there are mechanisms beyond the activation of OTR within the NAcc that have functional implications for selective affiliation in adulthood. Second, putatively inconsistent outcomes of studies that result from different perturbations over development must be placed in a broader context.

Different manipulations over the course of development can create different forces that operate on a suite of neural mechanisms in distinct ways. These differences can collectively affect each component of a behavioral system, such as propensity to form bonds in dramatically different ways depending on the type of developmental experience that occurred. In other words, a simple developmental alteration (i.e. 1 variable) of an operational network (built on the interactions of several different mechanisms operating on cell populations across a neural circuit) could result in

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neural and behavioral outcomes of one form, whereas complex (multivariate) manipulations of that same network are very likely to result in neural and behavioral phenotypes of another form. This is because more interdependent components of a network are potentially impacted in many more and several different ways when multiple factors that are certain to interact are manipulated in tandem. More simply stated, manipulating one important aspect of development might have caused a behavioral change (altered bonding), which could be potentially attributed to mechanisms other than NAcc OTR (e.g. CRF; see Ahern & Young 2009), whereas manipulating multiple aspects of development might induce changes in a broader set of neural areas (e.g. NAcc OTR among other potential mechanisms; see results) that collectively have the potential to negate or reverse the behavioral outcomes that animals are likely to produce. As mentioned above, our neural results would predict that single-raised animals would be more likely to form bonds, despite the contrary results of Ahern and Young (2009). Nevertheless, whatever the direction it might take, it is clear that early life social experiences hold great potential to impact motivated behaviors by operating on the OT system within the NAcc.

Effect of post-wean post-natal social experiences on nonapeptide receptor expression

We found that post-wean experience significantly impacts V1aR expression in the AH, such that Single-housed animals had higher V1aR expression compared to

Pair-housed animals. The AH is a region closely associated with rodent aggression

(Ferris et al. 1997; Albers 2012). Pair-bonded male prairie voles display aggression

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toward novel females (Winslow et al. 1993), and this selective aggression is mediated by VP activation of V1aR in the AH (Gobrogge et al. 2009). In addition, pair-bonded males show higher levels of AH V1aR compared to sexually naïve males.

Furthermore, naïve males show increased levels of aggression toward novel females after AH V1aR agonism, and overexpression of V1aR in the AH via viral vector- mediated gene transfer. Our data indicate that Single-housed animals have a neural profile that reflects particularly aggressive behavioral phenotypes in this species, such that postnatal social experience may shape adult aggression.

Indeed, a wealth of literature on rodent social isolation and adult levels of aggression supports the interpretation that early experiences impact aggression. In rats and mice, social isolation experienced during development or adulthood have both been found to lead to increased levels of adult aggression (Valzelli, 1973;

Wongwitdecha & Marsden 1996). In prairie voles, adult females that were isolated for

4 weeks show increased pup-directed aggression, and a greater likelihood of attacking an adult intruder, compared to non-isolated females (Grippo et al. 2008; Scotti et al.

2015). Sexually-naïve male prairie voles reared by low-contact parents exhibit more aggression towards a novel male compared to males reared by high-contact parents, providing further evidence that low levels of social exposure (either by social isolation or diminished parental care) during development can impact adult levels of aggression in this species (Perkeybile & Bales 2015). Although the neural and endocrinological mechanisms that underlie the connection between social isolation and increased adult aggression in rodents remain unclear, our neural data suggest a potential role for developmental plasticity of AH V1aR in adult aggression. Further work that explores

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the interface between AH V1aR and early-life social experiences at a developmental stage that is roughly equivalent with adolescence (Schneider 2013) is likely to provide important insight into the roots and labile nature of aggressive social behavior.

Nonapeptide receptor expression is influenced by developmental interactions

Unsurprisingly, the complexity of the social brain is obscured by the ways in which experiences and biology can interact to shape neural phenotypes across development. We found that males and females appear differentially susceptible to the influence of paternal presence. Specifically, we found that V1aR density in the VMH was greater in females than males, but only for offspring reared with a father present.

In addition, females with fathers present had greater V1aR density in the VMH than females reared by mothers alone, whereas males were not impacted by paternal presence when compared to each other in this region. The VMH is a sexually dimorphic structure (Matsumoto & Arai 2008) that is sensitive to the organizational effects of sex steroid exposure during development (Matsumoto & Arai, 1983), and is critical in rodent sexual behavior (Pfaff & Sakuma 1979; Wersinger et al. 1993).

Unfortunately, the role of V1aR in the VMH is not well characterized, largely because V1aR does not appear to be expressed in rat VMH. The majority of what we do know regarding V1aR VMH expression has been identified in hamsters. For example, male Siberian hamsters have higher VMH V1aR density than female

Siberian hamsters. Furthermore, castrated males have reduced V1aR density in the

VMH compared to intact males, and testosterone implants rescue this reduction, indicating that V1aR expression in the VMH is modulated by gonadal steroids in this

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species (Dubois-Dauphin et al. 1994). In addition, dominant male Syrian hamsters have increased V1aR density in the VMH compared to subordinate males (Cooper et al. 2005), indicating a potential role of VMH V1aR density in aggression and social dominance status. Considering the seemingly opposite direction of sex effects found between Siberian hamsters (where males have higher VMH V1aR density compared to females) and Prairie voles (where females have greater VMH V1aR density compared to males in the Father-present condition), it remains possible that V1aR has species- specific functions within the VMH, which require behavioral data for further elucidation.

Considering the roles that both the VMH and V1aR play in rodent reproductive behavior, we speculate that the presence of a father may shape the mechanisms that underlie adult sociosexual behaviors in a sex-specific manner. This result also highlights the need to include sex as an important biological variable. Without explicitly including both sexes as subjects, the nuanced heterogeneity of the ways in which the male and female social brains are differentially shaped by experience cannot be understood.

The pre-wean experience also interacts with social environments in later postnatal life. Oxytocin receptor density in the SHi (a region important for memory functioning: see Ophir 2017) varied across levels of both paternal presence and post- wean housing. Our lab previously demonstrated that OTR SHi density is susceptible to the interaction of pre-wean and post-wean social environments (Prounis et al. 2015). It is important to note that the current study included both sexes as a factor, whereas

Prounis et al. (2015) focused on just males but incorporated a behavioral study as

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well. It is unclear why the results from these 2 studies were inconsistent; however, the methodological and design differences between them might explain why animals without fathers in the current study demonstrated the opposite pattern of OTR expression from those in Prounis et al. (2015). Nevertheless, the results from both studies clearly indicate that OTR in the SHi is particularly sensitive to the interactive effects of social experiences in early and late development.

More broadly, our study reaffirms that nonapeptide systems that are shaped by social experiences in the natal nest can be pronounced or offset by social experiences later in development. It is imperative to consider developmental experiences along a continuum, in which there are multiple sensitive periods that may be both independently and/or synergistically influential in neural scaffolding (Fox et al. 2010).

This point is amplified when sex is considered as a variable. For example, OTR expression in the LS showed a significant 3-way interaction such that only males that were raised with fathers but that were housed singly after weaning showed an increase in septal OTR density compared to females. Considering the broad involvement of the

LS and oxytocin in social behavior, social memory and other behaviors that are relevant to social functioning (Francis et al. 2001; Guzmán et al. 2014), this unique sensitivity of males to a particular series of early life experiences might help explain how differences in complex behavioral phenotypes develop in some animals or sub- populations of animals and not others. We suggest that sexual dimorphisms in neurobiology and physiology may open the potential for offspring to be differentially sensitive to subsequent neural shaping by interacting perinatal experiences (Curley &

Champagne 2016; Moore & Depue 2016).

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Stability and variability among the nonapeptide systems

Childhood and adolescence are periods of time in development with obvious implications for shaping the adult phenotype. Our study is a relatively exhaustive foray into the potential impact of early life experience at the rodent equivalent of these

2 distinct developmental time points (see Spear 2000 and references therein) on forebrain nonapeptide receptor phenotype. Based on our results, we highlight 2 important overall patterns: (i) the discrepancy in the distribution of effects found in

V1aR and OTR; and (ii) the apparent robustness of these systems despite diversity in sex and social environments across development.

The first point speaks to the comparatively larger number of effects found for

V1aR expression compared to OTR expression in our results. Interestingly, all 3 variables analyzed yield the main effects on V1aR expression, and all the main effects of sex reported are in V1aR expression. This pattern of results draws attention to the contrast between the plasticity of V1aR compared to the consistency of OTR expression across our variables. Other studies have shown a similar pattern, in which

OTR expression appears to be less malleable compared to that of V1aR (Ophir et al.

2013; Prounis et al. 2018). For example, a comparison of OTR and V1aR densities across the forebrain of pregnant female prairie voles showed that females only differed in V1aR expression within the ventral pallidum and the PVN, a contrast to the striking stability of OTR forebrain pattering in the wake of large hormonal fluctuations across pregnancy (Ophir et al. 2013). A comparison of OTR and V1aR within the social decision-making network (O’Connell & Hofmann 2012) of female prairie voles living

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freely in outdoor enclosures showed that most structures expressing V1aR differed by the reproductive mating tactic, reproductive success or the interaction therein, whereas this was only true of 2 OTR expressing brain areas (Zheng et al. 2013). Indeed, a thorough analysis of the extant literature (for which there are fewer studies targeting

OT/OTR) reveals many more sex differences in VP/V1aR systems compared to

OT/OTR differences (Dumais & Veenema 2016). A systematic cross-species characterization of OT-ir and VP-ir across developmental time and between the sexes exhibited plentiful differences in VP-ir in the social behavior network (Newman 1999;

Goodson 2005), but none on OT-ir in rats (DiBenedictis et al. 2017). Taken together, there is substantial evidence indicating that the vasopressin system varies more than that of oxytocin, particularly as a function of sex and developmental experiences. Why the VP system is relatively more plastic than the OT system is a pressing and extremely interesting question, but currently remains unresolved. Still, this collection of evidence leads to testable predictions that differences in both plasticity and functional roles between the 2 nonapeptide systems should exist.

Our second point, in contrast, emphasizes how relatively few differences were found in either OTR or V1aR expression overall, despite the number of potential interactions between variables and the numerous regions we analyzed. Although we found clear effects of sex, pre-wean experiences and post-wean experiences on receptor density, a remarkable number of regions exhibited no differences in receptor expression. Thus, our data reveal a robust consistency in receptor profiles despite profound biological and environmental variation. These results fall in line with the paucity of structural sex differences found in monogamous species more generally.

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Considering the known changes in adult behavior that occur as a function of similar early life experiences and sex (Ahern & Young 2009), how do we reconcile this receptor stability with divergences in behavioral outcomes? One explanation for these results could be that early social experiences and sex have more substantial impacts on the density of nonapeptide-producing neurons or fiber innervation than on receptor turnover. If true, this explanation would have important implications for the modulation of social behavior. In any case, it is critical to bear in mind that these differences in receptor profiles or immunoreactivity may either drive sexual dimorphisms in behavior, or compensate for other structural and physiological sex differences to facilitate behavioral equifinality (De Vries 2004). This hypothesis cannot be tested without directly manipulating receptors within specific cell groups and systematically measuring behavioral outcomes (Kelly & Goodson 2014).

CONCLUSIONS

Our study demonstrates that both OTR and V1aR binding densities are sensitive to early life social experiences, biological sex, and the interactions between these variables. The direction of the effects is region-specific and receptor-specific; instances of low social exposure (Father-absent and/or Single-housed) or high social exposure (Father-present and/or Pair-housed) did not have uniform effects on nonapeptide expression across the social brain. Instead, regulation of receptor density as a function of experience was either enhanced or dampened in a manner specific to each area of interest, which likely has functional implications for the neuromodulatory effects of OT/VP systems in response to social stimuli. As such, sweeping

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generalizations regarding how experiential factors impact the development of adult sociality and neural functioning as they relate to the nonapeptides are ineffectual in working towards a systems-level understanding of the social brain. Our data reaffirm prior work that has demonstrated sex-specific effects on the impacts that interacting early life experiences have on nonapeptide systems (Perkeybile et al. 2015). To further elucidate these functional relationships, we should strive to incorporate factors across developmental time and biology to better reflect the complexity of the real world.

Acknowledgements

The authors would like to thank Johnna Graham, Ruth Witmer, Marcos Moreno,

Lauren Foley, Asad Rehman and George Prounis for their contributions to the data collection. The authors acknowledge the support from the National Institutes of Health

(Eunice Kennedy Shriver National Institute of Child Health and Human Development

HD079573 to A.G.O.) and the National Science Foundation Graduate Research

Fellowship Pro- gram (under DGE-1650441 to L.C.H.).

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FIGURES

Figure 3.1. Experimental design. Sample sizes for each group are indicated within the cells next to the symbols for female (♀) and male (♂). .

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Figure 3.2 Main effects of Sex on mean vasopressin 1a receptor (V1aR) binding density (dpm/mg TE) ± standard error bars for the main olfactory bulbs (MOB), accessory olfactory bulbs (AOB), lateral septum (LS), lateral bed nucleus of the stria terminalis (BSTl) and medial bed nucleus of the stria terminalis (BSTm). *P ≤ 0.05.

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Figure 3.3 Main effect of Pre-wean condition on mean vasopressin 1a receptor (V1aR) binding density (dpm/mg TE) ± standard error bars for the paraventricular nucleus of the hypothalamus (PVN). *P ≤ 0.05.

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Figure 3.4 Main effect of Pre-wean condition on mean oxytocin receptor (OTR) binding density (dpm/mg TE) ± standard error bars for the nucleus accumbens (NAcc). *P ≤ 0.05.

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Figure 3.5 Main effect of Post-wean condition on mean vasopressin 1a receptor (V1aR) binding density (dpm/mg TE) ± standard error bars for the anterior hypothalamus (AH). *P ≤ 0.05.

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Figure 3.6 Interaction effect of Pre-wean condition and Sex on mean vasopressin 1a receptor (V1aR) binding density (dpm/mg TE) ± standard error bars for the ventromedial hypothalamus (VMH). *P ≤ 0.05.

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Figure 3.7 Interaction effect of Pre-wean condition and Post-wean condition on mean oxytocin receptor (OTR) binding density (dpm/mg TE) ± standard error bars for the septohippocampal nucleus (SHi). *P ≤ 0.05.

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Figure 3.8 Interaction effect of Pre-wean condition, Post-wean condition and Sex on mean oxytocin receptor (OTR) binding density (dpm/mg TE) ± standard error bars for the lateral septum (LS). (a) shows the relationship within the Single-housed animals, and (b) shows the relationship within the Pair-housed subjects. *P ≤ 0.05.

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TABLES

Table 3.1 Vasopressin 1a receptor (V1aR) results from the omnibus linear-mixed model ANOVAs for each structure analyzed. Significant P-values (after corrections for multiple comparisons) are highlighted in bold and italic font. Values presented in each cell are F-values (degrees of freedom); P-values. Abbreviations for neural structures are: AOB, accessory olfactory bulb; AH, anterior hypothalamus; BSTl, lateral bed nucleus of the stria terminalis; BSTm, medial bed nucleus of the stria terminalis; BSTv, ventral bed nucleus of the stria terminalis; CeA, central amygdala; LDTh, latero-dorsal nucleus of the thalamus; LS, lateral septum; MDTh, medio-dorsal nucleus of the thalamus; MeA, medial amygdala; MOB, main olfactory bulb; PVN, paraventricular nucleus of the hypothalamus; RSC, retrosplenial cortex; VMH, ventromedial hypothalamus; VPall, ventral pallidum; VPTh, ventro-posterior nucleus of the thalamus.

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Main effects Two-way interactions Pre-wean Structure Sex Pre-wean Post-wean Sex X Pre- Sex X X Post- Three-way wean Post-wean wean F(1, 77.5) = F(1, 31.0) = F(1, 81.1) = F(1, 76.1) = F(1, 81.5) = F(1, 85.2) = F(1, 83.6) = MOB 8.6, 2.8, 3.3, 0.27, 0.45, 0.039, 0.3, P < 0.005 P = 0.1 P = 0.07 P = 0.6 P = 0.5 P = 0.8 P = 0.6 F(1, 80.4) = F(1, 33.7) = F(1, 82.4) = F(1, 79.3) = F(1, 85.1) = F(1, 88.4) = F(1, 86.4) = AOB 6.1, 2.5, 0.69, 0.65, 3.1, 0.35, 0.63, P < 0.05 P = 0.1 P = 0.4 P = 0.4 P = 0.08 P = 0.6 P = 0.4 F(1, 94.1) = F(1, 30.8) = F(1, 87.9) = F(1, 89.6) = F(1, 93.2) = F(1, 98.2) < F(1, 93.4) = LS 7.2, 0.56, 3.1, 0.017, 0.09, 0.01, 2.3, P < 0.01 P = 0.5 P = 0.08 P = 0.9 P = 0.8 P = 0.9 P = 0.1 F(1, 80.3) = F(1, 30.0) = F(1, 80.2) = F(1, 75.1) = F(1, 79.0) = F(1, 88.2) = F(1, 81.1) = VPall 0.82, 0.27, 2.2, 2.0, 0.2, 0.054, 0.13, P = 0.4 P = 0.6 P = 0.1 P = 0.2 P = 0.7 P = 0.8 P = 0.7 F(1, 100.5) = F(1, 99.8) = F(1, 100.0) = F(1, 99.8) = F(1, 100.0) < F(1, 100.1) = F(1, 100.6) = BSTl 11.0, 0.61, 0.15, 0.081, 0.01, 0.32, 1.9, P < 0.005 P = 0.4 P = 0.7 P = 0.8 P = 1 P = 0.6 P = 0.2 F(1, 98.8) = F(1, 23.8) = F(1, 100.0) < F(1, 97.4) = F(1, 99.8) = F(1, 99.6) = F(1, 100.9) = BSTm 4.6, 1.2, 0.01, 0.91, 0.84, 0.3, 1.0, P < 0.05 P = 0.3 P = 1 P = 0.3 P = 0.4 P = 0.6 P = 0.3

