The Pennsylvania State University. The Graduate School. Huck Institutes of Life Sciences

Transcription

1 The Pennsylvania State University The Graduate School Huck Institutes of Life Sciences SIBLING VARIANCE IN EARLY LIFE SOCIAL INTERACTIONS PREDICTS ADULT ANXIETY-RELATED BEHAVIOR AND PHYSIOLOGY IN RODENTS A Dissertation in Neuroscience by Christina M. Ragan 2011 Christina M. Ragan Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2011

2 The dissertation of Christina Marie Ragan was reviewed and approved* by the following: Sonia A. Cavigelli Associate Professor, Biobehavioral Health Dissertation Advisor Co-Chair of Committee Byron C. Jones Professor, Biobehavioral Health and Pharmacology Co-Chair of Committee David J. Vandenbergh Associate Professor, Biobehavioral Health Victoria Braithwaite Professor, Fisheries and Biology Tracy L. Langkilde Assistant Professor, Biology Ping Li Professor of Psychology and Linguistics Co-Chair, Huck Institutes of the Life Sciences, Neuroscience Graduate Program *Signatures are on file in the Graduate School

3 iii ABSTRACT Experiences early in infancy can influence offspring development of behavior and physiology in response to stress. During this early period, variance in maternal care has been shown to affect offspring stress responses in adulthood. In rodents, rat pups of dams categorized as high-licking, tend to be more exploratory and have a reduced stress hormone response to novel stimuli in adulthood compared to offspring of low-licking dams. In addition, pups will display maternal solicitation behaviors, such as perioral contact, that prompt the dam to initiate maternal behaviors. Several studies have examined the long-term effects of varying amounts of maternal-neonate interactions on offspring development across litters, but few have investigated these interactions and effects on siblings within a litter. In the work presented here, we found that interactions with a rodent dam during the first postnatal week vary within a litter and are related to sibling differences in stress and anxiety-related behavior and physiology in adulthood. In the first and second studies with outbred Sprague-Dawley rats and inbred Agouti viable yellow mice, respectively, we found that within a litter, high-licked pups received approximately twice as many licks as their low-licked, same-sex siblings. Pups that made more maternal solicitation behaviors received more licks than siblings that did not show these behaviors as often. In adulthood, offspring that had been frequently-licked as pups were slower to approach novelty than their low-licked littermates, which was the opposite of what previous research had shown between litters.

4 iv In the last study, we examined the relationships among infant-maternal interactions, adolescent and adult behavior, and behavioral and adult physiological responses to novelty and found sex-specific results. We measured mrna expression of glucocorticoid receptors (GR) in the prefrontal cortex and hippocampus and serotonin transporter (Sert) in the brainstem, both of which are involved in stress and anxiety. Adult females that received high amounts of maternal licking as neonates expressed higher GR mrna in the prefrontal cortex and lower corticosterone production in response to novelty compared to their low-licked sisters. Adult males that were slow to approach novelty at post-weaning and adulthood expressed lower Sert mrna compared to their more exploratory brothers. These data imply that the pathways involved in responding to novel stimuli may be sex-specific which is similar to the marked sexdependent responses to stress seen in humans. The studies using rodent models discussed in this dissertation have identified putative early-life predictors of adult sibling differences in responses to novel situations. Although our findings are correlational, they may shed light on possible causal relationships between the early environment and later anxiety behavior and physiology.

5 v TABLE OF CONTENTS LIST OF FIGURES... ix LIST OF TABLES... xi ACKNOWLEDGEMENTS... xii Dedication... xiv Chapter 1 Introduction and Significance... 1 Behavioral inhibition as a risk factor for anxiety disorders... 3 Behavioral inhibition is associated with maternal care... 4 Maternal care varies among siblings... 6 Neonate involvement in soliciting maternal attention... 7 Behavioral inhibition is associated with offspring HPA axis activity... 8 Behavioral inhibition and the serotonin transporter The serotonin transporter and early development Statement of the problem Methodology References Chapter 2 Within-litter variance in early rat pup-mother interactions and adult offspring responses to novelty Introduction Methods Animals Strain and Housing Maternal and Pup Behavior Offspring Responses to Novelty Statistical Analyses Results Within-litter variability in licking bouts received by pups Within-litter variability in neonate maternally-directed behavior Early postnatal maternal licking and neonate maternally-directed behavior and later offspring response to novelty Discussion Within-litter variance in maternal licking Within-litter variability in maternally-directed neonate behavior Within-litter variability in response to novel object and novel social arenas Processes involved in within- vs. between-litter variance Conclusion References Chapter 3 Maternal-neonate interactions and offspring responses to novelty and anxiety in Agouti viable yellow mice Introduction... 56

6 vi Between-litter differences in maternal-neonate interactions Within-litter differences in maternal-neonate interactions Methods Subjects Maternal-neonate observations Novel Object and Novel Social Arenas Elevated Plus Maze Statistical Analyses Results Within-litter variance in maternal licking received by neonates Within-litter variance in neonate maternally-directed behaviors Postnatal maternal licking, neonate maternally-directed behavior and offspring later exploratory and anxiety-related behavior Discussion Within-litter variance in maternal licking received by neonates Within-litter variance in neonate maternally-directed behaviors The relationship between maternal licking and neonate maternally-directed behavior and offspring exploratory and anxiety-related behavior References Chapter 4 Within-litter variance in physiological measures of responses to novelty and anxiety-related behaviors Introduction Methods Sprague-Dawley rat protocol Maternal-neonate interactions Offspring response to novelty Corticosterone measurements RNA isolation and Real-Time Polymerase Chain Reaction analysis of mrna expression Agouti mice protocol Statistical Analyses Results Postnatal maternal licking, and adult offspring Sert measures Sprague-Dawley maternal licking and offspring HPA-related mrna expression in adulthood Neonate maternally-directed behaviors and adult Sert expression Neonate maternally-directed behaviors and adult GR and Crhr1 mrna expression Rodent exploratory behavior at post-weaning and adult Sert expression Rodent exploratory behavior in adulthood and adult Sert expression Rodent exploratory behavior in adulthood predicts adult GR but not Crhr1 expression Discussion Postnatal maternal licking and adult offspring Sert mrna expression Maternal licking and offspring HPA mrna expression in adulthood Neonate maternally-directed behaviors and adult Sert expression Neonate maternally-directed behaviors and adult GR and Crhr1 expression

7 Rodent exploratory behavior at post-weaning and adult Sert expression Rodent exploratory behavior at post-weaning and adult GR and Crhr1 expression Rodent exploratory behavior in adulthood and adult Sert expression Rodent exploratory behavior in adulthood and adult GR and Crhr1 expression Sex differences in anxiety-related physiology Conclusion References Chapter 5 Synthesis and Conclusions Summary of findings Limitations Future Directions Conclusions References Appendix A IACUC Approval of Protocol # Appendix B IACUC Approval of Protocol # Appendix C Maternal/Pup Behavioral Observations Protocol and Descriptions of Behaviors Appendix D Exploration Arena Testing Protocol Appendix E Elevated Plus Maze Protocol Appendix F Pairwise comparisons of sex or genotype and licks received during the first half, second half, or total first postnatal week Appendix G Pearson correlations of the relationship between neonate maternally-directed behaviors and maternal licking during the first postnatal week Appendix H Linear regressions of the relationship between neonate maternal licking and maternally-directed behaviors and during the first postnatal week, and offspring exploratory behavior in adulthood Appendix I SERT mrna expression Protocol Appendix J Glucocorticoid and Crhr1 mrna expression Protocol Appendix K RNA gels to detect RNA quality Appendix L Pearson correlations of maternal licking during the first postnatal week and adult GR and Crhr1 expression vii

8 Appendix M Pearson correlations of neonate maternally-directed behaviors during the first postnatal week with adult Sert expression Appendix N Pearson correlations of neonate maternally-directed behaviors during the first postnatal week and adult GR and Crhr1 expression Appendix O ANOVA of exploratory behavior at post-weaning and adulthood and Sert expression in adulthood Appendix P Pearson correlations of exploratory behaviors at post-weaning and adulthood and adult GR and Crhr1expression viii

9 ix LIST OF FIGURES Figure 1-1. Conceptual Model Figure 2-1. Novelty Arenas Figure 2-2. Frequency of maternal licks received by individual pups (PND1-8) in four distinct litters Figure 2-3. Sex differences in total licking (body and anogenital) across PND Figure 2-4. Frequency of maternally-directed behavior by individual pups (PND 1-8) in four distinct litters Figure 2-5. Comparison of neonatal perioral contact to maternal licking rates during the first postnatal week Figure 2-6. Comparison of maternal licking rates and neonatal perioral contact during the first postnatal week to offspring latency to approach novelty in adulthood Figure 3-1. Novelty arenas Figure 3-2. Frequency of maternal licking for each pup during PND Figure 3-3. Frequency of maternal licking PND 2-8 in males vs. females Figure 3-4. Frequency of maternally-directed behavior performed by each pup during PND Figure 3-5. Linear regressions of anogenital licks and maternally-directed behaviors performed by neonates Figure 3-6. Approach latencies to novelty in adulthood and A) body licks PND 2-8; B) frequency of nipple attachment PND Figure 3-7. Approach latencies to enter the open arms of the elevated plus maze in adulthood relative to maternal and neonate behavior during PND Figure 3-8. Approach latencies to enter the open arms of on the elevated plus maze for black (a/a, black bars) vs. Agouti viable yellow siblings (Avy/a white bars) Figure 4-1. Novelty arenas Figure 4-2. Linear regression of postnatal maternal body licking and adult offspring Sert mrna expression

10 x Figure 4-3. Linear regression of maternal licking during the first postnatal week and glucocorticoid receptor mrna expression in adulthood Figure 4-4. Linear regression of maternal licks and CORT response to novelty in adulthood Figure 4-5. Linear regression of postnatal maternally-directed neonate behaviors and Sert expression in adulthood Figure 4-6 A,B. Linear regression of mean latency to approach novelty at post-weaning and adult Sert expression in adulthood Figure 4-6, C,D. Linear regression of mean latency to approach novelty at post-weaning and adult Sert expression in adulthood Figure 4-7 A,B. ANOVA of rat adult response to social novelty and adult Sert expression Figure 4-7 C,D. ANOVA of adult mouse response to social novelty and adult Sert expression Figure 4-8. Linear regression of adult mean response to novelty and adult hippocampal GR mrna expression Figure 4-9. Linear regression of adult latency to approach the open arm of the EPM and adult hippocampal GR mrna expression Figure 5-1. Modified Conceptual Model

11 xi LIST OF TABLES Table 4-1. Pearson correlations of maternal licking during the first postnatal week and adult Sert expression

12 xii ACKNOWLEDGEMENTS They say it takes a village to raise a child. I believe that it also takes a village to raise a graduate student. Here, I would like to thank the village that I am forever indebted to. Thank you to my intelligent, supportive, creative, ever-optimistic, tenured, adviser Dr. Sonia Cavigelli. You took me under your wing, and I thank you for giving me a chance. I could always count on you for a pep talk whenever I got into a funk. You challenged me intellectually, and I am a much stronger scientist for it. Thank you for spreading your enthusiasm and curiosity about animal behavior on to me. I d also like to thank my committee members: Dr. Byron Jones, Dr. David Vandenbergh, Dr. Victoria Braithwaite, and Dr. Tracy Langkilde. You all have given me great feedback over the years in your various areas of expertise and I thank you for your guidance. I d like to especially thank Dr. Byron Jones (yes, I remember that story) for his help over the past six years. You were my advocate from day 1 and I appreciate you believing in me. I also cannot thank you enough for bringing me to France to represent the Cavigelli lab after my third year. What a great experience that was, and I thank you for the opportunity. Dr. Vandenbergh, thank you for your endless amounts of knowledge of all things molecular. Thank you for always taking the time to talk with me about data, and I especially thank you for allowing me to use your lab for my assays and for sharing your animals with us. I could not have stuck it out this long without my graduate school friends. To my Neuro buddies, Alicia and Dave, I ve enjoyed being your mentor. Alicia, I ll miss our TV nights, and Dave, thank you for the comic relief. Lexan, I ve always enjoyed our Facebook chats, and I ll miss going to pizza seminar with you. Clayton, you provide much-needed snark, and Let us cling together as the years go by. To the ladies of GWIS, thanks for keeping science fun and being that extra bit of estrogen I needed. Finally, thank you to my Dinner Club buddies: Maryjo, Anne, Brian, Steve, Marcela, Charles, Josh, Matt, and Allison. We ve gone through a lot together, and it was always nice to be able to relax over one of my favorite things-free food! Anne, I especially appreciated our daily IM chats over coupons, grad school, and relationships. Maryjo, I m so thankful we ve grown so close, and I know we have a place to crash at each others houses if we ever need it. Karen and Lisa, you two have been a huge support for me through grad school. You have cheered for me along the way and it really helped me get through it all. Karen, you never fail to make me laugh, and I love that we can talk for hours (even when I m supposed to be working). Lisa, it s especially nice to have a close friend to laugh and cry about graduate school with. I know we ll always be there for each other. Thank you Cavigelli and Vandenbergh lab members past and present. I know that watching rodent mothers and her pups for hours might not have been how you envisioned doing science, but without your hours of dedication this dissertation would not have been possible. I d like to thank Mollie Woehling for her assistance, insight, and effervescent personality. I know

13 you ll make a great doctor! Joe Gyekis, thanks for your never-ending depth of knowledge and for providing assistance without hesitation. Kerry Michael, I m so glad that I had you by my side as my lab sibling in our cozy office space. I ll miss accidentally bumping our chairs together and our dissertation dance parties. Dr. Lori Francis and Dr. Sheila West, you two are my biggest advocates, besides my committee members, in Biobehavioral Health. Thank you for cheering me on when I got awards and thanks for coming to check on me every now and then as I worked on my big lab report. Thank you Mr. Gary Spinella, my 6th grade science teacher. When you met my mother on back to school night, you told her that the world is [my] oyster, and [I] can do anything. I ve never forgotten that and I thank you for those kind words. Thank you Pandora, Lady Gaga, and the Blackeyed Peas for the spontaneous afternoon dance parties to help get me out of a writing slump. I am very grateful for funding from: the Children Youth Family Consortium at Penn State, Women in Science and Engineering (WISE) Institute, Sigma Xi Scientific Research Society Grant-in-Aid, College of Health & Human Development Alumni Society Award, and the Doctoral Research Fellowship Application Incentive Award from the Graduate School. A huge thank you goes out to Tom McCrory. You were the unconditional support that was so greatly needed in graduate school. I can t thank you enough for being the voice of reason when times got tough. You encouraged me to stick with it, and I'm sure glad I did. I m so thankful we had each other to get through graduate school together, especially because we both could actually understand what the other was talking about (for the most part). Thank you for the laughter, tears, and love. And finally, thank you to my family. You always pushed me to pursue whatever I wanted to do. Mom, thank you for always saying, Things will work out for you. They always do. Dad, thank you for always giving me great advice whenever I felt stuck or needed to make a major decision. Thank you all for being proud of every accomplishment, no matter how big or small. You all supported me ever since I was little, and I cannot thank you enough for all of the love and encouragement. xiii

14 xiv Dedication I dedicate this dissertation to my nephews Luke Carlsen and Matthew Ragan. They bring out my maternal behavior in the best way, and I hope that they never lose their curiosity about the exciting world around them.

15 1 Chapter 1 Introduction and Significance Approximately 40 million Americans 18 and older suffer from anxiety disorders (Kessler, et al. 2005). Although there are genetic risk factors associated with anxiety disorders, siblings can greatly differ in their susceptibility to the disease (National Coalition for Health Professional Education in Genetics, 2004). Because of this discrepancy in concordance rates for anxiety, non-genetic influences, such as the early parental environment, must also play a role in the development of anxiety disorders (Stocker, Dunn, & Plomin, 1989; Eley, Liang, Plomin, Sham, Sterne, Williamson, & Purcell, 2004; Shanahan, McHale, Crouter, & Osgood, 2007a). In humans, there is strong evidence that the early developmental environment can heavily influence offspring susceptibility to anxiety and depression (Heim & Nemeroff, 1999; Heim & Nemeroff, 2001). Last century, seminal work determined that in mammals, including humans, early, secure maternal attachment is important for the emotional development of offspring (Harlow, Dodsworth, & Harlow, 1965; Bowlby, 1969). Another risk factor for adult anxiety that can be identified early in life is known as behavioral inhibition, or fearfulness in response to novel situations. The exact mechanisms that contribute to anxiety are not fully understood, and identifying the early influences behind the development of anxiety disorders is important for treatment of these disorders. Our research has several scientific goals to support the statement that experiences in early development predict sibling differences in adult exploratory behavior and physiology.

16 2 Here, we focus on individual differences in behavioral inhibition and anxiety-related behavior and physiology to determine relationships between early behavior and environment and adult anxiety-related physiology. Ultimately, we aim to clarify mechanisms behind sibling differences in behavioral inhibition and adult anxiety disorders. In this chapter, I will first review the literature on behavioral inhibition as a risk factor for anxiety disorders. Then I will present studies on maternal influences on offspring behavior and physiology related to anxiety. Next, I will review literature on stress and anxiety-related physiology including the hypothalamic-pituitary-adrenal (HPA) axis and serotonin transporter. Lastly, I will present a hypothesized model and highlight how my work will add to what is currently known about the relationships between early maternal-neonate interactions, subsequent offspring behavioral responses to novel situations, and physiology related to stress and responses to novelty. A behavioral trait during childhood associated with increased risk for adolescent and adult anxiety is behavioral inhibition (also known as shyness or fearfulness) indicated by withdrawal or avoidance in response to novelty (Kagan et al., 1999). Signs of inhibition are shown as early as 4 months of age (Kagan et al., 1994) and early interventions in the maternal environment may be effective in preventing anxiety disorders in adulthood (Tillfors, 2004). Several studies, with both humans and animals, have shown that early maternal interactions, particularly tactile stimulation, with young offspring are associated with later offspring behavior and physiology related to anxiety (human: Hane, Cheah, Rubin, & Fox, 2008; Rubin, Cheah, & Fox 2001; primate: Seay &

17 3 Harlow, 1965; Harlow, Dodsworth, & Harlow, 1965; rodent: Stanton & Levine 1990; Moore & Power 1992; Plotsky & Meaney, 1993; Caldji, Francis, Sharma, & Plotsky, 2000). In our laboratory, we have developed a rat model of behavioral inhibition (stable neophobia), and in this model have found significant within-family differences in the expression of inhibition and in maternal behavior toward siblings (Cavigelli & McClintock 2003; Ragan, Loken, Stifter, & Cavigelli, 2011). Behavioral inhibition as a risk factor for anxiety disorders Human studies suggest that behavioral inhibition in childhood is a strong risk factor of social anxiety in adulthood (Biederman et al. 1990; Kagan, 1994; Reeb- Sutherland et al., 2009). From previous studies, we know that human and primate behavioral inhibition can be a stable trait (humans: Kagan et al., 1987; Hirschfeld et al., 1992; primates: Kalin & Shelton, 2006), and is correlated with: (1) low or negative maternal care (human: Rubin et al. 2001; Hane et al. 2008; (2) increased stress hormone production (humans: Kagan et al. 1987; rat: Cavigelli and McClintock, 2003); and (3) specific serotonin transporter (SERT) alleles (human: Hariri et al., 2002; Fox et al. 2005). In a human study, 60% of children who were shy at age 1 were again categorized as behaviorally-inhibited at age 5 (Kagan et al., 1987). In addition, children who were stably behaviorally-inhibited at ages 21 months, 4 years, 5.5 years, and 7.5 years had higher rates of 2 or more anxiety disorders than unstably inhibited, stably uninhibited, or unstably uninhibited children (Hirshfeld et al., 1992). In 189 adults ranging from years, those who reported high childhood inhibition were more likely to meet the criteria

18 4 for a social phobia than those who had lower levels of childhood inhibition (Gladstone et al., 2005). Maternal reports of child stable behavioral inhibition are also a strong predictor of social anxiety later in life (Chronis-Tuscano et al., 2009). Kalin & Shelton (1989) found rhesus monkeys ages 5-7 months old showed similar levels of behavioral inhibition when presented with a strange human at two time points one month apart. In a previous study on Sprague-Dawley rats, we found that about 40% of our rats were either stably inhibited or stably uninhibited in response to novelty at two time points 4 months apart (Cavigelli et al., 2007). These studies of early behavioral inhibition as a predictor of later behavior towards novelty suggest that individuals that are susceptible to anxiety may be identified early in life. Behavioral inhibition is associated with maternal care Behavioral inhibition, in several species, is correlated with low or negative maternal care. In humans, low maternal care, defined as mothers who show low sensitivity (i.e. low awareness to, poor interpretation of, or inappropriate responses to infant signals) and are highly intrusive (pushing towards novelty), is associated with fearful responses and negative affect in infants compared to infants who received highquality maternal care (Ainsworth, 1976; Hudson & Rapee, 2005; Hane & Fox, 2006). In addition, maternal behavior that is unresponsive, intrusive, and derisive predicts shyness and behavioral inhibition in toddlers (Rubin, Cheah, & Fox, 2001). For juvenile capuchin monkeys, those that showed low play behavior expressed high corticosterone (CORT) and clung to their mother more compared to monkeys that showed more play

19 5 behavior (Byrne & Suomi, 2002). In rats, it has been well-documented that maternal care influences the development and behavior of the offspring and is associated with offspring HPA axis activity. Behaviorally, rat pups of mothers that show high licking/grooming and arched-back nursing (LG-ABN) show less anxiety-like behavior in novelty tests than pups of low LG-ABN mothers (Francis et al., 1999). Adult offspring of high LG-ABN dams spent more time exploring the inner area of an open field versus adult offspring of low LG-ABN mothers (Caldji et al. 1998). In addition to long-term behavioral consequences, adult offspring of high-licking mothers also show a decreased physiological response to stress than low-licked adult offspring (Francis et al, 2000). Physiologically, offspring of high LG-ABN mothers have increased glucocorticoid receptors in the hippocampus and prefrontal cortex (Kaffman and Meaney, 2007), decreased corticotropin releasing hormone (CRH) receptors in the locus coeruleus (Francis et al., 1999), lower CRH mrna expression in the central amygdala (Diorio and Meaney, 2007), and lower levels of plasma adrenocorticotropic hormone (ACTH) and CORT in adulthood than pups of low-licking mothers, which suggests that offspring of high-licking mothers have a more efficient negative feedback system in response to stress. Stanton and Levine (1990) reported that maternal presence affects HPA axis activity in developing pups with CORT levels significantly lowered in pups that had contact with an anesthetized dam in a novelty stimulus response test compared to those exposed to novelty without mother. Early maternal separation and neonate handling leads to both behavioral and physiological changes in rat pups. Behaviorally, pups become more fearful and less exploratory in novel environments after long periods of maternal separation ( min/daily for 14 days) (Caldji et al., 2000). In addition,

20 6 pups that experience long-term maternal separation, show increased hypothalamic CRF mrna expression, and decreased negative feedback resulting in a more enhanced stress response vs. non-separated pups (Plotsky & Meaney, 1993). On the other hand, daily, brief handling (15 min/day) is beneficial because handled pups develop long-term decreased stress reactivity into adulthood (Levine et al., 1967). Decreased CRF expression in the hypothalamus and the amygdala coupled with increased HPA negative feedback are all advantageous effects from handling (Meaney et al., 1988). These studies, as well as several others, stress that adequate maternal care is important for offspring stress-related behavior and physiology. Maternal care varies among siblings Mothers of several different species will invest attention in some offspring more than others (Stocker et al., 1989; Clutton-Brock, 1991; Moore, Cohn, & Campbell, 1997; Kivijärvi, Räihä, Kaljonen, Tamminen, & Piha, 2005; Eley, et al., 2004; Shanahan, McHale, Osgood, & Crouter, 2007). In primates, mothers will treat male and female offspring differently, especially in threatening situations. For instance, when presented an unfamiliar mother and infant of the same species, mothers of infant macaque females will shield the females away from threat, while mothers of infant males may threaten their own infants (Mitchell and Brandt, 1970). Additionally, depending on resources available, rhesus macaque mothers are known to invest more maternal attention in females rather than male offspring (Maestripieri, 2001). In outbred Sprague-Dawley rats, we observed within-litter differences in both maternal licking and in neonate behaviors