F(1, 100.3) = F(1, 99.7) = F(1, 99.9) = F(1, 99.7) = F(1, 99.9) = F(1, 100.0) = F(1, 100.5) = BSTv 0.79, 0.37, 0.18, 0.011, 0.08, 0.012, 0.5, P = 0.4 P = 0.5 P = 0.7 P = 0.9 P = 0.8 P = 0.9 P = 0.5 F(1, 98.4) = F(1, 20.7) = F(1, 98) = F(1, 97.1) = F(1, 97.9) = F(1, 96.2) = F(1, 98.9) = AH 2.9, 1.0, 3.9, 0.022, 0.95, 0.16, 0.3, P = 0.09 P = 0.3 P = 0.05 P = 0.9 P = 0.3 P = 0.7 P = 0.6 F(1, 98.7) = F(1, 30.6) = F(1, 98.0) = F(1, 96.0) = F(1, 97.7) = F(1, 98.9) = F(1, 99.6) = PVN 0.78, 4.0, 0.58, 0.35, 0.24, 0.69, 0.68, P = 0.4 P = 0.05 P = 0.4 P = 0.6 P = 0.6 P = 0.4 P = 0.4 F(1, 99.1) = F(1, 35.5) = F(1, 101.6) = F(1, 95.2) = F(1, 98.9) = F(1, 102.4) = F(1, 102.1) = MDTh 0.17, 1.8, 0.76, 1.1, 0.02, 0.13, 0.5, Vasopressin receptor (V1aR)Vasopressin receptor P = 0.7 P = 0.2 P = 0.4 P = 0.3 P = 0.9 P = 0.7 P = 0.5 F(1, 97.0) = F(1, 32.2) < F(1, 99.4) = F(1, 93.7) < F(1, 97.2) = F(1, 100.5) = F(1, 99.9) = LDTh 1.7, 0.01, 2.1, 0.01, 0.26, 0.84, 0.07, P = 0.2 P = 0.9 P = 0.1 P = 0.9 P = 0.6 P = 0.4 P = 0.8 F(1, 94.0) = F(1, 31.0) = F(1, 90.9) = F(1, 90.6) = F(1, 94.5) = F(1, 99.1) = F(1, 93.8) = VPTh 0.74, 1.9, 3.4, 0.022, 0.07, 0.01, 0.18, P = 0.4 P = 0.2 P = 0.07 P = 0.9 P = 0.8 P = 0.9 P = 0.7 F(1, 92.2) = F(1, 30.1) = F(1, 94.9) = F(1, 89.4) < F(1, 94.4) = F(1, 97.9) = F(1, 97.9) = CeA 3.9, 0.24, 0.31, 0.01, 0.62, 3.2, 3.3, P = 0.05 P = 0.6 P = 0.6 P = 1 P = 0.4 P = 0.08 P = 0.07 F(1, 90.4) = F(1, 23.7) = F(1, 88.3) = F(1, 87.2) = F(1, 90.3) = F(1, 94.6) < F(1, 90.4) = MeA 0.78, 0.02, 0.42, 0.13, 2.7, 0.01, 0.6, P = 0.4 P = 0.9 P = 0.5 P = 0.7 P = 0.1 P = 1 P = 0.4 F(1, 84.4) = F(1, 36.4) = F(1, 87.1) = F(1, 81.9) = F(1, 86.6) = F(1, 91.5) = F(1, 88.3) = RSC 0.85, 0.96, 2.6, 2.4, 0.01, 0.058, 0.25, P = 0.4 P = 0.3 P = 0.1 P = 0.1 P = 0.9 P = 0.8 P = 0.6 F(1, 99.2) = F(1, 31.7) = F(1, 99.5) = F(1, 97.6) = F(1, 99.3) = F(1, 100.3) = F(1, 100.6) < VMH 0.41, 0.54, 0.03, 4.6, 1.4, 0.92, 0.01, P = 0.5 P = 0.5 P = 0.9 P < 0.05 P = 0.2 P = 0.3 P = 0.9

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Table 3.2 Oxytocin receptor (OTR) results from the omnibus linear-mixed model ANOVAs for each structure analyzed. Significant P-values (after corrections for multiple comparisons) are highlighted in bold and italic font. Values presented in each cell are F-values (degrees of freedom); P-values. Abbreviations for neural structures are: BLA, basolateral amygdala; CeA, central amygdala; CPu, caudate-putamen; HPC, hippocampus; ICa, anterior portion of the insular cortex; ICm, medial portion of the insular cortex; IMD, intermediodorsal thalamic nucleus; LS, lateral septum; NAcc, nucleus accumbens; PFC, prefrontal cortex; Shi, septo-hippocampal nucleus.

Main effects Two-way interactions Sex X Pre-wean Structure Sex Pre- Post-wean Sex X Pre- Post- X Post- Three- wean wean way wean wean F(1, 94.6) = F(1, 99.6) = F(1, 88.8) = F(1, 97.7) = F(1, 97.9) = F(1, 103.3) = F(1, 100.6) = BLA 0.12, 3.4, 0.42, 2.5, 1.2, 0.55, 0.21, P = 0.7 P = 0.07 P = 0.5 P = 0.1 P = 0.3 P = 0.5 P = 0.6 F(1, 92.5) = F(1, 97.4) = F(1, 87.4) = F(1, 95.7) = F(1, 95.7) = F(1, 101.8) = F(1, 99.2) = CeA 2.1, 1.9, 1.6, 2.0, 0.12, 0.18, 0.18, P = 0.1 P = 0.2 P = 0.2 P = 0.2 P = 0.7 P = 0.7 P = 0.7 F(1, 103.4) = F(1, 102.2) = F(1, 100.5) = F(1, 101.1) = F(1, 102.1) < F(1, 106.1) = F(1, 108.5) = CPu 2.9, 3.0, 0.44, 0.3, 0.01, 0.16, 3.1, P = 0.09 P = 0.08 P = 0.5 P = 0.6 P = 0.9 P = 0.7 P = 0.08 F(1, 90.9) = F(1, 28.2) = F(1, 81.0) = F(1, 91.9) = F(1, 92.3) = F(1, 100.5) = F(1, 98.5) = HPC 0.29, 1.6, 0.032, 1.5, 0.13, 0.23, 1.3,

P = 0.6 P = 0.2 P = 0.9 P = 0.2 P = 0.7 P = 0.6 P = 0.3 F(1, 100.9) = F(1, 99.7) < F(1, 98.4) = F(1, 98.7) = F(1, 99.7) = F(1, 103.2) < F(1, 105.4) = ICa 0.17, 0.01, 2.8, 0.077, 0.4, 0.01, 0.03, P = 0.7 P = 1 P = 0.1 P = 0.8 P = 0.5 P = 1 P = 0.9 F(1, 90.3) = F(1, 95.9) = F(1, 84.9) = F(1, 95.7) = F(1, 94.4) = F(1, 100.3) = F(1, 98.7) = ICm 0.085, 1.7, 0.1, 1.6, 2.0, 0.54, 0.3, eceptor (OTR) eceptor

r P = 0.8 P = 0.2 P = 0.7 P = 0.2 P = 0.2 P = 0.5 P = 0.6 F(1, 91.8) = F(1, 17.7) = F(1, 92.6) = F(1, 94.5) = F(1, 101.2) = F(1, 108.4) = F(1, 100.8) = IMD 0.28, 0.02, 0.056, 0.85, 0.02, 0.2, 0.2, P = 0.6 P = 0.9 P = 0.8 P = 0.4 P = 0.9 P = 0.7 P = 0.7 Oxytocin Oxytocin F(1, 108.3) = F(1, 106.3) = F(1, 102.7) = F(1, 104.0) = F(1, 105.9) = F(1, 111.0) = F(1, 109.6) = LS 0.71, 0.45, 0.72, 0.6, 0.83, 0.11, 3.8, P = 0.4 P = 0.5 P = 0.4 P = 0.4 P = 0.4 P = 0.7 P = 0.05 F(1, 103.4) = F(1, 102.3) = F(1, 100.5) = F(1, 101.1) = F(1, 102.1) = F(1, 106.3) = F(1, 108.7) = NAcc 0.2, 6.8, 3.4, 0.82, 0.48, 0.039, 0.01, P = 0.7 P = 0.01 P = 0.07 P = 0.4 P = 0.5 P = 0.8 P = 0.9 F(1, 103.5) = F(1, 102.5) = F(1, 100.1) = F(1, 100.7) = F(1, 102.2) = F(1, 107.2) = F(1, 108.9) = PFC 1.2, 0.32, 1.1, 0.28, 0.25, 0.058, 0.02, P = 0.3 P = 0.6 P = 0.3 P = 0.6 P = 0.6 P = 0.8 P = 0.9 F(1, 94.7) = F(1, 37.0) = F(1, 86.0) = F(1, 96.4) = F(1, 96.7) < F(1, 103.6) = F(1, 98.5) = SHi 0.16, 0.11, 0.072, 0.47, 0.01, P = 9.4, 0.11, P = 0.7 P = 0.7 P = 0.8 P = 0.5 1 P < 0.005 P = 0.7

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CHAPTER 4

PATERNAL ABSENCE, FAMILIAL HANDLING, AND OFFSPRING AGE

INDEPENDENTLY AND INTERACTIVELY SHAPE THE DEVELOPMENT OF

PRAIRIE VOLES (MICROTUS OCHROGASTER)

In prep for submission: Lisa C. Hiura, Vanessa A. Lazaro, Alexander G. Ophir

ABSTRACT

In species that exhibit biparental care, the absence of a father during early life has profound implications for the development of offspring. In the present study, we investigated if behaviorally upregulating maternal care can offset the behavioral and neural consequences of paternal absence on pup development in the socially monogamous prairie vole (Microtus ochrogaster). We used an established handling manipulation to experimentally vary levels of parental care in single-mother and biparental families, then measured offspring behavioral outcomes at subadult and adult stages of life. We found that experimental handling appeared to rescue the effect of paternal absence on age-specific social dominance behavior, demonstrating that multiple factors of the early environment can interact to shape offspring development.

However, handling did not offset the impact of paternal absence on other types of social interactions, suggesting that there is heterogeneity in the developmental processes that shape distinct social behaviors. Father presence interacted with experimental handling to pattern the number of oxytocinergic, but not vasopressinergic, neurons in the hypothalamus. These results advance our

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understanding of the ways in which early experiences function to synergistically or antagonistically shape offspring phenotypes, and provide insights into the neural mechanisms that mediate developmental plasticity in social behavior.

INTRODUCTION

Early life experiences serve a crucial role in shaping the development of physiological, neural, and behavioral outcomes in offspring. Most notably, natural and experimentally induced variation in mother’s behaviors and their offspring-directed care has been repeatedly shown to direct developmental processes in rodents (Curley and Champagne, 2016). Maternal behaviors including licking and grooming provide pups with cues that signal the quality of the postnatal environment (Champagne and

Curley, 2005). When dams exhibit high levels of licking and grooming behaviors, their adult offspring displayed reduced levels of anxiety-like behaviors and attenuated physiological responses to stressors compared to adult offspring of dams who licked and groomed at low levels (Caldji et al., 1998; Liu et al., 1997). These outcomes are subserved by maternal care-dependent epigenetic modifications to several genes important for neuroendocrine signaling systems (Champagne, 2011). In this way, mothers behaviorally tune the transcriptional profiles off their offspring in early life, guiding their pups down distinct neurodevelopmental trajectories.

Given that variation in maternal behavior confers such powerful effects, it stands that the contributions of other neonatal caregivers would also be influential in offspring development. The majority of mammalian species are uniparental, with only

5-10% exhibiting biparental care (Kleiman and Malcolm, 1981). Of this subset, the

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socially monogamous prairie vole (Microtus ochrogaster) has served as an invaluable model system in which to investigate the impact that fathers exert in the shaping of offspring phenotypes. Prairie vole mothers and fathers display all of the same care behaviors towards their pups with the exception of nursing (Thomas and Birney,

1979). Prior studies have revealed that when fathers were removed from the natal nest, offspring were faster to begin consuming solid foods and egressing from the nest

(Wang and Novak, 1992), spent more time engaged in alloparental care as juveniles

(Wang and Novak, 1994), and had shorter latencies to form adult pair bonds (Ahern and Young, 2009). Additionally, pups that were reared by single mothers receive significantly less parental care than pups that were reared biparentally (Chapter 5,

Ahern and Young, 2009; Tabbaa et al., 2017). Together these results suggest that offspring phenotypic development may be mediated by the reduced levels of care that stem from the absence of a father.

Prairie vole mothers do not naturally upregulate their care to compensate for the absence of a father (Ahern et al., 2011; Ahern and Young, 2009; Bosch et al.,

2018; McGuire et al., 2007; Rogers and Bales, 2019; Tabbaa et al., 2017, but see Kelly et al. in press). We hypothesized that if mothers were induced to upregulate their caregiving behavior, they may be capable of offsetting the loss in care that results from a missing father. Experimental handling manipulations increase the levels of care that prairie vole offspring receive from their parents (Carter et al., 2008). Therefore, the present study leveraged handling manipulations to ask if handled, behaviorally upregulated parents could compensate for the developmental effects of a lack of a parenting partner in the natal nest. We examined behavioral outcomes in prairie vole

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offspring that were reared in families with and without their fathers, and with or without experimental handling. We also assessed behavioral profiles at two separate ages to determine if the effects of rearing condition were age specific.

The social experience-specific developmental plasticity of offspring is driven by alterations in the neural substrates underlying social behavior. Namely, the two nonapeptide neuromodulators oxytocin (OT) and arginine vasopressin (VP) are involved in the control of a variety of animal behaviors (including parental care, pair bonding, social recognition, aggression, and sexual behaviors) and their profiles are impacted by variation in the early environment (Bales and Saltzman, 2016). OT and

VP are primarily produced in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus (Gainer et al., 2002). These sites project to the posterior pituitary where they release hormones into the periphery, but they also have extensive extrahypothalamic efferents and can release peptides axonally to provide signals that act on oxytocin and vasopressin receptors throughout the forebrain

(Landgraf and Neumann, 2004). Smaller OT and VP cell groups are also found in the bed nucleus of the stria terminalis (BST), a region also heavily tied to social behavior

(Kelly and Goodson, 2014a). As such, variation in the numbers of OT and VP neurons in these cell groups may have significant implications for the volume of peptide available for release, and therefore, the neuroendocrine modulation of behavior. We investigated the impact of rearing experiences on the number of OT and VP cells within the PVN, SON, and BST to provide potential neural corollaries that map onto behavioral outcomes.

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MATERIALS AND METHODS

Experimental Animals:

We created F1 breeders derived from wild-caught animals originally trapped in

Champagne-Urbana, Illinois and reared at Cornell University. Only breeders that birthed a litter of three or more, and of which at least two were male, were used in this study. We bred families in cohorts to accommodate the number of animals to be used within the spatial constraints of the colony and testing rooms. From these breeders, 55

F2 offspring from 21 breeding cohorts served as subjects in this study. All animals were housed in standard polycarbonate rodent cages (29 × 18 × 13 cm) lined with

Sani-chip bedding and provided nesting material. We provided animals ad libitum access to water and food (Rodent Chow 5001, LabDiet, St. Louis, MO, USA).

Animals were kept under a 14:10 light-dark cycle with ambient temperature maintained at 20±2°C. No animals in this experiment were raised in isolation. All procedures used in this study were approved by the Institutional Animal Care and Use

Committee of Cornell University (ACUP 2013-0102).

Experimental conditions:

We created four rearing conditions that manipulated father presence or absence and parental caregiving (via experimental handling) during preweaning development of subjects. For most behavioral tests, we also conducted a repeated measures assessment of subjects by age at two stages of maturation (subadulthood and adulthood). Therefore, most comparisons followed a 2 x 2 x 2 design (exposure to fathers, manipulation of parents to modify caregiving, and subject age, see below).

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Exposure to Fathers - Prairie vole gestation is 21 days from fertilization to birth. To create a Father Absent treatment group, we removed fathers from home cages after 20 days of cohabitation with a partner to ensure that pups were never exposed to their fathers. For families in the Father Present condition, we removed fathers then immediately returned them to their home cages to account for the potential effect of nest disturbance. Nests were carefully monitored for the birth of pups, the number of pups was recorded at birth, and the litter was culled to three to standardize litter size across breeders. Males were preferentially spared for testing in the following experiments.

Modification of Parental Caregiving - By removing fathers, we reduced the total amount of care pups would receive (Chapter 5, Perkeybile et al. 2015, Kelly et al, in press). Our second factor aimed to increase caregiving by physically handling parents, which induces caregiving (Carter et al., 2008). On postnatal days (PND) 2, 9, and 16, families were transferred to a clean cage during weekly cage changes. Parents in the Handled condition were gently scruffed by a gloved hand, whereas parents in the Non-handled condition were scooped and moved with a plastic beaker (Bales et al., 2007). Young pups (PND 2 and PND 9) were typically latched to their mother’s nipples, in which case pups were moved by their mothers in the Handled condition or transferred with mother in the cup in the Non-handled condition. When pups were unattached to mothers, they were transferred in the same manner as their parent(s).

One-hour video recordings of the home cages were taken immediately following the cage transfers for a separate study.

At PND 21, all pups were weaned from their parents and moved to a new

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home cage with their same sex sibling(s). One male from each cage was randomly assigned to be the focal male for behavioral testing and histological analysis. Taken together we created four groups: Father Present / Non-handled (i.e., a control group,

N = 15), Father Present / Handled (N = 16), Father Absent / Non-handled (N = 13), and Father Absent / Handled (N = 11).

Behavioral Testing:

All focal animals underwent the same order of behavioral testing (Fig. 4.1).

After weaning, subjects were run though a fixed sequence of behavioral testing once as subadults (PND 24-31) and then again as adults (PND 60-67). The testing battery consisted of an open field test to evaluate anxiety/exploration, a social interaction test to assess pro-and anti-social behavior, a resident-intruder test to assess territorial behavior, and a tube test to assess dominance behavior. Finally, all subjects were housed with a sexually receptive female and then tested in a partner preference test to assess pair bonding. Animals always had one day between behavioral tests during the subadult and adult phases. The testing apparatuses were thoroughly cleaned between tests using 70% ethanol or soapy water.

Open Field Test

On PND 24/25 and PND 60/61, subjects underwent a 10-minute open-field test

(OFT). Subjects were transferred by cup to the center of a transparent Plexiglas arena

(57 cm x 57 cm) on top of a 4x4 grid of squares (14.25 cm x 14.25 cm). The centermost four squares comprised the arena “center” (28.5 cm2). We measured the

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duration of time spent in the center, frequency of visits to the center, and the total distance moved during the test.

Social Approach Test

On PND 26/27 and PND 62/63, subjects were placed under a plastic beaker on one side of a transparent Plexiglas arena (57 cm x 57 cm). Another plastic beaker on the opposite side held a novel adult male conspecific. Both beakers were simultaneously lifted, and animals were allowed to explore and interact for 10 mins.

We analyzed the total distance moved, the duration of body contact between animals, and the mean distance between animals.

Resident-Intruder Test

On PND 28/29 and PND 64/65, we removed focal subjects’ siblings from the home cage and allowed subjects to reacclimate for five minutes. After the acclimation period, we placed a novel male conspecific into the home cage and recorded their behaviors for five minutes. At the end of the test, we removed the stimulus animal and returned the sibling(s) that had been removed back to the home cage. We measured the latency to the first attack, and counted each time the subjected chased or attacked the stranger, fled, submitted (laid supine), and reared up on their hindlegs. We combined the counts of attacks and chases into a single outcome we called ‘offensive aggression’. We also collapsed submissions and flees into a measure we called

‘defense behavior’.