21 7 towards the dam (Cavigelli, Ragan, Barrett, & Michael, 2010). This variance in maternal care among offspring may better prepare offspring for highly variable and unpredictable environments, ultimately increasing maternal fitness (Crump 1981; McGinley et al. 1987; Phillipi & Seger, 1989; Dziminski & Roberts, 2006; Crean & Marshall, 2009). In addition, variance in maternal care can be adaptive when offspring may later be presented with stressful situations allowing offspring to be resilient to the potentially stressful environment (Boyce & Ellis 2005; Fries, Hesse, Hellhammer, & Hellhammer, 2005). Neonate involvement in soliciting maternal attention Research by Harlow, Levine, Bowlby, Hofer, and Stern have shown 1) the importance of maternal-infant attachment and its long-term effects on the developing offspring; and 2) that neonates perform specific behaviors to receive maternal attention (Harlow et al., 1965; Harlow & Suomi 1971; Stern, 1997; Polan & Hofer, 1999; Polan, Soo-Hoo & Hofer, 1997; Polan et al., 2002). The central issue that has yet to be addressed about maternal licking behavior is the extent to which the offspring and the mother influence the direction of these early interactions. Stern suggests that it is the pups that induce the dam s licking behavior (Stern, 1997). This is highly supported through experiments where virgin rats and male rats display maternal behavior, like pup retrieval and licking, despite any maternal hormonal prompts (Stern, 1997). Stern also notes that tactile contact seems to be one of the most important directors of maternal licking (Stern, 1996). Lesions of the trigeminal orosensory branches (which innervate the snout) indicate that the greater the amount of severed branches, the lower the amount of

22 8 mouth-related behaviors the dam will perform. Additionally, using a muzzle on the dam to block snout contact prevents nursing, although the dam will try to contact the pups (Stern and Johnson, 1989). Additional research also supports the idea that pups perform certain behaviors that elicit maternal attention. For instance, neonates will place themselves in positions that allow close maternal contact in order to thermoregulate and to nurse (Polan & Hofer, 1999; Soo-Hoo & Hofer, 1997; Polan et al., 2001). In addition, pups will make ultrasonic vocalizations to elicit maternal attention (Shair, Masmela, Brunelli & Hofer, 1997; McFarland, 2008). Because pups perform behaviors that can trigger maternal behavior onset, this suggests that both offspring and the mother may play a role in differences in maternal care. Behavioral inhibition is associated with offspring HPA axis activity The Hypothalamic-Pituitary-Adrenal (HPA) axis is activated during stress and is responsible for regulating the release of stress hormones. Normally, in response to stress, the HPA axis releases the stress hormones, Corticotropin Releasing Hormone (CRH), Adrenocorticotropic hormone (ACTH) and corticosterone (CORT), and stops producing them when CORT is detected via the negative feedback loop. However, anxious individuals show prolonged CORT production as a result of a dysfunctional negative feedback loop (Gómez et al., 1998; Plotsky et al., 1998). Once CORT binds to GR in the cytosol, the receptor undergoes a conformational change and is able to pass through the cell s nuclear membrane. After entering the nucleus, the complex can bind to DNA and/or proteins and triggers genomic action. The epigenetic interaction between stress

23 9 response genes and environment shape individual differences in behavior found in response to stressful situations (Sapolsky and Meaney, 1986; Gross and Hen, 2004; Fox et al. 2005). At the neuroendocrine level, behavioral inhibition in humans and primates and consistent low exploratory behavior in rats is associated with increased HPA axis and sympathetic activity (humans: Kagan et al., 1987; primates: Kalin et al., 2000; rats: Cavigelli & McClintock, 2003; Cavigelli et al., 2007). Behaviorally-inhibited children show elevated cortisol levels at baseline and in response to novelty and stress, and they also have overall higher heart rates, compared to behaviorally-uninhibited children (Fox et al., 2005; Kagan et al., 1987). Similarly, inhibited rhesus monkeys have higher basal CRF levels compared to non-inhibited monkeys (Kalin et al., 2000). Likewise, male rats that show low exploratory behavior in a complex novel setting also have higher CORT response at baseline and in to novelty compared to their neophilic brothers (Cavigelli &McClintock, 2003; Cavigelli et al., 2007). On the other hand, rat high responders (HR) that have been categorized as novelty-seekers when placed in a simple novel situation, surprisingly show higher, more prolonged levels of CORT and tend to be the most active, based on their locomotion, compared to low responders (LR) (Piazza et al., 1991; Dellu et al. 1996; Kabbaj, et al., 2000). It is thought that HRs are more aggressive and impulsive, whereas the LRs show more anxiety and depression-like behavior which may explain the differences in CORT secretion (Stedenfeld et al., 2011) Based on what is known about neuroendocrine responses and behavioral inhibition, I chose to investigate if early life responses to novelty relates to adult sibling differences in CORT production

24 and mrna expression of targets related to the stress response. Below is a description of the specific genes involved in stress-related processes that I examined. 10 Behavioral inhibition and the serotonin transporter In addition to the HPA axis physiology, behavioral inhibition is also associated with the serotonin transporter (Slc6a4, SERT). SERT is a protein responsible for reuptake of serotonin into the presynaptic terminal. Dysfunctional SERT can lead to excess serotonin in the synapse which is one hypothesis for the mechanisms that result in anxiety and depression behavior. Variations in the SERT promoter (5-HTTLPR) are involved in predicting vulnerability for depression, anxiety, and neuroticism in humans (Mann et al., 2000; Caspi et al., 2003; Schmitz et al., 2007; Dick et al., 2007; Canli and Lesch, 2007). The short (s) allele, which has fewer repeats (14) in the genetic sequence, results in lower gene transcription compared to the long (l) form (16 repeats). The first association between the SERT promoter polymorphism and reduced SERT function was shown in vitro using cells transfected with either the short or long form of the SERT promoter (Lesch et al., 1996). The cells transfected with the long form showed increased serotonin uptake compared to the cells transfected with the short form. Several researchers have found this association in vivo with affective disorders in humans and non-human primates, although the presence of the short allele alone was not enough to increase depression risk (primates: Suomi et al., 2000; humans: Anguelova et al., 2003; Caspi et al., 2003; Hoefgen et al. 2005). Because various research have reported mixed

25 11 findings regarding SERT and depression and anxiety, more research is required to understand the exact relationship between the two. In most studies that examine the SERT promoter polymorphism and depression outcomes, the short allele is most closely associated with depression risk. Individuals homozygous for the short alleles in the SERT promoter region with high exposure to stress during childhood/adolescence showed increased depression rates and increased stress hormone production to stress (Caspi et al., 2003). Furthermore, girls with two copies of the short SERT promoter alleles with a family history of depression, showed increased and prolonged cortisol production in response to stress vs. girls with at least one long allele (Gotlib et al., 2008). Children two short alleles had lower SERT transcription, lower serotonin uptake, and increased amygdala activation to fearful adult faces (Hariri et al., 2002). Additionally, children homozygous for the short allele, who also had low social support, had an increased risk for behavioral inhibition at age 14 months and 7 years (Fox et al., 2005). Although the connection between the short allele of the SERT promoter and its association with depression risk has been documented, several researchers have claimed that the connection between the two is modest and requires further investigation (Du, Bakish, & Hrdina, 2000; Caspi et al., 2003; Gillespie et al., 2005; Kendler et al., 2005; Kaufman et al., 2004; Lemogne et al., 2011). Although this polymorphism in the SERT promoter is not found in rodents, SERT knockout rodents show higher anxiety, decreased social behavior, and behavioral inhibition (Olivier et al, 2008; Kalueff et al., 2009). Male SERT knockout mice (-/-) that did not show initial behavioral differences from wild types on the elevated plus maze

26 12 (EPM) did spend less time in the open arms of the EPM and the center of the open field after the resident intruder test, suggesting that SERT knockouts show decreased exploratory behaviors compared to the wildtypes (+/+) and that these differences are related to a social context (Jansen et al., 2010). In the light/dark transition test, SERT knockouts had the longest latency to approach the lit compartment followed by SERT heterozygotes (+/-) and then wild types. Lastly, SERT -/- show higher thigmotaxis in the open field compared to the SERT wild types indicating that SERT knockout are less exploratory. Physiologically, male SERT knockout mice from a C57/BL6 background have alterations in their HPA function compared to wild type littermates. SERT -/- express lower baseline CRH in the periventricular nucleus of the hypothalamus, show higher baseline Crhr1 function and density in pituitary, and a lower baseline GR in the hypothalamus, pituitary, and adrenal cortex compared to their SERT +/- and SERT +/+ littermates. In response to the EPM, animals missing at least one SERT allele show an increase in GR in the hypothalamus compared to their siblings with both SERT alleles intact (Jiang et al., 2008). In this same study, the SERT knockout mice also showed increased ACTH production in response to the EPM vs. the wild types, yet they had lower baseline CORT than wild types suggesting that they might have a hypoactive HPA axis (Lanfumey et al., 2000). In other words, the SERT knockouts might not show higher CORT responses to stress compared to their SERT intact littermates because their baseline levels start much lower than their siblings. Because it is clear that Sert expression is related to phenotypes associated with behavioral inhibition, one goal of this

27 thesis was to determine if natural variance in maternal-neonate interactions or exploratory behavior relates to adult variance in Sert expression. 13 The serotonin transporter and early development SERT is associated with mechanisms related to offspring development early in life. Macaques that were peer-reared, as opposed to maternally-reared, and had the long/short genotype, had lower serotonin metabolite levels in the cerebrospinal fluid during early development compared to peer-reared macaques with two long alleles (Bennett et al., 2002). Maternally-reared macaques with the short allele showed no effect on serotonin metabolic levels, indicating a strong gene-environment interaction for this effect. It has become more acceptable that the short allele is associated with increased affective disorder risk, if paired with a stressful life event (Caspi et al., 2003; Gillespie et al., 2005; Kendler et al., 2005; Kaufman et al., 2004; Lemogne et al., 2011). Several researchers hypothesize that the connection between the SERT promoter polymorphism and anxiety and depression is due to low levels of serotonin in early development in individuals who carry the short allele which potentially alters later serotonin levels, and as well as the levels of other neurotransmitters involved in emotion regulation. Specifically, it is thought that the serotonin transporter gene is highly expressed in emotion centers of the brain early in life, and transiently later, suggesting that it has a more relevant role during early brain development (Hansson et al., 1998, 1999). Although SERT activity is not a direct part of the HPA axis, serotonergic neurons from the dorsal raphe nucleus project excitatory inputs to brain structures like the

28 14 hippocampus, amygdala, periventricular nucleus of the hypothalamus, and prefrontal cortex, which are involved in the stress response, thus playing a role in the initiation of the stress cascade (Feldman, Conforti, & Melamed, 1987; Chen et al. 1992; Hensler, Ferry, Labow, Kovachich, & Frazer, 1994; Dinan, 1996; Ziegler & Herman, 2002). In addition, experiments with serotonin agonists will also increase plasma CRH and ACTH levels (Siever, Murphy, Slater, de la Vega, & Lipper, 1984; Beavan & Scanlan, 1992). Additionally, corticosteroid receptors in serotonergic neurons in the raphe nucleus and glucocorticoid availability can modulate SERT function (Kinnally, Lyons, Abel, Mendoza, & Capitanio, 2008). Removal of the adrenal gland results in decreased serotonin levels in HPA and dorsal raphe, and injection of corticosterone restores this depletion (Gerlach & McEwen, 1972; de Kloet, Versteeg, & Kovacs, 1983) Serotonergic projections to HPA-related structures and corticosteroid receptors in serotonergic neurons suggest a bi-directionality of SERT activity and the HPA (Glatz, Mössner, Heils, & Lesch, 2003). Statement of the problem The research that I have conducted and that I will discuss in this dissertation used novel approaches to fill in the gaps in research on the long-term effects of maternalneonate experiences early in life. While most studies that observe the long-term consequences of maternal behavior have analyzed behavioral and physiological outcomes of litters as a whole, my work examines these effects within the litter taking each individual pup into account following them from postnatal day 1, continuing through

29 15 peri-adolescence, and ending in adulthood. Our longitudinal studies provide a more clear idea of the developmental trajectory associated with sibling differences in exploratory behavior and physiology, whereas most studies examine the neonatal environment, and then do not observe the animals again until adulthood. Additionally, researchers tend to examine either maternal behavior or pup behavior, however our research focuses on not only maternal behavior, but also behaviors that the pups performed that elicited maternal attention. In all of the studies reported here, we observe natural variations in maternalpup interactions closely without greatly manipulating the maternal environment conditions. The current rodent model of behavioral inhibition categorizes animals based on freezing and ultrasonic vocalizations (Takahashi, 1994), however we have a variety of longitudinal measures to classify animals as high vs. low exploratory. Lastly, here we report maternal-neonate interactions in both outbred Sprague-Dawley rats as well as, for the first time, inbred Agouti viable yellow mice. All together, our findings suggest a novel way to examine sibling differences in not only the early environment with the mother, but also throughout different stages of development, making a more complete model. Understanding causes and consequences of early behavioral inhibition may better provide insight for preventative tools for the treatment of anxiety disorders.

30 16 Pre-adult phenotypes and early-life experiences Maternal solicitation behavior Adult phenotype Sert, GR expression Maternal care Crhr1 expression CORT secretion Post-weaning/Adolescent Latency to novelty Anxiety-like behavior & Latency to novelty Environment Physiology Offspring Behavior Positive correlation Negative correlation Figure 1-1. Conceptual Model Methodology To observe mechanisms early in life that may relate to adult anxiety-related behavior we used several different methods. First, to examine early maternal experiences, we observed rodent offspring with their mothers during the first week of life and recorded both maternal and neonate behavior. Then we used our current rodent model for human behavioral inhibition to measure exploratory behavior in novel situations. These methods mimic those used to identify behaviorally-inhibited young

31 17 children in unfamiliar environments. To measure early life responses towards novelty, animals are exposed to either physical novelty (rodent-sized objects) or social novelty (an unfamiliar rodent of same sex and age) in a novel arena at different developmental stages of life. Later in adulthood, we exposed the animals to the novelty arenas again, and then tested them on the elevated plus maze which is a classic measure of anxiety-related behavior. After exposure to the novel object test in adulthood in rats, we collected blood to analyze the stress hormone corticosterone. Finally at sacrifice, we collected brain tissue to quantify mrna expression in the brainstem, prefrontal cortex and hippocampus, which are target brain regions that are associated with the stress response and emotion regulation. We then quantified glucocorticoid receptors (GR) and corticotrophin releasing hormone receptor 1 (Crhr1) in the hippocampus and prefrontal cortex (which are both associated with the stress response cascade); and serotonin transporter (Sert) expression in the brainstem which is associated with mood and anxiety and depression susceptibility. It is important to conduct these longitudinal studies that range from birth to adulthood to understand the development of adult anxiety-like behavior. Finding early predictors at the individual level, whether they are behavioral, environmental, or physiological, can help find potential risk factors for later development of anxiety disorders. This dissertation reports the investigation of putative behavioral (behavioral inhibition), physiological (CORT production, GR, Crhr1 and Sert expression), and environmental (neonate/maternal interactions) correlates of adult anxiety-like behavior in rodents. The first study (Chapter 2) measures the relationship between maternal behavior and early behavioral inhibition and later anxiety-like behavior and CORT production in

32 18 Sprague Dawley rats. The second study (Chapter 3) analyzes the relationship between maternal behavior, early behavioral inhibition and adult anxiety-like behavior in the inbred Agouti viable yellow mouse strain. The last study (Chapter 4) examines the relationship between maternal behavior, early behavioral inhibition and later anxiety-like behavior, and GR, Crhr1, and Sert expression in the Sprague-Dawley and Agouti viable yellow mice cohorts from the previous chapters. In this dissertation, I examine early environmental and behavioral processes associated with preweanling neophobia and adolescent/adult anxiety-related behavior and physiology in outbred Sprague-Dawley rats and inbred Agouti viable yellow mice. More specifically, I discuss the relationship between maternal behavior and behavioral inhibition among siblings and differences later in life in stress and anxiety-related physiological processes. This work contributes to understanding anxiety and its related behavior and physiology in three ways: 1) predicting adult anxiety-related behavior and physiology according to early experiences and phenotypes; 2) suggesting ways to experimentally manipulate early social interactions that may alter later anxiety; and 3) integrating brain chemistry and behavior to elucidate the mechanisms behind anxiety. Determining the relationships between early behavior and environment and adult physiology can help clarify the mechanisms underlying the relationship between early behavioral inhibition and adult-anxiety disorders.

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41 27 Chapter 2 Within-litter variance in early rat pup-mother interactions and adult offspring responses to novelty Introduction The underlying mechanisms of anxiety are currently unknown and animal models can provide valuable insight into the biological and environmental influences of this disease. Anxiety disorder concordance rates for monozygotic vs. dizygotic twins suggest that both genetic and non-genetic factors contribute to the disease (National Coalition for Health Professional Education in Genetics, 2004). Some known non-genetic risk factors that can alter an individual s susceptibility to anxiety disorders include differences in social interactions that occur early in development. For instance, studies have shown that siblings can be quite different in temperament and in their susceptibility to diseases like depression, and that parental behavior can play a key role in these differences (Stocker, Dunn, & Plomin, 1989; Eley, Liang, Plomin, Sham, Sterne, Williamson, & Purcell, 2004; Shanahan, McHale, Crouter, & Osgood, 2007). Many animal studies have investigated the influences of early maternal behaviors on adult offspring anxiety-related behaviors of litters as a whole, but few have quantified the amount of within-litter variance in motheroffspring interactions and the relation of this variance to behavioral variance among adult siblings. The goal of the current study was to test the hypotheses that, neonate-maternal interactions vary among rodent siblings, and that this variance is associated with adult sibling variance in fear-related exploratory behavior.

42 28 Several studies, with both humans and animals, have shown that early maternal interactions with young offspring are related to later offspring behavior and physiology (human: Hane, Cheah, Rubin, & Fox, 2008; Rubin, Cheah, & Fox 2001; primate: Seay & Harlow, 1965; Harlow, Dodsworth, & Harlow, 1965; rodent: Stanton & Levine 1990; Moore & Power 1992; Plotsky & Meaney, 1993; Caldji, Francis, Sharma, & Plotsky, 2000). In rats, mother-pup interactions during the first week of life predict adult offspring behavior and physiology, with maternal licking of pups being a highly-studied behavior. Maternal licking/grooming arched-back nursing (LG-ABN) peaks during the first few days after birth and then decreases after the first eight days (Fleming & Rosenblatt, 1974; Caldji et al., 1998). During this first week, differences between high and low LG-ABN mothers are prominent and emerge around postnatal days 3-4 (Fleming & Rosenblatt, 1974; Caldji et al., 1998). On average, adult offspring of high LG-ABN mothers show reduced physiological stress responses, more exploratory behavior, and less anxiety-like behavior compared to those of LG-ABN mothers (Caldji, Tannenbaum, Sharma, Francis, Plotsky, & Meaney, 1998; Francis, Caldji, Champagne, Plotsky, & Meaney, 1999; Weaver, Champagne, Brown, Dymov, Sharma, Meaney & Szyf, 2005). Experimental manipulations of maternal licking have led to similar results. Decreased rat maternal anogenital licking leads to decreased male offspring exploratory behavior and increased play behavior in social tests during adolescence, compared to adolescent males of control mothers (Birke & Sadler, 1987; Moore & Power, 1992). Female rat pups that were artificially-reared and received artificial anogenital licking by a paintbrush became low-licking dams as adults compared to control offspring (Gonzalez, Lovic, Ward, Wainwright, & Fleming, 2000; Lovic & Fleming, 2004). These previously-reported

43 29 long-term effects suggest further investigation of early maternal care and later offspring behavior is necessary to fully understand this relationship. This mother-offspring relationship works both ways. In addition to the evidence that maternal behavior can influence offspring behavior, there is ample evidence that neonate behavior directed towards the mother influences maternal behavioral responses (Rosenblatt, 1969; Stern, 1997). For example, tactile contact with active pups stimulates maternal behavior in virgin female rats and male rats (Stern, 1996). Furthermore, if pups are anesthetized and/or do not display appropriate solicitation behaviors, such as rooting and suckling, mothers will not engage in nursing-related behaviors like crouching (Stern & Johnson, 1990). Additionally, if a rat mother cannot sense pup contact on her snout either through lesions of the trigeminal orosensory branches (which innervate the snout), or a muzzle that blocks direct snout contact then she engages in less mouth-related maternal behaviors and nursing (Stern, 1996; Stern & Johnson, 1989; Polan, Milano, Eljuga, & Hofer, 2002). Thus, early natural variance in sibling neonate behavior directed toward the mother could potentially stimulate different levels of maternal attention from the same dam towards different offspring. In our laboratory, we have developed a Sprague-Dawley rat model of behavioral inhibition observed as stable neophobia. Previously, we reported relatively large withinlitter differences in neophobia between same-sex siblings a difference that remains stable into adulthood (Cavigelli & McClintock 2003; Cavigelli, Yee, & McClintock, 2006). Thus, these rats provide a good system to examine within-litter variance in early social interactions and its relationship to adult sibling behavioral variance. In the current study, we observed maternal licking and neonate behavior in Sprague-Dawley rat litters

44 30 to quantify the within-litter variability in maternal and pup behavior during the first week of life, and related these early behaviors to adult sibling variance in behavioral responses to both novel objects and social novelty. The aims of this study were to determine: (1) the variance in frequency of maternal licks toward pups within a litter during the first postnatal week, and if this within-litter variance is comparable to variance in licking rates documented among different dams (Caldji et al., 1998; Champagne et al., 2003), (2) the amount of variance within a litter in neonate initiated perioral contact with their mother, (3) if neonates that show more frequent perioral contact receive more maternal licking compared to siblings that show less contact, and (4) if within-litter variance in maternal licking and/or neonate maternally-directed behavior is predictive of later variance in adult sibling exploratory behavior. Methods Animals Strain and Housing Adult Sprague-Dawley rats (4 female, 4 male) were purchased from Charles River Laboratories (Wilmington, MA) and bred to produce 4 litters. All animals were maintained on a 12L:12D lighting schedule (lights on at 0800h) in a room maintained at 23 C and approximately 50% humidity, with food and water available ad libitum. Rats were housed in solid bottom cages (43.5 X 23.5 X 20.5 cm) that were cleaned weekly by trained animal facility personnel. To minimize intrusions, maternal cages were not

45 31 changed during the first postnatal week. Breeder rats were weighed and handled daily for at least one week after arrival in the laboratory and prior to breeding. The selection of mates was random and a male was placed with a female in a cage for at least 8 days for breeding, after which females were housed individually and monitored for birth. From the four resulting litters, 68 pups (33 females, 35 males) were born with the day of birth defined as postnatal day 0 (PND 0). Litters had pups each (n = 15, 17, 17, 19) and litters were not culled to observe normal within-litter variation in maternal and neonate behaviors. At PND 25, animals were weaned and placed in cages consisting of 2-3 samesex rats per cage. All methods used in this study were approved by the Pennsylvania State University Institute for Animal Care and Use Committee (Appendix A). Maternal and Pup Behavior To identify individual pups during the first week of life, pups were given a unique mark on their backs, head, and belly with a black Sharpie TM marker (Sanford Corporation, Oak Brook, IL) at the same time daily because maternal licking would fade the marks. These marks allowed us to identify individual pups as they received maternal licks and contacted the dam s snout. The procedure involved less than one minute of daily pup handling. During this time, the mother was placed in a new cage away from her pups for 15 minutes to ensure equal maternal separation time for all pups. This handling process increases mean anxiety-related behavior of offspring in adulthood, but it does not influence the variance in this behavior among siblings (Cavigelli, Ragan, Barrett, & Michael, 2010). We observed maternal and neonate behavior 8 times /day

46 32 across 7 consecutive days, from 1500h on PND 1 to 1300h on PND 8. Each maternal cage was located on an individual cart that allowed observers to clearly observe and record all maternal and neonate behaviors directed towards the dam as they occurred and to easily identify individual neonates by their unique marks. To increase accuracy, behavioral coding was conducted live rather than from videotapes. Thirty-minute observations were conducted primarily during the light phase (0900h, 1100h, 1300h, 1500h, 1700h, and 1900h) with 2 observations in the dark phase using red lights to allow visibility without providing an aversive stimulus to the rats (0700h, and 2100h). During each 30-minute observation, we recorded the frequency and type of licking (body vs. anogenital) the dam gave each neonate (Champagne et al., 2003; Appendix B). Each litter was observed for a total of hrs, with a total of 212 observations across all four litters. To track each offspring from birth to adulthood, each pup was given a unique ear punch after the maternal observations were complete, that corresponded to the individually-unique neonate marks. Offspring Responses to Novelty In adulthood (PND 68-82), we tested offspring responses to a novel object and a novel social arena that were designed to reduce anxiety-provoking stimuli such as white light and open space that are often found in tests of exploratory behavior (Cavigelli, Stine, Kovacsics, Jefferson, Diep, & Barrett, 2007, Appendix D). These arenas mimic tests used to identify behaviorally-inhibited, or shy, children (Kagan et al., 1987). The arenas are 120 cm X 120 cm (L x W) with 46 cm-high white polypropylene walls and a

47 33 clear plastic cover. To create the novel object arena, three of the four corners had a novel rat-sized object placed 13 cm from the arena walls (Fig. 2-1A). The novel social arena had the same dimensions as the novel object arena, but had 2 cages placed in the arena: one empty and one with an unfamiliar rat of equivalent age, sex, and size (Fig. 2-1B). Each litter was exposed to a different novel rat, however each rat within a litter experienced the same sex-matched novel animal. In both tests, animals were placed into a clean ceramic bowl with 5 cm high walls and lowered into the empty arena corner. Rats were videotaped for 5 minutes with a camera placed 1.5 m directly above the arena. Rats were removed immediately after testing, and the ceramic bowl rinsed with tap water and dried prior to introducing the next test animal. Test order in each arena was balanced, such that if a rat was one of the first of its littermates tested in the novel object arena, then it would have been one of the last tested in the novel social arena. In the novel object arena, we measured the latency to approach the first novel object, and in the novel social arena we measured latency to approach the novel rat. A B Figure 2-1. Novelty Arenas. (A). Novel object arena: novel object in 3 corners, home bowl in 1 corner. (B). Novel social arena: novel rat in one corner, empty cage in opposite corner, home bowl in third corner.