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Tube Test

Testing took place on PND 30/31 and PND 66/67. A transparent Plexiglas tube

(30 cm length x 3.15 cm diameter) was used in the testing process. In this test, animals are released into opposite ends of a tube, where they can interact at the point at which they meet. The more dominant animal forces its opponent out of the tube. Before initiating the test, the subject and an unfamiliar same same-sex, age-matched, and weight-matched conspecific were individually allowed to pass through the tube to acclimate to the procedure. The acclimation order was randomized. At the start of a test trial, both animals were simultaneously released by hand into opposite ends of the tube. An animal was declared the loser when it had all four paws out of the tube. A trial was ended as a draw if two mins elapsed without a loser. The starting side was randomly assigned at the beginning of the test and held constant across three consecutive test trials, with one min between each trial. The proportions of wins were counted as the dependent variable, and animals that won at least two trials were labeled as the winner of the test.

Partner Preference Test

Two unrelated sexually mature and inexperienced females were primed with dirty bedding from an unrelated male’s cage. The next day, one of these females was randomly assigned to be the cage mate and moved into a clean cage with the focal subject on PND 73/74. After 24 hours of cohabitation, the female was considered the

‘partner’. Prior work has demonstrated that 24 hours of cohabitation in the absence of mating is sufficient for voles to form a partner bond (Williams et al., 1992). After this

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cohabitation phase, on PND 74/75 subjects underwent a 3 h partner preference test in a

3-chamber apparatus (51 x 102 x 30.5 cm). The chambers were separated by opaque walls, but contained small access doors to the center chamber that were off-set in opposite directions. The subject’s partner was tethered in one side chamber (51 x 28 cm) and a sexually primed unfamiliar stimulus female was tethered in the other side chamber. The chamber that would contain each animal was determined randomly.

Tethering involved using a plastic zip-tie as a collar connected to a lightweight chain attached to the apparatus, allowing for normal activity (i.e., moving and mating).

Stimulus animals were allowed 30 min to acclimate to tethering in the testing apparatus. The focal male was then gently placed into the center chamber (51 x 46 cm) and allowed to freely explore for three hours. We measured the time spent in each chamber, and the duration of time spent in side-by-side contact with the cage mate and the stranger.

Behavioral quantification:

All tests were video recorded using a Sony HDR-CX330 camcorder (Sony,

New York City, NY, USA) or a GoPro HERO3 camera (GoPro Inc, San Mateo,

California, USA). EthoVision XT v13 software (Noldus Information Technology,

Leesburg, VA, USA) was used to score the open field test, social approach test, resident-intruder test, and partner preference test. The social dominance test was evaluated by hand.

Histology and Immunocytochemistry:

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Immediately following the partner preference test, subjects were sacrificed using isoflurane overdose and underwent transcardiac perfusion (0.1M phosphate- buffered saline [PBS, pH = 7.4], 4% paraformaldehyde in PBS). Brains were post- fixed in 4% paraformaldehyde (24 h) and sunk in 30% sucrose (48 h) before storage at

-80°C. Each brain was coronally sectioned into three series (40 µm slice) in cryoprotectant. One free-floating series from each subject was fluorescently double labeled for OT and VP immunoreactivity (-ir). Sections were rinsed in PBS (2 x 30 m), blocked (1 h, PBS + 10% normal donkey serum + 0.03% Triton-X-100), and incubated in primary antibodies (48 h, mouse anti-OT 3:1000, Millipore, Billerica,

MA; guinea pig anti-VP 1:1000, Peninsula Laboratories, San Carlos, CA). Sections were then rinsed in PBS (2 x 30 m), incubated in biotinylated donkey anti-Guinea pig

(1h, 1:8000, Jackson Immunoresearch West Grove, PA), and rinsed again in PBS (2 x

15 m). Sections were then incubated in secondary antibodies (2 h at room temp, streptavidin conjugated to Alexa Fluor 488 3:1000; donkey anti-mouse Alexa Fluor

594 5:1000, ThermoFisher Scientific, Waltham, MA). Sections were washed in PBS

(overnight at 4°C), mounted onto microscope slides, and cover-slipped with Prolong

Gold antifade + DAPI nuclear stain (ThermoFisher Scientific, Waltham, MA) before imaging.

Microscopy and quantification:

A Zeiss AxioImager II scope with an AxioCam MRm attachment, z-drive, and

Apotome optical dissector (Carl Zeiss Inc., Gottingen, Germany) was used to capture images of the PVN, BST, and SON at two coronal sections (separated by 240 µm

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along the rostral-caudal axis). Observers blind to the experimental conditions manually labeled OT-ir cells and VP-ir cells in the GNU Image Manipulation Program

(GIMP, 2.8.22). An ImageJ script (National Institutes of Health, Bethesda, MD) compiled counts of the labeled images, and these cell counts were combined within each brain region for statistical analysis.

Statistical Analysis:

All of the statistical analyses were calculated in R software v.3.6.2 (R Core

Team, 2013). Behavioral and neural data were analyzed using generalized linear mixed models (GLMM) in the glmmTMB package (Brooks et al., 2017). Models were constructed with Father Presence, Handling Condition, and Age as fixed factors (as well as their full interactions), and individual Subject ID nested within Cohort as a random effect. Inclusion of the random effect term was dropped from the model when it resulted in model convergence errors. The partner preference test data and the neural data both left out Age as a fixed factor, as these measures only took place once.

Stimulus was included as a fixed effect in the model when the total duration of huddling and the total duration in stimulus chambers was analyzed in the partner preference tests. The family function and link function for each GLMM was selected based upon the type and distribution of each dependent variable. Model selection was conducted by comparing Akaike’s Information Criterion values across potential models using the stats package. The DHARMa package (Hartig, 2019) was used to assess model fits with simulated residuals and regression diagnostic plots. Type II

Wald chi-squared tests were conducted via the Anova function of the car package to

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generate p-values and assess significant effects of independent variables. The emmeans package (Lenth et al., 2020) was used to conduct post-hoc tests with a

Bonferroni correction for multiple analyses. When interactions were significant, we report the highest order interaction and related post-hoc contrast(s) rather than lower order effects that include the same factors. For all tests, an alpha cutoff of 0.05 was used to determine statistical significance. In Table 4.1 we report the factors, families, and link functions that were specified in all of the final selected models.

RESULTS

Exploratory behaviors are higher in younger animals and in experimentally handled animals

Open field test - The number of visits that offspring made to the center of the open field apparatus varied by pup age (!! = 11, df = 1, p < 0.01), where animals visited the center more frequently when they were subadults compared to when they were adults (t(92) = 3.23, p= < 0.01; Figure 4.2A). The frequency of center visits did not differ as a function of father presence (!! = 0.0627, df = 1, p = 0.8), or by experimental handling condition (!! = 2.21, df = 1, p = 0.14). The duration of time spent in the center of the apparatus did not differ across any factors (Father Presence:

(!! = 0.0507, df = 1, p = 0.82); Handling Condition: (!! = 0.357, df = 1, p = 0.55);

Age: (!! = 0.201, df = 1, p = 0.65)). Offspring differed in the total distance they traveled in the OFT as a function of age (!! = 15, df = 1, p < 0.01), and experimental handling (!! = 4.02, df = 1, p = 0.045), but not by father presence (!! = 0.253, df = 1, p = 0.62). Animals traveled more when they were subadults compared to when they

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were adults (t(92) = 3.8, p < 0.01; Figure 4.2B), and Handled animals traveled more than Non-handled animals (t(92) = -2.11, p = 0.037; Figure 4.2C).

Offspring age, father presence, and experimental handling during the rearing period all impact permissive/aggressive/defensive behavioral phenotypes

Social interaction test – The mean distance between the subjects and the stimulus animals varied as a function of father presence (!! = 7.08, df = 1, p < 0.01), where animals reared in the Father Present condition kept a greater mean distance from the stimulus animals compared to animals reared without their fathers (t(96) = -

2.64, p < 0.01; Figure 4.3). The total distance moved during the social interaction test was not impacted by any of our experimental factors (Father Presence:(!! = 2.3, df =

1, p = 0.13); Handling Condition: (!! = 0.0189, df = 1, p = 0.89); Age: (!! = 0.635, df = 1, p = 0.43)). Furthermore, the experimental factors did not impact the duration of body contact between the subjects and the stimulus animals (Father Presence:(!! =

2.98, df = 1, p = 0.084); Handling Condition: (!! = 0.098, df = 1, p = 0.75); Age: (!!

= 1.18, df = 1, p = 0.28)).

Resident-Intruder test - Offensive aggression was not impacted by father presence (!! = 2.38, df = 1, p = 0.12) or by experimental handling condition (!! =

0.185, df = 1, p = 0.67). However, age was a significant factor in the number of offensive aggressive behaviors exhibited (!! = 13.4, df = 1, p < 0.01), where animals displayed a significantly greater number of aggressive behaviors when they were adults compared to when they were subadults (t(89) = -3.65, p < 0.01; Figure 4.4A).

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The latency to initiate the first attack did not vary as a function of father presence (!!

= 0.344, df = 1, p = 0.56), handling condition (!! = 0.172, df = 1, p = 0.68), or age

(!! = 2.65, df = 1, p = 0.1).

There were no main effects of father presence (!! = 1.69, df = 1, p = 0.19), handling condition (!! = 5.93 x 10-5, df = 1, p = 0.99), or age (!! = 0.745, df = 1, p =

0.39) on the number of defensive behaviors residents displayed toward the intruders.

However, father presence did interact with handling condition (!! = 3.88, df = 1, p =

0.049) and with age (!! = 4.38, df = 1, p = 0.036). When animals did not undergo handling by the experimenter, the presence of the father had no impact on the number of defensive behaviors exhibited toward the intruder (t(89) = -0.0403, p = 0.97; Figure

4.4C). Animals that underwent handling, in contrast, performed more defensive behaviors if they were reared without their fathers than if they were reared with their fathers (t(89) = 2.26, p = 0.027). Furthermore, when animals were subadults, the presence of the father had no impact on the frequency of observed defensive behaviors

(t(89) = -0.187, p = 0.85; Figure 4.4D). Once animals were adults, animals reared without their fathers exhibited more defensive behaviors than animals reared with their fathers (t(89) = 2.36, p = 0.021). Age also interacted with handling condition when analyzing defensive behaviors (!! = 3.89, df = 1, p = 0.048), but post-hoc contrasts did not withstand corrections for multiple comparisons (Adult Non-handled - Adult

Handled: t(89) = -1.6, p = 0.11); Subadult Non-handled- Subadult Handled: t(89) =

1.29, p = 0.2; Figure 4.4E). The number of rear ups performed by subjects varied as a function of father presence (!! = 4.78, df = 1, p = 0.029), where animals raised without their fathers reared up more frequently than animals raised with their fathers

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! (t(89) = 2.19, p = 0.031; Figure 4.4B). Neither handling condition (! = 0.0828, df =

1, p = 0.77) nor age (!! = 1.01, df = 1, p = 0.31) significantly affected the frequency of rear ups.

Father presence and experimental handling interact to impact social dominance in subadult, but not adult, animals

We found a three-way interaction between father presence, handling condition, and pup age on social dominance outcomes (!! = 3.74, df = 1, p = 0.053). When subjects were subadults, animals reared by biparentally Non-handled parents were more likely to be dominant than subjects reared without their fathers (t(98) = -2.37, p =

0.02; Figure 4.5). However, subadult animals reared with their fathers were not more dominant than animals reared by just mothers if their families had also been handled

(t(98) = -1, p = 0.32). When animals were adults, there were no interactions between father presence and handling condition (Non-handled, Father Absent-Father Present:

(t(98) = 1.07, p = 0.29); Handled, Father Absent-Father Present: (t(98) = -1.01, p =

0.31)).

Early-life experiences did not impact preferences for partners or total huddling behavior

The identity of the stimulus animal was a reliable predictor of the total time test animals spent huddling (!! = 20, df = 1, p < 0.01). Subjects spent significantly more time huddling with their partner than the opposite sex stranger (t(98) = 4.4, p <

0.01; Figure 4.6A). Stimulus ID also had an effect on the total time the focal animals

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spent in the corresponding stimulus chamber (!! = 22.2, df = 1, p < 0.01), where animals preferred spending time in the chamber of the partner over that of the stranger

(t(99) = 4.72, p < 0.01; Figure 4.6B). Neither huddling time nor the time spent in the chamber were impacted by father presence (Huddle: !! = 0.404, df = 1, p = 0.53;

Time in side: !! = 0.0119, df = 1, p = 0.91) or handling condition (Huddle: !! =

0.683, df = 1, p = 0.41; Time in side: !! = 0.00765, df = 1, p = 0.93), and there were no higher order interactions found for either response variable.

To assess if early social conditions impacted affiliation in general, we combined the time spent huddling with both the partner and the stranger. This total time spent huddling with a stimulus was not affected by father presence (!! = 2.27, df

= 1, p = 0.13), handling condition (!! = 0.285, df = 1, p = 0.59), or by an interaction between father presence by handling condition (!! = 0.141, df = 1, p = 0.71; Figure

4.6C). Finally, we calculated what proportion of the total huddling time subjects spent with their partners, but did not find an effect of father presence (!! = 0.0764, df = 1, p

= 0.78), handling condition (!! = 0.0131, df = 1, p = 0.91), or by an interaction between father presence by handling condition (!! = 0.0949, df = 1, p = 0.76) on partner huddling proportions (Figure 4.6D).

Counts of hypothalamic OT cell populations, but not VP cell populations, vary as a function of early-life handling experiences

We analyzed the number of OT-ir and VP-ir cells in the PVN, SON, and BST to investigate how early-life social experiences shape neuroendocrine cell populations that are implicated in social behaviors. Cell counts of specific neural populations were

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differentially impacted by our experimental conditions (Table 4.1). The number of

OT-ir cells in the PVN varied as a function of father presence (!! = 5.39, df = 1, p =

0.02), handling condition (!! = 11.6, df = 1, p < 0.01), and an interaction between father presence and handling condition (!! = 7.63, df = 1, p < 0.01). Post-hoc contrasts for the highest order interaction revealed that pups that were reared in

Handled families with their fathers had significantly more PVN OT-ir neurons relative to the other three groups (Handled/Father Present - Handled/Father Absent: t(48) =

3.57, p < 0.01; Handled/Father Present – Non-handled/Father Absent: t(48) = 4.05, p <

0.01; Handled/Father Present - Non-handled /Father Present: t(48) = 4.37, p < 0.01;

Figure 4.7A). There were no other significant pairwise comparisons in the post-hoc contrasts (Handled /Father Absent - Non-handled /Father Absent: t(48) = 0.385, p = 1;

Handled /Father Absent - Non-handled /Father Present: t(48) = 0.457, p = 1; Non- handled /Father Present - Non-handled /Father Absent: t(48) = -0.1, p = 1).

Handling condition had a significant effect on the number of OT-ir cells in the

SON (!! = 5.12, df = 1, p = 0.024), where subjects from Handled families had more

OT-ir neurons than those from Non-handled families (t(47) = -2.18, p = 0.034; Figure

4.7B). The number of SON OT-ir neurons was not impacted by father presence (!! =

0.21, df = 1, p = 0.65), or by an interaction between father presence and handling condition (!! = 0.324, df = 1, p = 0.57). No other cells populations were significantly affected by father presence, handling condition, or showed an interaction between father presence and handling condition (Table 4.2).

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DISCUSSION

The absence of a father in the natal nest alters the trajectory of developing prairie voles (Ahern and Young, 2009; Wang and Novak, 1992, 1994). The current study aimed to assess if offspring phenotypes that follow paternal absence could be rescued by behaviorally upregulating parental care. To accomplish this task, we reared animals with and without their fathers and under two different experimental handling conditions to measure behavioral outcomes at subadult and adult stages of life. We verified that our handling manipulation increased pup directed care behavior

(in the form of more pup retrievals, Chapter 5). Results from our behavioral tests revealed effects of age, father presence, and experimental handling conditions, as well as interactions among these factors on offspring behaviors. Specifically, we found that aggressive behaviors increased, whereas exploratory behaviors decreased, with offspring age. Paternal presence shaped how behaviorally tolerant and submissive offspring were to conspecific interactions in an age- and handling-specific manner, highlighting the importance of considering multiple dimensions of early experiences on animal behavior across the lifespan.

We also examined if variation in early experiences concurrently impacts nonapeptide cell populations that are extensively tied to social behavior. We analyzed the number of OT-ir and VP-ir cells in the PVN, SON, and the BST, then compared these totals across treatment conditions. We found that the number of BST nonapeptide neurons did not vary as a function of early experiences. We also revealed that the number of OT-ir cells in the PVN and SON were affected by handling condition (and father presence, for just the PVN), but the total number of VP-ir

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neurons did not differ across groups for any of the factors we manipulated. Below we discuss what is known about the functions of these cell populations in the regulation of social behavior and provide insight into the immense functional plasticity of oxytocin networks in the brains of developing animals.

The presence of fathers in the natal nest drives species-specific social behavioral phenotypes later in life

The absence of a father during the rearing period had a profound effect on an integrated suite of behaviors across our tests. Rearing up on hind legs has been considered a behavioral response to environmental novelty (Lever et al., 2006). The observation that single-mother raised voles reared up more than other groups in the resident-intruder test may indicate that these animals were more prone to gather information in the presence of a social stimulus before engaging in other behaviors.

Furthermore, animals raised by single mothers were (on average) closer to the stimulus animal in the social interaction test, and displayed more submissive behaviors in adulthood during the social interaction test. These outcomes did not interact with handling condition, suggesting that behaviorally upregulating maternal care did not offset the effects of single motherhood for these behavioral outcomes. However,

Father Absent subadult offspring were less likely than Father Present subadults to be the winner in the dominance tube test, unless their mothers had been handled to induce more caregiving. In this case, upregulating maternal care via handling appeared to rescue the submissive phenotype of single mother raised offspring so that they more closely resembled biparentally reared offspring. These results suggest that the capacity

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for increased care to offset the developmental consequences of no paternal care is trait-specific in prairie voles.

Although we did not find an influence of father presence on total distance traveled in the OFT, Ahern and Young (2009) found that single-mother reared offspring travelled more than biparentally-reared offspring in the first 5 m of an OFT test. It is possible that single-mother reared males are more active or exploratory than biparentally reared males. However we also note that Ahern and Young (2009) reported that this effect disappeared when the first 5 minutes of the test were excluded from the analysis, suggesting that exploratory behavior in the OFT is dynamic across the testing period. Because our analysis collapsed performance across the session, it is possible that we missed the nuances of temporal variation in exploratory patterns as a function of early-life experiences. Nevertheless, collectively these results suggest that male’s born to single mothers are generally more socially permissive, possibly more exploratory, and less aggressive in non-reproductive contexts.

Mounting work on behavioral plasticity has characterized a ‘bold’ and ‘shy’ continuum of animal personality, and our data are consistent with other studies that have suggested some elements of personality are linked to developmental experiences

(Sih et al., 2004). Indeed, lower levels of socialization during early prairie vole development may generate a more socially permissive behavioral phenotype in adulthood. We and others have demonstrated that prairie voles that are raised by single mothers receive less total parental care compared to biparentally reared offspring

(Chapter 5, Perkybile et al. 2015, Kelly et al. in press) establishing a disparity in caregiving in the natal nest of biparental and uniparental families. Moreover, several

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studies support the notion that caregiving leads to differences in social permissiveness.

For instance, single-mother reared offspring spent more time in side-by-side contact with a novel conspecific during a social interaction task compared to biparentally reared offspring (Tabbaa et al., 2017). Similarly, single-mother reared males spent more time in contact with a novel infant pup during an alloparental care test than did males reared by communal pairs of mothers and alloparents (Rogers and Bales, 2019).

Finally, males that were experimentally “socially limited” (i.e., singly reared, and then weaned to isolated housing conditions) spent more time in contact with a juvenile pup during an affiliation test compared to “socially enriched” males (i.e., biparentally reared, weaned with a sibling) (Prounis and Ophir, 2019). Thus, paternally deprived male prairie voles (or males that received relatively lower levels of socialization during the postnatal period) consistently demonstrated higher levels of prosocial and/or permissive behaviors compared to males that were reared by multiple caregivers.

We note that although this putative relationship between social enrichment in the natal nest and adult social permissiveness appears to be robust in prairie voles, the strength and direction of this relationship might be species-specific. For example, male socially monogamous Mandarin vole siblings (Lasiopodomys mandarinus) that were isolated from each other for six hours were less aggressive to, and displayed more contact with, their siblings upon reunion if they were raised without fathers compared biparentally reared sibling pairs (Wang et al., 2012). Conversely, the biparental and socially monogamous California mouse exhibits the opposite effect; when paternally deprived male and female mice were observed in a social interaction test, they had

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longer latencies to first contact a conspecific, and also spent less time investigating conspecifics, compared to the performance of biparentally reared mice (Bambico et al., 2015). Thus, although the impact of social experiences can have long lasting impacts on developing offspring, the consequences of early socialization on subsequent social behaviors appear to be species-specific, highlighting the need to take a comparative approach if we are to ever fully understand the evolution of developmental programming.

Pair bonding behavior was not impacted by father presence or by experimental handling

The capacity to form a pair bond is a key feature in the socially monogamous life history strategy, and marks a complex transition in the evolution of mammalian mating systems (Lukas and Clutton-Brock, 2013). Prairie voles are popularly known for their ability to form pair bonds (Young et al., 2011). However, there is incredible individual variation in the extent to which males demonstrate a partner preference

(Vogel et al., 2018), and this diversity is presumptively driven by environmental variation (Madrid et al., 2020). We asked if variation in parental care could account for the environmental variation that leads to differences in pair bonding behavior.

Bales et al. (2007) demonstrated that inducing increased parental care via experimental handling did not appear to impact partner preference behaviors in male prairie voles. However, Ahern and Young (2009) and Rogers and Bales (2019) independently demonstrated that male prairie voles that were reared biparentally showed a preference for a partner vs a stranger after 24 hours of cohabitation, but

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males that were reared by single mothers did not develop such a preference in the same time frame or at all. Thus, we were interested to know to what extent these two factors would interact, and more specifically, if boosting parental care (via experimental manipulation) could compensate for rearing effects of absent fathers on pair bond formation. When rearing males in the absence of fathers, males formed bonds at the same rate and strength as those raised with both parents, contrary to the bonding deficiencies that Ahern and Young (2009) or Rogers and Bales (2019) reported in uniparentally reared males. It is important to make clear that Ahern and

Young (2009) did not show that single-mother reared males were unable to form a pair bond; rather, they showed that these males took longer to display a partner preference

(i.e., 48 h of cohabitation was insufficient to form a bond, but one week was enough).

Therefore, in light of the fact that other adult traits including exploration and defensive behaviors were shaped by increased caregiving attributable to experimental handling of parents in our study, it is possible that we found no effects of bonding because the expression of pair bonding may be highly robust to complex environmental perturbations. This idea is not terribly surprising considering that bonding appears to be a deeply ingrained behavior, possibly with major implications for reproductive success among male prairie voles (Blocker and Ophir, 2016). However, we cannot discount the possibility that between-study differences in methodology (e.g., differences in testing procedures, or variation in the genetic makeup of independent lab colonies) could account for differences in outcomes attributable to the impact of father presence on male offspring pair bonding.

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Aggressive and exploratory behaviors shift over offspring age

Immature offspring often affiliate extensively with caregiving parents, then progress to interactions with siblings and peers during the juvenile stage, and finally transition to the expression of courtship and sexual behaviors with mates (Nelson et al., 2016). With this progression through different social contexts, we commonly observe age-related changes in animal aggression. Age-related increases in aggressive behaviors have been reported in a variety of species including zebrafish (Ricci et al.,

2013), yellowtail (Sakakura and Tsukamoto, 1999), flesh flies (Moore et al., 2014), rhesus macaques (Singh, 1989), and rats (Takahashi and Lore, 1982). The overarching pattern depicts an increase in aggression from immature offspring to reproductively mature adults. Prior work in prairie voles recapitulates this pattern, showing that between the subadult and adult stages of life, same-sex conspecific-directed prosocial behavior decreases while aggressive behavior increases with age (Kelly et al., 2018b).

The current results from our resident-intruder test replicate this developmental shift towards a more aggressive adult phenotype in prairie voles. The subadult age we tested corresponds to the period leading up to reproductive activation (around PND30) in prairie voles (Solomon, 1991). Higher levels of aggression as adults may serve as an adaptive behavioral trait by which to facilitate the defense of resources, mates, and territories, particularly at a sexually mature stage of life (Buss and Shackelford, 1997).

Interestingly, our OFT results depict a decrease in exploratory behavior from the subadult age to the adult age. The age of testing in the subadult animals map onto the weanling period in voles, during which juveniles are capable of independent living and soon begin dispersing from the nest (Arias del Razo and Bales, 2016; McGuire et

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al., 1993). One hypothesis is that a high level of exploratory behavior during this stage could enable weanlings to venture out to seek their own territories. However, an alternative hypothesis for these results stems from the experimental design: animals tested as adults had already experienced the OFT once, and therefore, may have demonstrated a change in behavior that is attributable to test experience rather than age. The effects of age can be challenging to dissociate from the confounds of experience in developmental research. Future work may benefit from using different measures to assess anxiety-like behavior across the lifespan to account for effects related to prior testing experience.

Early postnatal experiences shape oxytocin, but not vasopressin, cell groups in adult brains

Developmental shifts in behavior must be accompanied by changes in the underlying neural mechanisms. Previous work has demonstrated that oxytocin and vasopressin systems in prairie vole brains are susceptible to shaping by distinct early- life experiences (Ahern and Young, 2009; Bales et al., 2011; Bales and Perkeybile,

2012; Carter et al., 2008; Kelly et al., 2018a; Perkeybile et al., 2019; Perkeybile and

Bales, 2015a). Our lab has expanded upon this foundation by including the analysis of multiple dimensions of the early postnatal experience on nonapeptide system profiles

(Hiura and Ophir, 2018; Prounis et al., 2018, 2015; Prounis and Ophir, 2019).

Earlier work has characterized that prairie vole families that were manipulated by hand on PND 1 (and thus experienced more parental care) had male offspring with significantly more OT-ir positive cells in the SON compared to control groups that

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were handled in cups (Bales et al., 2011). We replicated this pattern in our study and found that the number of OT-ir positive cells in the SON were greater in animals whose parents were induced to provide care more (via three experimental handling bouts) compared to the families that were not experimentally handled. These corresponding findings suggest that early-life experience in the form of experimentally elevated caregiving (via handling) has a predictable effect on oxytocin cells in the

SON. We acknowledge that it is not directly clear if the differences we and others have reported are a function of the experimental handing itself, or of the consequences of the modifications to parental behavior that handling appears to cause (Bales et al.,

2011).

Animals that were designated to receive the most parental care (i.e., from biparental + handled families) had more OT-ir cells in the PVN than all other experimental groups. However, this same experimental group did not differ from the other three groups in any of the behavioral tests, making it difficult to ascertain the functional implications of changes in this cell group. Given that the PVN is the primary source of OT in the brain, and is also highly involved in modulating social behavior (Kelly and Goodson, 2014a), there is strong evidence that suggests that this neural phenotype has the potential to impact social behavior in a number of contexts beyond what was tested in the present study. PVN OT is involved in the modulation of the Hypothalamic-Pituitary-Adrenal (HPA) axis (Grippo et al., 2007; Neumann et al.,

2000), social grouping (Kelly and Goodson, 2014b), parental care (Da Costa et al.,

1996; Kelly et al., 2017), consolation behavior (Li et al., 2019), social reward (Hung et al., 2017), and of course bonding and partner loss (Hirota et al., 2020; Sun et al.,

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2014). In any case, our results provide direct evidence for the synergistic actions of two qualitatively distinct early experiences on the phenotypic profile of a cell group critical in the control of a variety of social behaviors.

Unfortunately, we did not find any effects of early-life experiences on nonapeptide cell counts in the BST, or group differences in VP-ir cell neurons overall.

These data are consistent with previous work that found prairie vole VP-ir did not differ in the PVN or in the SON as a function of early-life handling (Bales et al.,

2011). Our results are also similar to those published by Perkeybile and Bales (2015b) that reported that VP-ir did not differ in the PVN or the SON between males of high care and low care biparental families. Moreover, our results are consistent with the hypothesis that OT-ir neurons are highly plastic to the influence of variation in early- life experiences, and more plastic than are VP-ir hypothalamic cell groups.

Interestingly, although there appears to be a lot of developmental plasticity in the receptors for OT and VP, it is the VP receptors in males that appear to be most sensitive to environmental perturbations during postnatal development (Hiura and

Ophir, 2018; Prounis et al., 2018). Together, these results imply that the plasticity of

OT and VP systems may differ by which level of their signaling systems are influenced by developmental experiences (peptide-producing neurons for OT vs. receptor expression for VP).

Future work would greatly benefit from the study of simultaneous changes in both receptor densities and peptide containing cells to better understand the relationship between these systems over social development. Furthermore, few empirical studies have directly manipulated these cell groups or receptor profiles in

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early development, which will be necessary to functionally assess their roles in the ontogeny of social behavior (Kelly and Goodson, 2014a).

CONCLUSIONS

Distinct forms of early-life experiences are not homogenous in their effects on offspring development. We found evidence that multiple factors of the early environment synergistically shape both offspring behavioral phenotypes and related nonapeptide cell group profiles. Specifically, we found that father presence in the natal nest has lasting consequences on a suite of behaviors that determine a prairie vole’s social permissiveness, but that this effect is contingent upon enhanced parental care

(due to experimental handling of parents) and offspring age. Access to different forms of caregiving opportunities though natural (i.e., father presence) and synthetic (i.e., enhanced caregiving by experimenter handling) means also interacted to shape the number of oxytocin, but not vasopressin, immunoreactive cells in the PVN and SON, suggesting that early social experiences with parents are encoded in specific neuroendocrine profiles of adult animals. The experimental variation we implemented only captures a fraction of the complexity of offspring development in the natural world. However, incorporating the influence of variation in multiple dimensions of postnatal experiences will bring us closer to understanding and appreciating the mechanisms that promote the development of behavioral plasticity.

Acknowledgements

The authors would like to thank Mandy Chan and Sara O’Malley for their

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contributions to the data collection. The authors acknowledge the support from the

National Institutes of Health (Eunice Kennedy Shriver National Institute of Child

Health and Human Development HD079573 to A.G.O.) and the National Science

Foundation Graduate Research Fellowship Program (under DGE-1650441 to L.C.H.).

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FIGURES

Figure 4.1 Timeline of experimental treatments, behavioral testing schedules, and brain extractions for all subjects by postnatal day (p). Grey boxes group experimental phases together.

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Figure 4.2 Offspring performance in the OFT as a function of experimental conditions. A) The total number of visits to the center compared between offspring ages. B-C) The total distance traveled during the OFT test by offspring age (B) and Handling Condition (C). Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

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Figure 4.3 The mean distance in centimeters (cm) between the focal subject and the novel stimulus animal over the total duration of the Social Interaction test. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

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Figure 4.4 Offspring performance in the Resident-Intruder test. A) The total number of aggressive behaviors as a function of Age. B) The total frequency of rear ups by Father Presence. The variation in the total number of defensive behaviors between offspring in the Father Present condition compared to the Father Absent condition, split by C) families in the Non-handled and the Handled condition, and by D) offspring Age. E) The total number of defensive behaviors between offspring in the Non-handled and the Handled condition, split by Age. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

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Figure 4.5 The three-way interaction of the modeled probabilities of offspring being determined as a “Winner” in the social dominance test (i.e., winning the best out of three trials) between offspring reared by single mothers or biparentally, from the Control Handling condition and the Experimental Handling condition, split by Age. *p ≤ 0.05.

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Figure 4.6 Offspring performance in the partner preference test. A) The duration in minutes (min) that offspring spent huddling with each stimulus animal. B) The duration (min) that offspring spent within each stimulus chamber. C) The total duration (min) that subjects spent huddling with their partner and the stranger combined. D) The proportion of the composite huddling time that subjects spent huddling with their partner (duration with partner / duration with partner + duration with stranger). Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

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Figure 4.7 Plots of OT-ir cell counts that varied by experimental conditions. A) The total number of OT-ir neurons within the PVN was subject to an interaction between Father Presence and Handling Condition. B) SON OT-ir counts varied between subjects from the Non-handled condition and the Handled condition. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

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TABLES

Table 4.1 Final parameters of selected GLMM for behavioral and neural analyses.

Test Dependent Variable Fixed Effects Random Effects Family Link Function Center Frequency Father Presence X Handling X Age Cohort | Subject ID Negative Binomial Log OFT Center Duration Father Presence X Handling X Age Cohort | Subject ID Zero-inflated Gamma Log Total Distance Father Presence X Handling X Age Cohort | Subject ID Gamma Log Total Distance Father Presence X Handling X Age Cohort | Subject ID Gamma Log Social Duration Body Contact Father Presence X Handling X Age Cohort | Subject ID Zero-inflated Gamma Log Interaction Mean Distance Apart Father Presence X Handling X Age Cohort | Subject ID Gamma Log Offensive Aggression Father Presence X Handling X Age Cohort | Subject ID Negative Binomial Log

Resident- Defensive Behavior Father Presence X Handling X Age Cohort | Subject ID Negative Binomial Log Intruder Rear Up Father Presence X Handling X Age Cohort | Subject ID Negative Binomial Log Latency to First Attack Father Presence X Handling X Age Cohort | Subject ID Gamma Log Social Winner Father Presence X Handling X Age Cohort | Subject ID Binomial Logit Dominance Duration Huddling Stimulus Father Presence X Handling X Stimulus N/A Zero-inflated Gamma Log

Partner Duration in Stimulus Chamber Father Presence X Handling X Stimulus N/A Gamma Log Preference Total Huddling Duration Father Presence X Handling Cohort | Subject ID Gaussian Log Proportion Huddling Partner Father Presence X Handling Cohort | Subject ID Beta Logit PVN OT-ir Father Presence X Handling Cohort | Subject ID Poisson Log PVN VP-ir Father Presence X Handling Cohort | Subject ID Negative Binomial Log BST OT-ir Father Presence X Handling Cohort | Subject ID Poisson Log Cell Counts BST VP-ir Father Presence X Handling Cohort | Subject ID Poisson Log SON OT-ir Father Presence X Handling Cohort | Subject ID Poisson Log SON VP-ir Father Presence X Handling Cohort | Subject ID Poisson Log

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Table 4.2 Effects of experimental factors on hypothalamic cell groups.

Cell Population Factor χ2 df p-value Father Presence 5.39 1 0.020 PVN OT Handling Condition 11.58 1 0.001 Father Presence x Handling Condition 7.63 1 0.006 Father Presence 0.35 1 0.553 PVN VP Handling Condition 2.13 1 0.145 Father Presence x Handling Condition 0.44 1 0.506 Father Presence 0.07 1 0.792 BST OT Handling Condition 1.19 1 0.275 Father Presence x Handling Condition 0.73 1 0.392 Father Presence 0.07 1 0.792 BST VP Handling Condition 1.19 1 0.275 Father Presence x Handling Condition 0.73 1 0.392 Father Presence 0.21 1 0.647 SON OT Handling Condition 5.12 1 0.024 Father Presence x Handling Condition 0.32 1 0.569 Father Presence 0.09 1 0.765 SON VP Handling Condition 1.95 1 0.162 Father Presence x Handling Condition 1.80 1 0.180

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CHAPTER 5

PRAIRIE VOLE (MICROTUS OCHROGASTER) PARENTAL BEHAVIORS AND

VASOPRESSIN CELL GROUPS EXHIBIT PLASTICITY IN RESPONSE TO

EXPERIENCES AS A PARENT

In prep for submission: Lisa C. Hiura, Vanessa A. Lazaro, Alexander G. Ophir

ABSTRACT

The impact of variation in parental caregiving has lasting implications for the development of offspring. Experiences as a parent inherently vary as well, but the consequences of diversity in parenting experiences for adults are poorly understood.

Prairie voles (Microtus ochrogaster) are a wild rodent species that can rear their young biparentally, or with a mother alone. We administered a handling stressor on prairie vole families of single mothers and biparental pairs to investigate how variation in the experience of raising offspring (induced by the presence or absence of a parental partner and/or and acute stressor) impacts parents’ subsequent behaviors and neurobiology. Mothers and fathers exhibited robust behavioral plasticity in response to the age of their pups, but in sex-dependent ways. The behavior of mothers did not vary as a function of their partners’ presence but did covary with the number of hypothalamic vasopressin neurons they had in an experience-dependent manner. The relationship between vasopressin neuron numbers and fathers’ behaviors was also contingent upon the handling manipulation, indicating that brain-behavior associations are plastic in adulthood. These findings demonstrate that the behavioral and

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neuroendocrine profiles of adults are sensitive to distinct and interacting experiences as a parent, and extends our knowledge of the neural mechanisms that may facilitate parental behavioral plasticity.

INTRODUCTION

Parents adapt their behaviors in response to environmental contexts, and one of the most influential contexts that a parent will experience is the dynamic social environment comprised of familial interactions (Royle et al., 2014). All mammalian mothers interact with their offspring to varying degrees over the course of offspring development, but for the 5-10% of mammalian species that are biparental (Kleiman and Malcolm, 1981), partner interactions make up a substantial portion of the parent’s social environment. Several species (with a primary focus on birds) have been found to flexibly respond to the actions or presence of a parenting partner (Harrison et al.,

2009). Adapting parental behavior to variation in the social environment can increase reproductive fitness, but the consequences of social environmental variation on mammalian paternal and maternal brains and behaviors remains underexplored.

Prairie voles (Microtus ochrogaster) are small rodents that form socially monogamous pair bonds and exhibit biparental care (Madrid et al. 2020). With the exception of nursing, fathers exhibit all of the same care behaviors as mothers

(Thomas and Birney, 1979). Paternal care is not obligate for pup survival in prairie voles, and several studies have removed fathers from the natal nest to assess developmental consequences of variation in the early social environment on offspring.