48 34 Statistical Analyses Because rates of body and anogenital licking were relatively low in the litters (i.e. < 1 lick/ pup / hour of observation, and the number of body and anogenital licks to each pup were correlated (r = 0.36, n = 68, p < 0.01) we present the results of analyses on combined body and anogenital licks. We also conducted separate analyses for body and anogenital licks and present these results when they differ from those of the combined licks analyses. Paired t-tests were used to compare rates at which male and female pups received maternal licking, neonate perioral contact, and adult approach latencies. These analyses of maternal and neonate behavior were conducted separately for the first and second half of the week because of prior research reports changes in maternal behavior and neonate behavior across the first postnatal week (e.g. Rosenblatt, 1969; Champagne, Curley, Keverne, & Bateson, 2007). To control for litter effects, same-sex means of licking, perioral contact, and approach latencies were calculated for each litter and these means used in paired t-tests. To determine if neonate perioral contact related to the frequency of maternal licking, and to determine if licking or perioral contact predicted approach latencies in adulthood, we used regression analyses, including neonate weight on PND 1-2 as a predictor. Prior studies have shown that neonate weight is related to neonate huddling, (Bautista, García-Torres, Prager, Hudson, & Rödel, 2010) and may predict maternal licking rates (Cavigelli et al., 2010). Litter and sex effects were controlled by centering variables to the litter mean for same-sex siblings. Approach latencies were skewed right so we used log-transformed values in statistical analyses.

49 35 There were no systematic differences in maternal licking or neonate behavior rates relative to litter size. Results Within-litter variability in licking bouts received by pups During PND 1-8, there was considerable variance in the frequency with which dams licked each pup. Within sex in each litter, maternal licking was quite variable with the most-licked pups receiving 2-3 times as many licks as the least-licked pups (Fig. 2-2). In the first days after birth (PND 1-4), there were no differences in licking rates between male and female pups (anogenital licks: t 3 < 1, p < 0.62; body licks: t 3 < 1, p < 0.53; Fig. 2-3). During the latter half of the week (PND 5-8) males received significantly more anogenital licking, but not body licking, than female pups (anogenital licks: t 3 = 3.29, p < 0.05; body licks: t 3 = 2.23, p < 0.11; Fig. 2-3).

50 Sum of Licks / Hrs Observed Sum of Licks / Hrs Observed Sum of Licks / Hrs Observed Sum of Licks / Hrs Observed Litter A Females Males 1.8 Litter B Individual Pups 0 Individual Pups 1.8 Litter C 1.8 Litter D Individual Pups 0 Individual Pups Figure 2-2. Frequency of maternal licks received by individual pups (PND1-8) in four distinct litters. Light grey: females; Dark grey: males.

51 Mean Body and Anogenital Licks/Hrs. Observed PND1-2 PND2-3 PND3-4 PND4-5 PND5-6 PND6-7 PND Males Females Postnatal Day Figure 2-3. Sex differences in total licking (body and anogenital) across PND1-8. Light grey: females; Black: males Within-litter variability in neonate maternally-directed behavior Sibling maternal perioral contact was also variable in the four litters. During PND 1-8, pups that engaged in the most contact did so 3 times more often than pups that were less-frequently engaged in perioral contact (Fig. 2-4). This range between the pup that made the most perioral contact vs. the pup that made the least contact was evident in both sexes and across all four litters. There was no sex difference in rates of perioral contact behaviors during PND 1-4 or 5-8 (t 3 < 1, p < 0.97; t 3 = 0.74, p < 0.51, respectively). Female (but not male) pups that frequently made perioral contact with mother during PND 1-8 were licked more often than pups that made less contact, even

52 Perioral Contact/Hrs. Observed Perioral Contact/Hrs. Observed Perioral Contact/Hrs. Observed Perioral Contact/Hrs. Observed 38 after controlling for neonate weight (females: r = 0.51, n = 33, p < 0.003; males: r = 0.20, n = 35, p < 0.25; Fig. 2-5) Litter A Females Males 0.35 Litter B Individual Pups 0 Individual Pups 0.35 Litter C 0.35 Litter D Individual Pups 0 Individual Pups Figure 2-4. Frequency of maternally-directed behavior by individual pups (PND 1-8) in four distinct litters. Litter B: 3 female animals never made perioral contact during observations. Light grey: females; Dark grey: males.

53 Licks/Hrs. Observed Litter A Litter B Litter C Litter D Perioral Contact/Hrs. Observed Figure 2-5. Comparison of neonatal perioral contact to maternal licking rates during the first postnatal week. Pups that frequently displayed snout contact were licked more often by the mother than pups that displayed these behaviors less frequently. Symbols indicate litter and separate regression lines are plotted for each litter. Early postnatal maternal licking and neonate maternally-directed behavior and later offspring response to novelty There was no sex difference in adult offspring approach latencies in either novel arena (object: t 3 < 1, p < 0.39; social: t 3 < 1, p < 0.48). Within a litter, female (but not male) pups that were more-frequently licked by mother were slower to approach a novel object in adulthood than were pups that were less-frequently licked (females: r = 0.38, n = 33, p < 0.03; males: r = 0.16, n = 35, p < 0.35; sexes combined: r = 0.27, n = 68, p < 0.05 Fig. 2-6A). Pups that made frequent perioral contact with the mother were slower to approach a novel object in adulthood than pups that made less perioral contact (females:

54 40 r = 0.29, n = 33, p < 0.09; males: r = 0.27, n = 35, p < 0.12; sexes combined: r = 0.28, n = 68, p < 0.02; Fig. 2-6B). When maternal licking and perioral contact were simultaneously used as predictors of adult responses to novelty the standard coefficient for maternal licking was comparable to that for perioral contact (βs = 0.19 vs. 0.21). There was no significant relationships between maternal licking or perioral contact and adult offspring approach latencies in the novel social arena (r = 0.00, n = 68, p < 0.97; r = -0.02, n = 68, p < 0.91, respectively).

55 Latency to Physical Novelty in Adulthood Latency to Physical Novelty in Adulthood 41 A Litter A Litter B Litter C Litter D Licks/Hrs. Observed B Litter A Litter B Litter C Litter D Perioral Contact/Hrs. Observed Figure 2-6. Comparison of maternal licking rates and neonatal perioral contact during the first postnatal week to offspring latency to approach novelty in adulthood.

56 (A) Frequently-licked pups were slower to approach physical novelty in adulthood than pups that were less-frequently licked. (B) Pups that frequently displayed snout contact were slower to approach physical novelty in adulthood than pups that displayed these behaviors less frequently. 42 Discussion In this study, we documented variation in maternal- and pup-directed behavior within a litter, and found that these differences in the first week after birth carried over to affect the behavior of siblings when they reached adulthood. We found a 2-3 fold difference in the frequency that individual pups within a litter were licked by the dam, and in the frequency in which they engaged in maternally-directed behavior during the first week of life. Rat mothers differentially licked pups within the same litter, and this difference was differed with the sex of the pup with sons being licked more than daughters PND 4-8. In addition, we found that pups within a litter differed in their displays of perioral contact, and that pups that made frequent perioral contact with the dam were more likely to receive licks than pups that performed this behavior less often. Finally, both maternal licking and neonate perioral contact during the first week of life predicted offspring responses to novel objects in adulthood. Sibling neonates that received frequent licking from mother and made frequent perioral contact were slower to approach novelty (objects or animals) as adults than pups that received fewer licks or made less contact.

57 43 Within-litter variance in maternal licking There was a 2-3-fold difference in the frequency of maternal licking among samesex siblings within litters. This degree of licking variance within litters was similar to that previously documented across litters (Caldji et al. 1998; Champagne et al. 2003). In these prior studies, high-licking mothers licked pups an average of 6 times/hr (distributed over approximately 5-18 pups) while low-licking mothers licked an average of 3 times/hr. In the current study, the range of licking rates across mothers was times/hr across four females, and the range of licking rates across individual pups within litters ranged from licks/pup/hr for the least variable litter and licks/pup/hr for the most variable litter. Based on these initial examinations, it appears that the range of within-litter variance in maternal licking may be as great as the range among litters. This degree of variation within families suggests further examination of both maternal and neonate processes that drive this variation. Studies in humans and other species suggest that mothers differentially invest in their offspring (Stocker et al., 1989; Eley, et al., 2004; Clutton-Brock, 1991; Shanahan, et al., 2007a; ; Moore, Cohn, & Campbell, 1997; Kivijärvi, Räihä, Kaljonen, Tamminen, & Piha, 2005). From an evolutionary perspective, variation in maternal investment within families/litters/clutches may provide a means to increase phenotypic diversity within a family unit, where genotypic diversity is constrained and future environmental conditions for the offspring could be highly variable and unpredictable (Crump 1981; McGinley et al. 1987; Dziminski & Roberts, 2006; Crean & Marshall, 2009). Behaviorally-diverse offspring will increase the likelihood of surviving to reproductive age since, as a unit, the

58 44 litter will have a flexible response to environmental changes (Phillipi & Seger, 1989). This type of within-litter variance in behavior is known as diversified bet-hedging, which increases mean fitness in subsequent generations by using a don t put your eggs all in one basket approach within generations (Phillipi & Seger, 1989). Mothers may purposefully vary the amount of maternal care given to different offspring in order to ensure the reproductive success of as many of their offspring as possible. Alternatively, within-family variance in maternal behavior may not lead to a simple increase in phenotypic diversity in a reproductive unit (e.g. family), but may in fact also be a response to pre-existing phenotypic variance within the unit. In other words, the offspring may be pre-disposed to be more solicitous or less solicitous towards the mother and she is simply responding to their cues. Within-litter variability in maternally-directed neonate behavior Like maternal licking behavior, neonate perioral contact behavior directed towards the mother also varied within litters. The most-solicitous pups made perioral contact 3 times as often as the least-solicitous pups during PND 1-8. Furthermore, there was a positive linear relationship between the frequency that pups contacted their mother s snout and the frequency of maternal licking during the first week of life. These results suggest that maternal licking variance within a litter is related to within-litter variance in neonate behaviors that are thought to elicit maternal behavior in general (Stern & Johnson, 1989 & 1990). Individual differences in offspring maternally-directed behaviors might emerge relatively quickly after birth. Neonates do not simply respond to

59 45 mother s cues, but rather, bi-directional communication exists between mother and offspring in several different species through vocalization, touch, chemical release, or visual cues (reviewed in: Kilner & Johnstone, 1997; Stern, 1997). In primates, behaviors initiated by the offspring, like mutual gaze and reciprocal body contact between mother and offspring, suggest that neonates influence maternal behavior (Ferrari, Paukner, Ionica, & Suomi, 2009). There is variance in maternally-directed behaviors among siblings, and it may be driven by competition between siblings for maternal resources (Kilner & Johnstone, 1997). For example, in Pied Flycatchers, nestlings that were in close proximity to parents and begged more often were more likely to be fed than nestlings that were farther away (Gottlander, 1987). Mothers needing to spend as little energy as necessary when feeding offspring is one possible explanation to account for this pattern of mothers delivering more attention to offspring that are close to them or display more active solicitation behavior (Gottlander, 1987). Our data showing that pups with a high frequency of perioral contact are more likely to receive licks than pups that did not perform these behaviors provide further support for these ideas. Furthermore, based on our findings, proximity to mother was important in successfully receiving maternal care only if actual snout contact was made, whereas simply being close to the mother s snout may not have been enough to elicit a response from her thus requiring tactile stimulation but still conserving maternal energy.

60 46 Within-litter variability in response to novel object and novel social arenas It is well-known that maternal behavior during the first week of life affects offspring behavior later in life. In cases of extreme neglect, like experiments with socially-isolated infant rhesus monkeys, offspring had hypothalamic-pituitary-adrenal desensitization and social and cognitive impairments (Harlow et al., 1965; Harlow & Suomi 1971). Similar effects have been seen in rat pups reared completely isolated from their mothers from postnatal day 3 onward (Lovic & Fleming, 2004). Furthermore, naturally high licking/nursing mothers produce adult offspring that show less anxietyrelated behavior than low licking/nursing mothers (Caldji et al., 1998; Francis et al., 1999; Weaver et al., 2005). In the current study, we showed that natural variation in maternal care within families, independent from neglect and between litter variance in maternal behavior like the studies mentioned above, is also associated with adult offspring behavior. In response to novelty, high-licked pups in a litter were slower to approach novel objects as adults compared to low-licked pups. As previously mentioned, others have found between-litter effects of high levels of maternal licking with an increase in exploratory behavior in adulthood compared to offspring of low-licking dams (Caldji, et al., 1998), yet we found exactly the opposite within litters. Our findings indicate that rat mothers differentially lick their offspring and that this differential licking is related to differential sibling behavior towards the mother during the first week of life. The current results are comparable to those from a prior study (Cavigelli et al., 2010), and suggest that variance in maternal behavior may have different functional roles when considered at the within- vs. between-family levels. Within-litter variance in maternal

61 47 licking may reflect differential maternal responses to different neonate sibling needs, whereas between-litter variance in maternal licking may reflect different maternal strategies that lead to average differences of offspring across families. Early-life interactions between a dam and her pups were significantly related to later offspring behavioral responses to novel objects in adulthood. We found no relationship between maternal licking nor neonate behavior and offspring response to social novelty in adulthood, suggesting that the relationship between early motherneonate interactions and adult offspring response to novelty is context-specific. This relationship is similar to results of studies that examined the effect of artificially manipulating licking bouts pups received on adolescent social behavior (Birke & Sadler, 1987; Moore & Power, 1992). In the future, experimental manipulations of maternal interactions with her pups could determine whether this effect is context-specific, and the role of early social experiences in predicting adult responses to a novel object vs. social novelty. Processes involved in within- vs. between-litter variance It is important to note the processes involved in relating maternal-neonate interactions to adult offspring behavior may differ at the between-family versus withinfamily levels. We note that the different results are not necessarily a contradiction. The previous between-litter findings were based on litter-to-litter differences where the mother's average behavior was correlated with the litter's average behavior (Champagne et al., 2003). The results of the current study are based on within-litter variance,

62 48 comparing maternal behavior toward individual pup siblings with the pups' subsequent individual outcomes in adult responses to novelty. It is widely known that betweengroup correlations can be different from within group-correlations, and are often reflective of different processes driving these relationships. When comparing among groups, often the means of the group are compared and do not account for individual differences within the group, whereas in our analyses, we centered each variable for an individual to that animal s same-sex litter mean. In other fields, failing to distinguish between-group and within-group processes has been referred to as the ecological fallacy (Robinson, 1950). Molenaar (2004) has challenged psychologists to consider the different implications of between-person variance and within-person variance, and his work includes evidence that the co-variation of personality dimensions might differ at the population and individual levels. The results of the current study lend further support to the notion that within- vs. between-group behavioral variance is a result of different processes and that these different kinds of variance may also predict different behavioral and physiological consequences to stressful situations. Given the current and prior studies showing that within-family variance in maternal behavior is related to within-family variance in neonate and adult sibling behavior, further areas of investigation include determining the conditions that affect differential maternal investment in offspring of the same family and the consequences of this variance. Recent studies suggest that mothers with ample resources will invest more in her more needy offspring, whereas mothers with limited resources will preferentially invest in her more hardy offspring (e.g. Bugental & Beaulieu, 2003; Beaulieu & Bugental, 2008; Bugental et al. 2010; Gottlander, 1987). Given these prior findings and

63 49 theory, it may be that in the laboratory environment, where maternal resources are high (guaranteed food and water daily, comfortable temperatures, no exposure to predators, etc.), dams provide more support to the neediest pups (which may be indicated by the frequency of snout contact behavior) as compared to their less needy pups because they can afford to take this somewhat risky strategy to increase overall litter survival. In this case, maternal investment in needy offspring may diminish anxiety-related symptoms in these pups, but may not obliterate the behavioral tendency in these pups compared to pups that make less snout contact with the mother. Further study is required to determine the environmental influences on mother-infant dynamics within a family and the potential long-term consequences of these interactions. Conclusion Our findings show that rat pups and mothers vary in their behavior within a litter, and that this variation is related to variation in offspring anxiety-related behavior in adulthood, however, the driving forces behind these differences are unknown. Withinlitter variance in maternal and pup behavior could provide a system to study epigenetic factors involved in the development of anxiety-related behavior in adults. We know that maternal care is important for offspring development, although the degree of optimal care has not been fully determined. In humans, caregivers protective behavior (like shielding from novelty) and intrusive behavior (like pushing towards novelty) have been associated with the development of anxiety in behaviorally-inhibited children (Hudson & Rapee, 2005). For example, when two year olds were overprotected by mothers when exposed

64 50 to medium to high-threat novelty, expression of fear increased in these children compared to children of mothers who showed intrusive or no behaviors in response to their distress (Kiel & Buss, 2009). Additionally, higher maternal protectiveness during low threat predicted higher rates of childhood anxiety at ages 2 and 4 in these toddlers. Toddlers who showed more distress in response to low and medium threat were also at high risk for anxiety, especially if mothers showed intrusive or overprotective behaviors. The animal model described here provides a method to investigate within-family variance in early interactions between mother and offspring, and the processes that lead to and result from this variance. These mechanisms and processes may be quite different from those involved in the development of non-sibling behavioral variance. Investigating family social dynamics during early development may clarify mechanisms involved in differential sibling behavior in adulthood.

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70 56 Chapter 3 Maternal-neonate interactions and offspring responses to novelty and anxiety in Agouti viable yellow mice Introduction Siblings may be raised in similar environments and share several common genes, however their personalities can be very different. Interactions with parents can influence sibling differences not only in personality but also, in humans, susceptibility to mental health-related outcomes (Stocker, Dunn, & Plomin, 1989; Eley, Liang, Plomin, Sham, Sterne, Williamson, & Purcell, 2004; Shanahan, McHale, Crouter, & Osgood, 2007). Sibling differences in stress-related outcomes have also been shown in animal models. For example, Cavigelli & McClintock (2003) found significant sibling differences in exploratory behavior and corticosterone response to novelty in adult outbred Sprague- Dawley rats. The questions that remain involve whether sibling differences in behavior and physiology are due to parental interactions, intrinsic sibling differences present early in life, or even sibling interactions among themselves. Given these notable differences in sibling behavior and stress physiology, the aims of the current study were to examine early maternal-neonate interactions during the first postnatal week and the possible relationship between these interactions and later offspring exploratory behavior in adulthood using an inbred mouse strain to observe how much sibling variance results from environment vs. genetics.

71 57 Between-litter differences in maternal-neonate interactions Variance in maternal interactions in the early postnatal environment can influence offspring behavior and physiology in adulthood and this variance is evident across different mothers within multiple species. Primate studies have shown that male and female infants are treated differently by mothers. For example, when presented an unfamiliar mother-infant duo of the same species, mothers of infant macaque females tend to be the protectors shielding the females away from potential threat, while mothers of infant males are punishers threatening their own infants. These maternal differences towards the two sexes may influence how these animals behave as adults (Mitchell and Brandt, 1970). Additionally, rhesus macaque mothers will invest more maternal care in females rather than male offspring depending on resources available for infant survival (Maestripieri, 2001). Rodent studies have also show that the early maternal environment can influence offspring behavior and physiology later in life. In rodents, pups of high-licking moms tend to, as a whole, be more exploratory in an open field and secrete less corticosterone in response to stressors in adulthood compared to pups of low-licking dams (Francis, Caldji, Champagne, Plotsky, & Meaney, 1999; Diorio & Meaney, 2007). On the other hand, pups born of high-licking mothers that are crossfostered to low-licking mothers show increased anxiety-related behavior than compared to offspring cross-fostered to high-licking mothers (Francis et al., 1999). In addition to the early postnatal environment, the prenatal maternal environment can also influence

72 58 offspring behavior later in life. In one study, pregnant heterozygous serotonin transporter knockout (+/- 5-HTT) dams were repeatedly exposed to the bedding of an unfamiliar male which poses as a potential threat for offspring (Heiming, Janesen, Lewejohann, Kaiser, Schmitt, Lesch, & Sachser, 2009). In late adolescence, offspring were tested on anxiety-related behavioral tests, like the light-dark test, and showed more anxiety-related behavior compared to the offspring of +/- 5-HTT dams that were not exposed to male bedding during pregnancy. Clearly, the maternal environment has long-term effects on the stress-related behavior of their offspring. Significant differences observed in maternal care may be related to differences in sibling behavior during the first postnatal week and need to be investigated more closely. Specific pup behaviors like suckling and perioral contact can influence the onset of maternal licking and nursing behaviors by directing maternal attention towards the pups (Stern, 1996; Stern & Johnson, 1989; Polan Milano, Eljuga, & Hofer, 2002). Blocking tactile stimulation from the pups will prevent the initiation of these maternal behaviors (Stern & Johnson, 1990). Olfactory cues from the dam attract pups to the dams ventrum allowing pups to orient and attach to a nipple to nurse (Hofer, Shair & Singh, 1976). Furthermore, nipple attachment is important for the survival of the neonates, as Hofer and colleagues showed that if the infraorbital branch of the trigeminal nerve is lesioned in 7 day-old rat pups that had previously been nursing, they fail to attach to the nipple and ultimately die within 6 days of the procedure (Hofer, Fisher, & Shair, 1981). In-depth analyses of the relationship between maternal licking given by the dam, maternallydirected behaviors by the neonates, and offspring behavior later in life may elucidate the long-term consequences of these behaviors.