These studies have determined that the presence of a father during the rearing period

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has significant impacts on pups’ phenotypes, including rates of physical maturation

(Wang and Novak, 1992), alloparental care and pair bonding as adults (Ahern and

Young, 2009), parental behavior toward their own offspring (Ahern et al., 2011), and neuropeptide receptor binding densities and mRNA expression (Ahern and Young,

2009; Bales and Saltzman, 2016; Prounis et al., 2015). Interestingly, far fewer studies have investigated the role of father-presence on the phenotypes of mothers. One such recent study found that compared to mothers who remained paired with their mate, single mothers show significant increases in passive stress-coping and anxiety-like behaviors alongside greater mRNA expression of corticotropin-releasing hormone

(Bosch et al., 2018). When maternal behaviors were compared in the days following parturition, no group differences were found. These findings demonstrate that variation in parental experience induced by the removal of the parenting partner has substantial context-specific behavioral and neuroendocrinological consequences for prairie vole mothers during the perinatal period.

Here, we extend this line of inquiry to ask how environmental variation during parenting impacts the behaviors of prairie vole mothers and fathers across the pre- wean stage of offspring development. We used a social context manipulation (father removal) and a non-social stress induction paradigm (experimenter handling) to investigate the potential interactions among multiple environmental experiences.

Furthermore, we asked how these variable parental experiences impact the neural systems known to be involved in vertebrate parental behaviors. We focused this investigation on vasopressin (VP) and corticotropin releasing hormone (CRH), two hormones that have been implicated in parental care and anxiety-like behaviors (Kohl

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et al., 2017). VP plays a crucial role in the regulation of parental behaviors in both male and female monogamous rodents (Horrell et al., 2018). Hypothalamic levels of

VP mRNA in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus are greater in prairie vole parents compared to sexually naïve controls (Wang et al., 2000). Moreover, prairie vole mothers and fathers both show greater levels of VP-cFos colocalization in the PVN when they are with their pups compared to when they are separated from their litter, demonstrating that this cell population is functionally responsive to pup stimuli in both sexes (Kelly et al., 2017).

In contrast, pup exposure reduces cFos induction in PVN CRH neurons of male prairie voles (Kenkel et al., 2012), and intracerebroventricular (ICV) injection of CRH reduces maternal aggression in mice (Gammie et al., 2004), suggesting that lower levels of CRH activity may be conducive to adaptive rodent parental behaviors

(Slattery and Neumann, 2008). A variety of stressors induce the secretion of CRH and

VP from the PVN, and together these neuropeptides function as the key regulators of hypothalamic-pituitiary-adrenal (HPA)-axis responsivity (Herman and Tasker, 2016).

To assess how variation in parental experiences impact these hormonal systems, we immunolabeled the PVN (for VP and CRH neurons) and the SON (for VP neurons), as these are the primary cells groups that provide the bulk of VP and CRH peptide signals in the central and peripheral nervous systems (Brownstein et al., 1980). We ask if, and in what ways, variation in parental experiences and behaviors affects these cell populations in the brains of monogamous parents.

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MATERIALS AND METHODS

Experimental Animals:

All animals used in these experiments were sexually mature, virgin F1 progeny of wild-caught breeders and were reared in the laboratory at Cornell University. All animals had ad libitum access to water and food (Rodent Chow 5001, LabDiet, St.

Louis, MO, USA) and were housed in polycarbonate rodent cages (29 × 18 × 13 cm) lined with Sani-chip bedding under a 14:10 light-dark cycle with ambient temperature maintained at 20±2°C. No animals in this experiment were raised in isolation. All procedures used in this study were approved by the Institutional Animal Care and Use

Committee of Cornell University (ACUP 2013-0102).

Family conditions:

We created two experimentally designed factors to manipulate the animals:

Fathers Present/Absent, and Experimentally Handled or Non-handled. Twenty days after breeding pairs were formed, males in families assigned to the Father Absent condition were permanently removed from the home cage. Males from families assigned to the Father Present condition were briefly removed, then returned to the home cage to control for nest disturbances. Nests were monitored daily for the birth of pups. At birth, litters were culled to three pups if necessary to control for the effect of litter size. Male offspring were preferentially spared for use in a related but separate study (see Chapter 4). Weekly cage changes were conducted on postnatal days (PND)

2, 9, and 16. In the Experimentally Handled condition, parents were scruffed and transferred by a gloved hand to a clean cage. In the Non-handled condition, families

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were gently scooped into a plastic beaker and transferred to a clean cage. When pups were not attached to their mothers, they were transferred in the same manner as their parent(s). Altogether we created six groups: Mothers / Father Present / Non-handled

(N = 14), Mothers / Father Absent / Non-handled (N = 13), Mothers / Father Present

/Handled (N = 15), Mothers / Father Absent / Handled (N = 12), Fathers / Non- handled (N = 14), and Fathers / Handled (N = 15). All pups were weaned at PND 21.

Home Cage Analyses:

One-hour home cage videos were recorded on PND 2, PND 9, and PND 16 immediately following each cage change. We selected these postnatal days for observation because they map onto behaviorally relevant developmental timepoints

(Hiura et al., 2018; Kelly et al., 2018). Specifically, PND 2 pups are relatively immobile with closed eyes, and are entirely dependent on parental care for food and warmth. At PND 9, pups open their eyes and become more physically and socially exploratory. By PND 16, pups are capable of consuming solid foods and are very mobile. GoPro HERO3 video cameras (GoPro Inc, San Mateo, California, USA) filmed overhead views of each family. Technical issues with the cameras resulted in several shortened videos, and thus the first 55 minutes of each video was used for subsequent behavioral analyses. Raters blind to the experimental conditions scored the behaviors of parents (Table 5.1) using Observer XT v14 (Noldus Information

Technology, Leesburg, VA, USA).

Open-field Test:

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The day after pups were weaned, parents were run through a 10-minute open- field test (OFT). All tests were recorded overhead on video cameras (Sony HDR-

CX330 camcorder, Sony, New York City, NY, USA). At the beginning of each test, the subject parent was gently placed by cup into the center of the transparent Plexiglas arena (57 cm x 57 cm). The arena floor consisted of a 4x4 grid of squares (14.25 cm x

14.25 cm), and the centermost four squares were designated as the arena “center” region. Video recordings were scored using EthoVision XT v13 (Noldus Information

Technology, Leesburg, VA, USA) for time spent in the center, number of visits to the center, and total distance moved.

Histology and Immunocytochemistry:

Immediately following the OFT, parents were rapidly anesthetized by isoflurane and perfused with 0.1M phosphate-buffered saline (PBS, pH = 7.4) followed by 4% paraformaldehyde in PBS. Brains were post-fixed in 4% paraformaldehyde (24 h) then 30% sucrose (48 h) and stored at -80°C. Brains were coronally cryosectioned into three series of 40 µm slices and stored at -80°C in cryoprotectant. A single series of free-floating sections from each subject was fluorescently double labeled for VP and CRH immunoreactivity (-ir). Sections were rinsed twice in PBS (30m) and blocked (1h, PBS + 10% normal donkey serum +

0.03% Triton-X-100) before being incubated in primary antibodies (48 h, Guinea Pig anti-VP 1:1000; Rabbit anti-CRH 1:2000, Peninsula Laboratories, San Carlos, CA).

Sections were rinsed in PBS (2 x 30m), incubated in biotinylated donkey anti-Guinea pig (1h, 1:8000, Jackson Immunoresearch, West Grove, PA), rinsed in PBS (2 x 15m),

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incubated at room temp in secondary antibodies (2h, streptavidin conjugated to Alexa

Fluor 488 3:1000; donkey anti-rabbit Alexa Fluor 594, 5:1000, ThermoFisher

Scientific, Waltham, MA), and washed in PBS (overnight at 4°C). Sections were mounted onto microscope slides and cover-slipped with Prolong Gold antifade +

DAPI nuclear stain (ThermoFisher Scientific, Waltham, MA).

Microscopy and quantification:

Photomicrographs of the PVN and SON were taken at 10x on a Zeiss

AxioImager II scope with an AxioCam MRm attachment, z-drive, and Apotome optical dissector (Carl Zeiss Inc., Gottingen, Germany). Two sections (coronal separation of 240 µm) were monochromatically imaged and manually counted for VP- ir cells and CRH-ir cells using GNU Image Manipulation Program (GIMP, 2.8.22) and

ImageJ (National Institutes of Health, Bethesda, MD). Cell counts of each region were combined across rostral and caudal sections of each subject and total counts per region were statistically analyzed.

Statistical Analysis:

All analyses were conducted using R software v.3.6.2 (R Core Team, 2013).

Behavioral home cage and OFT duration data were assessed using linear mixed models (LMM) via the ‘lme4’ package (Bates et al., 2015), and p-values were derived from a likelihood ratio test within the ‘lmerTest’ package (Kuznetsova et al., 2017).

Behavioral home cage and OFT count data were analyzed using generalized linear mixed models (GLMM) assuming a negative binomial distribution with the

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glmmTMB package (Brooks et al., 2017), followed by type II Wald chi squared test for significant factors. Models comparing behaviors of mothers included the Father-

Present/Absent condition, Experimental Handling/Non-handling condition, and postnatal day as fixed factors, and animal ID and cohort as random factors. Models comparing behaviors between mothers and fathers included the Experimental

Handling/Non-handling condition and postnatal day as fixed factors, and animal ID and cohort as random factors. Models comparing behaviors between fathers included the Experimental Handling/Non-handling condition and postnatal day as fixed factors, and animal ID and cohort as random factors. Regression diagnostic plots were used to assess model fits, and GLMM residuals were checked using the ‘DHARMa’ package

(Hartig, 2019). Bonferroni corrected post-hoc contrasts and corresponding p-values of categorical factors for both LMMs and GLMMs were extracted from the ‘emmeans’ package (Lenth et al., 2020).

Using prcomp of the ‘stats’ package, principal component analyses (PCA) based on correlation matrices were used to reduce the number of measured behaviors to be used as predictors of neural data. Separate PCAs on centered and scaled data were run for male and female subjects due to sexually dimorphic behaviors and conditions. The female PCA excluded partner grooming and partner retrieval given that these measures were not applicable to single mothers. The Kaiser criterion

(eigenvalues > 1) determined which PCs to retain. Behavioral loadings greater than

0.4 were considered to contribute significantly to each retained PC. Varimax rotated

PCs were included as covariates in GLMMs to predict cell counts with handling condition (for maternal and paternal data) and father condition (for maternal data) as

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fixed effects and animal ID and cohort as random factors. For all statistical models, random effects were excluded in instances where models failed to converge with their inclusion. A 0.05 α-level cutoff was used to determine statistical significance. In cases where model interactions were significant, we report the highest order interaction and related post-hoc contrast(s) in lieu of lower order effects when they involve the same factors.

RESULTS

Parental and non-parental care in home cages

Total parental care pups receive from parents differs by pup age and the presence of a father

We first compared pup grooming and pup retrievals because these behaviors are exhibited by both mothers and fathers. We found a main effect for the presence of fathers (F(1,52.4) = 24.8, p < 0.01; Figure 5.1A) and for postnatal day (F(2,94.4) = 27.2, p < 0.01); Figure 5.1B), and a significant interaction between these two factors

(F(2,95.5) = 5.54, p < 0.01, Figure 5.1C). Our post-hoc contrasts indicated that Father

Present animals received significantly more grooming than Father Absent animals at

PND 2 (t(125) = -5.69, p < 0.01) and at PND 9 (t(124) = -3.29, p < 0.01), but not at PND

16 (t(125) = -1.76, p = 0.08; Figure 5.1C). Experimental handling of the animals did not have a significant effect on total pup grooming (F(1,43.1) = 1.6, p = 0.21).

The total number of pup retrievals did not differ when fathers were present or absent (!! = 0.985, df = 1, p = 0.32; Figure 5.2A). Our GLMM revealed a significant main effect for experimentally handling parents (!! = 3.92, df = 1, p = 0.048)

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indicating that experimental handling significantly impacted pup retrieval overall.

However, the non-significant post-hoc comparison showed that Handled parents only tended to retrieve their pups more than Non-handled control parents (t(141) = -1.88, p =

0.063; Figure 5.2B). Similarly, we found that pup retrieval was significantly impacted by the age of pups (!! = 51.8, df = 2, p < 0.01). Specifically, parents retrieved pups significantly more often at PND 16 (t(141) = -6.6, p < 0.01) or (t(141) = -5.35, p < 0.01) than at PND 2, but retrievals did not differ between PND 9 and PND 16 pups (t(141) =

-1.28, p = 0.61; Figure 5.2C).

Behaviors of mothers varied by age of pups and experimental handling, but not by the presence of the father.

Parental Behaviors.

Overall, maternal care was generally consistent, independent of our experimental manipulations. The presence of fathers and influence of experimental handling did not alter the duration of pup grooming, neutral nursing, or active nursing

(Table 5.2). However, we did find a significant main effect of pup age for these forms of maternal care, in which maternal care decreased as pups became older (Table 5.3).

The presence of fathers did not affect the number of pup retrievals mothers performed (!! = 3.03, df = 1, p = 0.082; Figure 5.3A). However, experimental handling did significantly impact pup retrieval by mothers (!! = 5.14, df = 1, p =

0.023), where handled mothers retrieved pups more than control mothers (t(141) = -

2.12, p = 0.036; Figure 5.3B).

Consistent with results reported above, maternal pup retrieval significantly

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differed across the age of pups (!! = 31.1, df = 2, p < 0.01), where mothers retrieved their pups more when they were 16 days old (t(141) = -5.05, p < 0.01) or 9 days old

(t(141) = -4.36, p < 0.01) than when pups were 2 days old (Figure 5.3C). Mothers did not differ in how often they retrieved pups at PND 9 or PND 16 (t(141) = -0.704, p=1).

Nonparental Behaviors.

Mothers’ non-parental behaviors (including exploration, trail building, and autogrooming) were unaffected by the presence or absence of fathers or experimental handling (Table 5.4).

The age of pups, did, however, significantly affect maternal trail building, autogrooming, and exploration. Post-hoc contrasts showed that compared to mothers of PND 16 pups, mothers of PND 9 pups showed increased levels of both trail building (t(94.8) = 2.66, p = 0.027); Figure 5.4A) and autogrooming (t(97.2) = 2.81, p =

0.018); Figure 5.4B). Conversely, mothers of PND 16 pups explored more than mothers of PND 9 pups (t(95.1) = -7.56, p < 0.01) and PND 2 pups (t(95.1) = -9.33, p <

0.01), whereas mothers of PND 9 and PND 2 pups did not differ in their amounts of exploratory behavior (t(94.1) = -1.7, p = 0.28, Figure 5.4C).

Bi-parental mothers and fathers differ in parental and non-parental behaviors

Parental Behaviors.

It is reasonable to expect that the nature or quality of parental care might differ between mothers and fathers. Focusing on just biparental family units, the amount of grooming significantly differed by parent (F(1,131) = 22.3, p < 0.01) with mothers

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spending significantly more time grooming pups than fathers (t(132) = 4.72, p < 0.01;

Figure 5.5A). We found that pup grooming declined as pups became older (F(2,137) =

18.8, p < 0.01). Specifically, biparental parents also groomed younger pups more than they groomed older pups (PND 2 > PND 9 (t(136) = 2.72, p = 0.022); PND 2 > PND 16

(t(139) = 6.1, p < 0.01); PND 9 > PND 16 (t(138) = 3.41, p < 0.01; Figure 5.5C). Pup grooming for biparental parents was unaffected by experimental handling (F(1,26) =

2.97, p = 0.097; Figure 5.5B).

Furthermore, when analyzing the total number of pup retrievals in biparental families, we found a significant main effect of parent (!! = 7.69, df = 1, p < 0.01;

Figure 5.6A), handling condition (!! = 6.49, df = 1, p = 0.011; Figure 5.6B), and postnatal day (!! = 74.4, df = 2, p < 0.01; Figure 5.6C), with a significant interaction between postnatal day and parent (!! = 10, df = 2, p < 0.01; Figure 5.6D). Post-hoc contrasts for the highest order interaction revealed that mothers retrieved pups more than fathers on PND 2 (t(157) = 3.99, p < 0.01), but not on PND 9 (t(157) = 1.35, p =

0.18) or on PND 16 (t(157) = -0.369, p = 0.71).

Nonparental Behaviors.

Biparental mothers and fathers did not differ in trail building duration (all p’s >

0.05). However, consistent with the results reported above, the time biparental parents spent exploring differed as a function of pup age (F(2,136) = 19.6, p < 0.01), where parents spent more time exploring when pups were 16 days old compared to when pups were 9 days old (t(135) = -5.2, p < 0.01) and 2 days old (t(136) = -5.59, p < 0.01;

Figure 5.7A). Parent exploration did not differ when pups were PND 2 and PND 9

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(t(134) = -0.449, p=1). Similarly, we found a significant main effect of parent (F(1,131) =

5.15, p = 0.025), and a significant interaction between parent and postnatal day

(F(2,131) = 4.81, p < 0.01) on the duration of autogrooming. Post-hoc contrasts for the interaction revealed that fathers spent significantly more time engaging in self-care via autogrooming than mothers when pups had reached PND 16 (t(131) = -3.78, p < 0.01), but not during PND 9 (t(131) = 0.466, p = 0.64) or PND 2 (t(131) = -0.641, p = 0.52;

Figure 5.7B).

Handling manipulations did not impact fathers’ behaviors in the home cage

Parental Behaviors.

Only pup age influenced paternal pup grooming (F(2,53.9) = 11.1, p < 0.01) and pup retrievals (!! = 0.781, df = 2, p = 0.68). Specifically, fathers groomed PND 2 pups significantly more than they groomed PND 9 pups (t(52.7) = 2.77, p = 0.023) or

PND 16 pups (t(53.9) = 4.67, p < 0.01), but they did not differ in their grooming of

PND 9 pups and PND 16 pups (t(53.5) = 1.93, p = 0.18; Figure 5.8A). Conversely, pup retrievals significantly increased with pup age (PND 9 > PND 2 (t(77) = -6.12, p <

0.01); PND 16 > PND 9 (t(77) = -2.41, p = 0.055); PND 16 > PND 2 (t(77) = -8.3, p <

0.01); Figure 5.8B).

Non-parental Behaviors

Paternal trail building was unaffected by experimental handling (F(1,29.4) =

0.0771, p = 0.78) or pup age (F(2,52.6) = 1.08, p = 0.35). Pup age did, however, significantly affect the duration of time fathers spent exploring (F(2,54.1) = 3.45, p =

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0.039), and post-hoc analysis showed that fathers tended to explore more when their pups were PND 16 compared to when they were PND 2 (t(53.9) = -2.43, p = 0.056);

Figure 5.9A). Pup age also affected the duration of autogrooming by fathers (F(2,52) =

5.15, p < 0.01). Fathers autogroomed significantly more when their pups were PND 16 compared to when pups were either PND 9 (t(52.9) = -2.64, p = 0.032) or PND 2 (t(53.1)

= -2.89, p = 0.017); Figure 5.9B).