73 59 Within-litter differences in maternal-neonate interactions In previous studies with outbred Sprague-Dawley rats, we observed within-litter differences in both maternal licking of the neonates and in neonate maternally-directed behaviors such as perioral contact (Cavigelli, Ragan, Barrett, & Michael, 2010; Ragan, Loken, Stifter, & Cavigelli, 2011). Specifically, we found that within a litter, the highestlicked rat pups received 2-3 more licks than the lowest-licked same-sex rat pups during the first postnatal week (Ragan et al., 2011). The highest-licked pups also made the most perioral contact with the dam compared to their same-sex siblings. Most notably, the high-licked pups were also the slowest to approach a novel object in adulthood. Our results had shown that within-litter measures were the opposite of what was found between litters, in that we had found that the animals that sought and received the most maternal attention, were the least exploratory animals in their litter. In previous experiments, high-licked litters, as a whole, were more exploratory than low-licked litters (e.g. Francis et al., 1999). Because we saw differences in the maternal environment across outbred siblings, we investigated variance in maternal-neonate interactions in inbred Agouti viable yellow (A vy ) mice to determine if similar variability in maternal licking and neonate behavior exists in inbred rodents. To our knowledge, this is the first documentation of maternal behavior in this strain. The A vy are unusual due to the fact that even on an inbred genetic background, several different fur phenotypes exist (Dickies, 1962). The A vy strain was

74 60 first observed as a spontaneous mutation in the C3H/HeJ mouse strain and later bred onto a C57BL/6 background. It was later found that a retrotransposon insertion near the Agouti gene responsible for coat color phenotype yields these different phenotypes (Argeson, Nelson, & Siracusa, 1996). This dominant mutation causes ectopic expression of agouti protein (an endogenous melanocortin receptor antagonist) resulting in yellow fur, as well as an increased risk for obesity, diabetes, and tumors (Duhl, Vrieling, Miller, Wolff, & Barsh, 1994). Depending on the patterns of expression regulated by methylation, coat colors can range from completely yellow, to mottled, to golden brown (pseudoagouti), yet they all have the A vy /a genotype, (Morgan, Sutherland, Martin, & Whitelaw, 1999). At all other loci, A vy /a mice are genetically identical to their black (a/a) littermates. Of particular relevance to this study is the action of the ectopic agouti product on receptors normally affected by a paralogue, called agouti-related protein (AgRP), which is expressed in the brain and adrenal cortex known areas involved in exploratory behavior (Harris, Zhou, Shi, Redmann, Mynatt, & Ryan, 2001). Previous studies have shown that the experimental overexpression of agouti protein in A vy mice causes a heightened stress response, which is an additional reason for selecting the Agouti mice for this study. Specifically, after restraint stress, mice over-expressing Agouti showed higher corticosterone levels, spent less time in the open arms of the elevated plus maze (EPM), and spent less time in the light side of the light-dark box compared to black mice, which suggests that the A vy mice express an anxious-like phenotype (Harris, et al., 2001; Bazhan, Shevchenko, Karkaeva, Yakovleva, & Makarova, 2004). Because of the unusual physical characteristics of the A vy mice and for the known differences in anxiety-related behavior within a litter of Agouti viable yellow

75 61 (A vy /a) and black (a/a) littermates, this rodent model was suitable for our analyses to observe within litter variance in both maternal-neonate interactions and offspring adult exploratory behavior in an inbred strain. In Chapter 2, we documented within-litter variance in maternal licking of neonates, neonate behavior towards the dam, and offspring behavior in adulthood in Sprague-Dawley rats. To determine if sibling variance in exploratory behavior is related to within-litter variance in maternal behavior, neonate behavior, and offspring behavior in adulthood in inbred Agouti mice, we collected data to answer the following questions: 1) Do mothers of inbred mice differentially lick their pups within a litter? (Do we see similar variation in inbred mice as we observed in Sprague-Dawley rats?) 2) How much do maternally-directed behaviors by mouse neonates vary within a litter of inbred pups? 3) Do maternally-directed behaviors by neonate siblings predict rates of individual maternal licking? 4) Do maternally-directed behaviors and/or maternal licking predict differential sibling exploratory behavior in adulthood? and 5) Does maternal licking, neonate behavior, and/or exploratory behavior differ between two Agouti phenotypes (yellow vs. black)? Understanding the variance in early life experiences and consequent adult exploratory behavior of inbred animal models may reflect the importance of environment and help to explain the differences in anxiety behavior reflected in humans.

76 62 Methods Subjects Heterozygote Agouti viable yellow mice (gift from the Jirtle Lab, Duke University to Dr. Vandenbergh at PSU) acclimated for 1 week and bred for two generations. The mice were housed in shoebox cages on a 12:12 hour light dark cycle (lights on at 0800h). Room conditions were 24ºC with 50% humidity and food and water were available ad libitum. Four distinct breeder pairs produced 4 litters of 36 Agouti mouse pups total (3 died before adolescence) for a total of 16 males, 17 females; 15 Agouti viable yellow, 18 black, 0 pseudo Agouti). The coat color phenotype is driven by mother s phenotype passed through the germline (e.g. a yellow dam is likely to have more yellow offspring than mottled or pseudoagouti). To control for maternal imprinting effects, all the mothers in this study were yellow and mated with black males (Argeson et al., 1996; Harris et al. 2001). Litters at birth had between 6-11 pups (n = 6, 9, 10, 11). To examine natural within-litter variance in pup-maternal interactions in the Agouti viable yellow mouse strain, litters were not culled. On postnatal day (PND) 28, pups were weaned and housed with same-sex littermates in groups of 2-4 animals per cage. All methods received approval from the Pennsylvania State University Institute for Animal Care and Use Committee (Appendix C).

77 63 Maternal-neonate observations 60-minute observations were conducted 4 times/day (0900h, 1300h, 1700h 2100h; 3 during light phase, 1 during the dark) during PND 2-8 (date of birth = PND 0). We included analyses from PND 2-8 because observations were missing from PND 1 from one litter. Each litter was observed for hours, with a total of 108 observations across all four litters. Between PND 4-6, the coat color emerges due to a switch from melanocytes producing eumelanin to producing phaeomelanin resulting in a yellow coat (Argeson et al., 1996). To identify individual pups within litters, pups were marked daily on their dorsum, head, and ventrum with a non-toxic silver Sharpie marker (Sanford Corporation, Oak Brook, IL) to ensure visibility for both black and yellow pups. After this marking process, each pup was weighed making the total time of handling less than 60 seconds. The mother was removed from pups for 15 minutes in a separate cage to ensure equal separation time for all pups. Brief maternal separation is a natural occurrence in the wild; for example, rat mothers will separate from the litter for brief periods to forage for food before returning to the pups (Leon, Croskerry, & Smith, 1978; Jans & Woodside, 1990). Daily, short-term handling of both mice and rats, increases maternal attention upon return (Fenoglio, Chen, & Baram, 2006; Cirulli, Capone, Bonsignore, Aloe, & Alleva, 2007), dampens offspring response to stressors, and decreases overall offspring anxiety-like behavior in adulthood (Bell, Nitschke, Gorry, & Zachman, 1971; Meaney, Diorio, Francis, Widdowson, LaPlante, Caldji, Sharma, Seckl, & Plotsky, 1996; Francis et al. 1999; Pryce, Bettschen, & Feldon, 2001), however we

78 64 have previously found that it does not affect within-litter variance observed in anxietyrelated behavior (Cavigelli et al., 2010). We conducted daily observations to record variance in pup-maternal interactions during the first week of life (Appendix B). For each 60-minute observation, we recorded the frequency of neonate licking (body and anogenital licking) from the dam (Champagne et al., 2003). We also recorded the frequency of the following neonate maternallydirected behaviors that are known to solicit maternal attention to document neonate behavioral variance as well as maternal variance within litters (Stern & Johnson, 1990): location related to dam s snout (either near the snout or far from the dam s snout, defined as anterior or posterior to dam s forelegs), individual pup ventral probing (pup pushes on maternal ventrum usually seen prior to nipple attachment to nurse), perioral contact (pup contacts dam s snout), and nipple attachment (how often a pup was seen attached to a nipple or unattached). Ventral probing and perioral contact were recorded as they occurred during the 60-minute observations. Pup location and nipple attachment were recorded at 3-minute intervals (point samples) throughout the 60-minute observations. Each observation had two observers, one primarily dictated the behaviors occurring and the other recorded those behaviors. Novel Object and Novel Social Arenas At postweaning (PND 30-34) and in young adulthood (PND 64-70), animals were tested on two novelty arenas to observe their exploratory behavior towards novelty during

79 65 the dark phase at 2100h (Appendix D). The arenas for mice are 50 cm X 50 cm with 24 cm-high white polypropylene walls and a clear plastic cover. In the novel object arena, three of the four corners had a novel mouse-sized object (a plastic tunnel, an upside down ceramic bowl, and a right side up plastic bowl) placed 13 cm from the arena walls (Fig. 3-1A). The novel social arena had the same dimensions as the novel object arena, but had 2 cages placed on their side in the arena: one empty and one with a vented top with an unfamiliar mouse of equivalent age, sex, and size (Fig. 3-1B). Because stimulus and test animals needed to be age-matched, each litter was exposed to a different novel mouse, however each mouse within a litter experienced the same sex-matched novel animal. In previous studies with rats, we have never found an effect of the stimulus animal, test day, or order of testing within each test day on the test animal s latency to explore the novel social animal (Cavigelli et al., 2007; Cavigelli et al., 2009). In both tests, animals were placed into a clean metal bowl and lowered into the empty arena corner. Mice were videotaped for 5 minutes with a camera placed 1.5 m directly above the arena. Mice were then removed immediately after testing, and the metal bowl rinsed with tap water and dried prior to introducing the next test animal. Test order in each arena was balanced, (i.e. if a mouse was one of the first of its littermates tested in the novel object arena, then it was one of the last tested in the social arena). In the novel object arena, we measured the latency to approach the first novel object as defined by nose touching object, and in the novel social test we measured latency to approach the novel mouse cage as defined by the first nose touch.

80 66 A B Figure 3-1. Novelty arenas. A) Novel object arena: novel object in 3 corners, home bowl in bottom left corner. B) Novel social arena: novel mouse in top left corner, empty cage in bottom right corner, home bowl in bottom left corner. Elevated Plus Maze In young adulthood (PND 60-61), mice were tested on the elevated plus maze to observe anxiety-related behavior (Appendix E). The elevated plus maze was made of Plexiglas and consisted of two open arms (30 cm X 5 cm X 15cm) and two closed arms covered in black construction paper (30 cm X 5 cm X 15 cm) and was raised 38.5 cm above the floor. Mice were placed on the center area facing a closed arm and were video recorded for 5 minutes with a camera placed 1.5 m directly above the apparatus. After testing, the maze was cleaned with 70% ethanol and allowed to dry. In the elevated plus maze, we observed the latency and frequency to enter the open arms relative to the closed arms. An entry was defined by the presence of all four paws on the arm. Exploratory behavior on the novelty arenas and anxiety-like behavior were coded using Noldus Observer version 5.0 (Wageningen, Netherlands).

81 67 Statistical Analyses The number of body and anogenital licks received by each pup were not correlated (r = 0.22, n = 36, p < 0.21), although we had found a correlation in the Sprague-Dawley rat study, so for our analyses, we presented outcomes with body and anogenital licks separate, as well as with two types of licks combined (Cavigelli et al., 2010). The lack of correlation between licks may be a result of small sample size and low power for this study. Paired t-tests were used to compare maternal licking and neonate behavior during the PND 2-8 observation period between male and female pups within each litter. Because maternal licking peaked around PND 5, we separated analyses between PND 2-4 and PND 6-8. To determine if the frequency of maternally-directed pup behavior related to the frequency of maternal licking and/or neonate behavior, and to determine if maternal licking predicted latency to novelty in adulthood, we used regression analyses, including neonate weight (PND 1&2) as a predictor variable because in previous studies neonate weight has been shown to be related to neonate behavior inside the huddle (Bautista, García-Torres, Prager, Hudson, & Rödel, 2010). In these regressions, litter and sex effects were controlled for by centering all variables to the family mean for same-sex siblings and maternal identity was included as an independent variable to control for litter effects. Because latencies to approach novelty and to approach the open arms of the elevated plus maze were skewed right, these values were log transformed to achieve normally-distributed data for statistical analyses, however in the figures we show the non-log transformed, uncentered values for clarity purposes. We did observe differences in licking rates between the litters, however there was no

82 68 significant difference in licking between the smallest litter and the largest. The number of subjects available for analyses of maternal-neonate interactions is 34 due to two animals dying during the first postnatal week. One additional animal died after the maternal-neonate interactions, but before weaning. Analyses of exploratory behavior on novelty arenas have a sample size of 29, due to a loss of data for 4 animals. For the elevated plus maze, data for all 33 remaining animals were available. Results Within-litter variance in maternal licking received by neonates We observed within-litter variance in total maternal licking received by the neonates during the first postnatal week. Across all four litters, the highest-licked pups within each mouse litter received times as many licks during the first postnatal week compared to the least-licked same-sex siblings (Fig. 3-2). The 3 pups that died received between 1.5 and 2 licks/hour observed (not shown). This variance in licking was not a result of pup color or sex (Appendix F). There was no difference in maternal licking received between black and yellow siblings within a litter, regardless of whether we analyzed licking during the first vs. the latter half of the week or the entire week, therefore our analyses do not control for pup color (PND 2-4: body licks: t 3 < 1, p < 0.94; anogenital licks: t 3 < 1, p < 0.70; PND 6-8: body licks: t 3 < 1, p < 0.50; anogenital licks: t 3 = 2.52, p < 0.08; body and anogenital licks PND 2-8: t 3 < 1, p < 0.84). Male pups did not receive more licks on average than female pups, (PND 2-4: body licks: t 3 <

83 69 1, p < 0.63; anogenital licks: t 3 = 1.80, p < 0.17; PND 6-8: body licks: t 3 < 1, p < 0.65; anogenital licks: t 3 = 1.43, p < 0.25; Appendix F, Fig. 3-3). Licking rates were highest during the light phase compared to the dark phase (not shown). Figure 3-2. Frequency of maternal licking for each pup during PND 2-8. The highest-licked pups within each mouse litter received approximately times as many licks during the first postnatal week compared to the same-sex lowest-licked pups (white: females; gray: males; striped pups died pre-weaning, but after the first week).

84 Mean Body & Anogenital Licks per Day Females Males Postnatal Day Figure 3-3. Frequency of maternal licking PND 2-8 in males vs. females (black: females; gray: males). Within-litter variance in neonate maternally-directed behaviors The range of within-litter variance in maternally-directed neonate behavior was similar to the range of maternal licking within litters; i.e. pups that performed maternallydirected behaviors most often did so times as frequently as their same-sex siblings that displayed them the least (Fig. 3-4). Certain maternally-directed neonate behaviors were predictive of receiving maternal licks during the first postnatal week (Appendix G). Pups that were more-frequently located in front of the dam s snout received more anogenitally-directed maternal licks than siblings that were less-frequently located near her snout (anogenital: males: r = 0.55, n = 16, p < 0.03; females: r = 0.63, n = 17, p < 0.01; males and females: r = 0.59, n = 34, p < 0.001; body and anogenital: males: r =

85 , n = 16, p < 0.13; females: r = 0.42, n = 17, p < 0.10; males and females: r = 0.42, n = 34, p < 0.01; Fig. 3-5A). The 3 pups that died were located close to the dam s snout between 0.5 and 1 times/hour observed (not shown). Neonate perioral contact was not a significant predictor of maternal licking, but ventral probing was a significant predictor of anogenital licking (males: r = 0.78, n = 16, p < 0.007; females: r = 0.19, n = 17, p < 0.48; males and females: r = 0.39, n = 34, p < 0.02 Fig. 3-5B). Increased nipple attachment was associated with a greater likelihood of receiving anogenitally licks during PND 2-8 (males: r = 0.41, n = 16, p < 0.13; females: r = 0.26, n = 17, p < 0.33; males and females: r = 0.34, n = 34; p < 0.05 Fig. 3-5C). There were no differences in any of these solicitation behaviors between yellow vs. black pups within a litter. Figure 3-4. Frequency of maternally-directed behavior performed by each pup during PND 2-8.

86 Total Anogenital LicksDays 2-8/Hrs. Observed Pups that performed maternally-directed behaviors most often did so about times as frequently as the pups that displayed them the least (white: females; gray: males; striped pups died pre-weaning, but after the first week). A Litter 55 Litter 63 Litter 87 Litter B Times located in front of mother/hrs. observed

87 Total Anogenital Licks Days2-8/Hrs. Observed Litter 55 Litter 63 Litter 87 Litter Total Ventral Probes/hrs. observed C

88 Figure 3-5. Linear regressions of anogenital licks and maternally-directed behaviors performed by neonates. Frequency of anogenital licks PND 2-8 and A) frequency of time points located in front of the dam s snout PND 2-8; B) frequency of ventral probes PND 2-8; and C) frequency of nipple attachment PND 2-8. Symbols indicate litter and separate regression lines are plotted for each litter. 74 Postnatal maternal licking, neonate maternally-directed behavior and offspring later exploratory and anxiety-related behavior Because we saw a relationship between the maternal licking and neonate solicitation behavior towards the dam, we investigated the association of these early measures and offspring behavioral responses to novelty. The early postnatal environment was predictive of offspring responses to novelty and anxiety-related behavior in adulthood (Appendix H). Pups that received more maternal body licks than their siblings were slower to approach novelty in adulthood than those that received fewer licks (body: males: r = 0.70, n = 16, p < 0.03; females: r = 0.47, n = 13, p < 0.20; males and females: r = 0.59, n = 29, p < 0.007, Fig. 3-6A; anogenital: males: r = 0.02, n = 16, p < 0.95; females: r = 0.34, n = 13, p < 0.31; males and females: r = 0.16, n = 29, p < 0.41; body and anogenital: males: r = 0. 32, n = 16; p < 0.24; females: r = 0.44, n = 13, p < 0.17; males and females: r = 0.37, n = 29, p < 0.05). Additionally, pups that were more frequently attached to a nipple were less exploratory in response to novelty in adulthood, (i.e. these animals had a long latency to approach both types of novelty) (males: r = 0.45, n = 16, p < 0.09; females: r = 0.25, n = 13, p < 0.50; males and females: r = 0.36, n = 29, p < 0.06, Fig. 3-6B).

89 Mean Latency to Novelty in Adulthood Mean Latency to Approach Novelty in Adulthood 75 A Litter 55 Litter 63 Litter 87 Litter Total Body Licks Day2-8/Hrs. Observed B 80.0 Litter 55 Litter 63 Litter 87 Litter % times attached to nipple/observations Figure 3-6. Approach latencies to novelty in adulthood and A) body licks PND 2-8; B) frequency of nipple attachment PND 2-8.

90 76 Pups that received more anogenital licks during the first postnatal week compared to their siblings were slower to enter the open arm of the EPM as adults (males: r = 0.09, n = 16, p < 0.75; females: r = 0.71, n = 17, p < 0.002; males and females: r = 0.47, n = 33, p < 0.005, Fig. 3-7A). We also found a trend that pups that were located in front of the mother more often than their siblings were slower to enter the open arm of the elevated plus maze (males: r = 0.03, n = 16, p < 0.91; females; r = 0.49, n = 17, p < 0.05; males and females: r = 0.32, n = 33, p < 0.06, Fig. 3-7B) and pups that were frequently attached to the dam s nipple were slower to enter the open arm of the EPM (males: r = 0.28, n = 16, p < 0.31; females: r = 0.51, n = 17, p < 0.04; males and females: r = 0.38, n = 33, p < 0.03, Fig. 3-7C) in adulthood compared to their same-sex siblings that were less-frequently attached. Maternal licking and frequency of time points located near the dam s snout were used as predictors of offspring response to novelty in adulthood in a multiple regression analysis with the following outcomes: (body licking, location: β = 0.53, n = 29, p < 0.003; β = 0.18, n = 29, p < 0.27; body and anogenital licking, location: β = 0.38; n = 29; p < 0.07; β = -0.30, n = 29 p < 0.88; nipple attachment, location: β = 0.12, n = 29, p < 0.52; β = 0.35, n = 29, p < 0.07). Out of all of these analyses, the frequency of body licks received during PND 2-8 was the strongest predictor of offspring response to novelty in adulthood.

91 Latency to enter open arm (s) Latency to enter open arm (s) A Litter 55 Litter 63 Litter 87 Litter Total Anogenital Licks Day2-8/Hrs. Observed B Litter 55 Litter 63 Litter 87 Litter Total times located in front of mom/hrs. observed

92 Latency to enter open arm (s) C 78 Litter 55 Litter 63 Litter 87 Litter % times attached to nipple/observations Figure 3-7. Approach latencies to enter the open arms of the elevated plus maze in adulthood relative to maternal and neonate behavior during PND 2-8. A) anogenital licks; B) frequency of time points located in front of the dam s snout; and C) frequency of nipple attachment. If mouse never entered open arm = 310s. The only significant genotype effects were that A vy /a siblings took approximately twice as long to enter the open arms of the EPM compared to their same-sex black siblings (t 3 = 4.13, p < 0.03; Fig. 3-8), which is consistent with what others had previously reported (Harris et al., 2001; Bazhan, et al., 2004).

93 Latency to enter open arms (s) * a/a Genotype Avy/a Figure 3-8. Approach latencies to enter the open arms of on the elevated plus maze for black (a/a, black bars) vs. Agouti viable yellow siblings (Avy/a white bars). +/- s.e.m. (* p < 0.05). Discussion Here, we documented maternal and neonate interactions in an inbred mouse strain during the first postnatal week. We found sibling differences in licking bout received, neonate behavior towards the dam, and offspring exploratory behavior in adulthood, when we did not expect to observe within-litter variance in these measures to be notable in a litter that is genetically similar. Specifically, we observed a fold difference in licking received in the highest-licked vs. the lowest-licked same-sex siblings and also in the frequency of being located close to the mother snout. Pups that were more frequently

94 80 observed close to the dam s snout, ventral probing, and/or attached to a nipple were observed receiving anogenital licks more often than their siblings that did not perform these behaviors as often. In adulthood, we saw that the highest-licked pups within a litter were slower to approach novelty and slow to approach the open arms of the elevated plus maze compared to their siblings that were licked the least. Similarly, pups that solicited maternal attention more often than their siblings were slower to approach novelty in adulthood, and slower to approach the open arms of the elevated plus maze. There were no observable genotype differences in maternal-neonate interactions early in life, however A vy siblings showed anxiety-related behavior on the elevated plus maze in adulthood compared to black siblings. Within-litter variance in maternal licking received by neonates Agouti mice had a narrower range of variance in within-litter differences in pupmaternal interactions during the first postnatal week than outbred Sprague-Dawley rats in the prior chapter. In all 4 litters, the highest-licked A vy pups received times more licks than the lowest-licked same-sex pups, whereas in Sprague-Dawley rats, the highestlicked rats received 3 times as many licks as the lowest-licked siblings. Importantly, the Sprague-Dawley litters were twice as large as the Agouti litters. Additionally, mothers did not preferentially-lick a specific Agouti genotype within the litter. The relationships among licking, maternally-directed behaviors, and exploratory behavior in adulthood were not as strong in litter 87 as in the other litters, however this litter came from the dam

95 81 that licked neonates the least (0.904 licks/hr. on average) and had the least variance in neonate behavior within the litter, which may explain these results. Because the mice were inbred, we originally hypothesized that there would not be as much variance in maternal or sibling behavior as seen in outbred rats due to reduced genetic heterogeneity, yet we observed a consistent degree of variance in maternal licking bouts and maternally-directed behaviors across all 4 litters. Some inbred strains are more susceptible to environmental changes affecting their phenotype while others are less so. Because there is no environmental push in a laboratory setting to influence phenotypic variance, we hypothesized that there would be more behavioral homogeneity in those litters than was observed (Ginsburg, 1967). Perhaps larger litter sizes, increased number of litters, and experimental manipulations of the maternal environment would achieve similar degrees of variance as shown in the previous rat study. In our previous study with rats, we had shown that males received more licks than did females of the same litter, similar to what was shown in early maternal behavior studies conducted by Moore & Morelli (1979). The Moore & Morelli study has four major differences from this current study: 1) the subjects were Long Evans rats; 2) litters were culled to even numbers of males and females, 3) pups were individually introduced to the dam resulting in handling and separation from the mother; and 4) observations were only conducted during the first 3 hours of the dark phase. Another study had shown that Sprague-Dawley male offspring receive higher amounts of anogenital licking compared to females, but only after the pups were handled briefly (Richmond & Sachs, 1984). Those authors also culled the litters, measured duration (not frequency) of licking from foster mothers rather than biological, and only conducted observations on PND 4, 7,

96 82 and 10 all during the light phase. Conversely, in a study with Long Evans rats performed by Champagne and colleagues (2003), no sex differences with respect to average licking/grooming received during PND 1-8 were observed when observations were conducted during the light and dark phases. In the current mouse study, we did not find a sex difference in the number of licking bouts received, however our litters were inbred, small in litter size (the smallest litter of 6 animals only had 2 males; and the largest litter of 10 animals had 4 males), and we conducted most observations during the light phase. The importance of parental effects on phenotypic variation within a species was noted by Charles Darwin in The Origin of the Species (1859) when he described the maternal behavior of the cuckoo. Common cuckoos are brood parasites that will lay their eggs in other birds nests allowing the potential to lay more eggs which can be then cared for by others. Several cuckoo egg color polymorphisms exist increasing the number of the eggs that assume the foster bird s egg color and preventing rejection. Maternal effects on offspring are critical in the development of offspring for several reasons including modulation of natural selection. Parental care towards offspring can change in response to unstable environments much faster than evolutionary changes can appear, suggesting that maternal behavior can, in fact, affect natural selection of the offspring quite rapidly (Cairns, Gariépy, & Hood, 1990). From an evolutionary perspective, ensuring that offspring are phenotypically variable increases reproductive fitness of the mother. For instance, within-litter variance in maternal licking may increase likelihood of offspring survival in unpredictable environments post-weaning (such as high vs. low predation), allowing more offspring to reach reproductive age (Philippi & Seger, 1989).

97 83 In other words, in environments of low predation, high-exploratory offspring are more likely to forage food and encounter mates, while in high predation, low-exploratory offspring are better protected from exposure to prey animals and aversive environments. This variance in maternal licking may not be a direct influence of later variance in offspring behavior, but may be a maternal response to varying neonate tactile behaviors, with more solicitous pups receiving more licking within litter. Experimental manipulations of pups behaviors as well as close temporal examinations of maternal and neonate maternally-directed behaviors will help to determine the directionality of these behaviors. For instance, experimenters could place certain pups close to the dam s snout at the beginning of each observation period and then maternal licking rates would again be recorded. To measure the direction of maternal and neonate behaviors, one could record a behavior and then measure the latency for either maternal licking or pup behavior onset. Within-litter variance in neonate maternally-directed behaviors Mouse pups that made the most maternally-directed contact did so times as often as the pups that performed them the least. In the prior study with Sprague-Dawley rats, the most active pups performed these behaviors 3 times as often as their least active siblings. A vy mouse pups that made more maternally-directed contact, specifically, locating themselves near the mother s snout, received more anogenital licks from the dam than did littermates that made fewer contacts. Mouse pups that were more often attached to the nipple were more likely to be anogenitally licked than their siblings that were

98 84 attached the least. Similarly, neonates that frequently ventral probed received more licking bouts than their siblings that did not stimulate the dam s ventrum as often. Ventral probing and attaching to the nipple may draw attention from the dam, resulting in more licking bouts received compared to siblings that do not as readily probe and attach. These nursing-related behaviors are specific to an increased likelihood of receiving anogenital licks, which may be related to the mother stimulating urination in pups that have recently nursed. Anogenital stimulation from the dam allows for the neonates to eliminate waste, and also provides fluid replenishment for the dam (Friedman & Bruno, 1976; Gubernick & Alberts, 1983). The pups that attach to a nipple often may also be the weaker, needier pups that require more maternal care. Prior studies also support this hypothesis that weaker pups get more maternal attention; pups that make more ultrasonic vocalizations (USVs), or cries to elicit maternal responses, tend to get licked more often than pups that do not make USVs as frequently, supporting the old adage, the squeaky wheel gets the grease (McFarland, 2008). In other words, to receive maternal licking, pups need to actively seek it. Analyses of neonate weight and frequency of nipple attachment (not shown) do not suggest that weight is a significant predictor of maternal care for this strain although it was true for the Sprague-Dawley rats. Weight may be more of an issue in larger litters, like in the rat study, where more sibling competition for maternal resources is present than in the small Agouti viable yellow litters that have easier access to maternal care (Stockley & Parker, 2002). In the Sprague-Dawley study in Chapter 2, those neonates that placed themselves close to the mother did not receive more maternal licks, but those that initiated perioral contact more often than their siblings received more maternal licking. In these A vy mice,

99 85 location close to the mother s snout was associated with maternal licking. Perhaps A vy dams are more responsive to neonate cues to initiate maternal behavior than Sprague- Dawley rat dams. Because maternal behavior decreases per pup with increasing litter size, a small litter size may allow the mother to distinguish individual pups that are actively seeking maternal attention vs. pups that are aimlessly moving about the nest (Champagne, Curley, Keverne, & Bateson, 2007). In a study that compared variance in maternal care in inbred 129Sv, C57BL/6J, and outbred Swiss mice, C57BL/6J (which are the background strain for the A vy mice in the current study), the smaller the litter size, the longer the duration of nursing during PND 1-6, which was an effect that was not evident in the other two strains (Champagne et al., 2007). Considering these differences in maternal behavior among rodents, successful neonate solicitation behaviors towards the dam may be dependent on strain and species. The relationship between maternal licking and neonate maternally-directed behavior and offspring exploratory and anxiety-related behavior Early maternal-neonate interactions predicted variance in offspring exploratory behavior in adulthood in a manner that replicates previous studies. Specifically, siblings that received more licks as neonates were slower to approach novelty in adulthood. In addition, siblings that made more frequent maternally-directed behaviors were slower to enter the open arms of the EPM compared to their same-sex siblings. Lastly, pups that were frequently-attached to the nipple showed a longer latency to enter the open arms of the EPM and to approach novelty in adulthood. Also consistent with previous research,

100 86 A vy mice were less exploratory on the EPM than their black littermates. The relationships between neonate maternally-directed behaviors during the first postnatal week and both sibling variance in exploratory behavior and anxiety-related behavior are congruent with the hypothesis from Chapter 2 that the pups that seek out mother s attention may be the needier pups that later become less exploratory and/or more anxious adults. For instance, pups that are frequently attached to a nipple, may seek out maternal contact more often that their less-frequently attached littermates. Similarly, pups that receive more body licks compared to their littermates may also tend to stay close to the dam. Previous studies have shown that neonate body licking by the dam serves to thermoregulate pups, specifically, helping to cool neonate body temperature when buried in a huddle or under the dam s ventrum (Sullivan, Shokrai, & Leon, 1988). Because these pups stay close to the dam, they may be prone to be less exploratory as adults preferring to stay closer to familiar objects than investigating novel ones. Because the mechanisms driving differences in maternal behavior are unknown, additional experiments would need to be conducted. To test the contributions of pup-driven maternal behavior, examinations of maternal-pup interactions in rodent lines selectively bred for high USV such as the N: NIH line vs. low USVs or manipulations of maternallydirected behaviors shown by the neonates could be performed (Brunelli, 2005). Alternatively, examining variance in maternal behavior on anesthetized pups using an agent like short-acting isofluorane could elucidate maternal contributions to within-litter differences in maternal behavior (Jans & Leon, 1983, Alberts, 2007). In inbred A vy mice, like in the Sprague-Dawley rats in the previous chapter, we observed opposite outcomes within-litters compared to the between-litter analyses in

101 87 other rodent models. To assume that within-litter analyses would provide the same conclusions as between-group an analysis is known as the ecological fallacy (Robinson, 1950). In other words, comparing group means may not achieve the same results as accounting for each individual would. In previous studies, researchers have examined variations in maternal care by comparing mean licking rates across litters and mothers without examining variation in maternal care individual pup s licking rates within litters. By marking the pups and tracking their individual licks as well as maternally-directed behaviors, we were able to do a longitudinal study following pups until adulthood and to develop a new paradigm to determine the long-term effects of early maternal-neonate interactions. To examine individual differences in behavior, conducting a within-litter approach should be strongly considered to account for the behavior of each individual in order to examine genetic as well as environmental influences on offspring outcomes. Future studies should examine maternal-neonate behavioral differences in A vy dams compared to other Agouti phenotypes and follow the offspring outcomes through adulthood. Our sample size may have been too small to distinguish genotype differences, however we did replicate the anxiety-related differences found in A vy mice compared to black mice. In this same A vy cohort, we analyzed variance in maternal behavior during the first postnatal week among four A vy dams and found that these dams spent similar amounts of time performing maternal behaviors like nursing and grooming, and that these durations were also comparable to a study that compared two inbred mouse strains (129Sv and C57BL/6J) and one outbred strain (Swiss) (Woehling, 2011; Champagne et al., 2007). We are curious to investigate if, in a larger sample, differences in maternal behavior within a litter of varying Agouti phenotypes exist. Because the A vy mice show

102 88 more anxiety-like behavior compared to the black phenotype, we may see more erratic maternal behavior in that phenotype which could have long-term consequences on developing offspring. On the other hand, highly anxious dams may be less interested in exploration and direct more of their attention on their pups (Neumann, Kromer, & Bosch, 2005; Bosch & Neumann, 2008; Lovic, Palombo, & Fleming, 2010). A larger study may be necessary for detecting phenotype differences in other behaviors, including a broader spectrum of maternal care and longitudinal statistical methods to determine the reliability of individual differences and the temporal dimension of pup and dam interactions. In this study, we found early environmental and behavioral relationships to sibling variance in exploration and anxiety-related behaviors in inbred mice that were similar to what we previously found in outbred rats, however the variance in the mice was not as large as the variance in rats. Because we see variance not only in genetically-variable outbred rats but also in genetically-similar inbred mice, this rodent model of exploratory and anxiety-related behavior provides further evidence that variance in early experiences may be associated with adult sibling behavioral variance above and beyond underlying genetic inheritance.

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109 95 Chapter 4 Within-litter variance in physiological measures of responses to novelty and anxietyrelated behaviors Introduction Behavioral inhibition, defined as shyness in humans, and stable neophobia, defined as fear of novelty in rats, are associated with increased hypothalamic-pituitary-adrenal (HPA) axis activity in humans, primates, and rats (Kagan, Reznick, & Snidman, 1987; Cavigelli et al., 2007). Normally, in response to stress, the HPA axis releases stress hormones and stops producing them when cortisol (CORT), or corticosterone in rodents, is detected by glucocorticoid receptors (GRs, Nr3c1) in the hippocampus via the negative feedback loop. However, there is evidence that anxious individuals seem to have a dysfunctional negative feedback loop that causes longer production of CORT (Gómez, De Kloet, & Armario et al., 1998; Plotsky, Owens, & Nemeroff, 1998) which suggests that this pathway is important to investigate in stress-related mechanisms. Early handling experiments have shown that this stress pathway can be affected by early environmental manipulations. Neonate rats that were handled from birth until weaning showed lower adrenal weights after injection stress in adulthood (which translates to less stress hormone release) compared to unhandled injected rats, suggesting that the handling procedure had longterm effects on the HPA axis and required further investigation (Levine, 1957). In addition, early-life handling of rodents also caused HPA-related effects in the brain, with handled animals expressing more hippocampal and prefrontal glucocorticoid receptors, compared to unhandled animals (Meaney & Aitken, 1985; Meaney, Aitken, Bodnoff, Iny, Tatarewicz, Sapolsky, 1985;

110 96 Meaney, Aitken, Viau, Sharma, & Sarrieau, 1989; O'Donnell, Larocque S, Seckl, & Meaney, 1994; Bhantnagar & Meaney, 1995). However, high levels of GR expression is not always beneficial. Experimentally, overexpression of GR in the forebrain leads to an increase in anxiety and depression-like behavior in male C57BL/6J mice (Wei, Qiang, Xin-Yun, Liu, Schafer, Shieh, Burke, Robinson, Watson, Seasholtz, & Akil, 2004). Additionally, in Sprague-Dawley rats categorized as high reactors vs. low reactors, the high reactors show: a shorter latency to enter the light compartment of light/dark box, more locomotion in a novel arena, lower hippocampal GR mrna expression, and higher CORT responses to the light-dark test (Kabbaj, Devine, Savage, & Akil, 2000). Experiences in early life can have a long-term influence on an offspring s behavior and physiology in response to stress and novel situations. Several studies have shown in humans, non-human primates, and rodents that maternal care is related to offspring exploratory behavior and expression of genes related to stress and the HPA axis in offspring (human: Hane et al. 2008; Rubin et al. 2001; primate: Seay & Harlow, 1965; Harlow et al., 1965; Kalin et al., 2000; rodent: Stanton and Levine, 1990; Plotsky and Meaney, 1993; Caldji et al., 2000). In rodents, one aspect of maternal care is maternal licking of pups. For instance, laboratories have shown offspring of these high-licking mothers as a whole show increased exploratory behavior on the open field test in adulthood compared to offspring of low-licking mothers. In addition, offspring of high-licking mothers have greater glucocorticoid receptor levels in the hippocampus than those of low-licking mothers (Liu et al., 1997; Weaver et al., 2005). This change in GR expression may provide one mechanism underlying behavioral differences between high-licked and low-licked siblings in adulthood

111 97 Like GRs, corticotropin releasing hormone (CRH) receptors are involved in the HPA stress response. Limbic Crhr1 is thought to be responsible for modulating anxiety-related behavior and Crhr2 receptors (which have a lower affinity for CRH) may be responsible for dampening responses to chronic stress (Chalmers, Lovenberg, & De Souza, 1995; Müller, Zimmermann, Sillaber, Hagemeyer, Deussing, Timpl, 2003; Bale & Vale, 2004). Knockout experiments have helped to determine the functions of Crhr1 and Crhr2 in regards to stress. For instance, Crhr1 knockout mice show less anxiety-related behavior on the elevated plus maze (EPM) and are more exploratory in the light/dark transition and open field tests than the wild types (specifically, spending more time spent in the lit compartment, entering the lit compartment faster, making more entries in lit side, and spending more time in the center of the open field) and physiologically, have a lower ACTH and CORT response to stress (Smith et al., 1998). Compared to wild type littermates, baseline adrenocorticotropin releasing hormone (ACTH) and CORT do not differ, however 30 minutes after restraint stress, the knockouts produce much lower levels of these hormones. The Crhr1 knockout physiological data suggest that animals with impaired stress response system express lower levels of Crhr1 receptors (Bale & Vale, 2004, Smith et al., 1998). Based on Crhr2 knockout studies, this receptor has a specific function that differs from Crhr1 receptors. Crhr2 knockouts show increased anxiety-related behavior vs. wild types (Bale et al., 2000). Specifically, Crhr2 knockouts are less exploratory on the EPM, but are not different behaviorally from wild types in the light/dark transition test which is reported to be more a test of responding to novelty than of exploration (Belzung & Le Pape, 1994). Physiologically, Crhr2 knockouts express more CRH mrna in the central amygdala and paraventricular nucleus of the hypothalamus (Bale et al., 2000). Crhr2 deficient mice show

112 98 more rapid rises in CORT levels, and earlier ACTH peaks in response to stress, but show normal baselines compared to the wild type. These Crhr2 knockout studies highlights that CRHR2 has functions specific to anxiety-related behavior. In addition to GRs, and CRHRs, the serotonin transporter (SERT) is involved in stress regulation and anxiety-related behavior. SERT is a monoamine transporter comprised of 12 transmembrane domain proteins and is responsible for serotonin reuptake from the synapse (Cooper, Bloom, & Roth, 2003). SERT (SLC6A4), a plasma membrane carrier, mobilizes serotonin in or out of the synapse depending on the concentration gradient. Because serotonergic cells project to several areas of the brain, SERT protein is found all over the central nervous system, however mrna is localized to the raphe nucleus where the serotonergic cell bodies that contain the genetic material are located (Hensler, Ferry, Labow, Kovachich, & Frazer, 1994). Another reason why SERT is of interest in this study is because ten SERT -/- Quantitative Trait Loci (QTL)s have been identified in rodent models that are associated with activity and exploratory behaviors (Homberg et al., 2010). SERT is the binding site for common antidepressants like fluoxetine (Prozac ). Recently, it has been determined that variations in the SERT promoter (5-HTTLPR) are involved in predicting vulnerability for depression (Caspi et al., 2003). The short allele has fewer repeats (14) in the genetic sequence, resulting in lower gene transcription compared to the long form (16 repeats). The associations between the short allele of the SERT promoter and depression susceptibility have been cited often, but the direct connection between the two has been questioned, suggesting further research is required (Caspi et al., 2003; Gillespie et al., 2005; Kendler et al., 2005; Kaufman et al., 2004; Lemogne et al., 2011).

113 99 In this longitudinal study, we examined early life interactions between the rodent dam and her offspring, the offspring response to novelty at post-weaning and adulthood, and adult offspring physiological measures related to stress, specifically, GRs in the hippocampus and PFC, Crhr1 in the hippocampus and PFC, and Sert in the raphe nucleus. Because the hippocampus and prefrontal cortex have a high concentrations of GRs and CRHRs and are involved in HPA negative feedback and anxiety disorders (Diorio, Viau, & Meaney, 1993; Anisman et al., 1998; Ziegler & Herman, 2002) we selected these areas to observe GR and Crhr1 mrna expression in rodents. We selected three targets rather than just one to a provide a more comprehensive approach to examine HPA activity in these animals. Because we found that, within a litter, pups that received more licks than their littermates were less exploratory in adulthood (Cavigelli & McClintock, 2003; Ragan et al., 2011), we wanted to examine if these differences were also reflected in physiological measures associated with respondes to novelty. Specifically, to determine if variability within a litter in Sert, GR, and Crhr1 mrna expression relates to the variability in behavioral responses to novelty, we tested the following hypotheses: 1) rodents that were licked more often than their same-sex littermates during the first postnatal week will express lower GR mrna and higher Crhr1 mrna in the hippocampus and PFC and will express less Sert mrna in the brainstem; 2) rodents that made more frequent maternally-directed solicitation behaviors compared to their littermates will express lower GR mrna and higher Crhr1 mrna in the hippocampus and PFC and will express less Sert mrna in the brainstem as adults; and 3) rodents that were slow to approach novelty compared to their same-sex siblings will express lower GR mrna and higher Crhr1 mrna in the hippocampus, PFC; and will express less Sert mrna in the brainstem. These hypotheses suggest that differences in early life experiences with the mother and variance in

114 100 early exploratory behavior within a litter will be reflected in sibling differences in stress physiology. Previous chapters highlighted behavioral consequences related to variance in maternalneonate interactions and early responses to novelty. This chapter will focus on the physiological sequelae related to both the early postnatal maternal-neonate relationship and early behavioral responses to novelty. Based on what is known about neuroendocrine responses, Sert, and behavioral inhibition, we have examined the relationship between early life responses to novelty and adult exploratory and HPA-related mrna expression. Because early interactions can alter later physiology, here I will report associations between exploratory behavior and adult GR, Crhr1, and Sert mrna expression levels in Sprague-Dawley rats and adult Sert mrna expression levels in Agouti viable yellow mice. As previously mentioned, changes in the expression of these targets can lead to a cascade of events in the HPA axis. Observing the connection between early behavior and adult physiology will allow us to better understand the mechanisms underlying the relationship between early behavioral inhibition and adult-anxiety disorders. Methods Hippocampal and PFC GR, Crhr1 and brainstem Sert mrna expression were measured in the Sprague-Dawley rat cohort described in Chapter 2 and the Agouti viable yellow mouse cohort from Chapter 3. All methods used in these studies were approved by the Pennsylvania State University Institute for Animal Care and Use Committee (Appendix A and C). The procedures for the separate cohorts and for certain genes were slightly different and were as follows:

115 101 Sprague-Dawley rat protocol Our breeder (4 female and 4 male) Sprague Dawley rats were purchased from Charles Rivers Laboratory (Wilmington, MA). One male and one female were housed together for approximately 8 days, then females were housed individually and checked for signs of pregnancy daily. All animals were maintained on a 12L:12D lighting schedule (lights on at 0800h) in a room maintained at 23 C with 50% humidity. Food and water were available ad libitum. Maternal-neonate interactions The breeders produced four separate litters, for a total of 68 pups (33 females, 35 males). The day of birth was defined as postnatal day 0 (PND 0). On postnatal day (PND) 1-8, live pup and maternal interactions were observed (Appendix B). The frequency of the following behaviors were recorded maternal licking: anogenital and body; and neonate behaviors: initiating perioral contact with the dam, probing the dam s ventrum, and placing themselves within close proximity to the snout of the mother easy licking distance. For individual pup observations during PND 1-8, pups were marked with a black Sharpie TM marker (Sanford Corporation, Oak Brook, IL) on their backs, head, and ventrum and then weighed at the same time each day. This procedure lasted less than 1 minute per day. On PND 21, offspring were weaned into groups of 2-3 same-sex littermates per cage, and continued to be handled daily. Offspring response to novelty At PND (post-weaning) and then later between PND (young adulthood), offspring were tested on two novel arenas to measure exploratory behavior at (Fig. 4-1). The

116 102 arenas measured 120 cm X 120 cm (LXW) with 46 cm-high white propylene walls. The novel object arena consisted of a rat-sized novel object placed in 3 of the 4 corners 13 cm away from the walls and the test animal was placed in the 4 th corner (Fig. 4-1A). The novel social arena consisted of an empty cage with vented walls in one corner adjacent to the test animal, and another cage adjacent to the test animal with a novel animal of approximate same age and size (Fig. 4-1B). For both novelty tests, animals were videotaped for 5 minutes with a camera placed 1.5m above the arena. The latency to approach the first novel object was recorded in the novel object test, and the latency to approach the novel animal was recorded (Appendix D). After each test, the objects were rinsed and placed back into the arena before the next test subject entered the arena. Figure 4-1. Novelty arenas. A) Novel object arena: novel object in 3 corners, home bowl in 1 corner. B) Novel social arena: novel mouse in one corner, empty cage in opposite corner, home bowl in third corner. In young adulthood (PND 80-85), rats were tested on the elevated plus maze to observe anxiety-related behavior (Appendix E). The elevated plus maze was made of Plexiglas and

117 103 consisted of two open arms (30 cm X 5 cm X 15cm) and two closed arms covered in black construction paper (30 cm X 5 cm X 15 cm) and was raised 38.5 cm above the floor. Rats were placed on the center area facing a closed arm and were video recorded for 5 minutes with a camera placed 1.5 m directly above the apparatus. After testing, the maze was cleaned with 70% ethanol and allowed to dry. In the elevated plus maze, we observed the latency to enter the open arms relative to the closed arms. An entry was defined by the presence of all four paws on the arm. Exploratory behavior on the novelty arenas and anxiety-like behavior were coded using Noldus Observer version 5.0 (Wageningen, Netherlands). Corticosterone measurements Plasma CORT (pcort) levels in the Sprague-Dawley rats were measured using methods used previously in this laboratory (Cavigelli et al., 2006). After behavioral tests on PND 70, we collected rapid serial blood samples (250μl) at 10, 40, and 120 minutes-post test to examine reactive CORT levels. To detect CORT levels, we conducted radio immuno assays (RIAs) using 125 I (MP Biomedicals, Solon, OH). RIAs have been used in several of our previous experiments, and the protocol for pcort has been optimized for plasma samples using 1:200 dilutions. The radioactive binding pcort levels were calculated using a Gamma Counter. For complex CORT measures, when taking blood samples, collections were taken within 3 minutes of starting to draw blood to minimize the stress of blood collection from interfering with the stress of the behavioral tests. Our samples were placed on ice immediately after collection and then stored at -80 C to ensure the integrity of our samples.

118 104 RNA isolation and Real-Time Polymerase Chain Reaction analysis of mrna expression On PND 120, animals were sacrificed and the brains were flash-frozen in ice-cold isopentane then stored at -80 C. Brainstems were dissected and stored in RNAlater ice (Ambion Inc., Austin, TX) at 4 C overnight, then stored at -80 C. RNA was isolated using Qiazol reagent (Qiagen, Valencia, CA, Appendix I). The concentration of samples were then quantified using the NanoDrop 2000 spectrophotometer (Thermoscientific. Wilmington, DE). We used samples that had a 260/230 ratio of 1.8 or higher. Due to costs, select rat samples were run on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA; Nucleic Acid Facility, PSU) and scored a RNA integrity number (RIN) value of around 9, confirming that our tissues were of high-quality for RNA experiments (a RIN of 8 or higher is considered acceptable). The samples from mouse brainstem were run on an RNA gel to ensure that that RNA was intact (Appendix K). To convert RNA into cdna, polymerase chain reactions (PCR) were conducted using the High Capacity cdna Reverse Transcription Kit (Applied Biosystems, Foster City, CA). To quantify Sert mrna expression, quantitative real-time PCR was then performed using Taqman reagents (Applied Biosystems, Foster City, CA). For GR and Crhr1 mrna expression, the prefrontal cortex and hippocampus were dissected and then RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA; Appendix J). To convert RNA into cdna, PCR was then conducted with the same kit as above. To quantify GR and Crhr1 mrna expression, quantitative real-time PCR was performed using SYBR green (Applied Biosystems, Foster City, CA). For all three gene targets, relative quantification of mrna expression was determined using the 2 -ΔΔCt method (comparative Ct method) with fold change relative to the median same-sex littermate ΔCt for each litter (Applied Biosystems, Foster City, CA).

119 105 For this study, we focused on Crhr1 receptors in the hippocampus and prefrontal cortex, which are two areas that contain a high density of these receptors and have been strongly implicated in HPA negative feedback regulation (dekloet, Joëls, Oitzl, & Sutanto, 1991; Diorio, Viau, & Meaney, 1993; Feldman, Conforti, Itzik, & Weidenfeld, 1994; Jacboson & Sapolsky, 1991). For instance, lesioning the medial prefrontal cortex results in both an increase in CORT and ACTH after restraint stress recovery compared to sham control animals (Diorio, Viau, & Meaney, 1993). Had we exposed animals to chronic stress, we would have measured Crhr2 receptors, as well, however, this study was conducted for the purposes of observations of gene expression in animals who had been exposed to acute, mildly stressful situations at only a few time points throughout the lifespan. Agouti mice protocol Maternal-neonate interactions were observed from PND 2-8 (data started on PND 2 for one litter, so all analyses are based on PND 2-8) using the same protocol as the Sprague-Dawley rats. The Agouti viable yellow mice were housed with 2-3 same-sex littermates after weaning at PND 28. At postweaning (PND 30-34) and in young adulthood (PND 64-70), animals were tested on the same two novelty arenas as described above to observe their exploratory behavior towards novelty at 2100 h during the dark phase on PND 72, animals were sacrificed, and then brainstems were placed into RNAlater (Ambion Inc., Austin, TX) at 4 C overnight, and then stored at -80 C. Sert measures were conducted using the same protocols as the Sprague-Dawley rat Sert procedure above.