In some cases, prairie voles will retrieve their partners, much in the way that they retrieve pups, but presumably for different reasons. We found that the age of the pups affected the number of partner retrievals fathers performed on mothers (!! = 10, df = 2, p < 0.01). Specifically, fathers retrieved their partners significantly more frequently when pups were PND 9 compared to PND 2 (t(77) = -3.07, p < 0.01; Figure

5.9C). Partner retrievals did not differ between the other pup age contrasts (PND 16 and PND 9: t(77) = 1.83, p = 0.21; PND 16 and PND 2: t(77) = -1.25, p = 0.65).

Parental performance in the Open Field Test

The open field test is commonly used to assess anxiety-like behaviors, and/or exploratory behaviors under laboratory conditions. We investigated the total distance traveled within an OFT arena, and the frequency and duration of time spent visiting the center of the arena to assess how experimental handling, and the presence of fathers in the home cage, impacted these behaviors in parents.

Handling manipulation and the presence of the father affects mothers’ behavior in the

OFT

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Experimental handling altered the total distance mothers traveled in the OFT arena (F(1,34) = 6.5, p = 0.015), where Handled mothers covered a greater total distance compared to Non-handled mothers (t(34) = -2.39, p = 0.023; Figure 5.10A).

In contrast, Father Presence/Absence did not impact the total distance mothers traveled in the OFT arena (F(1,33.2) = 1.52, p = 0.23; Figure 5.10B), and there was no interaction between these factors (F(1,32.8) = 0.0115, p = 0.92).

Similarly, experimental handling impacted the number of visits mothers paid to the arena center (!! = 3.99, df = 1, p = 0.046). However, after correction for multiple comparisons, the post-hoc analyses showed only a non-significant trend toward experimentally handled mothers visiting the center more than control mothers (t(32) = -

1.82, p = 0.078; Figure 5.10C). Moreover, the presence of the father altered the number of visits mothers paid to the arena center (!! = 8.5, df = 1, p < 0.01), and in this case, mothers that were co-housed with fathers visited the center significantly more frequently than those that were forced to rear their pups alone (t(32) = -3.03, p <

0.01; Figure 5.10D). Our model found no interaction between the presence of fathers and the experimental handling conditions on visits to the center (!! = 1.11, df = 1, p =

0.29). We also investigated the total duration of time that mothers spent in the center of the arena, but unlike the number of visits, we found no differences due to experimental handling (F(1,32.5) = 0.239, p = 0.63) or father presence (F(1,30.3) = 0.149, p = 0.7).

Mothers and fathers of bi-parental families do not differ in OFT performance

Consistent with data reported above, among just the biparental families,

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experimental handling significantly impacted the total distance traveled within the

OFT arena (F(1,36) = 7.37, p = 0.01) and handled individuals traveled significantly greater distances than control individuals (t(28.3) = -2.41, p = 0.023; Figure 5.11A).

However, we did not find a sex difference for distance traveled in the arena (F(1,36) =

0.0285, p = 0.87, Figure 5.11B). We again investigated the number of visits to the center of the arena and duration of time spent in the center of the arena. Although experimental handling (!! = 1.94, df = 1, p = 0.16) and parent sex (!! = 0.135, df = 1, p = 0.71) did not affect the duration of time in or the frequency with which biparental parents visited the arena center, we did find a significant interaction of parent sex and experimental handling on the duration of time spent in the center (F(1,36) = 4.13, p =

0.05). However, post-hoc contrasts did not withstand correction for multiple comparisons, and thus no significant group differences were revealed (Non-handled, father-mother: t(29.2) = 1.21, p = 0.24; Handled. father-mother: t(29.2) = -1.64, p = 0.11;

Figure 5.11C).

OFT performance of fathers is not impacted by experimental handling

Fathers did not significantly differ in their total distance traveled (F(1,18) =

3.39, p = 0.082), their number of visits to the arena center (!! = 0.00276, df = 1, p =

0.96), or in their total duration of time spent in the arena center as a function of handling condition (F(1,8.95) = 1.95, p = 0.2).

Relationships between brains and behaviors

Behavioral principal components analysis

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Parental care and anxiety-like behaviors have been linked to the neural activity of VP and CRH systems (Bales and Saltzman, 2016; Hostetler and Ryabinin, 2013).

We quantified the amount of VP-ir and CRH-ir cells in hypothalamic subpopulations to ask how their neural expression related to prior parental experiences and suites of behaviors. Before we could assess how the presence of the father or experimental handling manipulation impacted the relationship between VP or CRH cell counts and home cage behaviors detailed above, we had to consolidate the redundancy across correlated suites of behaviors into a more simplified form that would enable such an investigation. To that end, we used principal component analyses (PCA) to collapse the behavioral data into meaningful subsets. Separate PCAs were conducted for males and females to account for sex-specific behaviors. The female PCA excluded behaviors such as partner grooming and partner retrieval because these measures were not applicable to single mothers.

This analysis resulted in two retained principal components (PC) for females

(Table 5.5). PC1 of the female PCA (which we term “contact care”) accounted for

47% of the variation and included the positive loadings of pup grooming duration and neutral nursing duration, and the negative loading of trail building duration. PC2 of the female PCA (which we term “nonparental behavior”) accounted for 20% of the variation and includes the negative loadings of autogrooming duration and exploration duration.

Similar to the PCA for females, the PCA analysis for males resulted in two retained PCs (Table 5.6). PC1 of the male PCA accounted for 49% of the variation and included positive loadings of huddling duration and exploration duration, and a

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negative loading of trail building duration. Given that these behaviors encompass the transitions between immobility and movement, we term this component

“stationary/ambulatory phase”. PC2 of the male PCA (which we term “adult grooming score”) accounted for 17% of the variation and included the positive loadings of autogrooming duration and partner grooming duration.

Behavioral predictors of neural data

For mothers, VP-ir cell counts in the PVN were negatively related to contact care scores (PC1) (Table 5.7, Figure 5.12A). Decreases in nonparental behaviors

(PC2) were positively associated with increases in PVN VP-ir cell counts, although the slope of this relationship interacted with the father condition; mothers who reared their young with their partners had a steeper slope between decreases in parental care and VP-ir neurons in the PVN compared to single mothers (Figure 5.12B).

CRH-ir cell counts in the PVN were negatively associated with contact care

(Figure 5.12C), but not significantly associated with any other variables. SON VP-ir counts were negatively associated with contact care (Figure 5.12D) and associated with decreasing nonparental behavior, although the direction of the latter was dependent on handling condition (Figure 5.12E). As nonparental behaviors decreased, mothers who were experimentally handled had fewer VP-ir neurons in the SON, whereas control mothers had more VP-ir neurons in the SON.

For fathers, PVN VP-ir cell counts were negatively associated with adult grooming scores (PC2) (Table 5.8, Figure 5.13A). These cell counts were also associated with stationary/ambulatory phase (PC1), but the slope of the relationship

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was reversed depending on experimental handling (Figure 5.13B). As PC1 scores increased, fathers who were Handled had fewer PVN VP-ir neurons, whereas Non- handled fathers demonstrated increases in PVN VP-ir neurons. There were no other significant associations between any region’s cell counts and the other modeled variables (Table 5.8).

DISCUSSION

Our study examined 1) how variation in rearing experiences for mothers and fathers impact their behaviors inside and outside of the natal nest, and 2) how parental experiences shape the neural systems known to be important for parental behavior and anxiety-like traits. We found that both mothers and fathers adjust their caregiving behaviors across the developmental ages of the pups, demonstrating behavioral plasticity in the capacity to respond to the changing needs of the offspring. However, we found sex differences in the patterns of adjustments in pup age-related home cage behaviors, which suggests that mothers and fathers may exhibit differential sensitivities to offspring developmental stage. When experimental handling significantly affected parents’ behavior, it tended to increase their activity, exemplified by more pup retrievals in the home cage and more distance traveled in the

OFT. Somewhat surprisingly, the absence of the father had no observable effects on mother’s behaviors within the home cage but did lead to an increase in the number of times mothers visited the center of the OFT. These results suggests that the impact of prior parental experiences on a mother’s behavioral phenotype may be elicited in a context-specific manner. Beyond the behavioral results alone, we revealed that both

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mothers and fathers had significant relationships between their home cage behaviors and hypothalamic neuropeptide cell counts. The direction of these associations between brains and behaviors were generally dependent upon experimental condition, suggesting that brain-behavior correlations can exhibit experience-dependent plasticity. Below, we discuss in more detail i) the consequences of pup age and parental composition on specific parental behaviors, ii) the associations between parents’ behavioral phenotypes and hypothalamic cell counts, iii) and the evidence for the mediating role of parental experiences on neuroendocrine-behavior relationships.

Pup age determines parental and nonparental behaviors of parents

Many parents are tasked with balancing the energetic demands of rearing vulnerable offspring with ensuring their own survival and preserving energy for future reproductive interests (Trivers, 1974). This parent-offspring conflict forces parents to mitigate their energetic investment in individual offspring. One way this can be accomplished is to adjust the investment that is required to meet the needs of offspring by only dedicating effort based on the offspring’s current needs, while decreasing the effort dedicated to meeting needs that no longer stand to appreciably benefit the offspring. For example, zebra finch parents whose chicks were swapped with broods that were either older or younger than their own offspring adapted the duration of their caregiving period to match the needs of their foster brood (Rehling et al., 2012), indicating that parents can rapidly and flexibly adjust their caregiving behaviors.

The current study employed salient handling manipulations designed to impose a minor stressor and temporarily alter parental stimulation (Bales et al., 2007), and

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produced ecologically relevant and distinct rearing conditions (Getz and Carter, 1996) by manipulating the presence of a rearing partner. Despite these experimentally imposed factors, the factor that most frequently accounted for variation in parental and non-parental behaviors was the age of the offspring. Prior work has shown that prairie vole parents display different rates of parental behaviors when their pups are neonates compared to when they are 10-11 days old (Lonstein and De Vries, 1999), and our findings replicate this pattern of results in which parents adjust caregiving over pup development. We found that prairie vole parents decreased caregiving behaviors, including nursing and grooming, as the pups approached an age associated with the ability to self-thermoregulate and consume solid food. Conversely, pup retrievals by both parents increased as pups grew older, a result likely attributable to the refinement of motor skills and onset of exploratory behaviors of the pups over development. The ability to decrease the time allocated to intense offspring-directed care may also explain the relative increases in nonparental behaviors, including exploration and auto grooming, a trade-off that has been theoretically modeled and empirically tested in other species (Kacelnik and Cuthill, 1990; Whittingham, 1993; Williams, 1966).

Taken together, our data provide another example showcasing the incredible behavioral flexibility that parents are capable of exhibiting over developmental timescales.

Mothers and fathers differ in their behavioral adaptations to pup age

Male and female prairie voles engage in the same parental care behaviors, with the exception of nursing (Thomas and Birney, 1979). However, biparental care is rare

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among mammals (Kleiman and Malcolm, 1981) and maternal and paternal care have evolved under different selection pressures (Trivers, 1974). As a result, one might expect that even under the rare cases of biparental care, differences in selection pressures coupled with the inability for male mammals to nurse young offspring could bias biparental animals to demonstrate some degree of sex differences in parental care, and this might be seen most prominently as a function of pup age. Indeed, research into the genetic contributions to mammalian parental behavior suggests that the mechanisms that drive parental behavioral variation are sex-specific (Bendesky et al.,

2017). Our results are fairly consistent with previous work that has highlighted more similarities than differences in the regulation of parental care. However, two notable exceptions demonstrated differences between mothers and fathers as a function of pup age. In comparing pup retrievals, mothers retrieved pups significantly more frequently than fathers when pups were 2 days old, but this sex difference was not found when pups were older. This result is consistent with earlier work that found that prairie vole mothers spend more time in contact with pups than fathers do during the first week of postnatal life, but not beyond (Solomon, 1993). We also found that mothers and fathers engage in similar levels of selfcare until pups reach PND 16, at which point fathers significantly increase their autogrooming behavior, whereas mothers do not.

Interestingly, (Ahern et al., 2011) found that fathers autogroomed more than mothers during the postnatal period, although they employed different sampling methods and did not observe behavior after PND 12.

The initial greater effort in pup retrievals by mothers combined with a clear shift towards self-care (i.e., autogrooming) behavior in fathers suggests that, even if

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mothers and fathers engage in the same types of care behaviors in general, parents differ in the degree to which they engage in these behaviors. Moreover, fathers show nuanced differences in their patterns of offspring investment and rearing that changes prominently as a function of offspring developmental age. Indeed, our data support the hypothesis that parental sex differences exist and can be tracked to when sex-specific trade-offs occur between pup-directed vs self-directed care behaviors. These data are consistent with earlier studies that demonstrated that prairie vole mothers engage in more pup-directed care behaviors than fathers (McGuire et al., 2007; Solomon, 1993).

Likewise, Mongolian gerbils (Meriones unguiculatus) are also biparental and their parental behaviors also change with offspring age (Elwood, 1975). However, the ratio of time that Mongolian gerbil fathers spend on the nest relative to their female partners increases as pups develop, demonstrating that the direction of age-dependent sex differences in parental care is species-specific. Taken together, to best understand the evolution and adaptive significance of mammalian biparental care, it is crucial to consider the timing and dynamics of cooperative parental behaviors within each species, and the contexts in which their life history strategy has evolved.

Single mothers did not adjust maternal care due to the absence of fathers

For mammalian mothers who invest heavily in offspring, parental partners can offset the rearing costs by provisioning resources, defending territories against intruders and predators, providing thermoregulatory and social stimulation, and residing over the young so that mothers can engage in nonparental behaviors with lowered risks of offspring mortality (Woodroffe and Vincent, 1994). In some

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mammals, paternal care increases offspring maturation rates, litter sizes, body condition, and survival (Gubernick et al., 1993; Stockley and Hobson, 2016; Wang and Novak, 1992), often as a result of shared or added forms of paternal care.

Unsurprisingly, the presence or absence of prairie vole fathers substantially impacts the physical and social development of pups; pups reared in the presence of both parents developed faster than pups reared without their fathers (Wang and Novak,

1992) and had shorter latencies to form adult pair bonds compared to singly reared offspring (Ahern and Young, 2009). Although these studies reveal the critical impact of paternal presence on offspring outcomes, far fewer studies have assessed the potential interdependence of behaviors between mothers and fathers (Bales and

Saltzman, 2016), such that paternal contributions may dynamically shape the behaviors of mothers.

Despite the benefits of partner presence for reproductive fitness, our data suggest that prairie vole maternal behaviors are resilient to the influence of partner absence. We did not find any differences in the behaviors of paired or single mothers living in the home cage, regardless of pup age or handling manipulation. Furthermore, we found no evidence that mothers behaviorally upregulate their care to compensate for the absence or shortcomings of a father, which echoes the findings of previous studies of prairie vole parental care (Ahern et al., 2011; Ahern and Young, 2009;

Bosch et al., 2018; McGuire et al., 2007; Rogers and Bales, 2019; Tabbaa et al., 2017).

However, it is possible that paternal absence failed to induce changes in mother’s behaviors because mothers may be exhibiting ceiling-levels of care imposed by laboratory conditions that induce relatively low parental costs (e.g., see Kelly et al. in

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press). Indeed, implementing sufficiently harsh environmental conditions may force single mothers to tradeoff between pup care and survival, therefore revealing the full effects of paternal presence on mother and offspring behaviors (Wright and Brown,

2002).

Several models assessing the evolution of biparental care predict that single parents of biparental species will increase their caregiving behaviors to (at least partially) compensate for the absence of a mate (McNamara et al., 1999). Support for these models include degus (Octodon degus, Wilson, 1982), Mongolian gerbils

(Meriones unguiculatus, Elwood and Broom, 1978), rock cavies (Kerodon rupestris,

Tasse, 1986), and the Mexican volcano mouse (Neotomodon alstoni, Luis et al., 2004), in which single mothers show higher levels of pup directed behavior compared to partnered mothers (Elwood and Broom, 1978; Luis et al., 2004; Tasse, 1986; Wilson,

1982). Although we did not find evidence for maternal compensation for the absence of a father as mentioned just above, these studies indicate that mothers often compensate for the absence of fathers. In contrast, a recent study in biparental

California mice also found that single mothers do not differ in their maternal care compared to paired mothers (Zhao et al., 2019), mirroring the pattern documented for prairie voles living in the lab. Nevertheless, in any such studies, it remains unclear if mothers are responding to altered pup behaviors as a function of paternal presence or the direct absence of the fathers themselves when mothers do compensate (Elwood and Broom, 1978). Interestingly, prairie vole maternal care decreases when both fathers and juveniles are cohoused with the mother and pups, but is not impacted by the presence or absence of the father alone (Wang and Novak, 1992). This suggests

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that juvenile alloparents might play an important role in offloading demands from parents in prairie voles (Kenkel et al. 2012).

In our study, pups reared biparentally received more grooming relative to single-mother reared pups, but this difference was clearly due to an additive effect of care by fathers (as has been found elsewhere, Perkybile et al. 2015, Kelly et al. in press). So, although mothers did not appear to alter their caregiving, the pups did receive more care overall, which can account for differences in biparentally raised pups (see above). Altogether these findings suggest that offspring may benefit from the extra direct care provided by fathers, but the indirect effects of paternal presence on the variation in mother’s behaviors are, at best, species-specific. Furthermore, failure to induce maternal variation in care by manipulating paternal presence in a lab context may not reflect ethologically representative conditions analogous to survival in the wild.

Parental experiences alter their behavior in an open field test

We induced variation in mildly stressful experiences by either physically handling animals once a week during cage changes or passively transferring them to new cages in a cup. We tested our subjects in the open field test to assess any effects of any of our manipulations on this common laboratory test of anxiety and / or exploration. We found that parents in the handled condition traveled significantly more in the OFT compared to parents in the control condition, suggesting that the acute handling manipulation may have increased exploratory behaviors in parents. It is noteworthy to mention that these same two groups did not differ in exploratory

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behavior during the home cage recordings, which suggests that the effect of handling on exploration may be dependent upon environmental context. Mothers that reared their offspring with a partner also visited the center of the OFT chamber more frequently than single mothers, which could be interpreted as displaying a less

“anxious-like” phenotype. Because single and partnered mothers did not display other differences in behavior within the home cages, it is possible that anxiety-related behaviors outside of a parental context may be more susceptible to perturbation by experience during the parenting period. These results contradict earlier work in which single mothers show greater levels of anxiety-like and depressive-like phenotypes compared to paired prairie vole mothers in an elevated plus maze and forced swim test, respectively (Bosch et al., 2018). However, our OFT testing followed pup- weaning, whereas Bosch et al. (2018) measured behavior in the days following parturition. In both experimental designs, it is challenging to discern if the behavioral results are due to bond dissolution or to experience as a single parent. When taken together, these studies suggest that the interval of time between partner separation and testing for anxiety-like traits may influence the direction of the effects of single- motherhood. It would be useful to address the potential differential influences of partner loss vs. the demands of single parenthood to best understand how these experiences may independently or synergistically shape parental phenotypes.

Neuroendocrine mechanisms of parental behaviors

Up to 10% of mammalian genera exhibit paternal care (Kleiman and Malcolm,

1981). Not surprisingly, the majority of what is known about the

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neuroendocrinological basis of mammalian parental behavior has been characterized in females (Saltzman and Ziegler, 2014). From these studies, it is clear that a symphony of oscillating hormones that accompanies pregnancy and parturition is critical in priming primiparous mothers to behave maternally toward their newborn offspring. Mammalian fathers, on the other hand, do not gestate their young. Thus, the endocrine mechanisms that subserve care in males greatly differs from those of females, and might vary between species (Horrell et al., 2018). Nevertheless, male hormones known to play a functional role in the display of paternal behaviors do fluctuate and are susceptible to a number of factors, including mating and pair bonding experiences, exposure to pregnant females, and exposure to offspring (Saltzman and

Ziegler, 2014). Below we discuss our experimental results in the context of what is known about CRH and VP systems in rodent behavior.

Corticosterone-releasing hormone cell counts in the PVN correspond to maternal behavior

The role of CRH in anxiety-like behaviors has been well established in the mammalian literature, but much less is known about how CRH may be related to parental behaviors. Adult virgin prairie voles are known to exhibit spontaneous pup- directed care, and a previous study reported that males show significantly less cFos induction of CRH-ir neurons in the PVN when exposed to a pup stimulus compared to an object control (Kenkel et al., 2012). Our data indicate that lower numbers of PVN

CRH-ir positive neurons in mothers corresponds to higher levels of PC1 (“contact care”), and together these results hint that CRH may have an inhibitory function for

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caregiving in prairie voles. Likewise, a study in rats demonstrated that ICV injections of CRH reduced maternal caregiving and increased levels of infanticide (Pedersen et al., 1991), providing a functional example of the negative effects of CRH in rodent caregiving.

Decades of research in rodents (primarily rats) have characterized the relationship between pregnancy and the blunting of HPA activity. Both HPA basal activity and responsiveness to stressors are dampened in late pregnancy and during lactation, marked by lower general levels of PVN CRH-mRNA and decreased corticosterone release following exposure to a variety of stressors (Brunton et al.,

2008). Such a reduction in HPA activity coincides with the postpartum period, during which dams must provide intensive maternal care to ensure the survival of their offspring. Prairie vole males, conversely, do not show differences in circulating corticosterone levels (the downstream result of CRH signaling) after the birth of a litter (Campbell et al., 2009), highlighting that a shift away from nulliparity to parenthood may have greater consequences for the modification of CRH systems in mothers compared to fathers. Such a result may explain why we did not find a significant association between PVN CRH-ir cell counts and male behavior.

Investigation of the potential sex differences in the relationships between parental care and CRH activity would be of great value.

Vasopressin cell counts are associated with parents’ behavior in an experience- dependent manner

We found that the number of VP-ir neurons in the PVN and the SON were

189

significantly associated with PC1 in mothers. Increases in PC1 measures (to which we refer generally as “contact care”) corresponded to decreases in the number of VP-ir neurons in the PVN and the SON. Considering that VP is involved in parental status and behavior in prairie voles (Bamshad et al., 1994; Wang et al., 1994, 2000) and other rodents (Bayerl et al., 2016; Bosch and Neumann, 2008; Parker and Lee, 2001), it initially seems paradoxical that mothers that expressed higher levels of care had fewer VP-ir cells. Interpretations of cell count data are quite challenging because differences in immunoreactivity can represent either peptide production, accumulation due to blocked secretion, or changes in release (Goodson and Kabelik, 2009; Panzica et al., 2001). Under the latter interpretation, the decrease in PVN and SON VP-ir neurons that we found could indicate greater release of bioavailable VP to extrahypothalamic sites, which in turn facilitates increases in maternal care. Without acute sampling data to determine fluctuations in VP profiles, we are unable to determine the functional significance of these differences.

VP-ir counts in the PVN and the SON of mothers were also significantly associated with PC2 (on which nonparental behaviors are negatively loaded). A negative loading here means that mothers are less engaged in nonparental behavior

(they decrease autogrooming and exploration) as the score on the PC2 scale increases.

Intriguingly, the nature of the association between PC2 and VP-ir cells counts differed if the father was present or absent (for the PVN) or if the mother was handled or not handled (for the SON). In particular, the slopes between VP-ir positive cells in each brain region and PC2 were more positive for the standard housing conditions (either father present, or parents not handled), and the slopes decreased under the more

190

“stressful” conditions (father absent, or parents handled).

Fathers also showed a similar pattern of results, in which experimental condition altered the relationship between behavior and PVN VP-ir cell counts; the relationship between PC1 and VP-ir was negative for unhandled fathers, but positive for handled fathers. Although the specific meaning of what each PC truly represents and how these relate to the function of VP is difficult to ascertain, our results appear to reveal a general pattern in which (social and nonsocial) experiences have shaped the associations between behavioral suites and hypothalamic VP cell groups in complex ways. Similar patterns of experience-dependent mediation of brain-behavior relationships have been described for steroid hormones in other species. For example, a recent study of wire-tailed manakins (Pipra filicauda) found that the relationship between circulating testosterone and male social behaviors was inverted between territory-holders and non-territory-holders, indicating that social status dynamically modulates hormone-behavior relationships in this species (Ryder et al., 2020).

Furthermore, the association between baseline corticosterone levels and parental success (fledgling numbers) is positive prior to egg laying and negative during the subsequent parental provisioning phase in great tits (Parus major), and is therefore dependent on reproductive stage (Ouyang et al., 2013). Moreover, group-housed mice show a significant positive correlation between serotonergic activity and social investigation, but such a correlation was not found in isolate-housed mice, suggesting that social housing contexts coordinate relationships between serotonin and social behavior (Keesom et al., 2017). Our study supplements these examples supporting the hypothesis that social experiences are involved in contingently shaping the

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relationships between neuropeptides and behavioral phenotypes, even across neurotransmitter classes and taxa.

Our results highlight the dynamic, experience-dependent associations between vasopressin-producing cell groups and suites of behaviors in prairie vole mothers and fathers. These data reveal categorical differences in the slopes of the relationship between PCs and VP cell counts, in which manipulations as distinct as handling and paternal presence both appear to significantly alter brain-behavior relationships. We did not find such a pattern of results for CRH cell counts and behavior, suggesting that the consistency of CRH-behavior relationships is potentially less plastic than that of

VP-behavior relationships. Critically, our neural measures took place after the behavioral observation periods and are thus correlational in nature. The cell counts were regressed against orthogonally transformed sum totals of parental caregiving over the preweaning period, which precludes us from making functional interpretations of these data. Nevertheless, our findings extend what is known about the interconnections between parental contexts, behaviors of parents, and neuroendocrine phenotypes, and demonstrates the value of integrating behavioral and physiological measures to understand the longitudinal dynamics of brain-behavior relationships.

CONCLUSIONS

We investigated how social and non-social variation in parental experiences impact prairie vole parents’ behaviors across pup development, and how these experiences shape the hormonal systems that drive, and respond to, social behaviors.

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We revealed that parental behaviors shift according to offspring development, but that the patterns of behavioral adaptations are sex-dependent. Prairie vole maternal behaviors are only subtly affected by experimental manipulations, but the associations between behaviors and hypothalamic VP cell counts are significantly shaped by parental experiences. Future work should address the functional implications of alterations in hypothalamic neuroendocrine signaling for parental behavior, and search for the underlying mechanisms by which experiences organize the relationships between hormones and behavioral phenotypes in parental brains.

Acknowledgements

The authors would like to thank Mandy Chan and Sara O’Malley for their contributions to the data collection. The authors acknowledge the support from the

National Institutes of Health (Eunice Kennedy Shriver National Institute of Child

Health and Human Development HD079573 to A.G.O.) and the National Science

Foundation Graduate Research Fellowship Program (under DGE-1650441 to L.C.H.).

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FIGURES

A 15 B 15 * * 10 10 * 5 5 *

Pup grooming (m) grooming Pup 0 (m) grooming Pup 0 Father Father PND2 PND9 PND16 Absent Present Father C Mother PND2 PND9 PND16 15 * 10 *

5

Pup grooming (m) grooming Pup 0 Father Father Father Father Father Father Absent Present Absent Present Absent Present

Figure 5.1 Total time (in minutes, m) parents groomed pups as a function of A) Father-Presence/Absence, B) postnatal day of the pups (Father Present and Absent combined) and C) the interaction of postnatal day and Father-Presence/Absence. *p ≤ 0.05.

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A 40 B 40 C 40 p = 0.06 Father Mother 30 30 30 *

20 20 20 * Pup retrievals Pup retrievals Pup retrievals Pup

10 10 10

0 0 0 Father Father Non- Handled PND2 PND9 PND16 Absent Present Handled

Figure 5.2 Total number of pup retrievals as a function of A) Father- Presence/Absence and B) Handling Condition. C) The number of pup retrievals by both parents combined across pup ages. *p ≤ 0.05.

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A B C 125 125 125 * 100 100 * 100 *

75 75 75

50 50 50 Pup Retrievals Pup Retrievals Pup Retrievals Pup

25 25 25

0 0 0 Father Father Non- Handled PND2 PND9 PND16 Absent Present Handled

Figure 5.3 Raw data and box and whisker plots of the total number of pup retrievals by mothers as a function of A) Father-Presence/Absence, B) Handling Condition, and C) postnatal day (PND). Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

204

A B C 20 60 * * 80 * 15 60 * 40

10 40

20 5 20 Total Duration of Exploration (m) Exploration of Duration Total Total Duration of Trail Building (m) Building Trail of Duration Total Total Duration of Autogrooming (m) Autogrooming of Duration Total

0 0 0 PND2 PND9 PND16 PND2 PND9 PND16 PND2 PND9 PND16

Figure 5.4 Raw data and box and whisker plots across postnatal days (PND) for the duration (in minutes, m) of mothers’ A) trail building, B) autogrooming, and C) exploration. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

205

A B C 25 25 25

20 20 20 * * * 15 15 15

10 10 10 * Pup grooming (m) grooming Pup (m) grooming Pup (m) grooming Pup 5 5 5

0 0 0 Mother Father Non- Handled PND2 PND9 PND16 Handled

Figure 5.5 Pup grooming duration in minutes (m) for biparental families analyzed by A) parent sex, B) Handling Condition, and C) postnatal day (PND). Points are raw data, thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

206

A B C 100 * 100 * 100 * 75 75 75 *

50 50 50

Pup retrievals Pup 25 retrievals Pup 25 retrievals Pup 25

0 0 0 Mother Father Non- Handled PND2 PND9 PND16 Handled

D PND2 PND9 PND16 100

75

50

Pup retrievals Pup 25 *

0 Mother Father Mother Father Mother Father

Figure 5.6 Counts of pup retrievals as a function of A) parent sex, B) Handling Condition, C) postnatal day (PND), and D) the interaction between parent sex and postnatal day. Points are raw data, thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

207

A 80 * 60 *

40

Exploration (m) Exploration 20

0 PND2 PND9 PND16

B PND2 PND9 PND16

15 *

10

5 Autogrooming (m) Autogrooming

0 Mother Father Mother Father Mother Father

Figure 5.7 Raw data and box and whisker plots of biparental families A) across postnatal days (PND) for the duration of exploration (in minutes, m). B) Autogrooming duration (in minutes, m) by mothers and fathers for each postnatal day observed. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

208

A 25 B 100 * 20 * * * 75 15 * 50 10 Pup Retrievals Pup Pup grooming (m) grooming Pup 5 25

0 0 PND2 PND9 PND16 PND2 PND9 PND16

Figure 5.8 Raw data and box and whisker plots across postnatal days (PND) for A) the duration of fathers’ pup grooming, and B) the number of pup retrievals performed by fathers. Thick vertical bars represent median values, and boxes span interquartile ranges. *P ≤ 0.05.

209

A B C 60 30

20 * * *

40 15 * 20

10 Exploration (m) Exploration Partner retrievals Partner

20 (m) Autogrooming 10

5

0 0 0 PND2 PND9 PND16 PND2 PND9 PND16 PND2 PND9 PND16

Figure 5.9 Changes in nonparental behaviors across postnatal days (PND) for fathers’ A) exploration duration (in minutes, m), B) autogrooming duration (in minutes, m), and C) the number of partner retrievals observed. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

210

A B 10000 10000

7500 * 7500

5000 5000

2500 2500

Distance Moved (cm) Moved Distance 0 (cm) Moved Distance 0 Non- Handled Father Father handled Absent Present

C D 40 40 * 30 30

20 20

10 10 Visits to the Center the to Visits Center the to Visits 0 0 Non- Handled Father Father handled Absent Present

Figure 5.10 Total distance (in centimeters, cm) moved by mothers in the open field test as a function of A) Handling Condition, and B) Father-Presence/Absence. The number of visits to the center also varied by C) Handling Condition, and D) Father- Presence/Absence. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

211

A B

9000 * 9000

6000 6000

3000 3000

0 0

Total Distance Moved (cm) Moved Distance Total Non- Handled (cm) Moved Distance Total Father Mother handled

C Non-handled Handled

90

60

30

0

Total duration in center (s) center in duration Total Father Mother Father Mother

Figure 5.11 Total distance moved (in centimeters, cm) during the open field test by biparental families as a function of A) Handling Condition, B) parent sex, and C) the parent sex split by Handling Condition. Thick vertical bars represent median values, and boxes span interquartile ranges. *p ≤ 0.05.

212

Figure 5.12 Plots of marginal effects depicting the relationships between cell counts in mothers and behavioral principal components (PC) on standardized and centered scales. VP-ir cell counts in the PVN were significantly associated with A) contact care and B) nonparental behaviors. C) CRH-ir cells counts in the PVN decreased with contact care. VP-ir cell counts in the SON were associated with D) contact care and E) nonparental behavior. Shaded regions are 95% confidence intervals.

213

Figure 5.13 Plots of marginal effects depicting the relationships between cell counts in fathers and behavioral principal components (PC) on standardized and centered scales. VP-ir cell counts in the PVN were significantly associated with A) adult grooming and B) stationary/ambulatory phase. Shaded regions are 95% confidence intervals.

214

TABLES

Table 5.1 Ethogram of scored behaviors with operationalized definitions.

Behavior Description

Trail Moves litter around with mouth or Movement Build forepaws Moves around cage while sniffing, Explore rearing, or climbing

Stationary Remains in one spot

Direct Contact with pups, without vigorous Huddle contact movement Active Pup(s) attached to nipples while Nurse nurse mother is mobile Neutral Pup(s) attached to nipples while

nurse mother is stationary Pup Retrieve Picks up pup(s) with mouth retrieval Partner Picks up/drags other parent with

retrieval mouth Cleans or scratches itself with paws Groom Autogroom or mouth

Pup groom Cleans pups with paws or mouth

Partner Cleans partner with paws or mouth groom

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Table 5.2 Effects of conditions on parental behavior of mothers

Behavior Factor df F-value p-value Father Presence 1, 55.1 0.0038 9.51e-01 Experimental Handling 1, 44.7 0.18 6.74e-01 PND 2, 96.5 21.0 2.46e-08 Father Presence:Experimental 1, 45.3 2.50 1.20e-01 Pup Handling grooming Father Presence:PND 2, 97.6 0.46 6.32e-01 Experimental Handling:PND 2, 96.8 0.38 6.86e-01

Father Presence:Experimental 2, 97.3 0.15 8.63e-01 Handling:PND

Father Presence 1, 51.3 1.1 3.05e-01 Experimental Handling 1, 44.4 0.059 8.09e-01 PND 2, 97.3 14.0 6.68e-06 Father Presence:Experimental 1, 43.1 0.61 4.40e-01 Active Handling Nursing Father Presence:PND 2, 98.7 0.58 5.59e-01 Experimental Handling:PND 2, 97.5 0.55 5.79e-01

Father Presence:Experimental 2, 98.5 0.44 6.46e-01 Handling:PND

Father Presence 1, 49.3 0.16 6.93e-01 Experimental Handling 1, 49.2 0.063 8.03e-01 PND 2, 95.2 23.0 9.08e-09 Father Presence:Experimental 1, 52.6 0.055 8.16e-01 Neutral Handling Nursing Father Presence:PND 2, 95.5 1.5 2.39e-01 Experimental Handling:PND 2, 95.3 1.2 3.11e-01

Father Presence:Experimental 2, 95.5 2.0 1.40e-01 Handling:PND

216

Table 5.3 Post-hoc contrasts between pup ages on maternal behaviors

Behavior Contrast df T-value p-value PND 2 - PND 9 94.8 1.66 3.00e-01 Pup grooming PND 2 - PND 16 96.3 6.30 2.72e-08 PND 9 - PND 16 96.5 4.59 4.07e-05 PND 2 - PND 9 95.5 -0.46 1.00e+00 Active Nursing PND 2 - PND 16 97.2 4.3 1.23e-04 PND 9 - PND 16 97.7 4.69 2.63e-05 PND 2 - PND 9 94.1 2.15 1.03e-01 Neutral Nursing PND 2 - PND 16 95.2 6.61 6.53e-09 PND 9 - PND 16 95.2 4.43 7.50e-05

217

Table 5.4 Effects of conditions on non-parental behavior of mothers

Behavior Factor df F-value p-value

Father Presence 1, 55.2 0.82 3.70e-01

Experimental Handling 1, 48.3 0.74 3.94e-01

PND 2, 95.7 3.6 3.05e-02

Trail Building Father Presence:Experimental Handling 1, 52.8 0.017 8.96e-01

Father Presence:PND 2, 95.9 0.16 8.56e-01

Experimental Handling:PND 2, 95.8 0.18 8.34e-01

Father Presence:Experimental Handling:PND 2, 95.8 2.0 1.37e-01

Father Presence 1, 46.8 2.9 9.43e-02

Experimental Handling 1, 50.5 1.4 2.42e-01

PND 2, 95.7 49.0 2.44e-15

Exploration Father Presence:Experimental Handling 1, 54.1 0.0018 9.66e-01

Father Presence:PND 2, 95.9 0.97 3.82e-01

Experimental Handling:PND 2, 95.7 1.1 3.37e-01

Father Presence:Experimental Handling:PND 2, 95.9 2.1 1.25e-01

Father Presence 1, 56.1 0.53 4.70e-01

Experimental Handling 1, 52.8 2.9 9.61e-02

PND 2, 98.5 4.3 1.67e-02

Autogrooming Father Presence:Experimental Handling 1, 56.1 0.032 8.59e-01

Father Presence:PND 2, 99.2 1.3 2.68e-01

Experimental Handling:PND 2, 98.5 0.36 6.98e-01

Father Presence:Experimental Handling:PND 2, 99.2 1.8 1.70e-01

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Table 5.5 Female behavior PCA PC1 PC2