120 106 Statistical Analyses We used regression analyses to determine if the frequency of maternal licking related to adult expression levels of Sert, GR, or Crhr1. We also used regression models to determine if maternally-directed pup behavior and if exploratory behavior on the novelty arenas or elevated plus maze predicted adult mrna expression of these genes. To control for litter and sex effects, all behavioral variables for each animal were centered to their within-litter, within-sex means. Because our power was low due to small sample sizes, and because our data are nested, for the Sert mrna analyses, we categorized animals as fast or slow to approach novelty based on median splits within litter, within sex, and then used ANOVAs to analyze if latency to novelty at post-weaning or adulthood was related to gene expression in adulthood. The latencies to approach novelty and to approach the open arm of the elevated plus maze were skewed right, so these values were log transformed to achieve normally-distributed data for statistical analyses, however in the figures we show the non-log transformed values for clarity purposes. All 68 Sprague-Dawley rats were used for our statistical analyses regarding behavioral data except the EPM. One litter was not tested on the EPM in adulthood. For the Sert analyses, 39 of the 68 rat brainstems were available due to loss of some samples and low RNA concentrations of others. CORT samples were only available for females in two of the four litters, but were available for all 35 male rats. For the Agouti viable yellow mice studies, the number of subjects available for analyses of maternal-neonate interactions was 34 because two animals from the same litter died in the middle of the first postnatal week. One additional animal died from the first litter after the maternal-neonate interactions, but before weaning so the total number of animals available was 33. Analyses of exploratory behavior on novelty arenas have a sample size of 29, due to a loss

121 107 of data for 4 animals. For the elevated plus maze, data for all 33 remaining animals were available. For the Sert measures, 29 mice brainstems were available for analyses. Results Postnatal maternal licking, and adult offspring Sert measures The relationship between maternal licks received during the first postnatal week and offspring Sert expression in adulthood was dependent on sex, species, and licking type. In the Sprague-Dawley rats, there was no relationship between maternal licks received and adult offspring Sert expression (Table 4-1). Table 4-1. Pearson correlations of maternal licking during the first postnatal week and adult Sert expression SERT Sprague-Dawley Rats body licks anogenital licks body and anogenital licks MALES r = 0.20, p < 0.35 r = 0.04, p < 0.86 r = 0.03, p < 0.81 FEMALES r = 0.27, p < 0.33 r = 0. 37, p < 0.18 r = 0.10, p < 0.73 ALL r = 0.19, p < 0.24 r = 0.11, p < 0.51 r = 0.03, p < 0.85 Agouti viable yellow mice MALES r = 0.68, p < 0.01 r = 0.24, p < 0.43 r = 0.31, p < 0.30 FEMALES r = 0.25, p < 0.36 r = 0.13, p < 0.63 r = 0.20, p < 0.45 ALL r = 0.23, p < 0.22 r = 0.18, p < 0.35 r = 0.03, p < 0.86

122 108 In the Agouti viable yellow mice, however, male pups that received more body licks than their littermates expressed less Sert in adulthood, but there was no relationship between anogenital licking and offspring adult Sert expression (body: males: r = 0.68, n = 13; p < 0.01; females: r = 0.25, n = 16, p < 0.36; males and females: r = 0.23, n = 29, p < 0.22; Fig. 4-2; Appendix L). A SERT mrna Expression 2 -ΔΔCt Agouti Males Litter 55 Litter 63 Litter 87 Litter Total Body Licks/ Hrs.observed B

123 109 SERT mrna Expression 2 -ΔΔCt Agouti Females Litter 55 Litter 63 Litter 87 Litter Total Body Licks/ Hrs.observed Figure 4-2. Linear regression of postnatal maternal body licking and adult offspring Sert mrna expression. A) Agouti viable yellow males; B) Agouti viable yellow females. Sprague-Dawley maternal licking and offspring HPA-related mrna expression in adulthood In addition to adult Sert measures, we analyzed the relationship between maternal licking bouts received and GR and Crhr1 mrna expression in adulthood. The association between licking and adult HPA-related mrna expression was also sex-specific. Females that received more total licks than their same-sex siblings expressed more GR in the PFC in adulthood than females that were not licked as often (males: r = 0.09; n = 27, p < 0.69; females: r = 0.39; n = 28, p < 0.04; males and females: r = 0.22; n = 55, p < 0.11; Fig. 4-3). There was no relationship between licks received during the first postnatal week and offspring hippocampal GR, PFC Crhr, and hippocampal Crhr1 in adulthood (Appendix L).

124 110 PFC GR mrna Expression 2 -ΔΔCt B PFC GR mrna Expression 2 -ΔΔCt A Sprague-Dawley Males Litter A Litter B Litter C Litter D Total Licks/Hrs. Observed Sprague-Dawley Females Litter A Litter B Litter C Litter D Total Licks/Hrs. Observed Figure 4-3. Linear regression of maternal licking during the first postnatal week and glucocorticoid receptor mrna expression in adulthood. A) Sprague-Dawley males B) Sprague-Dawley females.

125 111 We found that Sprague-Dawley females that received more licks than their sisters had a much lower adult CORT response to a novel object than did their low-licked littermates, however we found no such relationship with the males. (males: r = 0.02, n = 35, p < 0.92; females: r = 0.57, n = 19, p < 0.01; males and females: r = 0.12, n = 54, p < 0.38; Fig. 4-4).

126 reactive CORT (ng/ml) reactive CORT (ng/ml) A Sprague-Dawley Males Litter A Litter B Litter C Litter D Total licks/hrs observed B Sprague-Dawley Females Litter C Litter D Total Licks/Hrs. Observed Figure 4-4. Linear regression of maternal licks and CORT response to novelty in adulthood. A) Sprague-Dawley males; B) Sprague-Dawley females.

127 113 Neonate maternally-directed behaviors and adult Sert expression Because some neonate behaviors performed during the first week of life were associated with sibling differences in adult behavior (Chapter 2 and 3), we examined if these behaviors predicted adult Sert mrnaexpression. Some neonate behaviors that solicit maternal attention during the pups first week of life were associated with adult Sert expression in Agouti viable yellow mice, but none were associated with Sert expression in Sprague-Dawley rats (Appendix M). There was no relationship between the frequency of perioral contact made with the dam, frequency of nipple attachment, times located in front of the mother s snout, and between the frequency of probing the dam s ventrum for either of the sexes in the Sprague-Dawley rats. There was a positive relationship between perioral contact and Sert expression in adulthood for the Agouti viable yellow male mice (males: r = 0.65, n = 13, p < 0.01; females: r = 0.28, n = 16, p < 0.30; males and females: r = 0.23, n = 29, p < 0.22; Fig. 4-5 A,B). Additionally, Agouti viable yellow male pups that were frequently attached to the dam s nipple expressed less Sert than their littermates that were not attached as often (males: r = 0.67, n = 13, p < 0.01; females: r = 0.46, n = 16 p < 0.07; males and females: r = 0.17, n = 29 p < 0.38; Fig. 4-5 C,D). We found no relationship between other neonate behaviors directed toward the dam in the Agouti viable yellow mice (Appendix M).

128 SERT mrna Expression 2 -ΔΔCt A Agouti Males Litter 55 Litter 63 Litter 87 Litter Total Snout Contact/Hrs. Observed 114 SERT mrna Expression 2 -ΔΔCt B Agouti Females Litter 55 Litter 63 Litter 87 Litter Total Snout Contacts /Hrs. Observed

129 SERT mrna Expression 2 -ΔΔCt SERT mrna Expression 2 -ΔΔCt C D Agouti Males Litter 55 Litter 63 Litter 87 Litter Proportion times attached to nipple/observation Agouti Females Litter 55 Litter 63 Litter 87 Litter Proportion times attached to nipple/observation 115 Figure 4-5. Linear regression of postnatal maternally-directed neonate behaviors and Sert expression in adulthood. Perioral contact: A) Agouti viable yellow males; B) Agouti viable yellow females; Nipple Attachment: C) Agouti viable yellow males; D) Agouti viable yellow females.

130 116 Neonate maternally-directed behaviors and adult GR and Crhr1 mrna expression In the Sprague-Dawley rats, maternally-directed neonate behaviors were not strong predictors of expression of adult GR or Crhr1, however we did find that in females, ventral probing was positively associated with Crhr1 in the prefrontal cortex (PFC Crhr1: males: r = 0.12, n = 27, p < 0.55; females: r = 0.42, n = 28, p < 0.02; males and females: r = 0.27, n = 55, p < 0.04; Appendix N). Rodent exploratory behavior at post-weaning and adult Sert expression Exploratory measures in response to novelty at post-weaning were predictive of adult Sert mrna expression in offspring. Rats that were slow to approach both a novel object and social novelty at post-weaning expressed less Sert compared to their same-sex siblings that were faster to approach novelty and males drove this relationship (males: F(2, 21) = 3.37, p < 0.02; females: F(2, 12) = 1.18, p < 0.17; males and females combined: F(2, 31) = 3.87, p < 0.01 Fig. 4-6 A, B; Appendix O).

131 A 117 Sprague-Dawley Males B SERT mrna Expression 2 -ΔΔCt Sprague-Dawley Females FAST MIXED SLOW Latency to approach novelty at postweaning Figure 4-6 A,B. Linear regression of mean latency to approach novelty at post-weaning and adult Sert expression in adulthood. A) Sprague-Dawley males; B) Sprague-Dawley females. +/- s.e.m.

132 118 Likewise, Agouti viable yellow mice that were slow to approach novelty at post-weaning expressed less Sert than their siblings that were faster to approach novelty, however, this relationship was only approached significance in males (males: F(2, 11) = 2.31, p < 0.07; females: F(2, 13) < 1, p < 0.88; males and females; F(2, 27) = 1.87, p < 0.07; Fig.4-6 C, D; Appendix O).

133 C 119 Agouti Males D SERT mrna Expression 2 -ΔΔCt Agouti Females FAST MIXED SLOW Latency to approach novelty post-weaning Figure 4-6, C,D. Linear regression of mean latency to approach novelty at post-weaning and adult Sert expression in adulthood. C) Agouti viable yellow males; D) Agouti viable yellow females. +/- s.e.m. Rodent exploratory behavior at post-weaning and adult GR and Crhr1 expression

134 120 Although we found strong associations between post-weaning exploratory behavior and adult Sert expression, we found no associations between exploratory behavior at post-weaning and HPA-related mrna expression in adulthood (Appendix P). Rodent exploratory behavior in adulthood and adult Sert expression In the rats, we found an association between offspring adult exploratory behavior on the social novelty arena and adult Sert mrna expression, but not for the novel objects arena. Offspring that were slow to approach social novelty in adulthood expressed less Sert as adults compared to their siblings that were faster to approach novelty: (novel social: males: F(1, 22) = 3.85, p < 0.06, females: F(1, 13) = 2.12, p < 0.17; males and females: F(1, 37) = 5.73, p < 0.02; Fig. 4-7 A,B). We found no association between the latency to enter the open arm of the EPM in adulthood and adult Sert expression (Appendix O).

135 121 A SERT mrna Expression 2 -ΔΔCt B Sprague-Dawley Males FAST SLOW Latency to approach social novelty in adulthood SERT mrna Expression 2 -ΔΔCt Sprague-Dawley Females FAST SLOW Latency to approach social novelty in adulthood Figure 4-7 A,B. ANOVA of rat adult response to social novelty and adult Sert expression. A) Sprague-Dawley males; B) Sprague-Dawley females. +/- s.e.m.

136 122 In Agouti viable yellow mice, we found a similar relationship between exploratory behavior on the novel social arena and Sert expression, however this relationship was only evident in males. Males that were slow to approach social novelty in adulthood expressed less Sert in adulthood than their littermates that were faster to approach novelty (novel social: males: F(2, 10) = 6.49, p < ; female: F(2, 9) < 1, p < 0.14; males and females: F(2, 22) = < 1, p < 0.26; Fig.4-7 C, D; Appendix O). We found no association between latency to approach a novel object in adulthood nor the latency to enter the open arm of the EPM in adulthood and adult Sert expression in the Agouti mice (Appendix O).

137 C 123 SERT mrna expression 2 - Ct Agouti Males ** FAST SLOW Latency to approach social novelty in adulthood D SERT mrna expression 2 - Ct Agouti Females FAST SLOW Latency to approach social novelty in adulthood Figure 4-7 C,D. ANOVA of adult mouse response to social novelty and adult Sert expression. C) Agouti viable yellow males; D) Agouti viable yellow females. +/- s.e.m. (** p < 0.01).

138 124 Rodent exploratory behavior in adulthood predicts adult GR but not Crhr1 expression Offspring exploratory behavior on the novelty arenas and elevated plus maze was associated with adult hippocampal GR, but not Crhr1 expression, and this relationship was driven by the females. Females that were more exploratory on both novelty arenas expressed less hippocampal GR in adulthood, although this relationship appears to be driven by one litter (Fig. 4-8; Appendix P). Additionally, females that were slow to approach the open arm of the EPM expressed less hippocampal GR mrna than their more exploratory littermates (Fig. 4-9; Appendix P).

139 125 hippocampal GR mrna Expression 2 -ΔΔCt A Sprague-Dawley Males 2.0 Litter A Litter B Litter C Litter D Mean latency to approach novelty in adulthood (s) hippocampal GR mrna Expression 2 -ΔΔCt B Sprague-Dawley Females 2.0 Litter A Litter B Litter C Litter D Mean latency to approach novelty in adulthood (s) Figure 4-8. Linear regression of adult mean response to novelty and adult hippocampal GR mrna expression. A) Sprague-Dawley males; B) Sprague-Dawley females.

140 126 hippocampal GR mrna Expression 2 -ΔΔCt A Sprague-Dawley Males Litter A Litter B Litter C Latency to open arm EPM in adulthood (s) hippocampal GR mrna Expression 2 -ΔΔCt B Sprague-Dawley Females Litter A Litter B Litter C Latency to open arm EPM in adulthood (s) Figure 4-9. Linear regression of adult latency to approach the open arm of the EPM and adult hippocampal GR mrna expression. A) Sprague-Dawley males; B) Sprague-Dawley females.

141 127 Discussion We found significant relationships between behavioral and environmental measures early in life and mrna expression in adulthood of targets associated with stress and anxiety-related behavior that were species and sex-specific in some cases. Although we found no relationship between maternal licking bouts received during the first postnatal week and Sert adult mrna expression in Sprague-Dawley rats, we found a strong negative relationship between body licking bouts received and adult Sert expression in Agouti viable yellow male mice. In the rats, we found that females that received more licks than their same-sex siblings also expressed more prefrontal glucocorticoid receptors. In addition, we found that the latency to approach the open arm of the elevated plus maze in adulthood was negatively associated with hippocampal GR mrna expression in the Sprague-Dawley rats, and this finding was driven by the females. We did not find a relationship between maternally-directed behaviors performed by the neonates during PND 1-8 and GR, Crhr1, and Sert mrna expression in adulthood in Sprague- Dawley rats, but observed a positive relationship between perioral contact and adult Sert mrna expression, and a negative relationship between frequency of nipple attachment during the first postnatal week and adult Sert mrna expression in Agouti viable yellow males. The behavioral measures that were the strongest predictors of adult Sert expression in both Sprague-Dawley rats and Agouti viable yellow mice were the offspring mean latency to approach social and physical novelty at post-weaning and the latency to approach social novelty in adulthood, and these negative relationships were most evident in males of both species. With several analyses conducted in this study, one caveat is that Bonferroni corrections indicate that our results may not be statistically significant. Over 300 analyses were conducted on these two rodent cohorts and about 20 were orginally found to be statistically significant

142 128 (which is about the likelihood we would expect to see simply by chance alone). The more convincing and robust findings were the negative relationships between latency to approach novelty and adult Sert mrna expression in both the male Sprague-Dawley rats and male Agouti viable yellow mice. Another caveat that must be noted is that mrna transcriptional abundance does not necessarily directly translate to protein expression levels due to post-transcriptional modifications. Future research which compares more functional analyses of Sert comparing either serotonin availability in the synapse, and/or protein levels of GR and Crhr1 receptors in HPA-related brain regions, would lead to a more accurate portrayal of mechanisms involved in the long-term physiological consequences of variance in maternal-infant interactions in vivo. In addition, we measured basal GR and Crhr1 mrna expression, rather than expression in response to a stressful situation. Others have shown that major differences in HPA measures between high- exploratory and low-exploratory animals are found in response to stress, rather than in basal levels of expression (Diorio, Viau, & Meaney, 1993; Smith et al., 1998; Pryce & Feldon, 2003; Salomé, Salchner, Viltart, Sequeira, Wigger, Landgraf, & Singewald, 2004; Tang, Akers, Reeb, Romeo, & McEwen, 2006; Enthoven, Mark, & de Kloet, 2008; Akers, Yang, DelVecchio, Reeb, Romeo, McEwen, & Tang, 2008). In the future, we would sacrifice animals immediately after exposing to novelty to examine HPA-related differences in response to mild stress instead of basal levels. Alternatively, we could examine another type of glucocorticoid receptor, known as the mineralocorticoid receptor, which is responsible for regulating basal corticosterone and has a higher affinity for corticosterone than do GRs (Joëls & DeKloet, 1994). Understanding both the environmental and physiological aspects involved in sibling variance in

143 129 response to novelty in an animal model will help to elucidate factors driving these differences and their consequences on mental health. Postnatal maternal licking and adult offspring Sert mrna expression The association between body licking received as a neonate and Agouti viable yellow Sert mrna expression in adulthood may be explained by the association between body licking and response to novelty in adulthood (Chapter 3). In this previous study, we found that the more body licks neonates received, the slower they were to approach novelty in adulthood compared to their siblings that received fewer body licks. Here, we found a strong connection between early behavioral measures, later behavioral measures, and later Sert expression in the Agouti viable yellow mice. Because we know that Agouti viable yellow mice are prone to be less exploratory than black mice (Harris, et al., 2001; Bazhan, Shevchenko, Karkaeva, Yakovleva, & Makarova, 2004), this may explain why the relationships are stronger in the Agouti viable yellow mice and not the Sprague-Dawley rats. In other words, knowing that between litters, Agouti viable yellow mice will show anxiety-like behavior a priori, we could expect variance in exploratory animals within a litter of both the yellow and black phenotypes. Also, small litters tend to stay within the huddle to keep warm, and C57BL/6 litters (the background strain of these mice) specifically have shown an increased amount of nursing and contact with the mother compared to other strains (Champagne et al., 2007; Bautista, García-Torres, Prager, Hudson, & Rödel, 2010). As mentioned in Chapter 3, body licking is thought to cool down the pups after being in a huddle or nursing, so this may explain why we see this association between body licking in the Agouti viable yellow mice and not the Sprague-Dawley rats that had many siblings

144 130 available to help with thermoregulation, but probably less of a need actively seek contact with others (Bautista et al, 2010). Maternal licking and offspring HPA mrna expression in adulthood In Sprague-Dawley rats, we found a notable sex difference in the relationship between maternal licks received during the first week of life and glucocorticoid receptor mrna expression in the prefrontal cortex in adulthood. High-licked females, within a litter, expressed more prefrontal GR mrna and secreted less CORT into circulation after exposure to novel objects than did their low-licked sisters. We did not, however, find this relationship between licking and CORT in the male rats. What is of particular interest to this study is that several of the between-litter analyses of the behavioral and physiological consequences of maternal-infant interactions done previously have primarily been conducted on male rats, but in our studies we included both sexes and found different effects based on sex (Liu et al., 1997; Plotsky et al., 2005). Our finding suggests that the high-licked females may have better negative feedback control of the HPA compared to the low-licked females, however this needs to be studied further. We also only had CORT data for females in two of the four rat litters, and no CORT or HPA mrna expression data for the mice cohort, so a larger study would be necessary to replicate and confirm these results. One hypothesis for female-specific long-term changes in HPA reactivity as a result of maternal licking could be that reduced HPA responses to novelty is important for female offspring when they become mothers to recognize and care for their own offspring. For instance, others have shown that maternal behavior passes on to offspring, (with daughters of high-licking mothers becoming high-licking mothers themselves) so this phenomenon may explain the relationship between high maternal licking and female offspring adult HPA responses

145 131 to novelty (Francis, Diorio, Liu, & Meaney, 1999). Conversely, artificially-reared rodents and rodents that were separated from the dam for long periods of time exhibit less maternal care towards their own pups (Gonzalez et al., 2001; Lovic, Gonzalez, & Fleming, 2001; Rees & Fleming, 2001). In novel situations, artificially-reared animals show hyperactivity compared to maternally-reared animals (Gonzalez & Fleming, 2002). As mentioned in Chapter 3, impulsive, highly exploratory mothers do not pay attention to pups compared to less exploratory dams (Neumann, Kromer, & Bosch, 2005; Bosch & Neumann, 2008; Lovic, Palombo, & Fleming, 2010). In fact, compared to maternally-reared animals, artificially-reared females animals spend more time investigating unfamiliar conspecifics suggesting that they may not pay attention to pups much like the impulsive rodent mothers showed (Gonzalez & Fleming, 2002). In Chapters 2 and 3, we noted that high-licked animals, within a litter, were slower to approach a novel object in adulthood compared to their high-licked littermates. Perhaps a new rodent mother responds to her novel pups quickly, but is less responsive to other types of novelty, keeping the focus on her pups (Fleming, Morgan, & Walsh, 1996). Physiologically, artificially-reared females express less c-fos, (an indicator of neuronal activity), in the medial preoptic area (an area thought to be involved in maternal behaviors) compared to maternally-reared pups (Gonzalez & Fleming, 2002). Future studies could expand on our findings, perhaps with artificial rearing experiments, to further investigate this possible sex-difference in HPA physiology as well as transgenerational effects of varying maternal care within a litter. Neonate maternally-directed behaviors and adult Sert expression We found a significant relationship between the frequency of neonate nipple attachment and adult Sert mrna expression in the male Agouti viable yellow mice. Male Agouti viable

146 132 yellow neonates were more frequently attached to the mother s nipples during the first postnatal week had less Sert mrna expression in the brainstem in adulthood compared to siblings that were less frequently attached. In Chapter 3, we found a positive association between frequency of nipple attachment during the first postnatal week and the latency to approach novelty in adulthood. In this current study, we found a negative relationship between frequency of nipple attachment and Sert expression in adulthood. We hypothesized that needy pups that require more maternal contact than their siblings may become less exploratory, or inhibited, adults (as evidenced in Chapters 2 and 3). Now, we have physiological evidence that may also support the hypothesis that needy pups that frequently seek maternal attention become low-exploratory adults. These needy pups resemble children with separation anxiety (whose parents enabled or exacerbated their anxiety based on their responses to the child s cues), who then develop anxiety as adults (Dallaire & Weinraub, 2005). Perhaps this relationship was evident in the Agouti viable yellow mice but not the Sprague-Dawley rats due to differences in litter size. With small litter sizes, maternal resources are more available and should not be as competitively sought out in the Agouti viable yellow mice compared to the large Sprague-Dawley litters (Champagne et al., 2007). Neonate maternally-directed behaviors and adult GR and Crhr1 expression In the rats, we did not find any relationships between neonate maternally-directed behaviors and adult HPA-related mrna levels. The lack of relationship between very early measures and later physiological measures within an individual suggests a putative developmental moderator that may also contribute to the mechanism, during a different developmental window. Based on our studies, it difficult to decifer whether this mechanism

147 133 underlying stress and anxiety-related physiology occurs early in utero or later in life, and further research investingating these mechanism is necessary. Measures of possible epigenetic effects of maternal licking on offspring HPA-related physiology have primarily been performed in adult animals without a clear temporal trajectory of when the effect of licking on offspring physiology occurs (Weaver et al., 2004; Weaver et al. 2003). For instance, pups of low-licking mothers showed increased methylation of the exon1 7 promoter of hippocampal GR in adulthood compared to pups of high-licking mothers suggesting a physiological mechanism for the decreased hippocampal GR expression in the pups of low-licking mothers, however we do not know how early this methylation occurs. Alternatively, physiological differences in HPA-related mrna expression may purely be maternally-driven with no contribution of early neonate behavior whereas Sert expression is more developmentally self-guided or determined very early in development. Rodent exploratory behavior at post-weaning and adult Sert expression We found a strong association between exploratory behavior on both novelty arenas at the time of post-weaning, and adult Sert mrna levels in offspring in both the Sprague-Dawley rats and Agouti viable yellow mice. As we had predicted, animals that were slow to approach novelty expressed less Sert mrna in adulthood compared to their more exploratory littermates. In previous studies, the strongest associations between Sert and vulnerability to mood disorders are found when both biology and environment are considered. For instance, the parental environment has been shown to influence offspring susceptibility. Children with the s/s SERT genotype, who also have low social support, have an increased risk for behavioral inhibition at age 14 months and 7 years (Fox et al., 2005). In addition, third trimester maternal anxiety is

148 134 associated with increased childhood anxiety in s/s children (Oberlander et al., 2010). Adult subjects who had the s/s phenotype were more likely to report perceived childhood neglect and were more likely to have high baseline CORT and ACTH levels than subjects with at least one long allele (Gerra et al., 2010). Although this polymorphism in the SERT promoter and its association with susceptibility to depression has gained popularity within the last ten years, opponents claim that the relationship is not strong when simply examining genotype and mental health related outcomes only (Caspi et al., 2003; Gillespie et al., 2005; Kendler et al., 2005; Kaufman et al., 2004; Lemogne et al., 2011). As previously mentioned, Sert levels early in life, perhaps as early as prenatally, may more accurately describe the influence of Sert function on later offspring behavior (Caspi et al., 2003). Rodent exploratory behavior at post-weaning and adult GR and Crhr1 expression We did not find any associations between offspring exploratory behavior at post-weaning and their subsequent HPA-related mrna expression in adulthood. When examining the data further, the differences in HPA-related mrna expression between littermates may not be as variant as the differences in Sert mrna expression, so we may not have enough power to show a strong relationship between early exploratory behavior and adult HPA mrna expression. Finally, Sert may already completely developed by birth, but HPA axis is more plastic, so this might also explain why these early measures are not predictive (Macrì & Würbel, 2006).