Autogrooming 0.18 -0.56

Pup Grooming 0.45 0.044

Neutral Nursing 0.52 0.13

Active Nursing -0.33 0.31

Trail Building -0.47 0.26

Exploration -0.19 -0.61

Pup Retrieval -0.35 -0.36

Eigenvalue 3.3 1.4

Percentage of 46.7 19.9

VarianceCumulative 46.7 66.6

Percentage

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Table 5.6 Male behavior PCA

PC1 PC2

Autogrooming 0.14 0.69 Pup Grooming 0.39 -0.32

Partner Grooming 0.25 0.57

Huddling 0.42 -0.11

Trail Building -0.46 -0.013

Exploration 0.43 -0.12

Pup Retrieval -0.29 0.23

Partner Retrieval -0.33 -0.13

Eigenvalue 3.9 1.4

Percentage of 49.1 17.2

VarianceCumulative 49.1 66.3

Percentage

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Table 5.7 GLMM results for predictors of cell counts in mothers

Cell Population Factor χ2 df p-value

Experimental Handling 1 2.39e-01 PC1 1 1.58e-03 PC2 1 4.00e-01 Father Presence 1 7.37e-02 PVN VP PC1:Experimental Handling 1 2.29e-01 PC2::Experimental Handling 1 2.95e-01 PC1:Father Presence 1 8.47e-01 PC2:Father Presence 1 4.37e-02 Father Presence:Experimental Handling 1 5.61e-01 Experimental Handling 1 6.31e-01 PC1 1 3.33e-02 PC2 1 2.04e-01 Father Presence 1 9.63e-01

PVN CRH PC1:Experimental Handling 1 7.53e-01 PC2::Experimental Handling 1 7.90e-01 PC1:Father Presence 1 1.09e-01 PC2:Father Presence 1 5.02e-01 Father Presence:Experimental Handling 1 1.40e-01 Experimental Handling 1 5.52e-03 PC1 1 7.53e-03 PC2 1 1.46e-02 Father Presence 1 4.30e-01 SON VP PC1:Experimental Handling 1 7.58e-01 PC2::Experimental Handling 1 3.76e-03 PC1:Father Presence 1 2.11e-01 PC2:Father Presence 1 4.35e-01 Father Presence:Experimental Handling 1 2.13e-01

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Table 5.8 GLMM results for predictors of cell counts in fathers

Cell Population Factor χ2 df p-value

Experimental Handling 1 0.47

PC1 1 0.001

PVN VP PC2 1 0.039

PC1:Experimental Handling 1 0.001

PC2::Experimental Handling 1 0.5

Experimental Handling 1 0.86

PC1 1 0.46

PVN CRH PC2 1 0.8

PC1:Experimental Handling 1 0.12

PC2::Experimental Handling 1 0.66

Experimental Handling 1 0.12

PC1 1 0.79

SON VP PC2 1 0.21

PC1:Experimental Handling 1 0.077

PC2::Experimental Handling 1 0.65

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CHAPTER 6

GENERAL DISCUSSION

Animals exhibit incredible behavioral variation in response to their everchanging environments. This behavioral plasticity can be observed within the same individual when comparing across repeated samples (e.g., exploratory behavior as a juvenile vs. sexually mature adult), or by analyzing average individual differences within a species (e.g., dominant vs subordinate animals of the same social group)

(Foster 2013). Early-life experiences have the capacity to moderate the development of behavioral phenotypes, and the neurobiological substrates that underlie behavioral variation. In this thesis, the developmental programming of offspring phenotypes was explored across multiple levels of analysis. The roles of parental care, housing conditions, experimental handling, sex, and developmental age were all considered as potential interacting factors that contribute to the composite behavioral and neural phenotype of the prairie vole. Importantly, because prairie voles are biparental, I was able to consider a relatively understudied element of parental care; the contributions of fathers to offspring development. In doing so, I was able to characterize and incorporate paternal impacts on offspring with that which is known about mothers’ roles. The work described in the previous chapters address one or more of the following questions:

1) How does the perinatal vole brain’s response to the environment change over

the course of development?

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2) How does variation within, and interactions across, multiple dimensions of an

animal’s early environment pattern OTR and V1aR densities, nonapeptide-

producing cell groups, and age-specific behavioral profiles of offspring?

3) How do early postnatal environments and diversity in rearing experiences

drive the subsequent behavioral and neurochemical profiles of parents

themselves?

In doing so, the present dissertation is organized in such a way that it enhances understanding of the impact that experience in the postnatal nest has on developmental trajectories into adulthood. My results demonstrated that multiple dimensions of the early environment can interact to synergistically shape offspring development.

However, it is just as critical to recognize that phenotypes are made up of a constellation of interdependent traits. Therefore, measuring trait outcomes of a single qualitative type independently from others (morphology vs. behavior vs. physiology) ignores potential experience-contingent trait covariation (Kasumovic, 2013). To address this point, I incorporated the analysis of cellular activity, receptor densities, cell group profiles, and behavior to extract broader patterns of plasticity as driven by variation in a complex developmental environment. Altogether, the work presented in this thesis expands upon what is known about the mechanistic causes and consequences of developmental plasticity in nonapeptide systems and social behaviors.

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Activity-dependent neurodevelopment and early life experiences

In chapter 2, I addressed the question of how perinatal brains develop in their functional response profiles to changes in the environment. I found that cell groups within subregions of the medial extended amygdala show age-dependent patterns of neural induction, in which context-specific discriminatory cell responding to isolation and to the presence of family members became evident at PND 9 and PND 21, respectively. Furthermore, cellular responses to changes between social contexts were subregion and cell-type specific, demonstrating that the functional ontogeny of social brain regions differs across the spatial and genetic identities of the cells.

But what are the implications of these cell groups being activated by specific contexts? Neuronal firing does not solely propagate signals through circuits. Activated neurons also stimulate intracellular biochemical cascades that can alter the connectivity or firing patterns of those cells (Yap and Greenberg, 2018). For example cFos, the immediate early gene marker commonly used to infer neuronal activity, is a transcription factor whose expression is upregulated in response to appropriate stimulation (Piechaczyk and Blanchard, 1994). It is one of hundreds of genes that are regulated by cellular activity, many of which are involved in dendritic and synaptic modification processes (Flavell and Greenberg, 2008). As such, when the subregions of the meEA came “online”, the social experiences that drove their response profiles initiated transcriptional changes within those cells.

Activity-dependent transcriptional regulation of cells contributes to the organization and stabilization of circuits, providing a mechanism by which early external experiences begin scaffolding the brain’s functional architecture (Flavell and

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Greenberg, 2008). Importantly, the functional profiles of separate meEA substructures did not mature in a homogenous manner. Accordingly, the experience-dependent scaffolding of these socially sensitive regions exhibit differential timing in the onset of their sensitive periods. In this way, the presence or absence of environmental cues at specific periods during brain maturation leave a molecular “trace” within the functional circuits that are tuned to those cues (Chaudhury et al., 2016). Characterizing the ages at which neural response profiles discriminate between social contexts provides novel evidence to better predict the developmental timepoints at which specific brain regions with the prairie vole are most susceptible to alteration of their developmental trajectories by particular social experiences.

Diversity in early experiences pattern nonapeptide systems

The previous section discussed how external cues can drive activity-dependent scaffolding of neural circuits. Correspondingly, I reported results on the early-life experience-dependent densities of nonapeptide receptors and nonpeptide-producing cell groups in both chapters 3 and 4. In chapter 3, I describe OTR and V1aR densities across subregions of the young adult forebrain as a function of perinatal paternal presence, post-wean housing condition, sex, and the interactions therein. Interestingly,

I found multiple higher-order interactions between the experimental factors of interest:

1) V1aR density in the ventromedial hypothalamus varied according to an interaction between sex and paternal presence, 2) OTR density in the septo-hippocampal nucleus varied by an interaction between paternal presence and post-wean isolate housing, and

3) OTR density in the lateral septum varied by a three-way interaction of sex, paternal

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presence, and post-wean housing. Together these findings provide evidence that biological sex, or prior social experiences, may predispose an individual to be differentially sensitive to the influences of subsequent social experiences.

In chapter 4, I characterized how paternal presence in the natal nest and experimental handling experiences independently and synergistically affected age- specific behavioral suites. The presence or absence of the father during the rearing period contributed to aggression-related behavioral phenotypes, where animals reared without fathers were more socially permissive than animals reared with their fathers.

However, this effect was contingent upon age and handling condition (a method intended to increase pup-directed care), demonstrating that the influence of paternal presence on offspring behavior is not independent of these other biological and experience-dependent factors. Furthermore, the brains of adult offspring from handled families had more oxytocinergic neurons in hypothalamic cell groups compared to the brains of offspring from control families. However, this handling effect interacted with paternal presence for the PVN phenotype, demonstrating that interactions among the early environment are capable of exerting heterogenous effects for specific cell groups.

Developmental events appear to organize the densities of nonapeptide- producing cells and their receptors, which has functional implications for the regulation of adult social behavior (Bales and Perkeybile, 2012; Hammock, 2015).

Prior studies have characterized the transient expression of OTR and V1aR in mouse offspring, leading to the discovery that activation of these receptors in early life exerts organizational effects on neocortical development (Hammock and Levitt, 2012; Zheng

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et al., 2014). The transient expression of nonapeptide receptors within regions of the growing brain are hypothesized to sensitize developing offspring to the influence of social cues, enabling nonapeptide-mediated plasticity in the structuring of the social brain at specific points during development (Hammock, 2017). If we consider the hierarchical assemblage of brain development (where the maturation of neural systems is contingent upon the components that came before), experience-dependent regulation of nonapeptide systems in early-life could bias the neurodevelopmental trajectory of an animal, such that these regions are differentially sensitized to the effects of subsequent experiences (Hammock and Levitt, 2006). More simply put, experiences organize developing social circuits, which can be further canalized by subsequent experiences. Such a perspective could provide an explanatory mechanism for the higher-order interactions observed in the neural and behavioral data described in chapters 3 and 4.

Experimental findings, such as those just discussed, fit well into an established framework for the “multi-hit” hypothesis for the programming of animal phenotypes.

The literature on early-life adversity has contributed to the extension of a powerful hypothesis addressing gene by environment interactions and the adaptive value of developmental programming known as the ‘multi-hit’ hypothesis. The “multi-hit” hypothesis posits that genetic predispositions (Hit 1) interact with adverse experiences in the early-life environment (Hit 2) to determine an organism’s sensitivity to subsequent experiences (Hit 3) and their resultant levels of vulnerability or resilience to the development of mental disorders (Daskalakis et al., 2013). This framework has substantial clinical value for understanding the etiology of neuropsychiatric disease

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but is also beneficial in characterizing the relationship between naturalistic levels of environmental variation and the phenotypic plasticity of behavior and physiology.

The multi-hit hypothesis can be extended to incorporate data reported here.

Specifically, the genetic programming that drives the functional ontogeny of neural responding (Hit 1) opens up temporally constrained sensitive periods that encode rearing experiences (Hit 2), in turn biasing the patterning of nonapeptide systems such that social brain regions are tuned to the effects of additional dimensions of the early environment (Hit 3). In this way, experience-dependent developmental plasticity in social behavior is facilitated by the organizational effects of the complex early environment on nonapeptide circuitry. This framework highlights the critical importance of considering that the combined effects of multiple events during development could reflect additive, synergistic, or antagonistic interactions (Mueller,

2018). Furthermore, the developmental timing of the exposure event and the developmental timing of when outcome variables are measured must both be taken into consideration if we wish to fully understand the complex relationship between developmental experiences on phenotypes across the lifespan (Mueller, 2018).

Experience-dependent nonapeptide plasticity in parents

One of the most fascinating aspects of socially mediated developmental plasticity is that offspring themselves are active agents within their ontogenetic niche

(Schwab and Moczek, 2017). Prairie vole pups vocalize, approach their caregivers once they are capable of independent movement, and solicit interactions with conspecifics, thereby modifying their own experiences in a way that has consequences

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for their neural development (Alberts, 2008). Given the reciprocal interactions between developing pups and their caregivers, it is informative to consider the ways in which the rearing environment shapes the phenotypes of parents.

Although experiences exert more powerful effects on neural systems that are still being organized, adult brains are also plastic and retain the ability to adapt to new experiences (Fox et al., 2010). In chapter 5, I found that offspring age was a powerful determinant of parental behavior for both mothers and fathers. However, pup age- dependent changes in parental and non-parental behaviors were sex-specific, suggesting that mothers and fathers may vary in their sensitivities to pup age. The presence of the father and experimental handling conditions had surprisingly few effects on parents’ behavior inside of the natal nest, but father presence did appear to significantly impact the relationships between suites of behaviors and nonapeptide cell counts in hypothalamic subregions. These results provide evidence for experience- dependent plasticity in the structuring of parents’ brain-behavior relationships.

Functional consequences of neuroendocrine-behavioral plasticity?

Together the studies contained within this dissertation demonstrate that the trajectories of nonapeptide systems are mediated by early development, and nonapeptide systems retain some degree of experience-dependent plasticity in adulthood. Unlike pup age, experimental variation in rearing experiences did not result in robust alterations of parental behavior. Instead, the relationships between levels of parental care and vasopressin cell counts were modified in mothers and fathers.

What does it mean that physiological changes took place, but behavioral suites

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remained consistent? In such a case, it is possible that nonapeptide plasticity enabled consistency in care because of shifts in neural phenotypes. For example, the “dual- function hypothesis” for sex-differences in neuroendocrinology argues that sex- differences in the brain may drive sexually dimorphic behaviors, or prevent sex- differences in behavior by compensating for divergences in other physiological processes (De Vries, 2004). In a canonical example, male prairie voles have a significantly greater density of VP fibers in the lateral septum compared to females.

Gonadal steroids regulate the onset of pup-directed care in females (cued by hormonal changes associated with pregnancy), but do not appear necessary for male care. Thus, the hypothesis presents these results as evidence that the VP system of prairie voles in this brain area have evolved to compensate for the absence of parallel pregnancy- related alternations in gonadal hormones to promote parental behaviors in males (De

Vries, 2004). The dual-function hypothesis provides us with an empirical example of divergent neural phenotypes that support convergence in behavior.

Along this train of thought, it may be possible that our results on the experience-dependent reconfiguration of the relationships between VP cell counts and parental behavior describe another compensatory mechanism by which divergent neural phenotypes produce the same behaviors. If the rearing manipulations we used caused alterations in the physiology of other peptide or hormonal systems, perhaps compensatory changes in the transcriptional regulation of hypothalamic VP groups could have preserved parental care, thereby altering the association between these cell groups and parental behavior.

An influential hypothesis on the species-specific neural substrates of vertebrate

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social behavior theorized that social behaviors are not likely to be under the modular control of independent brain structures (Goodson, 2005; Newman, 1999). Instead, social behaviors are tied to changes in the patterns of network activity across six reciprocally connected brain structures that are rich in steroid receptors, and have all been tied to the expression of multiple social behaviors (Newman, 1999). This “Social

Behavior Network” (SBN) serves as a useful framework to conceptualize the idea of experience-dependent compensatory mechanisms of vasopressin for parental behavior

(Fig 6.1). Rather than patterns of activity across a network, we can envisage patterns of cell populations that dynamically shift in response to experiences to support functional equifinality in behavior.

More empirical evidence is required to test the hypothesis that neuroendocrine cell populations can compensate for alterations in other peptide groups to maintain important behaviors. However, several previous studies have reported similar experience-dependent shifts in brain-behavior associations, although these groups report activational neural data (Keesom et al., 2017; Ouyang et al., 2013; Ryder et al.,

2020). Given that my study (Chapter 5) describes only cell counts rather than neural responses, it would be prudent to utilize a measure of cellular activity or sample neuroendocrine release in future work to validate if group differences in cell counts are functionally relevant for variation in neural signals, and consequently, for behavior.

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Concluding remarks and future directions

Developmental plasticity allows for organisms to alter their phenotypes based on ontogenetic cues. The present thesis provides novel data on the functional maturation of the prairie vole brain and indicates where and when during the course of postnatal development experiences might begin exerting their organizational effects on the maturation of socially sensitive neural structures. I then examined the individual and combinatorial contributions of biotic and abiotic features of the early environment on the phenotypic trajectories of nonapeptide cell groups and their receptors, providing candidate neural substrates that may drive the variation observed in offspring behaviors. Finally, I determined that nonapeptide systems remain plastic in adult animals by demonstrating that parents undergo rearing experience-dependent patterning of vasopressin-parental care associations. Altogether, I conclude that prairie voles integrate contextual information across many dimensions of the environment and across the course of their lifespan, and that these experiences are encoded in the ongoing patterning of nonapeptide systems.

A fundamental premise of this interpretation is that varying amounts of stimulation of cell groups in early development induces differential gene expression of nonapeptide cells and receptors. In this way, experiences are encoded as long-term changes in the brain’s nonapeptide patterning in a manner that alters the probability of certain behaviors being expressed later in life. Thus far, the molecular mechanisms that translate social experiences into altered gene expression of oxytocin and vasopressin systems is underexplored. A recent study reported that maternal behavior impacts the methylation of regulatory regions of the prairie vole OTR gene, resulting

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in differential transcript and protein expression (Perkeybile et al., 2019). This finding provided the first concrete mechanism of care-mediated molecular alterations in the oxytocin system. Similarly, Kelly et al. (2020) demonstrated a similar mechanism of action, linking paternal care with DNA methylation of the vasopressin receptor gene within the lateral septum and its resulting impact on male offspring social approach.

Subsequent work must look at the physiological and transcriptional profiles of cell groups to determine more causal relationships between various stimuli and cell phenotypes. In conjunction, virally-mediated or gene editing strategies could be utilized to recapitulate neurochemical profiles in developing animals to determine if specific patterns of nonapeptide densities (peptide-producing cells and/or receptors) are sufficient to drive predicted behavioral phenotypes. Historically, advanced molecular tools have been unavailable for use in non-model organisms. However, these technologies are being actively developed for usage in prairie voles, which will enable their application to a rich array of developmental and evolutionary questions

(Horie et al., 2019).

Finally, we acknowledge that our data do not speak to the adaptive value of plasticity in nonapeptide systems, which is a critical component of understanding the evolution of plasticity. To address this point, future studies could subject prairie voles to multiple early-life manipulations and assess their reproductive fitness within semi- naturalistic enclosures. If group differences in fitness and neural phenotypes arose as a result of their early environment, we could begin to explore the conditions under which variation in phenotypes would be subject to selection pressures. By combining these questions of adaptivity with neuromolecular and developmental approaches, we

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come closer to formulating an integrated understanding of social behavioral plasticity

(Tinbergen, 1963).

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FIGURES

Figure 6.1 Schematic representation of patterns of activity across network nodes that control the expression of different social behaviors (left recreated from Goodson, 2005). On right, compensatory patterns of nonapeptide cell groups that vary by experience to drive the same social behavior.

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