149 135 Rodent exploratory behavior in adulthood and adult Sert expression We found a strong relationship between offspring response to social novelty in adulthood and their Sert mrna expression levels in adulthood. Studies of animal models and humans have suggested a potential role of Sert on individual variance in the response to novelty. Mice with lower levels of Sert function show low exploration and low social interactions in novel situations (Holmes et al. 2002, 2003; Kalueff et al. 2007; Murphy & Lesch 2008). In humans, children who expressed the s/s SERT genotype, who also had low social support are reported to be more susceptible to behavioral inhibition at age 14 months and 7 years (Fox et al., 2005). This early behavioral inhibition has been strongly linked to social anxiety in adulthood (Biederman et al. 1990; Kagan, 1994; Reeb-Sutherland et al., 2009). In addition, autism studies also suggest that Sert is involved in social behavior. For example, Integrinβ3 interacts with Sert in the midbrain and is a marker for whole blood serotonin levels. Integrinβ3 gene knockout mice show no preference for social novelty in the 3-chambered social test compared to animals with intact Integrinβ3 (Carter et al., 2011). In humans, twins that are discordant for autism show differential SERT expression; the twin who has autism produces less SERT than the unaffected twin (Hu et al., 2006). While models of autism show more extreme cases of disrupted social behavior than in our rodents, it is interesting to note that SERT is involved in social responses to novelty and perhaps SERT may be a potential target for further examination of social avoidance behavior. Rodent exploratory behavior in adulthood and adult GR and Crhr1 expression In this study, we found a postive relationship between the latency to approach novelty in adulthood and adult basal hippocampal GR mrna expression in females. The exploratory animals that were fast to approach novelty, compared to their littermates, showed a similar

150 136 relationship with hippocampal GR expression levels as do High Responsive (HR) Sprague- Dalwey rats that are classified for their novelty-seeking behaviors compared to the Low Responsive (LR) rats (Kabbaj, et al., 2000). HR animals show higher hippocampal GR mrna levels, yet they have a prolonged CORT response in response to novelty than do LR animals (a physiological effect that we would expect to see from a less exploratory animal) (Kabbaj et al. 2000; Piazza et al. 1989; Dellu et al. 1996). Like female rats in our previous studies (Cavigelli & Michael, unpublished), HR show a higher CORT response to novelty, even though they show high levels of exploration. For these behavioral and physiological reasons, we hypothesize that the females respond to our novelty arenas as novelty-seekers, whereas the males may be avoiding harm, as evidenced by previous studies (Ray & Hansen, 2004). On the other hand, females with a long latency to approach the open arm of the EPM had lower hippocampal GR mrna expression in adulthood. These findings may indicate that the association between responses to novel situations are specific to how aversive the environment is. The novelty arenas were purposely designed to be less aversive than other behavioral tests like the EPM, which adds the factor of height (a naturally fear-provoking stimulus) into the environment. The EPM was specifically designed as a measure of anxiety-related behavior, whereas the novelty arenas measure exploratory behavior in a novel environment (Pellow Chopin, File, & Briley, 1985). Because of the different types of behaviors measured in these behavioral tests, we would not assume that similar results would be found in both. Sex differences in anxiety-related physiology We observed the strongest effects between adult Sert mrna measures and exploratory behavior in the males, yet we found the strongest HPA-associated relationships with exploratory

151 137 behavior in females. The sex differences that we observed in response to novelty support previous work showing that male and female rodents have differential responses to novel situations with females being more exploratory than males (Fernandes et al., 1999; Ray & Hansen, 2004). In humans, a longitudinal study found that behaviorally-inhibited girls show normal social development as adults, however behaviorally-inhibited boys were delayed socially (Caspi, Bem & Elder, 1989). Previous studies have shown that novelty-seeking and harmavoidance behaviors have different neurobiological mechanisms with harm-avoidance being linked to serotonergic and noradrenergic changes (Gerra et al., 2000), while novelty-seeking is associated with dopaminergic systems, specifically the DRD4*7R allele at the D4 dopaminereceptor locus (reviewed in Gerra et al; 2000: Cloninger, 1987; Bardo et al., 1996; Benjamin et al., 1996; Cloninger et al., 1996; Ebstein et al., 1996; Gelernter et al., 1997; Kotler et al., 1997; Noble et al., 1998), although this is not true for all populations (Vandenbergh et al., 1997). Harm avoidance has been linked to high levels of serotonin release from presynaptic neurons, which is congruent with the low levels of Sert mrna expression (i.e. if Sert is down-regulated, reuptake is not occuring as often, and more serotonin would be found in the synapse) (Ruegg et al., 1997). In addition, the human SERT promoter polymorphism has also be associated with harm avoidance and anxiety behavior (Mazzanti et al., 1998; Ricketts et al., 1998; Katsuragi et al., 1999). These within-litter sex differences in response to novelty and exploration should be investigated further, perhaps in the context of drug self-administration behaviors, as they are highly correlative with sensation-seeking in humans, although the sex differences are reversed in humans where males tend to be sensation-seekers rather than females (Zuckerman & Neeb, 1979; Zuckerman, 1994).

152 138 Conclusion In this study, we found physiological variance within litters that associated early-life measures with offspring adult gene expression that relate to the stress response. We found that some measures were species-dependent with the Agouti viable yellow mice showing stronger effects than the Sprague-Dawley rats. Factors like litter size and the previously-established propensity for the Agouti yellow phenotype to be less exploratory than their black littermates may be reasons why we saw differences between strains. We also found significant sex differences in sibling variance in physiological measures related to exploratory behavior. Our findings suggest that males and females may utilize different mechanisms in response to novelty with males using an indirect physiological method that effects the HPA downstream and females using a method that more directly affects the HPA. More research needs to be done to fully understand these sex differences in both behavioral and physiological responses to novel situations to understand the putative neurochemical mechanism that may drive these sex differences.

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161 147 Chapter 5 Synthesis and Conclusions In this final chapter, I will summarize the findings of the projects discussed in this dissertation, and then suggest potential future research to expand on this work. I will also highlight the potential relevance of these rodent studies to human mechanisms. The purpose of these studies was to elucidate putative early environmental and behavioral contributors to stress and anxiety-related behavior and physiology later in life, with a focus on the long-term effects of the maternal environment. Because the underlying etiology of anxiety disorders is not clearly defined, we investigated several potential predictors (i.e. maternal behavior, offspring maternal solicitation behavior, offspring response to novelty, and offspring physiological measures) that may clarify mechanisms that contribute to these mental health disorders that affect 40 million adult Americans (Kessler et al., 2005). Although we found interesting associations between early life measures and later anxiety-related behavior and physiology, our sample sizes in both the rats and mice were quite limited, so additional studies with more litters are required. While our results shed led on some of the mechanisms between siblings differences in anxiety-related behavior and physiology, several questions related to direct causation remain unanswered. Summary of findings To determine how neonate early life interactions with their mother relate to offspring anxiety-related behavior within a litter, we assessed the relationship between rodent maternal and neonate behavior during the first week of life and behavior on novelty and anxiety-related tests at

162 148 post-weaning and adulthood. In Chapter 2, we observed mother-pup interactions during postnatal days 1-8 in four Sprague-Dawley rat litters and measured adult offspring behavioral responses to social and physical novelty. Our results indicated that pup and maternal behavior varied by at least two-fold within each litter, and that specific pup behaviors within each litter (perioral contact) were associated with increased maternal licking. Furthermore, siblings that received more licks and made more perioral contact during postnatal days 1-8 had longer latencies to approach a novel object in adulthood than siblings that received less licking and made less perioral contact. This within-litter variance in postnatal mother and pup behavior and offspring adult behavior indicates that early social dynamics within families are an important area to examine to understand the development of sibling variance. To determine if similar within litter variance exists in genetically homogenous siblings with phenotypic diversity in appearance, in Chapter 3, we conducted maternal-neonate observations similar to the observations conducted in Chapter 2, however we used inbred Agouti viable yellow mice. The highest-licked pups within each litter received approximately times as many licking bouts during the first postnatal week compared to the lowest-licked pups of the same sex. Pups that performed maternally-directed behaviors most often did so about times as frequently as the pups that displayed them the least and the number of maternallydirected behaviors a pup made was positively correlated with the number of licks it received from its mother. The highest-licked pups within a litter were slower to approach both physical and social novelty and slower to enter the open arm of the elevated plus maze than the lowestlicked pups within a litter in adulthood compared to the lowest-licked pups. Mothers did not lick pups of a specific coat color more, and neither color displayed significantly more maternallydirected behaviors. Our results indicate that within-litter variance in maternal and neonate

163 149 behavior is not specific to genetically-variable outbred rodents but that it is also present in genetically-similar inbred rodents. Because previous studies have detected pronounced behavioral differences among samesex adult rat siblings living in well-controlled laboratory environments, in Chapter 4 we compared sibling mrna expression of genes related to the stress and anxiety: glucocorticoid receptors (GR) and the serotonin transporter (Sert). This work was conducted with the Sprague- Dawley rats and Agouti viable yellow mice we studied in Chapters 2 and 3. We found sexspecific differences in GR mrna expression, where high amounts of maternal licking during the first postnatal week predicted higher GR mrna expression in the prefrontal cortex, and lower corticosterone production in response to novelty in adult females rats. In the male rats and mice, slow latencies to approach novelty at post-weaning and adulthood were associated with lower Sert mrna expression in the brainstem. These findings suggest that mechanisms underlying behavioral and physiological responses to unfamiliar stimuli are sex-specific, which may extend to the pronounced sex differences in responses to stress and anxiety in humans. Limitations For this study, several limitations should be discussed. First, we are extremely limited in sample size of both the rat and Agouti viable yellow cohorts. Because of this constraint, our power is not high because rodent siblings are not independent of one another, however our replication of both behavioral and physiological data for the rats and mice strengthens our results. Although our sex ratios for the two cohorts are balanced when the litters are combined (Sprague-Dawley rats: 33 females, 35 males; Agouti viable yellow mice: 17 females; 16 males), within each litter some were heavily female-biased while others males were male-biased.

164 150 Considering that we found sex-effects in several of our measures, a future study using more litters, may better quantify long-tem maternal and neonate effects on litters with male vs. female biases. Second, we have measures of the frequency of maternal and neonate behaviors during the first week of life, but we do not know the exact temporal onset of these behaviors. From our observations, we do not know the latency for maternal behavior onset after a neonate solicits attention, and we do not necessarily know if neonate behavior always precedes maternal behavior. We also do not know whether the animals were predisposed to be low-exploratory or high-exploratory regardless of maternal intervention or if increased maternal care exacerbated the neonate behavior. The A vy /a (yellow) mice showed lower exploratory behavior on the elevated plus maze, as was expected from previous studies, yet this behavior was not driven by high or low maternal care of this genotype. Lastly, we did not have the opportunity to run HPArelated measures on the Agouti viable yellow mice. Because these mice were part of a larger study, we did not want to introduce another stressor, such as collecting blood for CORT analyses, to these animals. While whole brains were available to us for the Sprague-Dawley rat study, the brain dissections in the Agouti viable yellow mice were conducted before the idea to analyze GR and Crfr1 mrna expression was initiated. Ideally, in a new study, we would expose the animals to novelty in adulthood (with some groups exposed to physical novelty and some groups exposed to social novelty) and then sacrifice them immediately to measure GR and Crfr1 mrna expression because we essentially measured basal mrna expression levels of elements in the HPA axis rather than expression levels in response to mild stress. Several studies that examine the relationship between the early environment and individual differences in stress hormones have shown that differences found in HPA responses is only found in response to stress and not in basal levels (Diorio, Viau, & Meaney, 1993; Pryce & Feldon, 2003; Salomé,

165 151 Salchner, Viltart, Sequeira, Wigger, Landgraf, & Singewald, 2004; Tang, Akers, Reeb, Romeo, & McEwen, 2006; Enthoven, Oitzl, Koning, van der Mark, & de Kloet, 2008; Akers, Yang, DelVecchio, Reeb, Romeo, McEwen, & Tang, 2008). Considering these limitations, it is highly recommended to run a larger study expanding on our initial methodology tol strengthen the results from the original studies. Future Directions In our studies with outbred Sprague-Dawley rats and inbred Agouti viable yellow mice, neonate maternal solicitations behaviors were positively associated with maternal licking. Although we found correlations between pup and maternal behaviors, the direction and causality of the onset of these behaviors are unknown. To understand the underlying mechanisms that drive these neonate-maternal interactions, experimental manipulations should be conducted. For example, to examine the maternal driving forces behind licking and nursing behaviors, pups could be temporarily anesthetized using a short-acting agent like isofluorane (Jans & Leon, 1983, Alberts, 2007). While the pups are anesthetized, careful observations of which pup receives maternal attention without solicitation from pups, could be conducted. Prior work has shown that if the dam s snout is muzzled or anesthetized to exclude tactile stimulation from pups, then she does not engage in licking and nursing behaviors (however pup retrieval is not affected) (Beach & Jaynes, 1956; Stern & Johnson, 1990). Other retrieval studies have measured the latency for the dam to retrieve pups when they are away from the nest (Beach & Jaynes 1956; Ressler, 1962; Rosenblatt, Mayer, & Giordano, 1988; Bridges, 1996; Rosenblatt, Olufowobi, & Siegal 1998; Champagne, Curley, Keverne, & Bateson, 2007). These studies focus on the dam s behavior, but do not track the order in which pups were retrieved. Olfactory cues signal the dam

166 152 to retrieve her own pups faster than others, and male and female pups within a litter emit specific odors that attract maternal attention, so perhaps specific pups within the litter also have specific olfactory cues (Wallace, Owen, Thiessen, 1973; Moore, 1981; Moore, 1985; Moore, Wong, Daum, & Leclair, 1997). To control for pup odors, pups can be scented with odors like lavender or lemon or even male littermate urine (Beach & Jaynes 1956; Birke & Sadler, 1987; Sullivan, Stackenwalt, Nasr, Lemon, Wilson, 1994). Lastly, pups will emit ultrasonic vocalizations (USVs) when apart from the dam to elicit her attention and eventual retrieval (Shair, Masmela, Brunelli, & Hofer, 1997; Barron, Segar Yahr, Baseheart, & Willford, 2000; D Amato, Scalera, Sarli, Moles, 2005). Given all these factors that are involved in maternal behavior, future studies could involve briefly removing pups from the nest, recording each individual s USVs, followed by placing them on the opposite end of the cage, and recording retrieval order. During observations neonate solicitation behaviors towards the mother as well as maternal licking could be recorded. In addition, a second cohort of pups could be artificially-scented, so the dam count not use odor as a distinguishing cue. After the early postpartum period, the offspring would undergo behavioral studies in adulthood to measure their latency to approach novelty. We would hypothesize that the pups that were needy during the unmanipulated observations may be the pups first retrieved. On the other hand, the dam may select the strongest pups first. If in a stressful situation, the dam may select the pups that have the best chances of surviving to increase her reproductive fitness in case not all pups will be able to be retrieved (Gottlander, 1987). The results from this type of experiment would give an additional measure of maternal behavior that may explain how the division of maternal attention is influenced and if odor is the salient cue for differential pup retrieval by mother.

167 153 Another way to understand the role of maternal licking on later offspring behavior would be to conduct artificial licking experiments. In the Sprague-Dawley rat study, after postnatal day 4, males began to receive more maternal licking bouts than their female siblings, which was similar to what others had shown in rats and gerbils (Birke & Sadler, 1987; Moore & Morrelli, 1979; Richmond & Sachs, 1984; Clark & Galef, 1989). In one study, artificially-reared females who received surrogate licking via a paintbrush for 45 seconds, twice daily displayed more maternal behaviors like licking and retrieving pups compared to artificially-reared females that did not receive paintbrush stimulation, suggesting that this tactile stimulation was sufficient to stimulate mechanisms supporting future maternal behavior (Gonzalez, Lovic, Ward, Wainwright, & Fleming, 2001). Using a paintbrush to replace maternal licking, pups could be artificiallyreared and then all receive equal amounts of artificial licking stimulation. Their identities would then be maintained into adulthood and their behavior tested. If maternal behavior accentuates or drives sibling variance, then in those pups that received equal licking stimulation, sibling variance in response to novelty should be reduced compared to unmanipulated pups. In the current study, we recorded the frequency with which pups probed the dam s ventrum and the frequency that pups were attached to a nipple to nurse. These rooting behaviors signal the mother to become quiescent and assume a nursing position (Kenyon, Keeble, & Cronin, 1982; Stern & Johnson, 1990). In our studies, we hypothesized that the needy pups may be the ones that are frequently seeking maternal contact. One related question to answer would be How long does it take a pup to successfully attach to a nipple? In our study, the pups that were more frequently attached to a nipple than their littermates (at 3 minute time points) received more licks, but we do not know how long it took them to attach and if they were consistently attached. To answer this question, the latency for a pup to attach would be

168 154 measured. Placing the dam in a supine position while temporarily anesthetized, pups would be removed from the nest and then reintroduced individually (Henderson, 1989). The latency to attach to a nipple, without assistance from the dam, would then be recorded. Specifically, the stronger ones would be able to attach more quickly and stay on more successfully than the weaker pups that may get pushed aside by their littermates. These results may indicate which pups within in the litter are stronger or weaker and could provide more evidence to explain why within-litter variance in maternal care exists. In other words, maternal behavior may need to vary to accommodate different offspring needs. Another way to characterize pups as needy or strong would be to characterize huddling behavior (Alberts, 2007; Bautista, García-Torres, Prager, Hudson, & Rödel, 2010). For example, it may be the case that some pups are always on top of the nest or buried underneath or always moving about pushing siblings aside to thermoregulate. More needy pups may end up on top of the litter unable to stay warm and thus requiring more maternal contact to stay warm. Experimenters could interfere and place pups in certain positions in the huddle, to determine influence of pup location in the huddle on maternal licking. On the other hand, there is evidence that rodent dams body lick pups to cool them off after being at the bottom of a huddle or underneath her (Sullivan, Shokrai, & Leon, 1988). Examining huddle behavior will provide more information about the early postnatal period so that we can understand not only pupmaternal interactions but pup-pup interactions, as well. Uterine positions during gestation can affect mechanisms that influence later adult behavior of developing rodent offspring. For example, if a female embryo develops between two male embryos, then she will display more aggressive behaviors than a female that was not positioned between two males because of increased exposure to testosterone (Vom Saal & Dhar,

169 ; Clark & Galef, 1995; Clark, Karpiuk, & Galef, 1993). Female gerbils that developed prenatally between two males also received more maternal licks compared to females adjacent to one or no males (Clark, Bone, & Galef, 1989). Without having to surgically remove fetuses at birth, anogenital distance can be used as an indicator of uterine position (Vandenbergh & Huggett, 1995). A future study could measure the anogenital distances of the pups to examine whether the differences in maternal licking within a litter, within sex is associated with testosterone exposure in utero. We would hypothesize that the highest-licked females within a litter would have the longest anogenital distance indicating that they developed between two male fetuses and thus being more masculine. Conversely, the lowest-licked males may be more feminized and developed between two females. Examination of the uterine environment may identify early, intrinsic contributors to within-litter differences in maternal care. Although our observations of maternal and pup behavior during the first postnatal week were very detailed, we do not know the exact temporal patterns involved in these behaviors. Because of this limitation, this question remains: What comes first, the neonate behaviors or the maternal? If a pup makes a maternal solicitation behavior such as snout contact, how long does it take for the dam to respond to a pup s cues? Knowing the order of these events may provide additional information as to whether a pup drives the maternal behavior, or if it is maternallydecided. As mentioned in previous chapters, maternal behavior has transgenerational effects on offspring. For instance, daughters of high-licking mothers, as a whole, become high-licking mothers to their subsequent offspring (Francis, Diorio, Liu, & Meaney, 1999). In addition, rodents that have experienced prolonged maternal separation exhibit less maternal care on their own pups (Gonzalez et al., 2001; Lovic et al., 2001; Rees and Fleming, 2001). Maternal

170 156 stimulation experiments, such as those mentioned earlier that use a paintbrush to stimulate the pups, could help to rescue the long term-effects of low maternal care on females. The findings presented here expand on the general knowledge of the development of rodent behavior in two different species, but can also be applied to understanding maternal behavior in other mammalian species, including humans. Mother-infant contact typically occurs immediately after birth in most mammals and is important in establishing secure attachment in developing offspring (Seay & Harlow, 1965; Harlow, Dodsworth, & Harlow, 1965, Klaus & Kennell 1970; Kennell et al., 1974; Oppenheim, Koren-Karie, & Sagi-Schwartz, 2007). Pre-term and high-risk infants must undergo immediate maternal separation that disrupts early maternal contact, which can later affect maternal behaviors as well as the child s emotional development (Klaus & Kennell 1970; Wijnroks, 1999; Feeley, Gottlieb, & Zelkowitz, 2005; Hill, Aldag, Demirtas, Zinaman, & Chatterton, 2006). Increasing maternal-infant physical contact is not only beneficial for the infant, but can also soothe anxiety and depression symptoms in the mother partially due to the release of oxytocin and endogenous opioids (Nissen, Lilja, Widstrom, & Uvnas-Moberg, 1995; Peláez-Nogueras, Field, Hossain, & Pickens, 1996; Matthiesen, Ransjo- Arvidson, Nissen, & Uvnas-Moberg, 2001). Perhaps a moderate amount of positive contact with the infant that is maternally-determined (i.e. not responding to every cry immediately), may be what drives the benefits of maternal-infant contact. For instance, mothers with depression tend to contact their infants too much and in a more intrusive, rough manner (Malphurs et al, 1996; Fergus, Schmidt & Pickens, 1998; Wijnroks, 1999; Ferber, Feldman & Makhoul, 2008). Also, the most consistent behavioral predictor of child anxiety from the parents is overprotection and overcontrol (Krohne & Gutenberg, 1990; Hudson & Rapee, 2001; Kiel & Buss, 2010). Although researchers have some clues to the physiological and behavioral advantages of maternal contact

171 157 early in infant development, the optimal amount of maternal tactile stimulation necessary as well as the exact mechanisms have not been determined. Not all studies have reported that high amounts of tactile stimulation early in life are beneficial for offspring. Rats with hippocampal lesions induced at postnatal day 7 correlated with high levels of nursing, which was then associated with high locomotor activity after amphetamine administration in adulthood (Wood, Marcott, Quirion, & Srivastava, 2001). Another study suggested that consistency and reliability of maternal licking was more important for developing offspring than quantity of licking bouts (Akers, et al., 2008). Specifically, highlicking dams were quite variable in their licking from day-to-day upon reunion with brieflyseparated pups, and their offspring experienced less CORT habituation to social novelty at 24 months of age. In premature human infants less than 32 weeks gestational age, increased stimulation from hospital procedures was associated with higher stress-associated physiological and behavioral reactivity (Als, Lawhon, Duffy, McAnulty, Gibes-Grossman, & Blickman, 1994; Long, Philip, & Lucey, 1980; Zahr & Balian, 1995; Harrison & Bodin, 2004), however in preterm infants older than 32 weeks, maternal stimulation was associated with lower physiological responses to pain and other stressful procedures (Cignacco, Hamers, Stoffel, van Lingen, Gessler, McDougall, & Nelle, 2007; Feldman, Singer, & Zagoory, 2010). In adults who reported high or low levels of maternal care based on the Parental Bonding Instrument questionnaire, cortisol responses to the Trier Social Stress Test were surprisingly much lower than those who had medium level of maternal care, yet the low care groups experienced more anxiety and depression (Engert, Efanov, Dedovic, Duchesne, Dagher, Pruessner, 2010). To explain this outcome, some researchers have suggested that the effects of low maternal care are adaptive (Boyce & Ellis 2005; Fries, Hesse, Hellhammer, & Hellhammer, 2005). The HPA axis

172 158 in offspring who receive low amounts of maternal care may be primed early in life to adapt to later stressful situations, with the HPA initially hyperresponsive and later hyporesponsive due to downregulation of this pathway. The hyporeponsive HPA axis phenomenon may explain why our low-licked animals were more exploratory than their high-licked siblings in a novel situation in adulthood. In future studies, early HPA responses to novelty rather than adult responses could be measured and may provide evidence of HPA hyperresponsive in the low-licked animals early in life compared to their littermates. Our studies with rats and mice showed a robust relationship between behavioral responses to novelty and Sert mrna expression in the brainstem in adult males. There are two additional experiments that could expand on this finding. The first experiment would measure Sert mrna expression earlier in life, perhaps as early as immediately after testing the animals on the novelty arenas at post-weaning. These results may identify an earlier physiological predictor for later anxiety-related behavior. Because we found an early behavioral predictor of Sert mrna expression in adulthood, it would be interesting to test possible therapeutic effects that a Selective Serotonin Reuptake Inhibitor (SSRI), like fluoxetine, may have on anxietyrelated behavior later in life. Animals could be exposed to novelty at post-weaning and then given a 6-week fluoxetine treatment. Although SSRIs almost immediately prevent serotonin reuptake, it can take between two to four weeks to produce anxiolytic effects. While on the fluoxetine treatment, we would test the animals as adults on our novelty arenas. We would hypothesize that animals that were slow to approach novelty at post-weaning, compared to their littermates, may become more exploratory as adults through this treatment. Studies using human populations have found mixed results regarding the SERT polymorphism and mental health-related traits. For instance, neuroticism was associated with

173 159 the short alleles of the 5-HTTLPR in a male population only (Du, Bakish, & Hrdina, 2000). However, when a larger study examined this polymorphism in Caucasian females all born during the same year, four of five anxiety-related scales indicated a positive association between the short allele and anxiety (Lang, Undine, Bajbouj, Wernicke, Rommelspacher, Danker-Hopfe, & Gallinat, 2004). East Asians have higher short allele frequency, but lower depression rates, suggesting a role that culture might play on depression risk (Goldman, Glei, Lin, & Weinstein, 2010). With such differences in results regarding SERT and mental health disorder risk, it is apparent that studies using human populations must carefully control for variables like age, gender, and ethnicity.

174 160 Females Males Pre-adult phenotypes and early-life experiences Adult phenotype Pre-adult phenotypes and early-life experiences Adult phenotype Maternal solicitation GR expression Maternal solicitation Agouti Agouti Sert expression Maternal care CORT secretion Maternal care Post-weaning/Adolescent Latency to novelty Anxiety-like behavior & Latency to novelty Post-weaning/Adolescent Latency to novelty Anxiety-like behavior & Latency to novelty Environment Environment Physiology Physiology Offspring Behavior Offspring Behavior Positive correlation Positive correlation Negative correlation Negative correlation Figure 5-1. Modified Conceptual Model

175 161 Conclusions The results from this dissertation help to clarify the relationship that early life experiences have on developing offspring. It is important for future studies that examine individual differences in behavior and physiology related to anxiety and exploration to consider not only between litter or between family effects, but also to look within those groups. As we have shown, not all siblings experience the same early-life environment and these differences in experiences may be related to sibling variance later in life. It is also imperative to examine the long-term effects that the maternal environment can have on adult offspring behavior and stress reactivity to unfamiliar situations. Our findings that associate the maternal environment, early and adult behavioral responses to novelty, and adult Sert and GR mrna expression provide possible contributors to sibling differences in anxiety-related behavior and physiology but additional research is necessary to understand the exact mechanisms behind these sibling differences.

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182 168 Appendix A IACUC Approval of Protocol #25780 Section of the protocol pertaining to the present maternal behavior study: Maternal & Neonate Behavior Day of birth will be defined as Day 0 of life. Behavioral observations will begin on Day 1. On this day, mothers will be momentarily removed from their cages and each neonate tattooed with an individually distinct mark on both flanks. During initial use of this method, we will also mark pup with an individually specific mark using a non-toxic Sharpie pen to ensure individual pup identity is maintained. Pups from the center and the periphery of the nest will be selected alternately for sequential marking. For this marking procedure, each neonate will be handled for 1.5 minutes. During this time, we will also measure neonate weight, anogenital distance, and ultrasonic vocalizations during a 15-second interval. After all pups are marked, mother will be returned to the cage and the first behavioral observation will begin. Six 30-min observations will be conducted daily for the first 7 days of life. We will record maternal grooming (both body and anogenital licking), a behavior known to differ between families and associated with adult offspring behavior and stress physiology. Focal animal sampling will be used to record all grooming bouts and the identity of neonate recipients of each bout. We will also record active attention-seeking behavior in the neonates: maternal snout contact (which elicits maternal behavior), movement, and relative location within the litter at specified time intervals. 'Novel Object Behavioral Test' (at days and 2.5 months): This test examines a rat's willingness to explore a novel complex arena. Rats are tested individually during their active period - i.e. during the red-light phase of the light cycle. They are carried from their home cage, placed in the testing arena and their behavior video-recorded for 5 minutes while in the arena. They are returned to their home cage after the 5 minute test. The arena consists of a 122 x 122 cm enclosure with 46 cm walls, a Plexiglas cover, wood chip bedding on the floor, and four rat-sized objects placed 13 cm from each corner. Objects include a plexiglass tunnel, a metal food hopper, a ceramic bowl, a metal tunnel, a brick and a rock, all items that are regularly cleaned in the cage washer. The test arena is made of Plexiglas. When in use, the arenas are wiped down on a daily basis using a solution of Quatricide PV15 (1/2 ounce in 1 gallon water) and allowed to air dry. To ensure novelty of the experience at each trial, objects are replaced with new ones for repeat testing. The test arena will be in a room near the animal colony room, maintained at the same temperature and on the same lighting schedule as the colony room. The arena is indirectly illuminated with a 90-watt red light bulb reflected off the walls of the room. At testing, rats are placed in a body-surrounding ('safe') bowl, thoroughly rinsed with hot water and allowed to air dry. Behavioral video tapes are coded after all rats have been tested. The frequency and total duration of the following behavior is recorded: 'locomotion' (squares entered) and 'inspection' (nose on, touching, climbing on or into an object). Defecation, rearing and latency to leave the home bowl are also scored for comparison with similar behavioral tests conducted by other researchers. It should be noted that defecation, a classic index of anxiety in animals, is rarely seen in this testing arena.

183 'Novel Social Behavioral Test' (at days and 2.5 months): This test is similar to the Novel Object Test described above, but in this case, the arena contains a novel social partner. The testing arena and protocol are similar as those used above, except in this case, the arena contains two wire cages in opposite corners one with a novel social partner, the other empty. In this way, the test animal can approach the novel stimulus animal at its own pace, can have limited physical contact with the novel stimulus rat, but aggressive interactions cannot lead to injury. Test animals stay in the arena for 5 minutes during which time their behavior is video-recorded. Locomotion and latency to approach the novel social partner are coded from video recordings. 169

184 170 Appendix B IACUC Approval of Protocol #29898 Section of the protocol pertaining to the present maternal behavior study: Maternal and neonate behaviors: Day of birth will be defined as Day 0 of life. Behavioral observations will begin on Day 1. On this day, mothers will be momentarily removed from their cages and each neonate marked with an individually specific mark using a non-toxic Sharpie pen to ensure individual pup identity is maintained. Pups from the center and the periphery of the nest will be selected alternately for sequential marking. For this marking procedure, each neonate will be handled for 90 seconds. During this time, we will also measure neonate weight, and anogenital distance. After all pups are marked the mother will be returned to the cage and the first behavioral observation will begin. Six, 30-min observations will be conducted daily for the first 7 days of life. We will record maternal grooming (both body and anogenital licking), a behavior known to differ between families and associated with adult offspring behavior and stress physiology. Litters will be observed as a whole to record all grooming bouts and the identity of neonate recipients of each bout. We will also record active attention-seeking behavior in the neonates: maternal snout contact (which elicits maternal behavior), movement, and relative location within the litter at specified time intervals. Quantification of mothers as to the degree of arched-back nursing, licking, and grooming of their pups will be made by videotape recording. This measure only requires positioning of the cages on their racks to maximize visibility when watching the videotape.(the Cavigelli group will perform this data collection and train the Vandenbergh lab). Novel object behavioral test (at PND days and 2.5 months): This test examines a mouse's willingness to explore a novel complex arena. Mice are tested individually during their active period - i.e. during the red-light phase of the light cycle. They are carried from their home cage, placed in the testing arena and their behavior video-recorded for 5 minutes while in the arena. They are returned to their home cage after the 5-minute test. The arena consists of a 122 x 122 cm enclosure with 46 cm walls, a Plexiglas cover, wood chip bedding on the floor, and four rat-sized objects placed 13 cm from each corner. Objects include a plexiglass tunnel, a metal food hopper, a ceramic bowl, a metal tunnel, a brick and a rock, all items that are regularly cleaned in the cage washer. The test arena is made of Plexiglas. When in use, the arenas are wiped down on a daily basis using a solution of Quatricide PV15 (1/2 ounce in 1 gallon water) and allowed to air dry. To ensure novelty of the experience at each trial, objects are replaced with new ones for repeat testing. The test arena will be in a room near the animal colony room, maintained at the same temperature and on the same lighting schedule as the colony room. The arena is indirectly illuminated with a 90-watt red light bulb reflected off the walls of the room. At testing, mice are placed in a body-surrounding ( safe ) bowl, thoroughly rinsed with hot water and allowed to air dry. Behavioral videotapes are coded after all mice have been tested. The frequency and total duration of the following behavior is recorded: 'locomotion' (squares entered) and 'inspection' (nose on, touching, climbing on or into an object). Defecation, rearing and latency to leave the home bowl are also scored for comparison with similar behavioral tests conducted by other researchers. It should be noted that defecation, a classic index of anxiety in animals, is rarely seen in this testing arena. (Dr. Cavigelli and group will perform this data collection and train the Vandenbergh laboratory).

185 171 Appendix C Maternal/Pup Behavioral Observations Protocol and Descriptions of Behaviors Maternal / Pup Behavior Observations Protocol: S.A. Cavigelli, PSU Behavioral Neuroendocrinology Laboratory 1. Make sure cameras around cage is in proper positions (one facing into cage on each side of cage) and correctly focused. 2. One person is in charge of calling events and time, and one is the scribe to record all the information. 3. Record all maternal behavior as it happens (this is called focal sampling you are focusing on mother only), identifying time that each new behavior begins. You do not need to record the time licks and carries occur they happen too quickly. 4. Each row in the data sheet is for a new behavior. Only one behavior per row. a. Pup lick, carry, ventral probing, and snout contact are coded by recording the pup number in the column in the next available row. Write an A next to the pup ID when pup was licked in anogenital area, body lick is B. If the mom licks a pup for more than 3 seconds, mark a star next to the pup number. b. Nursing and contact is coded as a duration. Record a starting and ending time and connect them with an arrow. c. Nest building, eat/drink, self groom, and misc. can be recorded as durations or time points. If a behavior lasts more than 3 seconds. Otherwise record a time and underline it. 5. Every 3 minutes, record pups behavior. Record all pup numbers that you can see in the peripheral column, unless they are not in contact with the huddle, in which case record them in the away column. (This will give us a sense of whether a pup was at the center or periphery of the huddle.) Then circle the numbers of the pups that are squirming or moving. Underline pups that are in easy licking distance from mom. This is a semi-circle around her snout. 6. End observation at 30 minutes (60 minutes for mouse protocol).

186 172 Appendix D Exploration Arena Testing Protocol Testing rats on Exploration Arena S.A. Cavigelli, PSU Behavioral Neuroendocrinology Laboratory Test during dark phase of the light cycle. Illuminate test room with red light bulb(s) only, deflecting them so they do not cast a glare on the arena cover. Sprinkle the field with several handfuls of bedding collected from each rat cage in the test animal s colony room. Center test arena under camera. For Novel Object testing, place 3 novel objects in each corner, far enough away from the walls so that the rat may pass easily around all sides of the novel object. For Novel Social testing, place two wire cages in the corners closest to the start bowl. There is nothing to place in the fourth corner furthest from the start bowl. In one of the two cages, place another rat from the colony, being careful to control for estrous cycle if testing females (i.e. use a female in the same phase as test subjects), and control for body weight (use a subject that is close to the body weight of the test subjects. Be sure that the cage with the novel social partner is securely closed/latched! Always double check that you have enough space on DVD before starting new day of recordings. On data sheet, record amount of light in center and edge of arena (using light meter and recording lux), room temperature, date, time, cohort, and initialize. Rats are tested one at a time for 5 minutes each. Begin video recording before placing animal in arena. (Press REC on DVD recorder.) IMPORTANT: Make sure DVD light is illuminated on DVD recorder. Transport rat from colony room to testing arena using the familiar bowl in their home cage. Make sure to cover rat if traveling through areas with white light. Place rat & bowl into side-turned clear cage and place the rat, bowl and cage into the free corner in arena. Make sure the opening of the side-turned cage faces the arena corner. Do this as gently as possible. A rough landing for the rat will freak them out and really affect their behavior. Set timer for 5 minutes. Code 'lines crossed' during the testing session. Count one line crossed when all four of the rat s paws cross the line. Record the latency (seconds) required to first investigate a novel object or the latency to investigate the novel social partner. Finally, record each time the rat s nose touches

187 173 novel objects, places both paws on object, and climbs on top of objects. Code the number of times the animal rears as well. After an animal has been in arena for 5 minutes, turn off video recording, remove animal from arena and return to home cage. Turn lights on in testing room and count and remove fecal samples and record number on data sheet. Check for urine in test bowl and record whether there was urine or not (Y or N) on data sheet. Rinse larger bowl with tap water and dry for next rat. At the end of the day, burn data onto DVD and label DVD with date, test name, and animal IDs.

188 174 Appendix E Elevated Plus Maze Protocol Who Two experimenters are ideal for performing an EPM testing - one scribe o before testing begins record on data sheet: date (don t forget to put the year!) time (military time: 8:00pm = 20:00; 9:00pm = 21:00; etc.) temperature of testing room experimenter initials o when testing begins record on data sheet: animal ID (also indicate animal sex next to ID using for female and for male) time start (time animal is retrieved) time in (time animal is placed on maze) o o record behaviors and animal location for 5 minutes when testing ends indicate if animal urinated (i.e. yes/no) indicate number of fecal boli animal left behind on maze - one shout out behaviors/directionality o o o o assist scribe with transitioning from animal to animal clean EPM with 70% ETOH, 30% H 2 O always wipe down EPM before beginning testing wipe down EPM after each animal is tested return animal tested back to home cage if testing a cage of more than one animal, place tested animal in a clean new cage temporarily until whole cage has been tested retrieve next animal on testing list maintain attentive visual on animal on monitor and verbalize animals behavior and location for scribe to record sweep up testing room and hallway when finished testing leave no bedding behind on floors remember to shut off monitor and DVD recorder at end of testing day

189 175 What Elevated Plus-Maze - elevated maze is approximately 2 feet from ground - has four arms (two open arms perpendicular with two closed arms) o closed arms = arms enclosed by high walls (Fig.1) closed arms are the arms outlined in dark black Direction is up and down o open arms = arms without walls (Fig.1) open arms are the arms outlined in blue Direction is left and right o Neutral = middle of maze (Neutral is a location of the maze that is not required for you to code during testing. However, neutral will become important during behavioral coding from the DVD on The Observer) Why The EPM is a method of assessing anxiety-like behaviors - types of behaviors o escape behaviors these behaviors may also reveal a form of anxiety; discomfort with being in a new environment rearing animal stands on hind legs with fore paws in the air (or against wall) and nose is usually pointing upwards towards the ceiling jumping off arm when animal launches from the arms of the EPM (rarely occurs, but never say never) o anxiety-like behaviors when animal is fearful, nervous, or uncomfortable with environment it will engage in repetitive self-conscious behaviors freeze animal ceases all muscle movement (like a deer in headlights) sitting still when animal remains in one location for 2 seconds or more, slight head movements may occur grooming when animal brings fore paws to face in repetitive motion, or when animal turns to groom back. The animal may also use its hind legs to groom its back o ethological behaviors head dip animal peers over edge of arm, essentially dipping its head over the edge to investigate the side of the arm stretch attend animal engages in a forward stretch/lunge where the hind legs remain planted in place (like a cat stretching its back) sway animal remains in one location and rocks body side to side, almost like it is in an hypnotic trance (this could be animals technique for honing their visual acuity)

190 176 Appendix F Pairwise comparisons of sex or genotype and licks received during the first half, second half, or total first postnatal week maternal behavior sex genotype PND 2-4 body licks t 3 = 0.52, p < 0.63 t 3 = 0.08, p < 0.94 anogenital licks t 3 = 1.80, p < 0.17 t 3 = 0.30, p < 0.70 PND 6-8 body licks t 3 = 0.51, p < 0.65 t 3 = 0.76, p < 0.50 anogenital licks t 3 = 1.43, p < 0.25 t 3 = 2.52, p < 0.08 PND 2-8 body and anogenital licks t 3 = 0.08, p < 0.94 t 3 = 0.21, p < 0.84

191 177 Appendix G Pearson correlations of the relationship between neonate maternally-directed behaviors and maternal licking during the first postnatal week neonate behavior maternal behavior body licks anogenital licks body and anogenital licks location r = 0.12, p < 0.50 r = 0.59, p < r = 0.42, p < 0.01 ventral probing r = 0.26, p < 0.14 r = 0.39, p < 0.02 r = 0.16, p < 0.37 nipple attachment r = 0.10, p < 0.58 r = 0.34, p < 0.05 r = 0.27, p < 0.13

192 178 Appendix H Linear regressions of the relationship between neonate maternal licking and maternallydirected behaviors and during the first postnatal week, and offspring exploratory behavior in adulthood. adult exploratory behavior Mean latency to novelty EPM maternal behavior body licks r = 0.59, p < r = 0.04, p < 0.85 anogenital licks r = 0.16, p < 0.41 r = 0.47, p < body and anogenital licks r = 0.37, p < 0.05 r = 0.27, p < 0.12 neonate behavior location r = 0.14, p < 0.45 r = 0.32, p < 0.06 ventral probing r = 0.04, p < 0.82 r = 0.30, p < 0.09 nipple attachment r = 0.36, p < 0.06 r = 0.38, p < 0.03 genotype t 3 = 1.64, p < 0.20 t 3 = 4.13, p < 0.03

193 179 Appendix I SERT mrna expression Protocol Brain tissue preservation. At sacrifice, whole rat brains were flash-frozen in ice-cold isopentane then stored at -80 C. For RNA isolation, brainstems were dissected and placed in RNAlater ice (Ambion/ABI, Austin, TX) and stored at -80 C. RNA was isolated using the RNeasy Lipid Tissue midi kit (Qiagen, Germantown, MD). RNA concentration was measured using a Nanodrop apparatus (Thermo Scientific; Nucleic Acid Facility, PSU), and then RNA quality was detected using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA; Nucleic Acid Facility, PSU). RNA Isolation using RNeasy Lipid Tissue Minikits Ref: RNeasy Lipid Tissue Handbook, September 2006 (Qiagen) RNeasy Lipid Tissue Minikit, Qiagen, Catalog# Label tubes with sample number, date and brain region a. 1 x conical bottom eppendorf to homogenize per sample b. 1 x conical bottom eppendorf to collect RNA layer per sample c. 1 x Mini spin column per sample d. 1 x PCR tubes to read samples on NanoDrop per sample e. 1 x 1.5ml collection tube per sample 2. Prepare solutions and necessary items a. Add 4 volumes of 100% ethanol to Buffer RPE concentrate Be sure so mark the volume level on the bottle so that if evaporation occurs, more ethanol can be added b. Make 70% ethanol c. Autoclave mini homogenizers 3. Estimate tissue weight keeping brain samples on dry ice 4. Homogenize tissue with mini hand held homogenizers initially with 150μL Qiazol (from Dr. Vandenbergh, autoclaved) 5. Add 850μL Qiazol to adjust to 1ml Qiazol + homogenate 6. Let homogenate sit for 5 min 7. Add 200μL chloroform to homogenate and shake vigorously for 15 sec, let sit 2-3 min (done under a fume hood) 8. Centrifuge 12000x g for 15 min at 4C 9. Carefully transfer upper aq phase to 2 ml collection tube (Store bottom layer (DNA) samples at -80C for Dr. Vandenbergh s lab) 10. Add 1 volume of 70% ethanol and mix by vortexing 11. Transfer up to 700μL to RNeasy Mini spin column placed in a 2 ml collection tube 12. Centrifuge >10000rpm for 15 sec, discard flow through and repeat until all has passed through column. 13. Add 700μL Buffer RW1 to column and centrifuge >8000 for 15 sec and discard flow through (Be sure column does not contact flow through)

194 14. Add 500μL Buffer RPE to column and centrifuge 8000rpm for 15 sec. Discard flow through 15. Add 500μL Buffer RPE to column and centrifuge 8000rpm for 2 min and discard flow through 16. Place column in new 2ml collection tube and centrifuge full speed 1 min 17. Place column in 1.5 ml collection tube and add 40μL RNase-free water directly to spin column membrane and centrifuge 8000rpm for 1 min 18. Repeat step 17 and combine flow through 19. Remove 5μL of sample to read concentration on NanoDrop in Nucleic Acid Facility 20. Place samples in -20C 180 cdna preparation. 500ng of RNA was transcribed into cdna in 20 ul reactions, using the High Capacity cdna Reverse Transcription Kit (ABI, Foster City, CA) was performed. The 3-step reaction was as follows: 25 C for 10 min., 37 C for 120 min., 85 C for 5 min. Quantitative Polymerase Chain Reaction (qpcr). To quantify mrna expression, quantitative polymerase chain reactions (qpcr) were conducted. Reactions were prepared in triplicate 10-µl reactions in 96-well plates using the Step One Plus Real-Time PCR machine (ABI, Foster City, CA). Taqman Gene Expression Assay primers and probes established and optimized for SERT were used (ABI, assay #: Rn _m1; Gene ID: 25553) and to normalize relative SERT mrna expression, beta actin was used as an endogenous control (ABI, Foster City, CA assay Rn _m1; Gene ID: 81822). For data analysis, the Sequence Detection System software (ABI, Foster City, CA ) was used. Relative quantification of product was determined using the 2 -ΔΔCt method with fold change relative to the median same-sex littermate for each litter.

195 181 Appendix J Glucocorticoid and Crhr1 mrna expression Protocol Adapted from Sigma-Aldrich packet (St. Louis, MO) Sample Preparation 1A. Tissue: Homogenize tissue samples in TRI Reagent (1 ml per mg of tissue) in a Polytron or other appropriate homogenizer. The volume of the tissue should not exceed 10% of the volume of the TRI Reagent. The supernatant contains RNA and protein. If the sample had a high fat content, there will be a layer of fatty material on the surface of the aqueous phase that should be removed. Transfer the clear supernatant to a fresh tube and proceed with step 2. Recover the high molecular mass DNA from the pellet by following DNA Isolation, steps 2 and 3. B. After the cells have been homogenized or lysed in TRI Reagent, samples can be stored at 70 C for up to 1 month. 2. Phase Separation: To ensure complete dissociation of nucleoprotein complexes, allow samples to stand for 5 minutes at room temperature. Add 0.1 ml of 1-bromo-3-chloropropane or 0.2 ml of chloroform (see Phase Separation, notes a and b) per ml of TRI Reagent used. Cover the sample tightly, shake vigorously for 15 seconds, and allow to stand for 2 15 minutes at room temperature. Centrifuge the resulting mixture at 12,000 g for 15 minutes at 2 8 C. Centrifugation separates the mixture into 3 phases: a red organic phase (containing protein), an interphase (containing DNA), and a colorless upper aqueous phase (containing RNA). RNA Isolation 1A. Start with only 400 ul of trireagent, homogenize for 30 seconds then add 600 ul. (once the sample is in trireagent, it can be stored in wet ice) 2. Phase Separation: use 0.2 ml of Chloroform 1. Add 750 ul of 75% EtOH, centrifuge for 5 minutes at 12,000g and 4 degrees Celsius. Repeat two more time with 100% EtOH. Be careful not to lose your pellet. 2. Use Nanopure water. Resuspend the pellets in 20 ul of nanopure water. More water can be added later. Freeze the samples until you spec them.

196 Appendix K RNA gels to detect RNA quality 182